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  the Department of Infectious Diseases First Affiliated Hospital School of Medicine Zhejiang University


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  Protease Inhibitor Cocktail III Calbiochem San Diego USA and Benzonase 25 U ml Novagen Madison USA After 30 min incubation on ice


id: cord-254384-mwzz1db5
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  Peking Union Medical College PUMC Chinese Academy of Medical Sciences CAMS Peking Union Medical College Chinese Academy of Medical Sciences


id: cord-263469-2w26l80a
type: CHEMICAL
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  CAA61498 Cytorhabdovirus lettuce necrosis yellows virus glycoprotein LYP425091 Nucleorhabdovirus rice yellow stunt


id: cord-034467-jh9msz1c
type: ORG
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  the Departments of Transport and Main Roads and Environment and Heritage Protection of the Queensland Government


id: cord-031316-yvid6qps
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  cord 031316 yvid6qps authors Bisimwa Patrick N Ongus Juliette R Tiambo Christian K Machuka Eunice M Bisimwa Espoir B Steinaa


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  MagNa Pure LC Total Nucleic Acid Isolation Kit Roche Diagnostics Basel Switzerland The different gene segments


id: cord-048466-fj9l8che
type: DNA
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  disease associated HLA DQ alpha 1 0501 beta 1 0201 molecule HLA B 0702 transgenic H 2KbDb doubleknockout mice


id: cord-048466-fj9l8che
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  influenza A H3N2 viruses isolated worldwide from 1994 to 2005 a cause for concern Molecular basis of resistance of influenza A viruses


id: cord-261579-f0prnpsu
type: CHEMICAL
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  N linked oligosaccharide chains on the processing cell surface expression and function of the measles virus fusion protein Importance of hemagglutinin glycosylation


id: cord-269277-x5c5ogo7
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  Human embryonic kidney cells 293 T human cervical carcinoma cells HeLa human lung epithelial cells A549


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  UPR Plasmids psg5 EBNA2 psg 5 EBNA3C 11 992 psg 5 EBNA3C 11 992 509 DVIEVID 515 509 AVIAVIA 515 M2 and reporter plasmids 512 72 LMP 1 Luc


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  the Ethics and Protocol Review Committee of the College of Health Sciences University of Ghana Approval


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  HIV EuroCoord CHAIN joint project a European multicohort study Report on the global epidemic The epidemiology of antiretroviral drug resistance among drug naive HIV 1 infected


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  B cell marker CD79 mouse monoclonal antibodies clone HM57 M7051 Dako diluted 1 in 500 and vimentin mouse monoclonal antibodies


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  feline leukemia virus proviral and viral RNA loads in leukocyte subsets Quantitation of feline leukaemia virus viral and proviral loads


id: cord-273179-bpnak9ov
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  human coronaviruses HCo 30 7 HCo OC43 23 HCo NL63 7 7 human parainfluenza virus type 3 PIV3 15 4 and human bocavirus HBoV 7 7


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  Beijing Friendship Hospital China Capital Institute of Pediatrics China and Institute of Blood Transfusion Chinese Academy Medical Sciences


id: cord-273179-bpnak9ov
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  the Beijing Friendship Hospital China Capital Institute of Pediatrics China and Institute of Blood Transfusion Chinese Academy Medical Sciences


id: cord-273179-bpnak9ov
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  the National Major Science Technology Project for the Control and Prevention of Major Infectious Diseases of China


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  mouse anti respiratory syncytial virus fusion protein antibody Ab24011 Abcam Goat anti mouse IgG PE BD Pharmingen


id: cord-273122-w9hemznv
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  RSV infected airway epithelial cells RSVexposed monocytes and virus specific antibodies Respiratory syncytial virus infection


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  viruses PIVs 1 4 human metapneumovirus HMPV human rhinovirus HRV WU polyomavirus WUPyV respiratory syncytial virus


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  influenza A FluA and B FluB viruses parainfluenza viruses PIVs 1 4 human metapneumovirus HMPV human rhinovirus HRV WU polyomavirus WUPyV respiratory syncytial virus RSV


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type: CHEMICAL
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  3 R potato virus M 5 CGCT TCGCTGCTTTCG 3 F and 5 CGGACCATTCATAC CACCA 3 R Marseillevirus marseillevirus 5 AAAGTC CCAAGTTATCACAAGC 3 F


id: cord-277547-2vim1wno
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  Intracellular Accumulation of Quercetin by Protection of the Chemically or Metabolically Susceptible Hydroxyl Groups


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  human rhinoviruses paramyxoviruses coronaviruses and bocavirus during acute respiratory tract infections


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  cord 284608 ba7wq52t authors Sias Catia Salichos Leonidas Lapa Daniele Del Nonno Franca Baiocchini Andrea Capobianchi Maria Rosaria Garbuglia Anna Rosa title Alpha Beta gamma human PapillomaViruses


id: cord-284608-ba7wq52t
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  MY09 5 CGTCCMARRGGAWACTGATC3 and MY11 5 GCMCAGGGWCATAAYAATGG3 20 21 PCR and FastStart DNA polymerase Roche Diagnostics GmbH Mannheim Germany


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  HIV positive and HIV negative subjects 21 21 33 63 6 vs 12 12 33 36 3 respectively Chi square test p 0 005 HIV positive


id: cord-284608-ba7wq52t
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  squamous cell carcinoma of Waldeyer s tonsillar ring Incidence of human papillomavirus HPV positive tonsillar carcinoma


id: cord-286332-cdg4im5h
type: DNA
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  NGS rabbit anti MHV polyserum k134 diluted 1 400 and chicken anti IBV H120 serum diluted 1 400 or rabbit anti MHV polyserum k134 diluted 1 400 and mouse MAb Ch IBV 26


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  cord 291961 usl8z6ep authors Zheng Wen zhi Wei Tian li Ma Fen lian Yuan Wu mei Zhang Qian Zhang Ya xin


id: cord-291961-usl8z6ep
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  RNA Transcripts of Enterovirus Species A to D Rhinovirus Species A to C and Human Parechovirus Assessment of Assay Sensitivity and Specificity of Real Time Screening and Typing Methods WU


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  subconfluent A549 BHK 21 CV 1 HEK 293 LLC MK2 MDCK MDCK London Mv1 Lu NIH 3 T3 Vero E6 and WI 38 cells


id: cord-290976-dhwlr2ui
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  hematopoietic stem cell transplant patients Human cytomegalovirus Herpes simplex virus and human cytomegalovirus replication in WI 38 cells III


id: cord-292794-okh6i4l1
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  influenza A virus The relative immunogenicity of DNA vaccines delivered by the intramuscular needle injection electroporation and gene gun methods A DNA transfection system for generation of influenza A virus


id: cord-295207-0p6x4lwx
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  class III viral fusion proteins domain I green domain II yellow domain III blue domain IV stem domain indigo Figure 1


id: cord-295207-0p6x4lwx
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  herpes simplex virus type 1 gB 1 28 and Epstein Barr virus gB into class III viral fusion proteins VFP 29


id: cord-283689-dzin12qb
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  anti GAPDH mouse anti ATG5 Alexa Fluor 488 conjugated anti rabbit IgG Alexa Fluor 488 conjugated anti mouse IgG


id: cord-310372-qc6941pm
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  Pathogenicity of Infectious Bronchitis Coronaviruses Complicated Evolution and Epidemiology in China


id: cord-303313-b8lesr2g
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  g g t t a g g t g t t t c t g t a g c g g c t g t g c t g c t 1 8 2 S b j c t 2 6 8 4 7 g g t t t t a g g a g t g t t a g t a


id: cord-303313-b8lesr2g
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  c t a g c t t a g t c t t t c t g g t a g t a t a a g t t a g t c c 3 0 2 S b j c t 2 6 7 2 7 t c c c t c t


id: cord-308397-kqftn75t
type: DNA
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  5 5 GCTCTAGAATGTGGTACCTAT ACTTATATGAGAACAGC 3 and 3 5 GCTCTAGA TCCGTGTTCAAGCATTTTATTAAATCAG 3 secondary pair


id: cord-308397-kqftn75t
type: PROTEIN
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  purified primary mouse antibody CD71 H68 4 Santa Cruz 1 500 dilution or anti β actin antibody MBL 1 1


id: cord-305315-0qt7eth0
type: DNA
entity:
  murine leukemia virus reverse transcriptase GBI Labs Golden Bridge International Inc Mukilteo WA USA


id: cord-310536-u30cufg7
type: CHEMICAL
entity:
  MgCl 0 2 mM dNTPs 2 units of Taq DNA polymerase 1X reaction buffer 1 pmol of each primer and 5 M N N N trimethylglycine betaine


id: cord-314954-otc0pc09
type: DISEASE
entity:
  herpes simplex virus 1 HSV 1 homologues UL25 to UL30 in duck enteritis virus DEV The UL31 to UL35 gene sequences of duck enteritis


id: cord-315688-ba5dus2j
type: PROTEIN
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  nucleoprotein N and five nonstructural proteins replicase 1a 1b 3a 3b and protein 7 14 15 16 The genome


id: cord-321756-a7eh4dkb
type: ORG
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  The WHO Working Group on Guidelines for Integrated Management of the Sick Child Qiagen Protocol Purification


id: cord-321756-a7eh4dkb
type: ORG
entity:
  Tropical South India Etiology of Acute Respiratory Infections in Children in Tropical Southern India Review


id: cord-316234-vtjsfi2c
type: DISEASE
entity:
  influenza and respiratory syncytial viruses Rapid PCR based molecular pathotyping of H5 and H7 avian influenza viruses Rapid discrimination of H5 and H9 subtypes of avian influenza viruses and Newcastle disease


id: cord-318853-mxyxwkhx
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entity:
  lipoprotein LDL receptors 85 86 transferrin receptor TfR 87 asialoglycoprotein receptor ASGP R 88 89


id: cord-318853-mxyxwkhx
type: GGP
entity:
  acetylcholine receptors 76 serotonergic neurotransmitters 5HT 77 endothelial cell glycoproteins 78 insulin


id: cord-322937-lakdi3x8
type: DNA
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  B human coronavirus 229E human coronavirus OC43 influenza B virus human parainfluenza virus and human adenovirus All samples


id: cord-325555-be78qely
type: PROTEIN
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  transmembrane UPR transducers inositol requiring kinase 1 IRE1 double stranded RNAactivated protein kinase


id: cord-314201-6njwigco
type: DNA
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  DENV2 771 bp UGSV 742 bp BSQV 700 bp MVEV 684 bp USUV 675 bp TYUV 620 bp TABV 500 bp YOKV 380 bp cDNA products


id: cord-320617-ucm7wx8b
type: TAXON
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  parainfluenza viruses PIV 9 human metapneumovirus hMPV 9 human bocavirus BoV 7 human coronavirus HCoV 4


id: cord-322738-nvyknvc7
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  internal controls 17 RNA 20 Respiratory syncytial viruses A and B hRSV A hRSV B 21 Coronaviruses E229 OC43 hCoV E229 OC43 22 Rhinovirus hRV 23 Adenoviruses hAdV 24 Enteroviruses Parechoviruses hEVs hPeV 25 human metapneumoviruses A and B hMPV 26 human Bocaviruses hBoV 27


id: cord-330251-dwjijmwz
type: DISEASE
entity:
  influenza A virus NS1 protein striking differences between different virus subtypes Changes in nucleolar morphology


id: cord-323009-frej2qmb
type: DISEASE
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  influenza B viruses 11 3 3 contained parainfluenza viruses types 1 and 3 and 9 2 7 contained human respiratory syncytial virus


id: cord-329517-3yn80r9h
type: WORK_OF_ART
entity:
  The Diagnostic Method of PCR for Lymphocystis Disease LCD of Cultured Paralichthys olivaceus Development


id: cord-329517-3yn80r9h
type: DISEASE
entity:
  Blood heart liver spleen lung kidney BF thymus esophagus trachea brain Harder s glands duodenum jejunum ileum cecum and rectum


id: cord-322238-8iwljdoi
type: CL
entity:
  stem lengths and cauliflower like structures with multiple loops formed by annealing between alternately inverted repeats of the target sequence


id: cord-322238-8iwljdoi
type: DISEASE
entity:
  Gastroenteritis Coronavirus TGEV strain H Porcine Reproductive and Respiratory Syndrome Virus PRRSV Pesudorabies PRV Porcine Parvovirus


id: cord-341321-paucodwz
type: DNA
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  open reading 1a 787 28 28 NA 29 29 NA NA 29 9 9 NA 10 22 24 1b 511 54 54 NA 54 54 NA NA 54 36 35 NA 36 47 44 2 756 24 24 24 23 23 24 24 24 15 16 16 11 18 19 frames


id: cord-337003-7ygcfzii
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entity:
  Nile virus dengue virus rhinovirus corona virus HIV respiratory syncytial virus and influenza A virus 3


id: cord-339209-oe8onyr9
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  3C like serine protease 3CLP RNA dependent RNA polymerase RdRp zinc binding Z helicase Hel exoribonuclease ExoN N7 methyltransferase NMT and 2 O methyltransferase OMT domains


id: cord-339209-oe8onyr9
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entity:
  associated helicase ZnHel1 exoribonuclease ExoN N 7 methyltransferase NMT and 2 O methyltransferase OMT domains


id: cord-336510-qzm9wgde
type: ORG
entity:
  International Lymphokine Nomenclature Committee International Union of Immunological Societies Nomenclature Committee Working Group


id: cord-336510-qzm9wgde
type: ORG
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  The Department of Clinical Microbiology Aarhus University Hospital Skejby and Department of Medical Microbiology and Immunology University of Aarhus


id: cord-335865-pibq3iwh
type: PROTEIN
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  human metapneumovirus attachment glycoprotein Respiratory Syncytial Virus and Metapneumovirus In Fields Virology Analysis of the


id: cord-342157-qjyooq68
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  V V I I I I II II II II II III III III T E 148 V I I I I 115 V I I I I 190 L F F F F NS4B 116 V I I I I 191 L


id: cord-348204-365z3qxz
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  TNF α and IFN γ expressions 9 26 Based on previous studies on the growth of FIPV 79 1146 in CRFK cells


id: cord-352361-jh31omg2
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  genus Orthobornavirus Mammalian 1 orthobornavirus Mammalian 2 orthobornavirus Passeriform 1 orthobornavirus Passeriform 2 orthobornavirus Psittaciform 1 orthobornavirus Psittaciform 2 orthobornavirus and Waterbird 1


id: cord-343893-sophqqne
type: DNA
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  human coronavirus strain OC43 phylogenetic relationships to bovine and murine coronaviruses and influenza C virus Sialic acids


id: cord-343893-sophqqne
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  coronaviruses and influenza C virus The hemagglutinin esterase gene of human coronavirus strain OC43 phylogenetic


id: cord-348876-v55piprx
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  matrix protein 2 Synthetic peptide vaccine design synthesis and properties of a high density multiple antigenic peptide system A novel method for producing antipeptide antibodies


id: cord-355489-tkvfneje
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  primer DEN1S2133 5 GGAAAATGTTYGAAGCAACYG CCC 3 DEN1AS2553 5 TCCTCCCATGCCTTCC CRATGG 3 and 2 5 U of Taq DNA Polimerase Invotrogene


id: cord-355489-tkvfneje
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  Tree View Program 58 In addition estimated rate of evolutionary change nucleotide substitutions per site


id: cord-355489-tkvfneje
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  Mosquitoes Put the Brake on Arbovirus Evolution Experimental Evolution Reveals Slower MutationAccumulation


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  American Type Culture Collection ATCC Manassas VA USA HEK293T human kidney epithelial cells CRL 11268 Raji Burkitt s lymphoma B lymphoblast ST486 Burkitt s lymphoma B lymphoblast


id: cord-351942-u9zpyy29
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  A549 human lung ATCC CRM CCL 185 HEK 293 T human embryonic kidney ATCC CRL 11268 NCI H292 human lung ATCC CRL 1848 LLC MK2 Macaca mulatta kidney ATCC CCL 7 MsIn Miniopterus schreibersi intestine RlKi Rousettus leschenaulti kidney


id: cord-349287-mwj2qby4
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  Interim Guidelines for Collecting Handling and Testing Clinical Specimens from Patients Under Investigation


id: cord-349287-mwj2qby4
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  the Epidemiology of MERS CoV Infection and Guidance for the Public Clinicians and Public Health Authorities Followup of Contacts of Middle East Respiratory Syndrome Coronavirus


id: cord-349287-mwj2qby4
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  human herpesvirus HHV rhinoviruses HRV enteroviruses EV respiratory syncytial virus RSV parainfluenzavirus types 1 2 and 3 PIVs influenzaviruses IFVs endemic HCoVs adenoviruses AdVs metapneumovirus MPV


id: cord-355906-yeaw9nr8
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  human leukocyte antigen class I and class II genes tumor necrosis factor alpha FcGRIIA vitamin D receptor transporters


id: cord-355906-yeaw9nr8
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  NS3 helicase nucleoside triphosphatase NTPase RNA 5 triphosphatase RTPase NS5 methyl transferase RNA


id: cord-355906-yeaw9nr8
type: DISEASE
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  respiratory syncytial virus hepatitis viruses influenza virus poliovirus and HIV 109 110 Low molecular weight phenolic


# bib

id: cord-000315-rfwzj1at
title: Hepatitis G Virus associated aplastic anemia: A recent case from Pakistan
author: Riaz Shah, Shahida Amjad
date: 2011-01-21
txt: ./txt/cord-000315-rfwzj1at.txt
cache: ./cache/cord-000315-rfwzj1at.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3032715/
doi: 10.1186/1743-422x-8-30
summary:
  title: Hepatitis G Virus associated aplastic anemia: A recent case from Pakistan One of 93 samples from patients with HA-aplastic anemia has hepatitis G associated aplastic anaemia with positive HGV RNA. Zaidi et al, reported a 19 year male before blood transfusion with positive HGV by RT-PCR and suggested that in the absence of any other clincial manifestions the possible infectious agent may be HGV for hepatitis G virus associated aplast anaemia [8] . In the list of studies of Hepatitis G Virus associated aplastic anaemia before blood transfusion we report a case of 11 years female girls. The ideal case regarding Hepatitis G associated aplastic anaemia are pre blood transfusion. Hepatitis GB virus C genome in the serum of aplastic anaemia patients receiving frequent blood transfusions Hepatitis G Virus associated aplastic anemia: A recent case from Pakistan

abstract:
  BACKGROUND: Aplastic anemia (AA) is a serious and rare disorder characterized by a hypocellular bone marrow. Hepatitis associated aplastic anemia (HAAA) is a variant of aplastic anemia in which aplastic anemia follows an acute attack of hepatitis. Several reports have noted an association between HGV and hepatitis-associated aplastic anemia besides other hepatitis causing viruses. CASE PRESENTATION: A female girl of age 11 year with a history of loose motion for one month, vomiting for last 15 days and poor oral intake for last few days is reported here. The physical examination presents fever, pallor whereas bleeding, hepatomegaly, Splenomegaly and bruising were absent, abdominal ultrasonography confirmed the absence of hepatomegaly, Splenomegaly and lymphodenopathy. The laboratory investigation parameters were: haemoglobin 6.2 g/L, total leucocytes count 1.51, neutrophils 0.47%, absolute reticulocyte count 0.5%, Monocytes 0.16%, red cell count 3.2 mil/uL, Picked cell volume (PCV) 30.13%, Mean Corpuscular Volume (MCV) 78 fL, Mean Corpuscular Hemoglobin (MCH) 26.3 pg. The liver enzymes were alanine aminotransferease (ALT) 98 IU/L, aspartate aminotransferase (AST) 114 IU/L. Serologic and molecular tests for hepatitis A, B, C, D, E, TTV, B19 were negative, whereas HGV RNA PCR test was found positive for hepatitis G virus. The bone marrow aspirate and trephine biopsy examination revealed hypo- cellularity, erythropoiesis, myelopoiesis and megakaryopoiesis. CONCLUSION: HAAA is an uncommon but severe condition, which may occur following idiopathic cases of acute hepatitis. Our finding suggests the involvement of HGV in the development of aplastic anemia. In patients presenting with pancytopenia after an episode of acute hepatitis, the definitive diagnosis should be considered and confirmed by RT-PCR and if possible by bone marrow biopsy.


id: cord-000389-9vnthmfn
author: Guo, Xichao
date: 2011-05-10
txt: ./txt/cord-000389-9vnthmfn.txt
cache: ./cache/cord-000389-9vnthmfn.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3104371/
doi: 10.1186/1743-422x-8-215
summary:
  The aim of this study was to investigate the dynamic fluctuations of the peripheral blood lymphocyte subgroups in patients infected with H1N1 swine-origin influenza A virus (S-OIV). The aim of this study is to analyze the dynamic fluctuations of T cells, B cells, natural killer (NK) cells, and regulatory T cells in patients infected with novel influenza H1N1, as well as serum cytokines and C-reactive protein (CRP). The serum cytokines of patients, specifically IL-2, IL-4, IL-6, IL-10, and IFN-γ, whether the H1N1 infection was severe or moderate, showed no significant changes during the whole monitoring process (data not shown). Our clinical data also showed that the most severe infections occur in individuals younger than 25 years old or in middle-aged patients Figure 1 Analysis of the dynamic changes in the lymphocyte subgroup and C-reactive protein (CRP) in the peripheral blood of patients with Influenza A (H1N1) 2009.

title:
  Dynamic variations in the peripheral blood lymphocyte subgroups of patients with 2009 pandemic H1N1 swine-origin influenza A virus infection

abstract:
  BACKGROUND: Novel Influenza A (H1N1) is an acute respiratory infectious disease. Animal experiments indicated that when H1N1 virus infected early hosts, it showed strong CD4(+), CD8(+), and CD4(+)CD25(+ )T cell reactions. The aim of this study was to investigate the dynamic fluctuations of the peripheral blood lymphocyte subgroups in patients infected with H1N1 swine-origin influenza A virus (S-OIV). METHODS: The frequency of T cells, B cells, natural killer (NK) cells, and regulatory T cells (Treg) in 36 severe H1N1 and 40 moderate H1N1 patients were detected at different periods by flow cytometry. In parallel, serum cytokines were detected by enzyme-linked immunosorbent assay and C-reactive protein (CRP) was analyzed through an image-type automatic biochemical analyzer. In addition, 20 healthy volunteers, who were not infected with 2009 H1N1 virus, were selected as controls. RESULTS: The frequency of NK cells were decreased in all cases and CD19(+ )B cells were increased in severe cases than those of the controls. At 1-2d from onset, the frequency of CD4(+ )and CD4(+)CD25(+ )T cells in moderate cases was higher than in the severe cases. Serum cytokines, specifically IL-2, IL-4, IL-6, IL-10, and IFN-γ exhibited no significant change both in the moderate and the severe cases during the whole monitoring process. In the early stage of the disease, serum CRP levels in the severe and moderate groups were significantly higher than that in the control group. CONCLUSIONS: Patients showed different lymphocyte subgroup distributions between mild and severe cases, which might affect the incidence and development of 2009 H1N1.


id: cord-000403-vzbh457k
author: Zhang, Weijun
date: 2011-05-30
txt: ./txt/cord-000403-vzbh457k.txt
cache: ./cache/cord-000403-vzbh457k.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3126774/
doi: 10.1186/1743-422x-8-263
summary:
  Twenty-seven nanopeptides derived from the matrix (M) protein of porcine reproductive and respiratory syndrome virus (PRRSV) were screened for their ability to elicit a recall interferon-γ (IFN-γ) response from the splenocytes of BALB/c mice following DNA vaccination and a booster vaccination with recombinant vaccinia virus rWR-PRRSV-M. We screened peptides derived from the PRRSV M protein for their ability to induce interferon (IFN)-γ in splenocytes harvested from BALB/ c mice following DNA vaccination and a booster vaccination with recombinant vaccinia virus expressing M protein. In contrast, control mice vaccinated with pVAX1-only or PBS-only did not show significant increases in M protein-specific antibody titers after the booster vaccination with rWR-PRRSV-M (P > 0.05, Additional file 5, Fig. S5 ). Specific increases in the number of cells producing IFN-γ following stimulation with the peptides "K 93 FITSRCRL" and "F 57 GYMTFVHF" was observed by day 3 after the booster vaccination with rWR-PRRSV-M (Figure 1 and 2) .

title:
  Identification of CD8(+ )cytotoxic T lymphocyte epitopes from porcine reproductive and respiratory syndrome virus matrix protein in BALB/c mice

abstract:
  Twenty-seven nanopeptides derived from the matrix (M) protein of porcine reproductive and respiratory syndrome virus (PRRSV) were screened for their ability to elicit a recall interferon-γ (IFN-γ) response from the splenocytes of BALB/c mice following DNA vaccination and a booster vaccination with recombinant vaccinia virus rWR-PRRSV-M. We identified two peptides (amino acid residues K(93)FITSRCRL and F(57)GYMTFVHF) as CD8(+ )cytotoxic T lymphocyte (CTL) epitopes. These peptides elicited significant numbers of IFN-γ secreting cells, compared with other M nonapeptides and one irrelevant nonapeptide. Bioinformatics analysis showed that the former is an H-2K(d)-restricted CTL epitope, and the latter is an H-2D(d)-restricted CTL epitope. Multiple amino acid sequence alignment among different PRRSV M sequences submitted to GenBank indicated that these two CTL epitopes are strongly conserved, and they should therefore be considered for further research on the mechanisms of cellular immune responses to PRRSV.


id: cord-000640-t0y0b0gb
author: Sumibcay, Laarni
date: 2012-01-26
txt: ./txt/cord-000640-t0y0b0gb.txt
cache: ./cache/cord-000640-t0y0b0gb.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3331809/
doi: 10.1186/1743-422x-9-34
summary:
  Following numerous failed attempts, hantavirus RNA was detected in ethanol-fixed liver tissue from two banana pipistrelles (Neoromicia nanus), captured near Mouyassué village in Côte d''Ivoire, West Africa, in June 2011. Phylogenetic analysis of partial L-segment sequences using maximum-likelihood and Bayesian methods revealed that the newfound hantavirus, designated Mouyassué virus (MOUV), was highly divergent and basal to all other rodentand soricomorph-borne hantaviruses, except for Nova virus in the European common mole (Talpa europaea). After innumerable failed attempts, hantavirus RNA was detected by RT-PCR in ethanol-fixed liver tissues from two of 12 banana pipistrelles (Neoromicia nanus Peters 1852), captured during June 2011 near Mouyassué village (5°22''07"N, 3°05''37"W) in Aboisso District, 130 km from Abidjan, in the extreme southeastern region of Côte d''Ivoire in West Africa ( Figure 1 ). The newfound hantavirus, designated Mouyassué virus (MOUV), exhibited low nucleotide and amino acid sequence similarity of less than 69% to all representative soricomorph-and rodent-associated hantaviruses, except for the 76.3% sequence similarity with Nova virus (NVAV), previously reported in the European common mole (Talpa europaea) [12] .

title:
  Divergent lineage of a novel hantavirus in the banana pipistrelle (Neoromicia nanus) in Côte d''Ivoire

abstract:
  Recently identified hantaviruses harbored by shrews and moles (order Soricomorpha) suggest that other mammals having shared ancestry may serve as reservoirs. To investigate this possibility, archival tissues from 213 insectivorous bats (order Chiroptera) were analyzed for hantavirus RNA by RT-PCR. Following numerous failed attempts, hantavirus RNA was detected in ethanol-fixed liver tissue from two banana pipistrelles (Neoromicia nanus), captured near Mouyassué village in Côte d'Ivoire, West Africa, in June 2011. Phylogenetic analysis of partial L-segment sequences using maximum-likelihood and Bayesian methods revealed that the newfound hantavirus, designated Mouyassué virus (MOUV), was highly divergent and basal to all other rodent- and soricomorph-borne hantaviruses, except for Nova virus in the European common mole (Talpa europaea). Full genome sequencing of MOUV and further surveys of other bat species for hantaviruses, now underway, will provide critical insights into the evolution and diversification of hantaviruses.


id: cord-000988-79fp75u3
title: A cost effective real-time PCR for the detection of adenovirus from viral swabs
author: Al-Siyabi, Turkiya
date: 2013-06-07
txt: ./txt/cord-000988-79fp75u3.txt
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extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3679997/
doi: 10.1186/1743-422x-10-184
summary:
  Twentyseven virus culture-positive specimens and 169 virus culture-negative specimens were randomly selected and tested for the presence of HAdV using a well established in-house real-time PCR assay [18] following recovery of viral DNA was recovered by homogenization with heat treatment or automated nucleic acid extraction. This internally controlled quantitative real-time PCR assay targets the hexon gene of adenovirus, and is validated for detection Table 1 Nucleotide sequences of primers and probes used in this study The analytical sensitivity (or limit of detection, LoD) of the homogenization with heat treatment or nucleic acid extraction, in combination with the real-time PCR, was determined using 10-fold serial dilutions (in UTM) of a cultured HAdV-C type 6. Virus stock dilutions were quantified using commercial real-time PCR assay, and the LoD for homogenization or nucleic acid extraction-based protocols were shown to be approximately equivalent (Figure 2) .

abstract:
  Compared to traditional testing strategies, nucleic acid amplification tests such as real-time PCR offer many advantages for the detection of human adenoviruses. However, commercial assays are expensive and cost prohibitive for many clinical laboratories. To overcome fiscal challenges, a cost effective strategy was developed using a combination of homogenization and heat treatment with an “in-house” real-time PCR. In 196 swabs submitted for adenovirus detection, this crude extraction method showed performance characteristics equivalent to viral DNA obtained from a commercial nucleic acid extraction. In addition, the in-house real-time PCR outperformed traditional testing strategies using virus culture, with sensitivities of 100% and 69.2%, respectively. Overall, the combination of homogenization and heat treatment with a sensitive in-house real-time PCR provides accurate results at a cost comparable to viral culture.


id: cord-000990-ci6db90d
author: Guo, Ling
date: 2013-05-26
txt: ./txt/cord-000990-ci6db90d.txt
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extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3680276/
doi: 10.1186/1743-422x-10-163
summary:
  BACKGROUND: In this study, we sequenced and phylogenetic analyses of the VP2 genes from twelve canine parvovirus (CPV) strains obtained from eleven domestic dogs and a giant panda (Ailuropoda melanoleuca) in China. Substitution of Gln for Arg at the conserved 370 residue in CPV presents an unusual variation in the new CPV-2a amino acid sequence of the giant panda and is further evidence for the continuing evolution of the virus. Canine parvovirus type 2a/2b having mutation at 297 residue (Ser→Ala) is designated as new CPV-2a/2b [8, 9] , residue 297 is located in a minor antigenic site close to epitope B and substitutions at this position may be responsible for changes in antigenicity of CPV variants [10] . In comparison to prototype new-CPV-2a (AY742953), the samples under this study had amino acid residue variations at Tyr324Ile caused by mutation TAT →ATT at nt 3756-3758 of the VP2 gene.

title:
  Identification of canine parvovirus with the Q370R point mutation in the VP2 gene from a giant panda (Ailuropoda melanoleuca)

abstract:
  BACKGROUND: In this study, we sequenced and phylogenetic analyses of the VP2 genes from twelve canine parvovirus (CPV) strains obtained from eleven domestic dogs and a giant panda (Ailuropoda melanoleuca) in China. A novel canine parvovirus (CPV) was detected from the giant panda in China. RESULTS: Nucleotide and phylogenetic analysis of the capsid protein VP2 gene classified the CPV as a new CPV-2a type. Substitution of Gln for Arg at the conserved 370 residue in CPV presents an unusual variation in the new CPV-2a amino acid sequence of the giant panda and is further evidence for the continuing evolution of the virus. CONCLUSIONS: These findings extend the knowledge on CPV molecular epidemiology of particular relevance to wild carnivores.


id: cord-003855-so8xl199
author: Ebert, Gregor
date: 2019-09-02
txt: ./txt/cord-003855-so8xl199.txt
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extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6720860/
doi: 10.1186/s12985-019-1217-6
summary:
  The bat innate immune response appears to be ''pre-activated'' with higher basal levels of type I interferon expression, in contrast to humans, who are very quick responders to viral infections, but require a lot more dampening of their immune signals afterwards to get back to basal levels. demonstrated that bats'' response to stress in form of viral infections is more targeted and thus potentially more effective by numerous adaptions and modifications of the innate immune system. In the ''Pathogenesis and Prevention of Infection'' session, Rosa Coldbeck-Shackley working with Michael Beard at the University of Adelaide, Australia, and also colleagues at the Hudson Institute, presented findings on the importance of interferon-epsilon (IFN-ɛ) in the innate immune response to ZIKV infection. Also in the ''Pathogenesis and Prevention of Infection'' session, Allison Abendroth (University of Sydney) presented ''Disarming the killer: targeting of natural killer cells by varicella zoster virus''.

title:
  Virology Downunder, a meeting commentary from the 2019 Lorne Infection and Immunity Conference, Australia

abstract:
  The aim of this article is to summarise the virology content presented at the 9th Lorne Infection and Immunity Conference, Australia, in February 2019. The broad program included virology as a key theme, and the commentary herein highlights several key virology presentations at the meeting.


id: cord-010336-xfzf7ath
title: H2 influenza A virus is not pathogenic in Tmprss2 knock-out mice
author: Lambertz, Ruth Lydia Olga
date: 2020-04-22
txt: ./txt/cord-010336-xfzf7ath.txt
cache: ./cache/cord-010336-xfzf7ath.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7178614/
doi: 10.1186/s12985-020-01323-z
summary:
  For in vivo studies, female Tmprss2 −/− (B6.129S1-Tmprss2tm1Tsyk) [12, 14] and C57BL/6 JRj wild type (WT) mice (Janvier, 8-12 weeks old) were infected intranasally with 2 × 10 4 ffu PR8_HA(H2) as described before [12] and changes in body weight and survival were monitored for the next 14 days. Viral replication in lungs of infected (dose of 2 × 10 4 ffu) female Tmprss2 −/− and WT mice (8-12 weeks old) revealed no detectable virus replication in Tmprss2 −/− mice, whereas WT mice showed increased lung titers at day 2 and 4 post infection (dpi) (Fig. 2c) . Since no viral replication and only minor immune responses were detected in the studies described above, we confirmed that mice had indeed been infected with the PR8_HA(H2) virus by determining the presence of H2-specific antibodies at 14 dpi in the serum.

abstract:
  The host cell protease TMPRSS2 cleaves the influenza A virus (IAV) hemagglutinin (HA). Several reports have described resistance of Tmprss2(−/−) knock-out (KO) mice to IAV infection but IAV of the H2 subtype have not been examined yet. Here, we demonstrate that TMPRSS2 is able to cleave H2-HA in cell culture and that Tmprss2(−/−) mice are resistant to infection with a re-assorted PR8_HA(H2) virus. Infection of KO mice did not cause major body weight loss or death. Furthermore, no significant increase in lung weights and no virus replication were observed in Tmprss2(−/−) mice. Finally, only minor tissue damage and infiltration of immune cells were detected and no virus-positive cells were found in histological sections of Tmprss2(−/−) mice. In summary, our studies indicate that TMPRSS2 is required for H2 IAV spread and pathogenesis in mice. These findings extend previous results pointing towards a central role of TMPRSS2 in IAV infection and validate host proteases as a potential target for antiviral therapy.


id: cord-011880-qlutgfu2
author: Barberis, Abdelheq
date: 2020-07-17
txt: ./txt/cord-011880-qlutgfu2.txt
cache: ./cache/cord-011880-qlutgfu2.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7366561/
doi: 10.1186/s12985-020-01377-z
summary:
  In addition, different studies, showed that circulating H9N2 strains have acquired affinity to mammalian like-receptors and gained high virulence and pathogenicity through substitutions in their viral proteins [13, 14] ; the most known substitutions are in the HA protein that promotes virus binding to cellular receptors. While the substitution 627 K that confers high pathogenicity, virulence and increased replication in mice [63] , was not detected in our Algerian viruses, three substitutions 318R, 590S and 661 T, associated with mammalian adaptation, were observed [71, 72] . The PB1 substitutions N105S, K577E/M and 578Q, known to be associated with increased polymerase activity, H9N2 pathogenicity in mice as well as adaptation to mammalians [61, 64, 78] , were not observed in the currently circulating Algerian strains, which however shared 105 N, 577 K and 578 K. Amino acids analysis showed that the Algerians H9N2 strains carried out different molecular markers associated with affinity to human-like receptors and increased virulence.

title:
  Full-length genome sequences of the first H9N2 avian influenza viruses isolated in the Northeast of Algeria

abstract:
  BACKGROUND: H9N2 avian influenza viruses (AIV) has a worldwide geographic distribution and affects poultry of different types of production. H9N2 AIV was first reported in the Northeast of Algeria in April 2017, following an outbreak associated with high mortality, in broiler flocks. In the present study, we report full-length genome sequences of AIV H9N2, and the detailed phylogeny and molecular genetic analyses. METHODS: Ten AIV H9N2 strains, collected in broiler flocks, were amplified in 9-day-old embryonated specific pathogen free (SPF) chicken eggs. Their full-length genomes were successfully sequenced and phylogenetic and molecular characterizations were conducted. RESULTS: Phylogenetic analysis showed that the isolates were monophyletic, grouped within the G-1 lineage and were very close to Moroccan and Algerian strains identified in 2016 and 2017, respectively. The low pathogenicity of the strains was confirmed by the sequence motif (335RSSR/GLF341) at the hemagglutinin (HA) cleavage site. An exclusive substitution (T197A) that had not been previously reported for H9N2 viruses; but, conserved in some pandemic H1N1 viruses, was observed. When compared to the G1-like H9N2 prototype, the studied strains showed one less glycosylation site in HA, but 2–3 additional ones in the stalk of the neuraminidase (NA). The HA protein harbored the substitution 234 L, suggesting binding preference to human-like receptors. The NA protein harbored S372A and R403W substitutions, previously detected in H9N2 from Asia and the Middle East, and especially in H2N2 and H3N2 strains that caused human pandemics. Different molecular markers associated with virulence and mammalian infections have been detected in the viral internal proteins. The matrix M2 protein possessed the S31N substitution associated with drug resistance. The non-structural 1 (NS1) protein showed the “GSEV” PDZ ligand (PL) C-terminal motif and no 80–84 deletion. CONCLUSION: Characterized Algerian AIV isolates showed mutations that suggest increased zoonotic potential. Additional studies in animal models are required to investigate the pathogenicity of these H9N2 AIV strains. Monitoring their evolution in both migratory and domestic birds is crucial to prevent transmission to humans. Implementation of adequate biosecurity measures that limit the introduction and the propagation of AIV H9N2 in Algerian poultry farm is crucial.


id: cord-031316-yvid6qps
author: Bisimwa, Patrick N.
date: 2020-09-03
txt: ./txt/cord-031316-yvid6qps.txt
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extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468181/
doi: 10.1186/s12985-020-01398-8
summary:
  Sequences of p72 and p54 amplicon were compared with 25 other p72 and p54 ASFV sequences retrieved from the Gen-Bank database and the phylogenetic analysis revealed that the South Kivu ASF virus strains analyzed clustered with p72 genotype X including strains reported in previous studies in Burundi (AF449463), Kenya (AY261360) and Tanzania (JX403648, AF301546, MF437291) ( Fig. 2a and b) . Sequences of African swine fever virus (ASFV) strains from the South Kivu province, eastern DRC, showing tetrameric repeats of representative genotypes, including a reference sequence of a virus isolated in 1950 in Kenya (Kenya 1950; GenBank accession no.

title:
  First detection of African swine fever (ASF) virus genotype X and serogroup 7 in symptomatic pigs in the Democratic Republic of Congo

abstract:
  BACKGROUND: African swine fever (ASF) is a highly contagious and severe hemorrhagic viral disease of domestic pigs. The analysis of variable regions of African swine fever virus (ASFV) genome led to more genotypic and serotypic information about circulating strains. The present study aimed at investigating the genetic diversity of ASFV strains in symptomatic pigs in South Kivu province of the Democratic Republic of Congo (DRC). MATERIALS AND METHODS: Blood samples collected from 391 ASF symptomatic domestic pigs in 6 of 8 districts in South Kivu were screened for the presence of ASFV, using a VP73 gene-specific polymerase chain reaction (PCR) with the universal primer set PPA1-PPA2. To genotype the strains, we sequenced and compared the nucleotide sequences of PPA-positive samples at three loci: the C-terminus of B646L gene encoding the p72 protein, the E183L gene encoding the p54 protein, and the central hypervariable region (CVR) of the B602L gene encoding the J9L protein. In addition, to serotype and discriminate between closely related strains, the EP402L (CD2v) gene and the intergenic region between the I73R and I329L genes were analyzed. RESULTS: ASFV was confirmed in 26 of 391 pigs tested. However, only 19 and 15 PPA-positive samples, respectively, were successfully sequenced and phylogenetically analyzed for p72 (B646L) and p54 (E183L). All the ASFV studied were of genotype X. The CVR tetrameric repeat clustered the ASFV strains in two subgroups: the Uvira subgroup (10 TRS repeats, AAAABNAABA) and another subgroup from all other strains (8 TRS repeats, AABNAABA). The phylogenetic analysis of the EP402L gene clustered all the strains into CD2v serogroup 7. Analyzing the intergenic region between I73R and I329L genes revealed that the strains were identical but contained a deletion of a 33-nucleotide internal repeat sequence compared to ASFV strain Kenya 1950. CONCLUSION: ASFV genotype X and serogroup 7 was identified in the ASF disease outbreaks in South Kivu province of DRC in 2018–2019. This represents the first report of ASFV genotype X in DRC. CVR tetrameric repeat sequences clustered the ASFV strains studied in two subgroups. Our finding emphasizes the need for improved coordination of the control of ASF.


id: cord-034467-jh9msz1c
author: Olagoke, Olusola
date: 2020-10-31
txt: ./txt/cord-034467-jh9msz1c.txt
cache: ./cache/cord-034467-jh9msz1c.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7602773/
doi: 10.1186/s12985-020-01442-7
summary:
  In the present study, we examined the expression of important koala cytokines, immune markers and host restrictions factors to determine their pre-and post-vaccination levels in northern koalas harbouring endogenous KoRV. Genes targeted included nine cytokines (CCL4L, Interleukin Open Access *Correspondence: oolagoke@usc.edu.au Genecology Research Centre, University of the Sunshine Coast, Sunshine Coast, QLD, Australia (IL)-1β, IL-4, IL-6, IL-8, IL-10, IL-17A, IL-18 and Interferon gamma (IFN-γ), four host restriction factors (BST2, ISG15, RSAD2 and TRIM1) and two T-cell markers (CD4 and CD8β). In addition, none of the host restriction factors tested (BST2, ISG15, RSAD2 and TRIM1) had significant changes in their expression either at 4-or 8-weeks post-vaccination when compared to prevaccination levels. In this cohort of KoRV vaccinated and endogenously infected koalas, a small but significant increase in the expression of IFN-γ at both 4-and 8-weeks post-vaccination was observed, compared to pre-vaccination levels. Our study investigated the expression of four host restriction factors in koalas harbouring endogenous KoRV: BST2, ISG15, RSAD2 and TRIM1.

title:
  Koalas vaccinated against Koala retrovirus respond by producing increased levels of interferon-gamma

abstract:
  Koala retrovirus (KoRV) is believed to be in an active state of endogenization into the koala genome. KoRV is present as both an endogenous and exogenous infection in all koalas in northern Australia. KoRV has been linked to koala pathologies including neoplasia and increased susceptibility to Chlamydia. A KoRV vaccine recently trialled in 10 northern koalas improved antibody response and reduced viral load. This communication reports the expression of key immune genes underlining the innate and adaptive immune response to vaccination in these northern koalas. The results showed that prior to vaccination, IL-8 was expressed at the highest levels, with at least 200-fold greater expression compared to other cytokines, while CD8 mRNA expression was significantly higher than CD4 mRNA expression level. Interferon-γ was up-regulated at both 4- and 8-weeks post-vaccination while IL-8 was down-regulated at 8-weeks post-vaccination.


id: cord-048229-ajlctjeb
author: Golden, Joseph W
date: 2005-05-31
txt: ./txt/cord-048229-ajlctjeb.txt
cache: ./cache/cord-048229-ajlctjeb.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1180477/
doi: 10.1186/1743-422x-2-48
summary:
  To identify the protease(s) responsible, U937 cells were treated with phorbol 12-myristate 13-acetate (PMA), an agent that induces cellular differentiation and results in decreased expression of acid-independent serine proteases, including NE and cathepsin (Cat) G. Experiments described in this report demonstrate that reovirus infection in U937 cells does not require cysteine protease activity and is not blocked in the presence of agents that raise vesicular pH. To examine the effect of the NE inhibitor on reovirus replication in U937 cells, we pre-treated them for 3 h with E64 in the presence or absence of the NE inhibitor, infected them with Lang virions or ISVPs at an MOI of 3, and quantified viral yields at 2 d p.i. A representative experiment is shown in Fig. 4 . To determine if NE-generated SVPs required further proteolytic processing of σ3, L929 cells were pre-treated with E64 to block cysteine protease activity and infected at an MOI of 3 with Lang virions, ISVPs or NE-generated subviral particles (NE-SVPs).

title:
  Neutrophil elastase, an acid-independent serine protease, facilitates reovirus uncoating and infection in U937 promonocyte cells

abstract:
  BACKGROUND: Mammalian reoviruses naturally infect their hosts through the enteric and respiratory tracts. During enteric infections, proteolysis of the reovirus outer capsid protein σ3 is mediated by pancreatic serine proteases. In contrast, the proteases critical for reovirus replication in the lung are unknown. Neutrophil elastase (NE) is an acid-independent, inflammatory serine protease predominantly expressed by neutrophils. In addition to its normal role in microbial defense, aberrant expression of NE has been implicated in the pathology of acute respiratory distress syndrome (ARDS). Because reovirus replication in rodent lungs causes ARDS-like symptoms and induces an infiltration of neutrophils, we investigated the capacity of NE to promote reovirus virion uncoating. RESULTS: The human promonocyte cell line U937 expresses NE. Treatment of U937 cells with the broad-spectrum cysteine-protease inhibitor E64 [trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane] and with agents that increase vesicular pH did not inhibit reovirus replication. Even when these inhibitors were used in combination, reovirus replicated to significant yields, indicating that an acid-independent non-cysteine protease was capable of mediating reovirus uncoating in U937 cell cultures. To identify the protease(s) responsible, U937 cells were treated with phorbol 12-myristate 13-acetate (PMA), an agent that induces cellular differentiation and results in decreased expression of acid-independent serine proteases, including NE and cathepsin (Cat) G. In the presence of E64, reovirus did not replicate efficiently in PMA-treated cells. To directly assess the role of NE in reovirus infection of U937 cells, we examined viral growth in the presence of N-Ala-Ala-Pro-Val chloromethylketone, a NE-specific inhibitor. Reovirus replication in the presence of E64 was significantly reduced by treatment of cells with the NE inhibitor. Incubation of virions with purified NE resulted in the generation of infectious subviron particles that did not require additional intracellular proteolysis. CONCLUSION: Our findings reveal that NE can facilitate reovirus infection. The fact that it does so in the presence of agents that raise vesicular pH supports a model in which the requirement for acidic pH during infection reflects the conditions required for optimal protease activity. The capacity of reovirus to exploit NE may impact viral replication in the lung and other tissues during natural infections.


id: cord-048368-wm4c7rk6
title: Experimental infection of H5N1 HPAI in BALB/c mice
author: Evseenko, Vasily A
date: 2007-07-27
txt: ./txt/cord-048368-wm4c7rk6.txt
cache: ./cache/cord-048368-wm4c7rk6.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1973068/
doi: 10.1186/1743-422x-4-77
summary:
  Serological analysis showed wide cross-reactivity of this virus with sera produced to H5N1 HPAI viruses isolated earlier in South-East Asia. Earlier HPAI viruses were investigated in mice [4, 5] and murine models were successively used for reverse genetics made influenza vaccines [6] . Sequence comparison of the NA protein of A/duck/Tuva/01/06 aligned with the NA of N2 subtype of A/Wuhan/359/95 (H3N2) influenza virus showed phenotype potentially sensitive to neuraminidase inhibitors. Despite powerful anti-influenza virus effects of TNF-α in lung tissue, as it was described previously [28] , we consider that elevated production of the cytokines seems to be crucial in the pathogenesis of HPAI infection. Summing up, in our study BALB/c mice infected with HPAI, strain A/duck/Tuva/01/06, appeared to be able to produce the innate immune response, which culminated to the development of shock and subsequent multiple organ failure.

abstract:
  BACKGROUND: In 2005 huge epizooty of H5N1 HPAI occurred in Russia. It had been clear that territory of Russia becoming endemic for H5N1 HPAI. In 2006 several outbreaks have occurred. To develop new vaccines and antiviral therapies, animal models had to be investigated. We choose highly pathogenic strain for these studies. RESULTS: A/duck/Tuva/01/06 belongs to Quinghai-like group viruses. Molecular markers – cleavage site, K627 in PB2 characterize this virus as highly pathogenic. This data was confirmed by direct pathogenic tests: IVPI = 3.0, MLD(50 )= 1,4Log10EID(50). Also molecular analysis showed sensivity of the virus to adamantanes and neuraminidase inhibitors. Serological analysis showed wide cross-reactivity of this virus with sera produced to H5N1 HPAI viruses isolated earlier in South-East Asia. Mean time to death of infected animals was 8,19+/-0,18 days. First time acute delayed hemorrhagic syndrome was observed in mice lethal model. Hypercytokinemia was determined by elevated sera levels of IFN-gamma, IL-6, IL-10. CONCLUSION: Assuming all obtained data we can conclude that basic model parameters were characterized and virus A/duck/Tuva/01/06 can be used to evaluate anti-influenza vaccines and therapeutics.


id: cord-048466-fj9l8che
title: The evolution of human influenza A viruses from 1999 to 2006: A complete genome study
author: Bragstad, Karoline
date: 2008-03-07
txt: ./txt/cord-048466-fj9l8che.txt
cache: ./cache/cord-048466-fj9l8che.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2311284/
doi: 10.1186/1743-422x-5-40
summary:
  BACKGROUND: Knowledge about the complete genome constellation of seasonal influenza A viruses from different countries is valuable for monitoring and understanding of the evolution and migration of strains. The influenza virus evades host immunity by accumulation of point mutations (drift) in the major surface glycoproteins, haemagglutinin (HA) and neuraminidase (NA) or by reassortment of segments from different viruses co-infecting the same cell leading to a new stain with a HA (and NA) not seen in the population before (shift). The A/Fujian/411/02(H3N2)-like clinical Danish viruses had several substitutions in HA at sites that might influence the virus'' capability for egg growth [10, 37] . Substitutions at antigenic site B and the predicted N-glycosylation at position 144 in HA antigenic site A together with a stronger NA might have contributed to the increased infectivity of the reassorted Fujian-like viruses of the 2003-2004 season, causing an epidemic in Denmark. Positive selection on the H3 hemagglutinin gene of human influenza virus A

abstract:
  BACKGROUND: Knowledge about the complete genome constellation of seasonal influenza A viruses from different countries is valuable for monitoring and understanding of the evolution and migration of strains. Few complete genome sequences of influenza A viruses from Europe are publicly available at the present time and there have been few longitudinal genome studies of human influenza A viruses. We have studied the evolution of circulating human H3N2, H1N1 and H1N2 influenza A viruses from 1999 to 2006, we analysed 234 Danish human influenza A viruses and characterised 24 complete genomes. RESULTS: H3N2 was the prevalent strain in Denmark during the study period, but H1N1 dominated the 2000–2001 season. H1N2 viruses were first observed in Denmark in 2002–2003. After years of little genetic change in the H1N1 viruses the 2005–2006 season presented H1N1 of greater variability than before. This indicates that H1N1 viruses are evolving and that H1N1 soon is likely to be the prevalent strain again. Generally, the influenza A haemagglutinin (HA) of H3N2 viruses formed seasonal phylogenetic clusters. Different lineages co-circulating within the same season were also observed. The evolution has been stochastic, influenced by small "jumps" in genetic distance rather than constant drift, especially with the introduction of the Fujian-like viruses in 2002–2003. Also evolutionary stasis-periods were observed which might indicate well fit viruses. The evolution of H3N2 viruses have also been influenced by gene reassortments between lineages from different seasons. None of the influenza genes were influenced by strong positive selection pressure. The antigenic site B in H3N2 HA was the preferred site for genetic change during the study period probably because the site A has been masked by glycosylations. Substitutions at CTL-epitopes in the genes coding for the neuraminidase (NA), polymerase acidic protein (PA), matrix protein 1 (M1), non-structural protein 1 (NS1) and especially the nucleoprotein (NP) were observed. The N-linked glycosylation pattern varied during the study period and the H3N2 isolates from 2004 to 2006 were highly glycosylated with ten predicted sequons in HA, the highest amount of glycosylations observed in this study period. CONCLUSION: The present study is the first to our knowledge to characterise the evolution of complete genomes of influenza A H3N2, H1N1 and H1N2 isolates from Europe over a time period of seven years from 1999 to 2006. More precise knowledge about the circulating strains may have implications for predicting the following season strains and thereby better matching the vaccine composition.


id: cord-253615-qylm0koe
author: Müller, Marcel A
date: 2010-01-15
txt: ./txt/cord-253615-qylm0koe.txt
cache: ./cache/cord-253615-qylm0koe.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/20078868/
doi: 10.1186/1743-422x-7-6
summary:
  In-silico analysis of potential glycosylation sites and membrane topology suggest properties similar to SARS-CoV ORF 3a protein ( Figure 1B and Table 1 ). To analyze the expression of ORF 3 protein during viral replication, colon carcinoma cells (CaCo-2) and Rhesus monkey kidney cells (LLC-MK2) cells were infected with hCoV-NL63 and an immunofluorescence assay (IFA) was done after two and four days, respectively. In contrast to virus-infected cells, cells overexpressing ORF 3 protein from plasmid with an N-terminal FLAG epitope showed only a single band in Western blot whose migration was consistent with the hypothetical unglycosylated form ( Figure 5B, left panel) . Severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice Severe acute respiratory syndrome coronavirus 3a protein is released in membranous structures from 3a protein-expressing cells and infected cells

title:
  Human Coronavirus NL63 Open Reading Frame 3 encodes a virion-incorporated N-glycosylated membrane protein

abstract:
  BACKGROUND: Human pathogenic coronavirus NL63 (hCoV-NL63) is a group 1 (alpha) coronavirus commonly associated with respiratory tract infections. In addition to known non-structural and structural proteins all coronaviruses have one or more accessory proteins whose functions are mostly unknown. Our study focuses on hCoV-NL63 open reading frame 3 (ORF 3) which is a highly conserved accessory protein among coronaviruses. RESULTS: In-silico analysis of the 225 amino acid sequence of hCoV-NL63 ORF 3 predicted a triple membrane-spanning protein. Expression in infected CaCo-2 and LLC-MK2 cells was confirmed by immunofluorescence and Western blot analysis. The protein was detected within the endoplasmatic reticulum/Golgi intermediate compartment (ERGIC) where coronavirus assembly and budding takes place. Subcellular localization studies using recombinant ORF 3 protein transfected in Huh-7 cells revealed occurrence in ERGIC, Golgi- and lysosomal compartments. By fluorescence microscopy of differently tagged envelope (E), membrane (M) and nucleocapsid (N) proteins it was shown that ORF 3 protein colocalizes extensively with E and M within the ERGIC. Using N-terminally FLAG-tagged ORF 3 protein and an antiserum specific to the C-terminus we verified the proposed topology of an extracellular N-terminus and a cytosolic C-terminus. By in-vitro translation analysis and subsequent endoglycosidase H digestion we showed that ORF 3 protein is N-glycosylated at the N-terminus. Analysis of purified viral particles revealed that ORF 3 protein is incorporated into virions and is therefore an additional structural protein. CONCLUSIONS: This study is the first extensive expression analysis of a group 1 hCoV-ORF 3 protein. We give evidence that ORF 3 protein is a structural N-glycosylated and virion-incorporated protein.


id: cord-254384-mwzz1db5
author: Lu, Guilan
date: 2011-02-10
txt: ./txt/cord-254384-mwzz1db5.txt
cache: ./cache/cord-254384-mwzz1db5.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/1743-422x-8-62
doi: 10.1186/1743-422x-8-62
summary:
  To assess the seroprevalence of hMPV infection in China, we tested a total of 1,156 serum specimens for the presence of anti-hMPV IgG antibody in children and adults free of acute respiratory illness in Beijing, China by using hMPV nucleocapsid (N) protein as an antigen. To assess the seroprevalence of hMPV infection in China, we used hMPV N protein as an antigen to test serum samples for the presence of anti-hMPV IgG antibody in children and adults free of acute respiratory illness in Beijing, China. Lower seropositive rates and geometric mean titer (GMT) of anti-hMPV IgG were observed in children aged six months to six years when compared to hRSV. To test the specificity of the ELISA methods established in this study, the reactions of mouse sera against influenza virus A (subtypes H1-H16), human coronaviruses (229E, HKU1 and NL63), and polyomavirus JC against hMPV and hRSV N protein were evaluated.

title:
  Large-scale seroprevalence analysis of human metapneumovirus and human respiratory syncytial virus infections in Beijing, China

abstract:
  BACKGROUND: Human metapneumovirus (hMPV), a recently identified virus, causes acute respiratory tract infections (ARTIs) in infants and children. However, studies on the seroepidemeology of hMPV are very limited in China. To assess the seroprevalence of hMPV infection in China, we tested a total of 1,156 serum specimens for the presence of anti-hMPV IgG antibody in children and adults free of acute respiratory illness in Beijing, China by using hMPV nucleocapsid (N) protein as an antigen. As a control, we used the human serum antibody against the N protein of human respiratory syncytial virus (hRSV), the most important viral agent responsible for ARIs in children. RESULTS: The seropositive rate for hMPV increased steadily with age from 67% at 1-6 mo to 100% at age 20. However, the rate dropped slightly between 6 mo and 1 yr of age. The seropositive rate for hRSV also increased steadily with age from 71% at 1-6 mo to 100% at age 20. In children aged six months to six years, the seropositive rates for the anti-hRSV IgG antibody were significantly higher than those for hMPV. Additionally, IgG antibody titers to hMPV and hRSV were significantly higher in adults than in young children. Consistent with the seropositive rates, the geometric mean titer of anti-hMPV IgG antibody was lower than that of anti-hRSV IgG antibody in children aged six months to six years. CONCLUSIONS: Our results indicate that similar to hRSV, exposure to hMPV is ubiquitous in the Beijing population. However, the seroprevalence of anti-hMPV IgG antibody is lower than that of hRSV in children between six months and six years old, which suggests a different number of repeat infections or a different response to infections.


id: cord-254713-ghcwfcx2
title: Detection of new genetic variants of Betacoronaviruses in Endemic Frugivorous Bats of Madagascar
author: Razanajatovo, Norosoa H
date: 2015-03-12
txt: ./txt/cord-254713-ghcwfcx2.txt
cache: ./cache/cord-254713-ghcwfcx2.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/s12985-015-0271-y
doi: 10.1186/s12985-015-0271-y
summary:
  RESULTS: From 351 frugivorous bats, we detected 14 coronaviruses from two endemic bats species, of which 13 viruses were identified from Pteropus rufus and one from Eidolon dupreanum, giving an overall prevalence of 4.5%. Studies which aimed to identify potential reservoirs of emerging human CoVs have revealed that the Betacoronavirus SARS-CoV was closely related to CoVs detected in bats, specifically members of the genus (Rhinolophus), which brought the hypothesis of a spillover of this virus to several animal species (including civet cats and raccoons) sold in Chinese markets as bushmeat for human consumption [9] [10] [11] . A total of 351 bats belonging to 3 endemic bat species of the family Pteropodidae were captured and sampled: Rousettus madagascariensis (n = 179), Pteropus rufus (n = 76) and Eidolon dupreanum (n = 96) ( Table 1) . In the context of this study, we detected 14 coronaviruses forming nine genetically distinct strains in two endemic Malagasy frugivorous bat species.

abstract:
  BACKGROUND: Bats are amongst the natural reservoirs of many coronaviruses (CoVs) of which some can lead to severe infection in human. African bats are known to harbor a range of pathogens (e.g., Ebola and Marburg viruses) that can infect humans and cause disease outbreaks. A recent study in South Africa isolated a genetic variant closely related to MERS-CoV from an insectivorous bat. Though Madagascar is home to 44 bat species (41 insectivorous and 3 frugivorous) of which 34 are endemic, no data exists concerning the circulation of CoVs in the island’s chiropteran fauna. Certain Malagasy bats can be frequently found in close contact with humans and frugivorous bats feed in the same trees where people collect and consume fruits and are hunted and consumed as bush meat. The purpose of our study is to detect and identify CoVs from frugivorous bats in Madagascar to evaluate the risk of human infection from infected bats. METHODS: Frugivorous bats belonging to three species were captured in four different regions of Madagascar. We analyzed fecal and throat swabs to detect the presence of virus through amplification of the RNA-dependent RNA polymerase (RdRp) gene, which is highly conserved in all known coronaviruses. Phylogenetic analyses were performed from positive specimens. RESULTS: From 351 frugivorous bats, we detected 14 coronaviruses from two endemic bats species, of which 13 viruses were identified from Pteropus rufus and one from Eidolon dupreanum, giving an overall prevalence of 4.5%. Phylogenetic analysis revealed that the Malagasy strains belong to the genus Betacoronavirus but form three distinct clusters, which seem to represent previously undescribed genetic lineages. CONCLUSIONS: Our findings suggest that CoVs circulate in frugivorous bats of Madagascar, demonstrating the needs to evaluate spillover risk to human populations especially for individuals that hunt and consume infected bats. Possible dispersal mechanisms as to how coronaviruses arrived on Madagascar are discussed.


id: cord-258935-tatae3hs
author: Lin, Wenyao
date: 2011-06-04
txt: ./txt/cord-258935-tatae3hs.txt
cache: ./cache/cord-258935-tatae3hs.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/21639929/
doi: 10.1186/1743-422x-8-273
summary:
  title: A baculovirus dual expression system-based vaccine confers complete protection against lethal challenge with H9N2 avian influenza virus in mice To evaluate the potency of the recombinant baculovirus BV-Dual-HA against lethal influenza virus challenge, the immunized mice were challenged with 50 MLD 50 of H9N2 V strain on day 42. Most mice vaccinated with pc-HA, AcMNPV-WT, or PBS had detectable lung virus titers A B Figure 3 Antibody responses in immunized mice with BV-Dual-HA. From these results, it is obvious that immunization with BV-Dual-HA can induce a robust antibody response and confer complete protection against lethal virus challenge in a mouse model, indicating that BV-Dual-HA is potential candidate vaccine that can prevent and control the pandemic spread of the H9N2 influenza virus. Baculovirus induces an innate immune response and confers protection from lethal influenza virus infection in mice A pseudotype baculovirus-mediated vaccine confers protective immunity against lethal challenge with H5N1 avian influenza virus in mice and chickens

title:
  A baculovirus dual expression system-based vaccine confers complete protection against lethal challenge with H9N2 avian influenza virus in mice

abstract:
  BACKGROUND: Avian influenza viruses of H9N2 subtype have become highly prevalent in avian species. Although these viruses generally cause only mild to moderate disease, they can infect a wide variety of species, including chickens, quail, turkeys, ducks, geese, pheasant, partridge, and pigeon, even transmitted to mammalian species, including humans, accelerating the efforts to devise protective strategies against them. RESULTS: The results showed that stronger immune responses were induced in a mouse model immunized with BV-Dual-HA than in those vaccinated with a DNA vaccine encoding the same antigen. Moreover, complete protection against lethal challenge with H9N2 virus was observed in mice. CONCLUSION: BV-Dual-HA could be utilized as a vaccine candidate against H9N2 virus infection.


id: cord-261579-f0prnpsu
author: Eichler, Robert
date: 2006-05-31
txt: ./txt/cord-261579-f0prnpsu.txt
cache: ./cache/cord-261579-f0prnpsu.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/16737539/
doi: 10.1186/1743-422x-3-41
summary:
  title: The role of single N-glycans in proteolytic processing and cell surface transport of the Lassa virus glycoprotein GP-C In this report, we investigated the effect of each N-glycan on proteolytic cleavage and cell surface transport by disrupting the consensus sequences of eleven potential N-glycan attachment sites individually. To address this question, we investigated in the present report each potential N-glycosylation site of the Lassa virus glycoprotein concerning N-glycan maturation, cleavage of GP-C and glycoprotein transport to the cell surface. In order to investigate the importance of the individual N-glycosylation sites of Lassa virus GP-C for intracellular trafficking we analysed cell surface expression of the mutated glycoproteins using a biotinylation approach. Taken together, our study suggest that individual N-linked oligosaccharides of the Lassa virus glycoprotein differ greatly in terms of their importance for correct protein folding which seems to be important for activation cleavage by SKI-1/S1P.

title:
  The role of single N-glycans in proteolytic processing and cell surface transport of the Lassa virus glycoprotein GP-C

abstract:
  Lassa virus glycoprotein is synthesised as a precursor (preGP-C) into the lumen of the endoplasmic reticulum. After cotranslational cleavage of the signal peptide, the immature GP-C is posttranslationally processed into the N-terminal subunit GP-1 and the C-terminal subunit GP-2 by the host cell subtilase SKI-1/S1P. The glycoprotein precursor contains eleven potential N-glycosylation sites. In this report, we investigated the effect of each N-glycan on proteolytic cleavage and cell surface transport by disrupting the consensus sequences of eleven potential N-glycan attachment sites individually. Five glycoprotein mutants with disrupted N-glycosylation sites were still proteolytically processed, whereas the remaining N-glycosylation sites are necessary for GP-C cleavage. Despite the lack of proteolytic processing, all cleavage-defective mutants were transported to the cell surface and remained completely endo H-sensitive. The findings indicate that N-glycans are needed for correct conformation of GP-C in order to be cleaved by SKI-1/S1P.


id: cord-261634-vfe1lawl
title: The effect of temperature on persistence of SARS-CoV-2 on common surfaces
author: Riddell, Shane
date: 2020-10-07
txt: ./txt/cord-261634-vfe1lawl.txt
cache: ./cache/cord-261634-vfe1lawl.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/33028356/
doi: 10.1186/s12985-020-01418-7
summary:
  Currently, there are conflicting reports on the survivability of SARS-CoV-2, with data ranging from 3 to 14 days at room temperature for a single surface type, stainless steel Open Access *Correspondence: Shane.Riddell@csiro.au Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australian Centre for Disease Preparedness, Geelong, VIC, Australia [5, 11] . At 20 °C, infectious SARS-CoV-2 virus was still detectable after 28 days post inoculation, for all non-porous surfaces tested (glass, polymer note, stainless steel, vinyl and paper notes). The present study has demonstrated that in controlled conditions, SARS-CoV-2 at a starting viral load and in a fluid matrix equivalent to that typically excreted by infected patients, remains viable for at least 28 days when dried onto non-porous surfaces at 20 °C and 50% relative humidity. It is important to note that after 28 days, infectious SARS-CoV-2 was also recovered from stainless steel, vinyl and glass, suggesting survivability on paper or polymer banknotes was not very different from the other non-porous surfaces studied.

abstract:
  BACKGROUND: The rate at which COVID-19 has spread throughout the globe has been alarming. While the role of fomite transmission is not yet fully understood, precise data on the environmental stability of SARS-CoV-2 is required to determine the risks of fomite transmission from contaminated surfaces. METHODS: This study measured the survival rates of infectious SARS-CoV-2, suspended in a standard ASTM E2197 matrix, on several common surface types. All experiments were carried out in the dark, to negate any effects of UV light. Inoculated surfaces were incubated at 20 °C, 30 °C and 40 °C and sampled at various time points. RESULTS: Survival rates of SARS-CoV-2 were determined at different temperatures and D-values, Z-values and half-life were calculated. We obtained half lives of between 1.7 and 2.7 days at 20 °C, reducing to a few hours when temperature was elevated to 40 °C. With initial viral loads broadly equivalent to the highest titres excreted by infectious patients, viable virus was isolated for up to 28 days at 20 °C from common surfaces such as glass, stainless steel and both paper and polymer banknotes. Conversely, infectious virus survived less than 24 h at 40 °C on some surfaces. CONCLUSION: These findings demonstrate SARS-CoV-2 can remain infectious for significantly longer time periods than generally considered possible. These results could be used to inform improved risk mitigation procedures to prevent the fomite spread of COVID-19.


id: cord-262599-19aj551d
author: Fongaro, Gislaine
date: 2013-05-28
txt: ./txt/cord-262599-19aj551d.txt
cache: ./cache/cord-262599-19aj551d.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/1743-422x-10-166
doi: 10.1186/1743-422x-10-166
summary:
  This study aimed to assess the integrity and viability of human adenovirus (HAdV) in environmental water and evaluate circulating strains by molecular characterization in three sites of the water supply in Florianópolis, Santa Catarina Island, Brazil: Peri Lagoon water, spring source water, and water from the public water supply system. ICC-RT-qPCR, a very sensitive and rapid technique, was able to detect as low as 1 × 10(2) HAdV genome copies per milliliter of infectious viral particles in the environmental water samples. We suggest that HAdV can be efficiently used as a marker of environmental and drinking water contamination and ICC-RT-qPCR demonstrated greater sensitivity and speed of detection of infectious viral particles compared to PA. In this study, we aimed to assess the integrity and viability of human adenovirus (HAdV) detected in environmental water samples and also to use molecular characterization to evaluate circulating strains.

title:
  Evaluation and molecular characterization of human adenovirus in drinking water supplies: viral integrity and viability assays

abstract:
  BACKGROUND: Human adenoviruses (HAdVs) are the second-leading cause of childhood gastroenteritis worldwide. This virus is commonly found in environmental waters and is very resistant to water disinfection and environmental stressors, especially UV light inactivation. Molecular techniques, such as PCR-based methods (Polymerase Chain Reaction), are commonly used to detect and identify viral contamination in water, although PCR alone does not allow the discrimination between infectious and non-infectious viral particles. A combination of cell culture and PCR has allowed detection of infectious viruses that grow slowly or fail to produce cytopathic effects (CPE) in cell culture. This study aimed to assess the integrity and viability of human adenovirus (HAdV) in environmental water and evaluate circulating strains by molecular characterization in three sites of the water supply in Florianópolis, Santa Catarina Island, Brazil: Peri Lagoon water, spring source water, and water from the public water supply system. METHODS: Water samples were collected, concentrated and HAdV quantified by real-time PCR. Viral integrity was evaluated by enzymatic assay (DNase I) and infectivity by plaque assay (PA) and integrated cell culture using transcribed mRNA (ICC-RT-qPCR). Samples containing particles of infectious HAdV were selected for sequencing and molecular characterization. RESULTS: The analyzed sites contained 83, 66 and 58% undamaged HAdV particles (defined as those in which the genetic material is protected by the viral capsid) at Peri Lagoon, spring source water and public supply system water, respectively. Of these, 66% of the particles (by PA) and 75% (by ICC-RT-qPCR) HAdV were shown to be infectious, due to being undamaged in Peri Lagoon, 33% (by PA) and 58% (by ICC-RT-qPCR) in spring source water and 8% (by PA) and 25% (by ICC-RT-qPCR) in the public water supply system. ICC-RT-qPCR, a very sensitive and rapid technique, was able to detect as low as 1 × 10(2) HAdV genome copies per milliliter of infectious viral particles in the environmental water samples. The molecular characterization studies indicated that HAdV-2 was the prevalent serotype. CONCLUSIONS: These results indicate a lack of proper public health measures. We suggest that HAdV can be efficiently used as a marker of environmental and drinking water contamination and ICC-RT-qPCR demonstrated greater sensitivity and speed of detection of infectious viral particles compared to PA.


id: cord-262904-0b0ljjq1
title: Prediction and evolution of B cell epitopes of surface protein in SARS-CoV-2
author: Lon, Jerome Rumdon
date: 2020-10-29
txt: ./txt/cord-262904-0b0ljjq1.txt
cache: ./cache/cord-262904-0b0ljjq1.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/s12985-020-01437-4
doi: 10.1186/s12985-020-01437-4
summary:
  It is worth mentioning that all 6 identified epitopes were conserved in nearly 3500 SARS-CoV-2 genomes, showing that it is helpful to obtain stable and long-acting epitopes under the condition of high frequency of amino acid mutation, which deserved further study at the experiment level. On this basis, we predicted the linear and conformational B cell epitopes, analyzed the conservation of the epitopes, the adaptability and other evolutionary characteristics of the surface protein, which provided a theoretical basis for the vaccine development and prevention of SARS-CoV-2. With the amino acid sequences of the surface protein of SARS-CoV-2 of NC_045512.2 as templates, we predicted the 3D structure of E and M protein through the online server SWISS-MODEL [10] based on homology modeling method, selected the optimal structure based on the template identity and GMQE value [10] , and the rationality of the structure was evaluated by Ramachandran plot [11] with PDBsum server.

abstract:
  BACKGROUND: In order to obtain antibodies that recognize natural proteins, it is possible to predict the antigenic determinants of natural proteins, which are eventually embodied as polypeptides. The polypeptides can be coupled with corresponding vectors to stimulate the immune system to produce corresponding antibodies, which is also a simple and effective vaccine development method. The discovery of epitopes is helpful to the development of SARS-CoV-2 vaccine. METHODS: The analyses were related to epitopes on 3 proteins, including spike (S), envelope (E) and membrane (M) proteins, which are located on the lipid envelope of the SARS-CoV-2. Based on the NCBI Reference Sequence: NC_045512.2, the conformational and linear B cell epitopes of the surface protein were predicted separately by various prediction methods. Furthermore, the conservation of the epitopes, the adaptability and other evolutionary characteristics were also analyzed, the sequences of the whole genome of SARS-CoV-2 were obtained from the GISAID. RESULTS: 7 epitopes were predicted, including 6 linear epitopes and 1 conformational epitope. One of the linear and one of the conformational consist of identical sequence, but represent different forms of epitopes. It is worth mentioning that all 6 identified epitopes were conserved in nearly 3500 SARS-CoV-2 genomes, showing that it is helpful to obtain stable and long-acting epitopes under the condition of high frequency of amino acid mutation, which deserved further study at the experiment level. CONCLUSION: The findings would facilitate the vaccine development, had the potential to be directly applied on the prevention in this disease, but also have the potential to prevent the possible threats caused by other types of coronavirus.


id: cord-263469-2w26l80a
author: Garry, Courtney E
date: 2009-09-18
txt: ./txt/cord-263469-2w26l80a.txt
cache: ./cache/cord-263469-2w26l80a.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/1743-422x-6-145
doi: 10.1186/1743-422x-6-145
summary:
  RESULTS: Class III viral fusion proteins (VFP) encoded by members of the Rhabdoviridae, Herpesviridae and Baculoviridae have an internal fusion domain comprised of beta sheets, other beta sheet domains, an extended alpha helical domain, a membrane proximal stem domain and a carboxyl terminal anchor. Structural models were established for BDV G based on the post-fusion structure of a prototypic class III VFP, vesicular stomatitis virus glycoprotein (VSV G). Members of the Flavivirus genus of the Flaviviridae and the Alphavirus genus of the Togaviridae encode class II viral fusion proteins (class II or β-penetrenes) possessing three domains (I-III) of mostly antiparallel β-sheets, a membrane proximal α-helical stem domain and a carboxyl terminal anchor [24] [25] [26] . The three VFP classes for enveloped virus membrane glycoproteins were established based on structural similarities in the post-fusion configurations.

title:
  Proteomics computational analyses suggest that the bornavirus glycoprotein is a class III viral fusion protein (γ penetrene)

abstract:
  BACKGROUND: Borna disease virus (BDV) is the type member of the Bornaviridae, a family of viruses that induce often fatal neurological diseases in horses, sheep and other animals, and have been proposed to have roles in certain psychiatric diseases of humans. The BDV glycoprotein (G) is an extensively glycosylated protein that migrates with an apparent molecular mass of 84,000 to 94,000 kilodaltons (kDa). BDV G is post-translationally cleaved by the cellular subtilisin-like protease furin into two subunits, a 41 kDa amino terminal protein GP1 and a 43 kDa carboxyl terminal protein GP2. RESULTS: Class III viral fusion proteins (VFP) encoded by members of the Rhabdoviridae, Herpesviridae and Baculoviridae have an internal fusion domain comprised of beta sheets, other beta sheet domains, an extended alpha helical domain, a membrane proximal stem domain and a carboxyl terminal anchor. Proteomics computational analyses suggest that the structural/functional motifs that characterize class III VFP are located collinearly in BDV G. Structural models were established for BDV G based on the post-fusion structure of a prototypic class III VFP, vesicular stomatitis virus glycoprotein (VSV G). CONCLUSION: These results suggest that G encoded by members of the Bornavirdae are class III VFPs (gamma-penetrenes).


id: cord-263976-b9shffb3
author: Shaukat, Shahzad
date: 2014-08-12
txt: ./txt/cord-263976-b9shffb3.txt
cache: ./cache/cord-263976-b9shffb3.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/25112200/
doi: 10.1186/1743-422x-11-146
summary:
  One sequence independent method, Virus Discovery based on cDNA Amplified Fragment Length Polymorphism (VIDISCA) is capable of identifying viruses that would have remained unidentified in standard diagnostics or cell cultures. Therefore, a simplified VIDISCA protocol was developed, which lacks the last amplification round, and evaluated using viruses that were cultured from stool samples of acute flaccid paralysis children, and which had remained unrecognized on both cell culture and enterovirus specific real-time PCR. On the other hand, genetic analysis in ORF2 gene (capsid protein) showed that PAK-NIH-VS908 shared 99.7% nucleotide and 100% amino acid similarities with HAstV type 3 isolate IDH2211 (AB54844), a finding which matches with the results from VIDISCA and we conclude that the astrovirus is a recombinant, with a recombination site between ORF1a and ORF2.

title:
  Identification and characterization of unrecognized viruses in stool samples of non-polio acute flaccid paralysis children by simplified VIDISCA

abstract:
  BACKGROUND: The use of sequence independent methods combined with next generation sequencing for identification purposes in clinical samples appears promising and exciting results have been achieved to understand unexplained infections. One sequence independent method, Virus Discovery based on cDNA Amplified Fragment Length Polymorphism (VIDISCA) is capable of identifying viruses that would have remained unidentified in standard diagnostics or cell cultures. METHODS: VIDISCA is normally combined with next generation sequencing, however, we set up a simplified VIDISCA which can be used in case next generation sequencing is not possible. Stool samples of 10 patients with unexplained acute flaccid paralysis showing cytopathic effect in rhabdomyosarcoma cells and/or mouse cells were used to test the efficiency of this method. To further characterize the viruses, VIDISCA-positive samples were amplified and sequenced with gene specific primers. RESULTS: Simplified VIDISCA detected seven viruses (70%) and the proportion of eukaryotic viral sequences from each sample ranged from 8.3 to 45.8%. Human enterovirus EV-B97, EV-B100, echovirus-9 and echovirus-21, human parechovirus type-3, human astrovirus probably a type-3/5 recombinant, and tetnovirus-1 were identified. Phylogenetic analysis based on the VP1 region demonstrated that the human enteroviruses are more divergent isolates circulating in the community. CONCLUSION: Our data support that a simplified VIDISCA protocol can efficiently identify unrecognized viruses grown in cell culture with low cost, limited time without need of advanced technical expertise. Also complex data interpretation is avoided thus the method can be used as a powerful diagnostic tool in limited resources. Redesigning the routine diagnostics might lead to additional detection of previously undiagnosed viruses in clinical samples of patients. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1743-422X-11-146) contains supplementary material, which is available to authorized users.


id: cord-267736-rya9w6sh
title: Development of an ELISA-array for simultaneous detection of five encephalitis viruses
author: Kang, Xiaoping
date: 2012-02-27
txt: ./txt/cord-267736-rya9w6sh.txt
cache: ./cache/cord-267736-rya9w6sh.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/22369052/
doi: 10.1186/1743-422x-9-56
summary:
  The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus. When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens.

abstract:
  Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.


id: cord-268560-pwps783y
title: EBNA3C interacts with Gadd34 and counteracts the unfolded protein response
author: Garrido, Jose L
date: 2009-12-29
txt: ./txt/cord-268560-pwps783y.txt
cache: ./cache/cord-268560-pwps783y.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/20040105/
doi: 10.1186/1743-422x-6-231
summary:
  In B-cells, Gadd34, and EBNA3C are present in a complex with PP1a using microcystin sepharose affinity purification, Using a lymphoblastoid cell line in which EBNA3C protein levels are conditional on hydroxytamoxifen, surprisingly, we found that (i) EBNA3C maintains phosphorylation of eIF2α at serine 51, and (ii) protects against ER stress induced activation of the unfolded protein response as measured by XBP1 (u) versus XBP1(s) protein expression and N-terminal ATF6 cleavage. Recombinant EBV containing a stop codon in the EBNA3C ORF is able to cause B-cell transformation only when transcomplemented for wildtype EBNA3C either in cis or trans, and LCLs immortal-ized by recombinant EBV containing a conditional EBNA3C gene, undergo growth arrest when EBNA3C expression is turned off [5] [6] [7] EBNA3C co-activates transcription with EBNA2 at the viral LMP-1 promoter, as well as heterologous reporter systems designed to test p300 function.

abstract:
  EBNA3C is an EBV-encoded nuclear protein, essential for proliferation of EBV infected B-lymphocytes. Using EBNA3C amino acids 365-545 in a yeast two hybrid screen, we found an interaction with the Growth Arrest and DNA-damage protein, Gadd34. When both proteins are overexpressed, Gadd34 can interact with EBNA3C in both nuclear and cytoplasmic compartments. Amino acids 483-610 of Gadd34, including the two PP1a interaction, and the HSV-1 ICPγ34.5 homology domains, are required for the interaction. Furthermore, interaction is lost with a mutant of EBNA3C ((509 )DVIEVID (515)→AVIAVIA), that abolishes EBNA3C coactivation ability as well as SUMO interaction[1]. In B-cells, Gadd34, and EBNA3C are present in a complex with PP1a using microcystin sepharose affinity purification, Using a lymphoblastoid cell line in which EBNA3C protein levels are conditional on hydroxytamoxifen, surprisingly, we found that (i) EBNA3C maintains phosphorylation of eIF2α at serine 51, and (ii) protects against ER stress induced activation of the unfolded protein response as measured by XBP1 (u) versus XBP1(s) protein expression and N-terminal ATF6 cleavage. In reporter assays, overexpression of Gadd34 enhances EBNA3C's ability to co-activate EBNA2 activation of the LMP1 promoter. Collectively the data suggest that EBNA3C interacts with Gadd34, activating the upstream component of the UPR (eIF2α phosphorylation) while preventing downstream UPR events (XBP1 activation and ATF6 cleavage).


id: cord-269277-x5c5ogo7
title: Key elements of the human bocavirus type 1 (HBoV1) promoter and its trans-activation by NS1 protein
author: Li, Jingjing
date: 2013-10-27
txt: ./txt/cord-269277-x5c5ogo7.txt
cache: ./cache/cord-269277-x5c5ogo7.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/24161033/
doi: 10.1186/1743-422x-10-315
summary:
  The pGL3-Bov-EGFP vector containing nt 1-252 was transfected into 293 T, A549, HeLa and WI-38 cell lines to assay the activity of the promoter of HBoV1 in these mammalian cells. Here, the activity of the HBoV1 promoter in cells co-transfected with P1 and pGL3-pCMV-NS1mut vector (no NS1 protein expression) was used as a control. In our recent study, NS1 transcripts from the left-hand of the viral genome were detected in pWHL-1 transfected 293 T cells and other mammalian cells including HeLa, A549, WI-38 cell lines (data not shown), suggesting that this promoter is unique and active in most mammalian cells. For detection of HBoV1 promoter activity, mammalian cell lines including 293 T, A549, HeLa and WI-38 were transfected with the pGL3-Bov-EGFP construct and the pEGFP-N1 control vector in 24-well plates. Key elements of the human bocavirus type 1 (HBoV1) promoter and its trans-activation by NS1 protein

abstract:
  BACKGROUND: Human bocavirus (HBoV), a parvovirus, is suspected to be an etiologic agent of respiratory disease and gastrointestinal disease in humans. All mRNAs of HBoV1 are transcribed from a single promoter. METHODS: In this study, we constructed EGFP and luciferase reporter gene vectors under the control of the HBoV1 full promoter (nt 1–252) and its mutated variants, respectively. Fluorescence microscopy was used to observe expression activities of the EGFP. Dual-luciferase reporter vectors were employed in order to evaluate critical promoter elements and the effect of NS1 protein on promoter activity. RESULTS: The HBoV1 promoter activity was about 2.2-fold and 1.9-fold higher than that of the CMV promoter in 293 T and HeLa cells, respectively. The putative transcription factor binding region of the promoter was identified to be located between nt 96 and nt 145. Mutations introduced in the CAAT box of the HBoV1 promoter reduced promoter activity by 34%, whereas nucleotide substitutions in the TATA box had no effect on promoter activity. The HBoV1 promoter activities in 293 T and HeLa cells, in the presence of NS1 protein, were 2- to 2.5-fold higher than those in the absence of NS1 protein. CONCLUSION: The HBoV1 promoter was highly active in 293 T and HeLa cell lines, and the sequence from nt 96 to nt 145 was critical for the activity of HBoV1 promoter. The CAAT box, in contrast to the TATA-box, was important for optimum promoter activity. In addition, the transcriptional activity of this promoter could be trans-activated by the viral nonstructural protein NS1 in these cells.


id: cord-271130-6s79q1c1
author: Filoni, Claudia
date: 2017-11-17
txt: ./txt/cord-271130-6s79q1c1.txt
cache: ./cache/cord-271130-6s79q1c1.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/29149857/
doi: 10.1186/s12985-017-0889-z
summary:
  title: Putative progressive and abortive feline leukemia virus infection outcomes in captive jaguarundis (Puma yagouaroundi) Thus, the aim of this study was to perform additional serological and molecular tests and monitor the population of jaguarundis at FPZSP for FeLV infection and development of FeLV-related diseases for 5 years (2003) (2004) (2005) (2006) (2007) . Two captive-born male jaguarundis, the geriatric #1 and the mature adult #4, presented serological and molecular FeLV test results similar to the progressive FeLV infection outcome in domestic cats [25] . Moreover, consistent with findings in domestic cats with a progressive FeLV infection, no antibodies to FeLV antigens were detected in jaguarundis #1 and #4. Two captive-born jaguarundis, #2 and #22, presented test results similar to those reported for domestic cats with abortive FeLV infection and seroconversion as the only marker of FeLV exposure [28] .

title:
  Putative progressive and abortive feline leukemia virus infection outcomes in captive jaguarundis (Puma yagouaroundi)

abstract:
  BACKGROUND: Feline leukemia virus (FeLV) is an exogenous gammaretrovirus of domestic cats (Felis catus) and some wild felids. The outcomes of FeLV infection in domestic cats vary according to host susceptibility, virus strain, and infectious challenge dose. Jaguarundis (Puma yagouaroundi) are small wild felids from South and Central America. We previously reported on FeLV infections in jaguarundis. We hypothesized here that the outcomes of FeLV infection in P. yagouaroundi mimic those observed in domestic cats. The aim of this study was to investigate the population of jaguarundis at Fundação Parque Zoológico de São Paulo for natural FeLV infection and resulting outcomes. METHODS: We investigated the jaguarundis using serological and molecular methods and monitored them for FeLV-related diseases for 5 years. We retrieved relevant biological and clinical information for the entire population of 23 jaguarundis held at zoo. Post-mortem findings from necropsies were recorded and histopathological and immunohistopathological analyses were performed. Sequencing and phylogenetic analyses were performed for FeLV-positive samples. For sample prevalence, 95% confidence intervals (CI) were calculated. Fisher’s exact test was used to compare frequencies between infected and uninfected animals. P-values <0.05 were considered significant. RESULTS: In total, we detected evidence of FeLV exposure in four out of 23 animals (17%; 95% CI 5–39%). No endogenous FeLV (enFeLV) sequences were detected. An intestinal B-cell lymphoma in one jaguarundi was not associated with FeLV. Two jaguarundis presented FeLV test results consistent with an abortive FeLV infection with seroconversion, and two other jaguarundis had results consistent with a progressive infection and potentially FeLV-associated clinical disorders and post-mortem changes. Phylogenetic analysis of env revealed the presence of FeLV-A, a common origin of the virus in both animals (100% identity) and the closest similarity to FeLV-FAIDS and FeLV-3281 (98.4% identity), originally isolated from cats in the USA. CONCLUSIONS: We found evidence of progressive and abortive FeLV infection outcomes in jaguarundis, and domestic cats were probably the source of infection in these jaguarundis.


id: cord-271868-giea69b5
author: Firth, Andrew E
date: 2009-09-08
txt: ./txt/cord-271868-giea69b5.txt
cache: ./cache/cord-271868-giea69b5.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/1743-422x-6-136
doi: 10.1186/1743-422x-6-136
summary:
  These viruses have a linear positive-sense ssRNA genome of ~25-30 kb, encoding a large polyprotein that is expressed from the genomic RNA, and several additional proteins expressed from a nested set of 3''-coterminal subgenomic RNAs. In this brief report, we describe the bioinformatic discovery of a new, apparently coding, ORF that overlaps the 5'' end of the polyprotein coding sequence, ORF1a, in the +2 reading frame. As with other members of the order Nidovirales, these viruses have a linear positive-sense ssRNA genome encoding a large replicase polyprotein that is expressed from the genomic RNA (ORF1a and, via ribosomal frameshifting, an ORF1a-ORF1b fusion product), and a number of other proteinsincluding the structural proteins -which are translated from a nested set of 3''-coterminal sub-genomic RNAs ( Figure 1A ) [1] [2] [3] [4] [5] [6] .

title:
  A case for a CUG-initiated coding sequence overlapping torovirus ORF1a and encoding a novel 30 kDa product

abstract:
  The genus Torovirus (order Nidovirales) includes a number of species that infect livestock. These viruses have a linear positive-sense ssRNA genome of ~25-30 kb, encoding a large polyprotein that is expressed from the genomic RNA, and several additional proteins expressed from a nested set of 3'-coterminal subgenomic RNAs. In this brief report, we describe the bioinformatic discovery of a new, apparently coding, ORF that overlaps the 5' end of the polyprotein coding sequence, ORF1a, in the +2 reading frame. The new ORF has a strong coding signature and, in fact, is more conserved at the amino acid level than the overlapping region of ORF1a. We propose that the new ORF utilizes a non-AUG initiation codon - namely a conserved CUG codon in a strong Kozak context - upstream of the ORF1a AUG initiation codon, resulting in a novel 258 amino acid protein, dubbed '30K'.


id: cord-271948-iq29xqrn
author: Obeng, Billal Musah
date: 2020-07-24
txt: ./txt/cord-271948-iq29xqrn.txt
cache: ./cache/cord-271948-iq29xqrn.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32709248/
doi: 10.1186/s12985-020-01386-y
summary:
  title: Transmitted drug resistance mutations and subtype diversity amongst HIV-1 sero-positive voluntary blood donors in Accra, Ghana BACKGROUND: Detection of HIV-1 transmitted drug resistance (TDR) and subtype diversity (SD) are public health strategies to assess current HIV-1 regimen and ensure effective therapeutic outcomes of antiretroviral therapy (ART) among HIV-1 patients. In this study, drug resistance mutations (DRMs) and SD amongst HIV-1 sero-positive blood donors in Accra, Ghana were characterized. The data obtained would inform the selection of drugs for ART initiation to maximize therapeutic options in drug-naïve HIV-1 patients in Ghana. This study found major drug resistance mutations, E138A and K65R that respectively confer high level resistance to NNRTIs and NRTIs. Although, CRF02_AG was most predominant, the recorded percentage of subtype B and the evolutionary relationship inferred by phylogenetic analysis may suggest possible subtype importation. The data obtained is useful for the selection of drugs for ART initiation to maximize therapeutic outcomes in drug-naïve HIV-1 patients in Ghana.

title:
  Transmitted drug resistance mutations and subtype diversity amongst HIV-1 sero-positive voluntary blood donors in Accra, Ghana

abstract:
  BACKGROUND: Detection of HIV-1 transmitted drug resistance (TDR) and subtype diversity (SD) are public health strategies to assess current HIV-1 regimen and ensure effective therapeutic outcomes of antiretroviral therapy (ART) among HIV-1 patients. Globally, limited data exist on TDR and SD among blood donors. In this study, drug resistance mutations (DRMs) and SD amongst HIV-1 sero-positive blood donors in Accra, Ghana were characterized. METHODS: Purposive sampling method was used to collect 81 HIV sero-positive blood samples from the Southern Area Blood Center and confirmed by INNO-LIA as HIV-1 and/or HIV-2. Viral RNA was only extracted from plasma samples confirmed as HIV-1 positive. Complementary DNA (cDNA) was synthesized using the RNA as a template and subsequently amplified by nested PCR with specific primers. The expected products were verified, purified and sequenced. Neighbour-joining tree with the Kimura’s 2-parameter distances was generated with the RT sequences using Molecular Evolutionary Genetic Analysis version 6.0 (MEGA 6.0). RESULTS: Out of the 81 plasma samples, 60 (74%) were confirmed as HIV-1 sero-positive by INNO-LIA HIVI/II Score kit with no HIV-2 and dual HIV-1/2 infections. The remaining samples, 21 (26%) were confirmed as HIV sero-negative. Of the 60 confirmed positive samples, (32) 53% and (28) 47% were successfully amplified in the RT and PR genes respectively. Nucleotide sequencing of amplified samples revealed the presence of major drug resistance mutations in two (2) samples; E138A in one sample and another with K65R. HIV-1 Subtypes including subtypes A, B, CRF02_AG and CRF09_cpx were found. CONCLUSION: This study found major drug resistance mutations, E138A and K65R in the RT gene that confer high level resistance to most NNRTIs and NRTI respectively. CRF02_AG was most predominant, the recorded percentage of subtype B and the evolutionary relationship inferred by phylogenetic analysis may suggest possible subtype importation. However, a more prospective and detailed analysis is needed to establish this phenomenon. The data obtained would inform the selection of drugs for ART initiation to maximize therapeutic options in drug-naïve HIV-1 patients in Ghana.


id: cord-273122-w9hemznv
author: Jans, Jop
date: 2016-03-22
url: https://www.ncbi.nlm.nih.gov/pubmed/27004689/
doi: 10.1186/s12985-016-0506-6
title:
  Actin- and clathrin-dependent mechanisms regulate interferon gamma release after stimulation of human immune cells with respiratory syncytial virus

abstract:
  BACKGROUND: Respiratory syncytial virus (RSV) can cause recurrent and severe respiratory tract infections. Cytoskeletal proteins are often involved during viral infections, either for cell entry or the initiation of the immune response. The importance of actin and clathrin dynamics for cell entry and the initiation of the cellular immune response against RSV in human immune cells is not known yet. The aim of this study was to investigate the role of actin and clathrin on cell entry of RSV and the subsequent effect on T cell activation and interferon gamma release in human immune cells. METHODS: Peripheral blood mononuclear cells and purified monocytes were isolated from healthy adults and stimulated in vitro with RSV. Actin and clathrin dynamics were inhibited with respectively cytochalasin D and chlorpromazine. T cell receptor signaling was inhibited with cyclosporin A. Flow cytometry was used to determine the role of actin and clathrin on cell entry and T cell activation by RSV. Enzyme-linked immunosorbent assays were used to investigate the contribution of actin and clathrin on the release of interferon gamma. RESULTS: Cell entry, virus gene transcription and interferon gamma release are actin-dependent. Post-endocytic processes like the increased expression of major histocompatibility complex II on monocytes , T cell activation and the release of interferon gamma are clathrin-dependent. Finally, T cell receptor signaling affects T cell activation, whereas soluble interleukin 18 is dispensable. CONCLUSION: Analysis of cell entry and interferon gamma release after infection with RSV reveals the importance of actin- and clathrin-dependent signaling in human immune cells. Insights into the cellular biology of the human immune response against respiratory syncytial virus will provide a better understanding of disease pathogenesis and may prove useful in the development of preventive strategies.


id: cord-273179-bpnak9ov
author: Ma, Fen-lian
date: 2017-08-14
txt: ./txt/cord-273179-bpnak9ov.txt
cache: ./cache/cord-273179-bpnak9ov.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/28806976/
doi: 10.1186/s12985-017-0817-2
summary:
  METHODS: We used real-time TaqMan-based PCR to detect MWPyV in the feces (n = 174) of children with diarrhea, nasopharyngeal aspirates (n = 887) from children with respiratory infections, and sera (n = 200) from healthy adults, and analyzed its clinical characteristics statistically. Therefore, in this study, we used realtime qPCR and DNA sequencing to investigate the presence of MWPyV in fecal samples from 174 children with diarrhea, nasopharyngeal aspirate (NPA) samples from 887 children with acute respiratory tract infections (ARIs), and sera from 200 healthy adults in China. In brief, the analysis of 1261 clinical samples only detected MWPyV in respiratory and fecal specimens from children, suggesting that the establishment of the primary infection occurs at an early age, and that the gastrointestinal and respiratory tracts are sites of viral persistence.

title:
  Quantitative detection of human Malawi polyomavirus in nasopharyngeal aspirates, sera, and feces in Beijing, China, using real-time TaqMan-based PCR

abstract:
  BACKGROUND: Human Malawi polyomavirus (MWPyV) was discovered in 2012, but its prevalence and clinical characteristics are largely unknown. METHODS: We used real-time TaqMan-based PCR to detect MWPyV in the feces (n = 174) of children with diarrhea, nasopharyngeal aspirates (n = 887) from children with respiratory infections, and sera (n = 200) from healthy adults, and analyzed its clinical characteristics statistically. All the MWPyV-positive specimens were also screened for other common respiratory viruses. RESULTS: Sixteen specimens were positive for MWPyV, including 13 (1.47%) respiratory samples and three (1.7%) fecal samples. The samples were all co-infected with other respiratory viruses, most commonly with influenza viruses (69.2%) and human coronaviruses (30.7%). The MWPyV-positive children were diagnosed with bronchopneumonia or viral diarrhea. They ranged in age from 12 days to 9 years, and the most frequent symptoms were cough and fever. CONCLUSIONS: Real-time PCR is an effective tool for the detection of MWPyV in different types of samples. MWPyV infection mainly occurs in young children, and fecal–oral transmission is a possible route of its transmission. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-017-0817-2) contains supplementary material, which is available to authorized users.


id: cord-273711-bxijla09
author: Zhao, Zhixun
date: 2012-02-17
txt: ./txt/cord-273711-bxijla09.txt
cache: ./cache/cord-273711-bxijla09.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/22340205/
doi: 10.1186/1743-422x-9-48
summary:
  title: RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells When Vero cells were transfected with shRNA expression vectors (p61/GFP, p70/GFP, p165/GFP and p296/GFP) and then infected with GTPV, GTPV-ORF095-70 was found to be the most effective inhibition site in decreasing cytopathic effect (CPE) induced by GTPV. These results suggest that the siRNA generated by in vitro transcription effectively and specifically inhibit the expression of GTPV ORF095 conserved regions in BHK-21 cells. To investigate whether or not knockout of ORF095 relieves cytopathic effect (CPE) induced by GTPV, Vero cells were transfected by plasmids expressing ORF095 protein-targeted shRNAs (p61/GFP, p70/GFP, p165/GFP and p296/GFP), respectively. Therefore, ORF095 gene is a good target to suppress GTPV replication by RNAi. In conclusion, this study demonstrates that vector-based shRNA methodology can effectively inhibit GTPV replication on Vero cells. RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells

title:
  RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells

abstract:
  BACKGROUND: Goatpox is an economically important disease in goat and sheep-producing areas of the world. Many vaccine strategies developed to control the disease are not yet completely successful. Hairpin expression vectors have been used to induce gene silencing in a large number of studies on viruses. However, none of these studies has been attempted to study GTPV. In the interest of exploiting improved methods to control goat pox, it is participated that RNAi may provide effective protection against GTPV. In this study we show the suppression of Goatpox virus (GTPV) replication via knockdown of virion core protein using RNA interference. RESULTS: Four short interfering RNA (siRNA) sequences (siRNA-61, siRNA-70, siRNA-165 and siRNA-296) against a region of GTPV ORF095 were selected. Sense and antisense siRNA-encoding sequences separated by a hairpin loop sequence were designed as short hairpin RNA (shRNA) expression cassettes under the control of a human U6 promoter. ORF095 amplicon was generated using PCR, and then cloned into pEGFP-N1 vector, named as p095/EGFP. p095/EGFP and each of the siRNA expression cassettes (p61, p70, p165 and p296) were co-transfected into BHK-21 cells. Fluorescence detection, flow cytometric analysis, retro transcription PCR (RT-PCR) and real time PCR were used to check the efficiency of RNAi. The results showed that the ORF095-specific siRNA-70 effectively down-regulated the expression of ORF095. When Vero cells were transfected with shRNA expression vectors (p61/GFP, p70/GFP, p165/GFP and p296/GFP) and then infected with GTPV, GTPV-ORF095-70 was found to be the most effective inhibition site in decreasing cytopathic effect (CPE) induced by GTPV. The results presented here indicated that DNA-based siRNA could effectively inhibit the replication of GTPV (approximately 463. 5-fold reduction of viral titers) on Vero cells. CONCLUSIONS: This study demonstrates that vector-based shRNA methodology can effectively inhibit GTPV replication on Vero cells. Simultaneously, this work represents a strategy for controlling goatpox, potentially facilitating new experimental approaches in the analysis of both viral and cellular gene functions during of GTPV infection.


id: cord-274375-a11ztdpg
title: Analysis of synonymous codon usage in Hepatitis A virus
author: Zhang, Yiqiang
date: 2011-04-16
txt: ./txt/cord-274375-a11ztdpg.txt
cache: ./cache/cord-274375-a11ztdpg.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/1743-422x-8-174
doi: 10.1186/1743-422x-8-174
summary:
  In surprise, only A 3 % has a significant correlation with both principle axes which represent the major trend in codon usage variation, suggesting that nucleotide A is the major factor influencing the synonymous codon usage pattern of HAV genome. Despite the ratio of U 3 % was the highest, the major compositional constraint, which shaping the synonymous codon usage pattern of HAV genome, was from the percent of nucleotide A on the third codon position (Table 4) . This discovery was different from many reports which suggest that C+G compositional constraints were the major factor influencing codon usage bias in virus genome [18, 29, 30] . Mutational pressure and natural selection are generally thought to be the main factors that account for codon usage variation between genes in different Table 3 Summary of correlation analysis between the A, U, C, G contents and A 3 , U 3 , C 3 , G 3 organisms [14] [15] [16] [17] [18] [19] [20] [21] .

abstract:
  BACKGROUND: Hepatitis A virus is the causative agent of type A viral hepatitis, which causes occasional acute hepatitis. Nevertheless, little information about synonymous codon usage pattern of HAV genome in the process of its evolution is available. In this study, the key genetic determinants of codon usage in HAV were examined. RESULTS: The overall extent of codon usage bias in HAV is high in Picornaviridae. And the patterns of synonymous codon usage are quite different in HAV genomes from different location. The base composition is closely correlated with codon usage bias. Furthermore, the most important determinant that results in such a high codon bias in HAV is mutation pressure rather than natural selection. CONCLUSIONS: HAV presents a higher codon usage bias than other members of Picornaviridae. Compositional constraint is a significant element that influences the variation of synonymous codon usage in HAV genome. Besides, mutation pressure is supposed to be the major factor shaping the hyperendemic codon usage pattern of HAV.


id: cord-276550-1in7m56w
author: Abdel-Moneim, Ahmed S
date: 2006-09-20
txt: ./txt/cord-276550-1in7m56w.txt
cache: ./cache/cord-276550-1in7m56w.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/16987422/
doi: 10.1186/1743-422x-3-78
summary:
  title: S1 gene sequence analysis of a nephropathogenic strain of avian infectious bronchitis virus in Egypt RESULTS: Infectious bronchitis virus (IBV) strain closely related to Massachusetts (Mass) serotype was isolated from broiler chickens suffering from severe renal and respiratory distresses. Protection based criteria were: virus re-isolation attempts from trachea, tracheal and renal histopathology as well as IBV antigens detection by immunofluorescent antibody technique in kidney sections. In the present study, Egypt/F/03 was isolated from 25day-old broiler chickens in Fayoum Governorate, identified by Dot-ELISA, RT-PCR and sequenced to determine its serotype. In this study, an Egyptian IBV strain; Egypt\F/03 was isolated from a tissue pool of kidney and trachea from unvaccinated broiler flock with a history of respiratory and renal disease. Challenge experiments to evaluate cross-protection induced at the trachea and kidney level by vaccine strains and Belgian nephropathogenic isolates of avian infectious bronchitis virus

title:
  S1 gene sequence analysis of a nephropathogenic strain of avian infectious bronchitis virus in Egypt

abstract:
  BACKGROUND: Infectious bronchitis is highly contagious and constitutes one of the most common and difficult poultry diseases to control. IBV is endemic in probably all countries that raise chickens. It exists as dozens of serotypes/genotypes. Only a few amino acid differences in the S1 protein of vaccine and challenge strains of IBV may result in poor protection. Tropism of IBV includes the respiratory tract tissues, proventriculus and caecal tonsils of the alimentary tract, the oviduct and the kidney. RESULTS: Infectious bronchitis virus (IBV) strain closely related to Massachusetts (Mass) serotype was isolated from broiler chickens suffering from severe renal and respiratory distresses. The isolate was serologically identified by Dot-ELISA and further characterized by RT-PCR then genotyped using S1 gene sequence analysis. Alignment of the S1 sequence of the isolate with 16 IBV strains revealed high homology to isolates related to Mass serotype. Inoculation with the strain reproduced the disease in experimental 1-day-old chickens and resulted in 20% mortality, severe renal and moderate respiratory distresses. Marked histopathological changes in both kidney and trachea were observed in experimentally infected chickens. A protection study using the H120 live attenuated vaccine showed low protection rate in spite of high S1 sequence homology (97%). Protection based criteria were: virus re-isolation attempts from trachea, tracheal and renal histopathology as well as IBV antigens detection by immunofluorescent antibody technique in kidney sections. CONCLUSION: Periodical evaluation of cross-protective capabilities of IBV vaccine(s) versus recently recovered field isolates should be performed to ensure optimum control of IBV.


id: cord-277547-2vim1wno
title: Antiviral activity of four types of bioflavonoid against dengue virus type-2
author: Zandi, Keivan
date: 2011-12-28
txt: ./txt/cord-277547-2vim1wno.txt
cache: ./cache/cord-277547-2vim1wno.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/22201648/
doi: 10.1186/1743-422x-8-560
summary:
  In the present study, antiviral activity of four types of bioflavonoid against dengue virus type -2 (DENV-2) in Vero cell was evaluated. Daidzein showed a weak anti-dengue activity with IC(50 )= 142.6 μg mL(-1 )when the DENV-2 infected cells were treated after virus adsorption. Although there was no significant direct virucidal activity against DENV-2 by quercetin, continuous treatment of cells from 5 h before virus infection up to 4 days post-infection exhibited anti-dengue activity with IC 50 = 28.9 μg mL -1 (Figure 3a) . There was no significant change in the antiviral activity of daidzein when cells were treated continuously from 5 h before virus infection up to 4 days post infection comparing to its anti-dengue activity for postadsorption treatment (Figure 1 ). To investigate which of the many flavonoids could affect DENV infection, in the present study, we examined the potential effects of quercetin, naringin, hesperetin and daidzein on dengue virus infection of Vero cells.

abstract:
  BACKGROUND: Dengue is a major mosquito-borne disease currently with no effective antiviral or vaccine available. Effort to find antivirals for it has focused on bioflavonoids, a plant-derived polyphenolic compounds with many potential health benefits. In the present study, antiviral activity of four types of bioflavonoid against dengue virus type -2 (DENV-2) in Vero cell was evaluated. Anti-dengue activity of these compounds was determined at different stages of DENV-2 infection and replication cycle. DENV replication was measured by Foci Forming Unit Reduction Assay (FFURA) and quantitative RT-PCR. Selectivity Index value (SI) was determined as the ratio of cytotoxic concentration 50 (CC(50)) to inhibitory concentration 50 (IC(50)) for each compound. RESULTS: The half maximal inhibitory concentration (IC(50)) of quercetin against dengue virus was 35.7 μg mL(-1 )when it was used after virus adsorption to the cells. The IC(50 )decreased to 28.9 μg mL(-1 )when the cells were treated continuously for 5 h before virus infection and up to 4 days post-infection. The SI values for quercetin were 7.07 and 8.74 μg mL(-1), respectively, the highest compared to all bioflavonoids studied. Naringin only exhibited anti-adsorption effects against DENV-2 with IC(50 )= 168.2 μg mL(-1 )and its related SI was 1.3. Daidzein showed a weak anti-dengue activity with IC(50 )= 142.6 μg mL(-1 )when the DENV-2 infected cells were treated after virus adsorption. The SI value for this compound was 1.03. Hesperetin did not exhibit any antiviral activity against DENV-2. The findings obtained from Foci Forming Unit Reduction Assay (FFURA) were corroborated by findings of the qRT-PCR assays. Quercetin and daidzein (50 μg mL(-1)) reduced DENV-2 RNA levels by 67% and 25%, respectively. There was no significant inhibition of DENV-2 RNA levels with naringin and hesperetin. CONCLUSION: Results from the study suggest that only quercetin demonstrated significant anti-DENV-2 inhibitory activities. Other bioflavonoids, including daidzein, naringin and hesperetin showed minimal to no significant inhibition of DENV-2 virus replication. These findings, together with those previously reported suggest that select group of bioflavonoids including quercetin and fisetin, exhibited significant inhibitory activities against dengue virus. This group of flavonoids, flavonol, could be investigated further to discover the common mechanisms of inhibition of dengue virus replication.


id: cord-278250-dwok857k
author: Li, Heng
date: 2019-08-19
txt: ./txt/cord-278250-dwok857k.txt
cache: ./cache/cord-278250-dwok857k.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/s12985-019-1211-z
doi: 10.1186/s12985-019-1211-z
summary:
  We performed metagenomic sequencing of fecal samples to detect the bacterial microbiome and virome composition of healthy one-year-old rhesus monkeys housed at the IMBCAMS (Fig. 1) . We comprehensively analyzed the interactions among the gut virome, bacterial microbiome and metabolomes based on the above results there were noticeable differences in bacterial β-diversity between control and experimental animals, as determined using principal component analysis (PCA), and the results showed good repeatability within a single group (Additional file 2: Figure S2F ). Briefly, the fecal virome composition was noticeably altered after depletion of the bacterial microbiome, and the abundances of many DNA viruses, bacteriophages and RNA viruses in the gut were clearly decreased. As expected, the whole gut bacterial microbiome, including gram-positive and gram-negative bacteria (Additional file 2: Figure S2E ), was depleted after treatment with the antibiotic cocktail, except for Escherichia-Shigella species belonging to Proteobacteria, which were resistant to the cocktail.

title:
  The altered gut virome community in rhesus monkeys is correlated with the gut bacterial microbiome and associated metabolites

abstract:
  BACKGROUND: The gut microbiome is closely associated with the health of the host; although the interaction between the bacterial microbiome and the whole virome has rarely been studied, it is likely of medical importance. Examination of the interactions between the gut bacterial microbiome and virome of rhesus monkey would significantly contribute to revealing the gut microbiome composition. METHODS: Here, we conducted a metagenomic analysis of the gut microbiome of rhesus monkeys in a longitudinal cohort treated with an antibiotic cocktail, and we documented the interactions between the bacterial microbiome and virome. The depletion of viral populations was confirmed at the species level by real-time PCR. We also detected changes in the gut metabolome by GC-MS and LC-MS. RESULTS: A majority of bacteria were depleted after treatment with antibiotics, and the Shannon diversity index decreased from 2.95 to 0.22. Furthermore, the abundance-based coverage estimator (ACE) decreased from 104.47 to 33.84, and the abundance of eukaryotic viruses also changed substantially. In the annotation, 6 families of DNA viruses and 1 bacteriophage family were present in the normal monkeys but absent after gut bacterial microbiome depletion. Intriguingly, we discovered that changes in the gut bacterial microbiome composition may promote changes in the gut virome composition, and tryptophan, arginine, and quinone may play roles in the interaction between the bacterial microbiome and virome. CONCLUSION: Our results indicated that the clearly altered composition of the virome was correlated with depletion in the bacterial community and that metabolites produced by bacteria possibly play important roles in the interaction. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12985-019-1211-z) contains supplementary material, which is available to authorized users.


id: cord-278324-eqqvwwh6
author: Wang, Huanan
date: 2018-09-21
txt: ./txt/cord-278324-eqqvwwh6.txt
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extension: .txt
mime: text/plain
url: https://doi.org/10.1186/s12985-018-1048-x
doi: 10.1186/s12985-018-1048-x
summary:
  BACKGROUND: A serological method to simultaneously detect antibodies against infectious laryngotracheitis virus (ILTV) and infectious bronchitis virus (IBV) is imperative for the differential diagnosis and evaluation of antibodies titers after vaccination. Therefore, it is important to establish a method to simultaneously detect ILTV and IBV antibodies for the differential diagnosis and immune response evaluation after vaccination. In this study, a method employing Luminex xMAP technology to simultaneously detect ILTV and IBV antibodies in serum was established, optimized and used for the differential diagnosis of IBV and ILTV. 2 and 3 demonstrated that xMAP duplex assay for ILTV and IBV has a high specificity since there were no cross-reactions with serum positive for other avian diseases, such as avian influenza virus To compare duplex xMAP assay with ELISA for ILTV/IBV detection, 90 chicken serum samples from a chicken farm in Huizhou, China were used and the results are shown in Table 7 .

title:
  Development of a sensitive and specific xMAP assay for detection of antibodies against infectious laryngotracheitis and bronchitis viruses

abstract:
  BACKGROUND: A serological method to simultaneously detect antibodies against infectious laryngotracheitis virus (ILTV) and infectious bronchitis virus (IBV) is imperative for the differential diagnosis and evaluation of antibodies titers after vaccination. METHOD: The microspheres coated with purified recombinant glycoprotein D (gD) of ILTV or nucleocapsid (N) protein of IBV were incubated with serum samples. The simultaneous quantification of ILTV and IBV antibodies were achieved through the interrogation of microspheres by Luminex 200 detection system. . RESULTS: This xMAP detection demonstrated no nonspecific reactions with avian influenza virus (AIV), avian leukosis virus (ALV), newcastle disease virus (NDV), and Marek’s disease virus (MDV). The results also demonstrated that the xMAP assay was four times more sensitive than the enzyme-linked immunosorbent assay (ELISA) for ILTV detection and two times more sensitive for IBV detection. A total of 90 chicken serum samples from a chicken farm were tested by xMAP and ELISA assays. The results showed that the coincidence rates were 84.44 and 100% for ILTV and IBV detection, respectively. CONCLUSION: This study exhibited an opportunity for the differential diagnosis through simultaneous detection of multiplex antibodies in serum and can be used for the multiplex antibodies evaluation after vaccination.


id: cord-279784-o80x8nj7
author: Wu, Yu
date: 2019-10-28
txt: ./txt/cord-279784-o80x8nj7.txt
cache: ./cache/cord-279784-o80x8nj7.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/s12985-019-1232-7
doi: 10.1186/s12985-019-1232-7
summary:
  title: Characterization and pathogenicity of Vero cell-attenuated porcine epidemic diarrhea virus CT strain METHODS: In this study, the highly virulent epidemic virus strain CT was serially passaged in Vero cells for up to 120 generations (P120). Previous studies conducted at Fig. 1 Biological characteristics of porcine epidemic diarrhea virus strains after 10, 64, or 120 passages. A newly isolated Chinese virulent genotype GIIb porcine epidemic diarrhea virus strain: biological characteristics, pathogenicity and immune protective effects as an inactivated vaccine candidate Oral efficacy of Vero cell attenuated porcine epidemic diarrhea virus DR13 strain Attenuation of an original US porcine epidemic diarrhea virus strain PC22A via serial cell culture passage Comparative genomic analysis of classical and variant virulent parental/attenuated strains of porcine epidemic diarrhea virus Preparation and characterization of an attenuated porcine epidemic diarrhea virus strain by serial passaging

title:
  Characterization and pathogenicity of Vero cell-attenuated porcine epidemic diarrhea virus CT strain

abstract:
  BACKGROUND: Porcine epidemic diarrhea virus (PEDV) has caused enormous economic losses to the global pig industry. Currently available PEDV vaccine strains have limited protective effects against PEDV variant strains. METHODS: In this study, the highly virulent epidemic virus strain CT was serially passaged in Vero cells for up to 120 generations (P120). Characterization of the different passages revealed that compared with P10 and P64, P120 had a higher viral titer and more obvious cytopathic effects, thereby demonstrating better cell adaptability. RESULTS: Pathogenicity experiments using P120 in piglets revealed significant reductions in clinical symptoms, histopathological lesions, and intestinal PEDV antigen distribution; the piglet survival rate in the P120 group was 100%. Furthermore, whole-genome sequencing identified 13 amino acid changes in P120, which might be responsible for the attenuated virulence of P120. CONCLUSIONS: Thus, an attenuated strain was obtained via cell passaging and that this strain could be used in preparing attenuated vaccines.


id: cord-282343-cko4curf
author: Cheng, Han
date: 2015-11-24
url: https://www.ncbi.nlm.nih.gov/pubmed/26596270/
doi: 10.1186/s12985-015-0420-3
title:
  A parallel genome-wide RNAi screening strategy to identify host proteins important for entry of Marburg virus and H5N1 influenza virus

abstract:
  BACKGROUND: Genome-wide RNAi screening has been widely used to identify host proteins involved in replication and infection of different viruses, and numerous host factors are implicated in the replication cycles of these viruses, demonstrating the power of this approach. However, discrepancies on target identification of the same viruses by different groups suggest that high throughput RNAi screening strategies need to be carefully designed, developed and optimized prior to the large scale screening. METHODS: Two genome-wide RNAi screens were performed in parallel against the entry of pseudotyped Marburg viruses and avian influenza virus H5N1 utilizing an HIV-1 based surrogate system, to identify host factors which are important for virus entry. A comparative analysis approach was employed in data analysis, which alleviated systematic positional effects and reduced the false positive number of virus-specific hits. RESULTS: The parallel nature of the strategy allows us to easily identify the host factors for a specific virus with a greatly reduced number of false positives in the initial screen, which is one of the major problems with high throughput screening. The power of this strategy is illustrated by a genome-wide RNAi screen for identifying the host factors important for Marburg virus and/or avian influenza virus H5N1 as described in this study. CONCLUSIONS: This strategy is particularly useful for highly pathogenic viruses since pseudotyping allows us to perform high throughput screens in the biosafety level 2 (BSL-2) containment instead of the BSL-3 or BSL-4 for the infectious viruses, with alleviated safety concerns. The screening strategy together with the unique comparative analysis approach makes the data more suitable for hit selection and enables us to identify virus-specific hits with a much lower false positive rate.


id: cord-283333-u3r1usfs
author: Yao, Li-hong
date: 2019-06-13
txt: ./txt/cord-283333-u3r1usfs.txt
cache: ./cache/cord-283333-u3r1usfs.txt
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mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/31196108/
doi: 10.1186/s12985-019-1185-x
summary:
  However, the genotype diversity and epidemiological information relating to HAdVs among hospitalized children with respiratory tract infections (RTIs) is limited. Therefore, the aim of this study was to evaluate the epidemiological, clinical, and molecular characteristics of HAdV infections occurring among children with RTIs in a Chinese tertiary hospital from April 2017 to March 2018. Herein, we describe (i) the prevalence of HAdVs in hospitalized children with RTIs presenting at Beijing Friendship Hospital during a one-year period; (ii) the clinical spectrum of the HAdV-positive RTI patients; (iii) the viral co-pathogens present in the HAdV infections; and (iv) the genetic diversity of the HAdVs. The clinical characteristics of the RTIs caused by HAdV are very similar to those of influenza, PIV and other respiratory tract pathogens, making it difficult to clinically diagnose this type of infection. This 1-year study documented the prevalence, age distribution, seasonality and molecular epidemiology of HAdV infections among children hospitalized with RTIs at Beiing Friendship Hospital.

title:
  Human adenovirus among hospitalized children with respiratory tract infections in Beijing, China, 2017–2018

abstract:
  BACKGROUND: Human adenoviruses (HAdVs) cause a wide range of diseases. However, the genotype diversity and epidemiological information relating to HAdVs among hospitalized children with respiratory tract infections (RTIs) is limited. Here, we describe the epidemiology and genotype distribution of HAdVs associated with RTIs in Beijing, China. METHODS: Nasopharyngeal aspirates (NPA) were collected from hospitalized children with RTIs from April 2017 to March 2018. HAdVs were detected by a TaqMan-based quantitative real-time polymerase chain reaction (qPCR) assay, and the hexon gene was used for phylogenetic analysis. Epidemiological data were analyzed using statistical product and service solutions (SPSS) 21.0 software. RESULTS: HAdV was detected in 72 (5.64%) of the 1276 NPA specimens, with most (86.11%, 62/72) HAdV-positives cases detected among children < 6 years of age. HAdV-B3 (56.06%, 37/66) and HAdV-C2 (19.70%, 13/66) were the most frequent. Of the 72 HAdV-infected cases, 27 (37.50%) were co-infected with other respiratory viruses, most commonly parainfluenza virus (12.50%, 9/72) and rhinovirus (9.72%, 7/72). The log number of viral load ranged from 3.30 to 9.14 copies per mL of NPA, with no significant difference between the HAdV mono- and co-infection groups. The main clinical symptoms in the HAdV-infected patients were fever and cough, and 62 (86.11%, 62/72) were diagnosed with pneumonia. Additionally, HAdVs were detected throughout the year with a higher prevalence in summer. CONCLUSIONS: HAdV prevalence is related to age and season. HAdV-B and HAdV-C circulated simultaneously among the hospitalized children with RTIs in Beijing, and HAdV-B type 3 and HAdV-C type 2 were the most frequent.


id: cord-283689-dzin12qb
title: Involvement of PRRSV NSP3 and NSP5 in the autophagy process
author: Zhang, Wei
date: 2019-01-28
txt: ./txt/cord-283689-dzin12qb.txt
cache: ./cache/cord-283689-dzin12qb.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/30691473/
doi: 10.1186/s12985-019-1116-x
summary:
  RESULTS: Autophagy was activated by porcine reproductive and respiratory syndrome virus (PRRSV) NSP3, NSP5 and NSP9, which are two transmembrane proteins and an RNA-dependent RNA polymerase, respectively. Based on the data presented in Fig. 2c , immunoblotting and immunofluorescence assay showed that the expression of the p62 protein was increased, indicating that NSP3 and NSP5 of PRRSV induced the formation of autophagosomes. As shown in Fig. 3a , ATG5 puncta were arranged in reticular structures and colocalized with calnexin in the Fig. 1 The distribution of autophagy proteins in PRRSV-infected Marc-145 cells. As shown in the present study, the induction of autophagy by PRRSV NSP3 and NSP5 contributed to the formation of autophagosomes derived from the ER, and the mature autophagosomes were not degraded by fusion with lysosomes.

abstract:
  BACKGROUND: Autophagy is an essential process in eukaryotic cells in which autophagosomes form to deliver cellular organelles and long-lived proteins to lysosomes for degradation. Many studies have recently identified the regulatory mechanisms involved in the interaction between viral infection and autophagy. METHODS: LC3 turnover and the proteins in the endoplasmic reticulum (ER) stress pathway were investigated using western blot analysis. The formation and degradation of autophagosomes were detected using immunofluorescence staining. RESULTS: Autophagy was activated by porcine reproductive and respiratory syndrome virus (PRRSV) NSP3, NSP5 and NSP9, which are two transmembrane proteins and an RNA-dependent RNA polymerase, respectively. The formation of autophagosomes was induced by NSP3 and NSP5 and developed from the ER; the fusion of these autophagosomes with lysosomes was limited. Although NSP3 and NSP5 are ER transmembrane proteins, these proteins did not activate the ER stress signaling pathways. In addition, the cytoplasmic domain of NSP3 plays a pivotal role in activating autophagy. CONCLUSIONS: The data presented in this study reveal an important relationship between PRRSV NSPs and autophagy and provide new insights that improve our understanding of the involvement of PRRSV NSPs in the autophagy process. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12985-019-1116-x) contains supplementary material, which is available to authorized users.


id: cord-284608-ba7wq52t
author: Sias, Catia
date: 2019-03-04
url: https://www.ncbi.nlm.nih.gov/pubmed/30832688/
doi: 10.1186/s12985-019-1132-x
title:
  Alpha, Beta, gamma human PapillomaViruses (HPV) detection with a different sets of primers in oropharyngeal swabs, anal and cervical samples

abstract:
  BACKGROUND: Recent studies have shown a 13-fold increase of oropharyngeal cancer in the presence of HPV, while α-HPV detection seems to be rare in oral cavity in comparison to anal or cervical district, many novel β and γ types have been isolated in this anatomical site suggesting a wide tropism range. Currently, there are no guidelines recommending HPV oral cavity screening as a mandatory test, and it remains unknown which HPV types should be included in HPV screening programs. Our goal was to assess HPV prevalence in oropharyngeal, anal, and cervical swabs using different sets of primers,which are able to amplify α, β, γ HPV types. METHODS: We analysed the presence of HPV DNA in oropharyngeal (n = 124), anal (n = 186), cervical specimens (n = 43) from HIV positive and negative patients using FAP59/64 and MY09/11 primers. All untyped strains were genetically characterized through PCR amplification and direct sequencing of partial L1 region, and the resulting sequences were classified through phylogenetic analysis. RESULTS: HPV prevalence was 20.9% in 124 oropharyngeal swab samples, including infections with multiple HPV types (5.6%). HPV prevalence in this anatomical site was significantly associated with serostatus: 63.3%in HIV positive and 36.3% in HIV negative patients (p < 0.05). Unclassified types were detected in 6 specimens. In our analysis, we did not observe any difference in HPV (α, β, γ) prevalence between men and women. Overall, β species were the most frequently detected 69.7%. When using anal swabs, for HIV positive patients, β genus prevalence was 1% and γ genus was 3.7% including 6 unclassified types. In cervical samples from 43 HIV positive women (18 HPV negative and 25 positive by MY09/11 PCR), only one sample was positivite for β(1) species (2.4%) using FAP primers. Six of the untyped strains clustered with sequences from species 7, 9, 10, 8,12 of γ genus. Four sequences remained unclassified. Finally, β and γ HPV prevalence was significantly lower than their respective HPV prevalence as identified by the Luminex system in all anatomical sites that were analyzed in previous studies. CONCLUSION: This study provides new information about viral isolates present in oropharyngeal site and it will contribute to improve the monitoring of HPV infection. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12985-019-1132-x) contains supplementary material, which is available to authorized users.


id: cord-284630-l9ghggu7
title: Molecular epidemiology of canine parvovirus type 2 in Vietnam from November 2016 to February 2018
author: Hoang, Minh
date: 2019-04-27
url: https://doi.org/10.1186/s12985-019-1159-z
doi: 10.1186/s12985-019-1159-z
abstract:
  BACKGROUND: Canine parvovirus type 2 (CPV-2) was first identified in the late 1970s; it causes intestinal hemorrhage with severe bloody diarrhea in kennels and dog shelters worldwide. Since its emergence, CPV-2 has been replaced with new genetic variants (CPV-2a, CPV-2b, and CPV-2c). Currently, information about the genotype prevalence of CPV-2 in Vietnam is limited. In the present study, we investigated the genotype prevalence and distribution of CPV-2 in the three regions of Vietnam. METHODS: Rectal swabs were collected from 260 dogs with suspected CPV-2 infection from northern, central, and southern Vietnam from November 2016 to February 2018. All samples were identified as parvovirus positive by real-time PCR, and further genotyping was performed using a SimpleProbe® real-time PCR assay. RESULTS: Of the 260 Vietnamese CPV-2 isolates, 6 isolates (2.31%) were identified as CPV-2a, 251 isolates (96.54%) were identified as CPV-2c and 3 isolates (1.15%) were untypable using the SimpleProbe® real-time PCR assay. In northern Vietnam, the percentages of CPV-2a and CPV-2c were 2.97% (3/101) and 97.3% (98/101), respectively. In central Vietnam, the percentages of CPV-2a and CPV-2c were 1.11% (1/90) and 98.89% (89/90), respectively. In southern Vietnam, the percentages of CPV-2a and CPV-2c were 3.03% (2/66) and 96.97% (64/66), respectively. CPV-2b was not observed in this study. The VP2 genes of CPV-2c in Vietnam are more genetically similar to those of CPV-2c strains in China and Taiwan than to those of prototype CPV-2c strains (FJ222821) or the first Vietnamese CPV-2c (AB120727). CONCLUSION: The present study provides evidence that CPV-2c is the most prevalent variant in Vietnam. Phylogenetic analysis demonstrated that the recent Vietnamese CPV-2c isolates share a common evolutionary origin with Asian CPV-2c strains. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12985-019-1159-z) contains supplementary material, which is available to authorized users.


id: cord-285253-ik5w4t5e
title: Real-world comparison of two molecular methods for detection of respiratory viruses
author: Ali, S Asad
date: 2011-06-29
txt: ./txt/cord-285253-ik5w4t5e.txt
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mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/21714915/
doi: 10.1186/1743-422x-8-332
summary:
  METHODS: We tested nasal and throat swab specimens obtained from 225 infants with respiratory illness for 11 common respiratory viruses using both a multiplex assay (Respiratory MultiCode-PLx Assay [RMA]) and individual real-time RT-PCR (RT-rtPCR). The RMA assay detected significantly more human metapneumovirus (HMPV) and respiratory syncytial virus (RSV), while RT-rtPCR detected significantly more influenza A. Restricting the analysis to the 11 viruses tested with both methods, at least one virus was detected in 174 (78.3%) samples by RMA and in 163 (73.4%) samples by RT-rtPCR. In the second round using redesigned RT-rtPCR HMPV primers and probes, HMPV was detected in 7 samples, 5 of which were concordant with the results of the first RMA assay. Comparison of the Eragen Multi-Code Respiratory Virus Panel with conventional viral testing and real-time multiplex PCR assays for detection of respiratory viruses

abstract:
  BACKGROUND: Molecular polymerase chain reaction (PCR) based assays are increasingly used to diagnose viral respiratory infections and conduct epidemiology studies. Molecular assays have generally been evaluated by comparing them to conventional direct fluorescent antibody (DFA) or viral culture techniques, with few published direct comparisons between molecular methods or between institutions. We sought to perform a real-world comparison of two molecular respiratory viral diagnostic methods between two experienced respiratory virus research laboratories. METHODS: We tested nasal and throat swab specimens obtained from 225 infants with respiratory illness for 11 common respiratory viruses using both a multiplex assay (Respiratory MultiCode-PLx Assay [RMA]) and individual real-time RT-PCR (RT-rtPCR). RESULTS: Both assays detected viruses in more than 70% of specimens, but there was discordance. The RMA assay detected significantly more human metapneumovirus (HMPV) and respiratory syncytial virus (RSV), while RT-rtPCR detected significantly more influenza A. We speculated that primer differences accounted for these discrepancies and redesigned the primers and probes for influenza A in the RMA assay, and for HMPV and RSV in the RT-rtPCR assay. The tests were then repeated and again compared. The new primers led to improved detection of HMPV and RSV by RT-rtPCR assay, but the RMA assay remained similar in terms of influenza detection. CONCLUSIONS: Given the absence of a gold standard, clinical and research laboratories should regularly correlate the results of molecular assays with other PCR based assays, other laboratories, and with standard virologic methods to ensure consistency and accuracy.


id: cord-286256-yol03hid
author: Xu, Tian-min
date: 2020-06-19
txt: ./txt/cord-286256-yol03hid.txt
cache: ./cache/cord-286256-yol03hid.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32560646/
doi: 10.1186/s12985-020-01354-6
summary:
  title: Non-optimal effectiveness of convalescent plasma transfusion and hydroxychloroquine in treating COVID-19: a case report BACKGROUND: Convalescent plasma (CP) transfusion was reported to be effective in treating critically ill patients with COVID-19, and hydroxychloroquine could potently inhibit SARS-CoV-2 in vitro. CASE PRESENTATION: Laboratory findings showed high lactic acid level (2.1 mmol/L) and C-reactive protein (CRP, 48.8 mg/L), and low white blood cell count (1.96 × 10(9)/L) in a 65-year-old Chinese man, who was diagnosed with severe COVID-19. The lactic acid and C-reactive protein levels remained high (2.1 mmol/L and 73.23 mg/L, respectively), while the arterial oxyhemoglobin saturation decreased to 86% with a low oxygenation index (OI, 76 mmHg) on day 4 after CP transfusion. Few studies have reported the effectiveness of convalescent plasma (CP) transfusion in treating critically ill patients with COVID-19 [2] [3] [4] . Herein, we administered CP transfusion and hydroxychloroquine to a patient with severe COVID-19, and analyzed their clinical symptoms, oxygenation index (OI), and dynamics of viral load.

title:
  Non-optimal effectiveness of convalescent plasma transfusion and hydroxychloroquine in treating COVID-19: a case report

abstract:
  BACKGROUND: Convalescent plasma (CP) transfusion was reported to be effective in treating critically ill patients with COVID-19, and hydroxychloroquine could potently inhibit SARS-CoV-2 in vitro. Herein, we reported a case receiving combination therapy with CP transfusion and hydroxychloroquine for the first time. CASE PRESENTATION: Laboratory findings showed high lactic acid level (2.1 mmol/L) and C-reactive protein (CRP, 48.8 mg/L), and low white blood cell count (1.96 × 10(9)/L) in a 65-year-old Chinese man, who was diagnosed with severe COVID-19. CP was intravenously given twice, and hydroxychloroquine was orally administrated for a week (0.2 g, three times a day). The lactic acid and C-reactive protein levels remained high (2.1 mmol/L and 73.23 mg/L, respectively), while the arterial oxyhemoglobin saturation decreased to 86% with a low oxygenation index (OI, 76 mmHg) on day 4 after CP transfusion. His temperature returned to normal and the OI ascended above 300 on day 11. Moreover, the RNA test remained positive in throat swab, and computed tomography revealed severe pulmonary lesions on day 11 after admission. CONCLUSION: These findings suggested that the effectiveness of combination therapy with CP and hydroxychloroquine may be non-optimal, and specific therapy needs to be explored.


id: cord-286332-cdg4im5h
author: van Beurden, Steven J.
date: 2017-06-12
url: https://www.ncbi.nlm.nih.gov/pubmed/28606144/
doi: 10.1186/s12985-017-0775-8
title:
  A reverse genetics system for avian coronavirus infectious bronchitis virus based on targeted RNA recombination

abstract:
  BACKGROUND: Avian coronavirus infectious bronchitis virus (IBV) is a respiratory pathogen of chickens that causes severe economic losses in the poultry industry worldwide. Major advances in the study of the molecular biology of IBV have resulted from the development of reverse genetics systems for the highly attenuated, cell culture-adapted, IBV strain Beaudette. However, most IBV strains, amongst them virulent field isolates, can only be propagated in embryonated chicken eggs, and not in continuous cell lines. METHODS: We established a reverse genetics system for the IBV strain H52, based on targeted RNA recombination in a two-step process. First, a genomic and a chimeric synthetic, modified IBV RNA were co-transfected into non-susceptible cells to generate a recombinant chimeric murinized (m) IBV intermediate (mIBV). Herein, the genomic part coding for the spike glycoprotein ectodomain was replaced by that of the coronavirus mouse hepatitis virus (MHV), allowing for the selection and propagation of recombinant mIBV in murine cells. In the second step, mIBV was used as the recipient. To this end a recombination with synthetic RNA comprising the 3′-end of the IBV genome was performed by introducing the complete IBV spike gene, allowing for the rescue and selection of candidate recombinants in embryonated chicken eggs. RESULTS: Targeted RNA recombination allowed for the modification of the 3′-end of the IBV genome, encoding all structural and accessory genes. A wild-type recombinant IBV was constructed, containing several synonymous marker mutations. The in ovo growth kinetics and in vivo characteristics of the recombinant virus were similar to those of the parental IBV strain H52. CONCLUSIONS: Targeted RNA recombination allows for the generation of recombinant IBV strains that are not able to infect and propagate in continuous cell lines. The ability to introduce specific mutations holds promise for the development of rationally designed live-attenuated IBV vaccines and for studies into the biology of IBV in general. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-017-0775-8) contains supplementary material, which is available to authorized users.


id: cord-286842-04cuk2cn
author: Plyusnina, Angelina
date: 2005-02-22
txt: ./txt/cord-286842-04cuk2cn.txt
cache: ./cache/cord-286842-04cuk2cn.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/15725355/
doi: 10.1186/1743-422x-2-12
summary:
  title: Recombinant Tula hantavirus shows reduced fitness but is able to survive in the presence of a parental virus: analysis of consecutive passages in a cell culture Tula hantavirus carrying recombinant S RNA segment (recTULV) grew in a cell culture to the same titers as the original cell adapted variant but presented no real match to the parental virus. Using the two specific RT-PCR conditions, the presence of V-type and REC-type S RNA was monitored on ten sequential passages of the mixture of TULV02 and RecTULV5 variants (Fig. 2 ). Although relatively short, the observed survival time of the recTULV in the presence of the original variant TUL02 seems to be sufficient for transmission of a recombinant virus, in a hypothetical in vivo situation, from one rodent to another. The data presented in this paper show that the recTULV presents no real match to the original cell adapted variant and that the lower fitness of the recombinant virus can not be increased by pre-passaging in cell culture.

title:
  Recombinant Tula hantavirus shows reduced fitness but is able to survive in the presence of a parental virus: analysis of consecutive passages in a cell culture

abstract:
  Tula hantavirus carrying recombinant S RNA segment (recTULV) grew in a cell culture to the same titers as the original cell adapted variant but presented no real match to the parental virus. Our data showed that the lower competitiveness of recTULV could not be increased by pre-passaging in the cell culture. Nevertheless, the recombinant virus was able to survive in the presence of the parental virus during five consecutive passages. The observed survival time seems to be sufficient for transmission of newly formed recombinant hantaviruses in nature.


id: cord-289081-9v04gzhf
author: Aponte, Fernando E
date: 2015-02-26
txt: ./txt/cord-289081-9v04gzhf.txt
cache: ./cache/cord-289081-9v04gzhf.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/s12985-015-0262-z
doi: 10.1186/s12985-015-0262-z
summary:
  In this study we determined the frequency and diversity of RV strains associated with upper and lower respiratory tract infections (URTI, LRTI) in Mexico, and describe the clinical characteristics of the illness associated with different RV species. Most studies have described the presence of RV genotypes in hospitalized patients with severe respiratory illness, and only a few studies have described the prevalence of virus genotypes in URTIs. In this work, we carried out a prospective multicenter study of two children populations having either URTI or LRTI. The phylogenetic trees in Figure 2 depict the wide distribution of 5′-UTR sequences of the RV-A and RV-C Table 1 Frequency of RV infections in children with upper and lower respiratory tract infections strains isolated from URTI and LRTI patients. This study describes the frequency of detection of rhinovirus species in children with upper and lower respiratory tract infections in Mexico and their genetic diversity, determined by sequencing the 5′ UTR region of the viral genome.

title:
  Rhinovirus is an important pathogen in upper and lower respiratory tract infections in Mexican children

abstract:
  BACKGROUND: Most of the studies characterizing the incidence of rhinovirus (RV) have been carried out in hospitalized children and in developed countries. In those studies, RV-C has been associated with more severe respiratory tract infections than RV species A and B. In this study we determined the frequency and diversity of RV strains associated with upper and lower respiratory tract infections (URTI, LRTI) in Mexico, and describe the clinical characteristics of the illness associated with different RV species. METHODS: A prospective surveillance of 526 and 250 children with URTI and LRTI was carried out. Respiratory samples were analyzed by RT-PCR for viruses. The 5′ untranslated region of the RV genome was amplified and sequenced. RESULTS: In the case of URTI, 17.5% were positive for RV, while this virus was found in 24.8% of LRTI. The RV species was determined in 73 children with URTI: 61.6% were RV-A, 37% RV-C and, 1.4% RV-B; and in 43 children with LRTI: 51.2% were RV-A, 41.8% RV-C, and 7% RV-B. No significant differences in clinical characteristics were found in patients with RV-A or RV-C infections. A high genetic diversity of RV strains was found in both URTI and LRTI. CONCLUSIONS: Both RV-A and RV-C species were frequently found in hospitalized as well as in outpatient children. This study underlines the high prevalence and genetic diversity of RV strains in Mexico and the potential severity of disease associated with RV-A and RV-C infections.


id: cord-289397-a1cuq29o
title: Lycorine reduces mortality of human enterovirus 71-infected mice by inhibiting virus replication
author: Liu, Jiangning
date: 2011-10-27
txt: ./txt/cord-289397-a1cuq29o.txt
cache: ./cache/cord-289397-a1cuq29o.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/22029605/
doi: 10.1186/1743-422x-8-483
summary:
  Lycorine treatment of mice challenged with a lethal dose of EV71 resulted in reduction of mortality, clinical scores and pathological changes in the muscles of mice, which were achieved through inhibition of viral replication. To study the inhibitory mechanism of lycorine against EV71 infection, the synthesis of several typical viral proteins were detected by western blotting in 1.5 hours periods following lycorine treatment, when the effects of inhibition was just appeared ( Figure 3A and 3B) . Consistent with the results of the clinical scores and survival rates, virus replication in the muscle of lycorine-treated mice were inhibited by 10-100 folds at different time points compared to that of the saline control as detected by qRT-PCR and semi-quantitative RT-PCR ( Figure 5A and 5B). The virus replication in muscle tissues of lycorine-treated (0.4 mg/kg) mice was significantly inhibited by more than 100-fold compared to the saline control as detected by qRT-PCR and semiquantitative RT-PCR ( Figure 7A and 7B) .

abstract:
  Human enterovirus 71 (EV71) infection causes hand, foot and mouth disease in children under 6 years old and this infection occasionally induces severe neurological complications. No vaccines or drugs are clinical available to control EV71 epidemics. In present study, we show that treatment with lycorine reduced the viral cytopathic effect (CPE) on rhabdomyosarcoma (RD) cells by inhibiting virus replication. Analysis of this inhibitory effect of lycorine on viral proteins synthesis suggests that lycorine blocks the elongation of the viral polyprotein during translation. Lycorine treatment of mice challenged with a lethal dose of EV71 resulted in reduction of mortality, clinical scores and pathological changes in the muscles of mice, which were achieved through inhibition of viral replication. When mice were infected with a moderate dose of EV71, lycorine treatment was able to protect them from paralysis. Lycorine may be a potential drug candidate for the clinical treatment of EV71-infected patients.


id: cord-290798-5ca2e6wm
author: Malhotra, Bharti
date: 2016-06-06
txt: ./txt/cord-290798-5ca2e6wm.txt
cache: ./cache/cord-290798-5ca2e6wm.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/27267595/
doi: 10.1186/s12985-016-0549-8
summary:
  title: Evaluation of custom multiplex real time RT PCR in comparison to fast track diagnostics respiratory 21 pathogens kit for detection of multiple respiratory viruses Therefore the aim of the present study was to develop a cost effective multiplex real time RT-PCR for the detection of 18 respiratory viruses and compare it with an in-vitro diagnostics approved Fast Track Diagnostic Respiratory Pathogens 21 Kit (FTD). METHODS: Nasopharyngeal aspirates and throat swabs were collected and processed for extraction of nucleic acid using an automated extraction system and multiplex real time RT-PCR was performed using the FTD kit and a custom assay on 356 samples. The present study compares custom real-time multiplex PCR primers and probes for the simultaneous detection of 18 respiratory viruses with an in-vitro diagnostics (IVD) approved fast track diagnostics (FTD) kit.

title:
  Evaluation of custom multiplex real - time RT - PCR in comparison to fast - track diagnostics respiratory 21 pathogens kit for detection of multiple respiratory viruses

abstract:
  BACKGROUND: Severe acute respiratory infections in children can be fatal, rapid identification of the causative agent and timely treatment can be life saving. Multiplex real time RT-PCR helps in simultaneous detection of multiple viruses saving cost, time and labour. Commercially available multiplex real time RT-PCR kits are very expensive. Therefore the aim of the present study was to develop a cost effective multiplex real time RT-PCR for the detection of 18 respiratory viruses and compare it with an in-vitro diagnostics approved Fast Track Diagnostic Respiratory Pathogens 21 Kit (FTD). METHODS: Nasopharyngeal aspirates and throat swabs were collected and processed for extraction of nucleic acid using an automated extraction system and multiplex real time RT-PCR was performed using the FTD kit and a custom assay on 356 samples. RESULTS: Custom and FTD assays detected one or more respiratory viruses in 268 (75.29 %) and 262 (73.60 %) samples respectively. The concordance between the custom assay and the FTD assay was 100 % for HCoV OC43, HCoV 229E, HPIV-1, HPIV-2, HBoV, HPeV, Flu A, and Influenza A(H1N1)pdm09 and 94.66 – 99.71 % for the remaining viruses; Flu B (99.71 %), HRV (99.71 %), HPIV-3 (98.87 %), HPIV-4 (99.43 %), HCoV NL63 (99.71 %), HMPV A/B (99.71 %), RSV A/B (94.66 %), EV (98.31 %), HCoV HKU1 (99.71 %), HAdV (99.71 %). Major discrepancy was observed for RSV A/B, which was over detected in 18 samples by the custom assay as compared to the FTD assay. The custom assay was much cheaper than the FTD assay and the time taken was only 29 min more. CONCLUSION: The custom primer and probe mix was found to be comparable to the FTD assay with good concordance but was much cheaper and the time taken for reporting was only 29 min more. The low cost custom multiplex RT-PCR can be a useful alternative to the costly FTD kit for rapid identification of viral aetiology in resource limited settings.


id: cord-290976-dhwlr2ui
author: Lednicky, John A
date: 2013-06-27
url: https://doi.org/10.1186/1743-422x-10-213
doi: 10.1186/1743-422x-10-213
title:
  Isolation and genetic characterization of human coronavirus NL63 in primary human renal proximal tubular epithelial cells obtained from a commercial supplier, and confirmation of its replication in two different types of human primary kidney cells

abstract:
  BACKGROUND: Cryopreserved primary human renal proximal tubule epithelial cells (RPTEC) were obtained from a commercial supplier for studies of Simian virus 40 (SV40). Within twelve hrs after cell cultures were initiated, cytoplasmic vacuoles appeared in many of the RPTEC. The RPTEC henceforth deteriorated rapidly. Since SV40 induces the formation of cytoplasmic vacuoles, this batch of RPTEC was rejected for the SV40 study. Nevertheless, we sought the likely cause(s) of the deterioration of the RPTEC as part of our technology development efforts. METHODS: Adventitious viruses in the RPTEC were isolated and/or detected and identified by isolation in various indicator cell lines, observation of cytopathology, an immunoflurorescence assay, electron microscopy, PCR, and sequencing. RESULTS: Cytomegalovirus (CMV) was detected in some RPTEC by cytology, an immunofluorescence assay, and PCR. Human Herpesvirus 6B was detected by PCR of DNA extracted from the RPTEC, but was not isolated. Human coronavirus NL63 was isolated and identified by RT-PCR and sequencing, and its replication in a fresh batch of RPTEC and another type of primary human kidney cells was confirmed. CONCLUSIONS: At least 3 different adventitious viruses were present in the batch of contaminated RPTEC. Whereas we are unable to determine whether the original RPTEC were pre-infected prior to their separation from other kidney cells, or had gotten contaminated with HCoV-NL63 from an ill laboratory worker during their preparation for commercial sale, our findings are a reminder that human-derived biologicals should always be considered as potential sources of infectious agents. Importantly, HCoV-NL63 replicates to high titers in some primary human kidney cells.


id: cord-291961-usl8z6ep
author: Zheng, Wen-zhi
date: 2015-10-13
txt: ./txt/cord-291961-usl8z6ep.txt
cache: ./cache/cord-291961-usl8z6ep.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/s12985-015-0390-5
doi: 10.1186/s12985-015-0390-5
summary:
  METHODS: The VP1 gene of HPyV6 was detected with an established TaqMan real-time PCR from nasopharyngeal aspirate specimens collected from hospitalized children with respiratory tract infections. All 15 HPyV6-positive patients were diagnosed with lower respiratory tract infections, and their viral loads ranged from 1.38 to 182.42 copies/μl nasopharyngeal aspirate specimen. CONCLUSIONS: The prevalence of HPyV6 was 1.7 % in nasopharyngeal aspirate specimens from hospitalized children with respiratory tract infections, as analyzed by real-time PCR. Previous studies have indicated that a number of HPyVs are associated with human diseases, such as progressive multifocal leukoencephalopathy (JCPyV), hemorrhagic cystitis (BKPyV), Merkel cell carcinoma (MCPyV), and trichodysplasia spinulosa (TSPyV) [3, 7, 9, [17] [18] [19] . Because initial infections with most HPyVs occur in infancy, the prevalence of HPyV6 in NPAs from children was detected with real-time PCR. The detection rate for HPyV6 by real-time PCR assay was 1.7 % in 887 NPA samples collected from hospitalized children with RTI.

title:
  Human polyomavirus type six in respiratory samples from hospitalized children with respiratory tract infections in Beijing, China

abstract:
  BACKGROUND: HPyV6 is a novel human polyomavirus (HPyV), and neither its natural history nor its prevalence in human disease is well known. Therefore, the epidemiology and phylogenetic status of HPyV6 must be systematically characterized. METHODS: The VP1 gene of HPyV6 was detected with an established TaqMan real-time PCR from nasopharyngeal aspirate specimens collected from hospitalized children with respiratory tract infections. The HPyV6-positive specimens were screened for other common respiratory viruses with real-time PCR assays. RESULTS: The prevalence of HPyV6 was 1.7 % (15/887), and children ≤ 5 years of age accounted for 80 % (12/15) of cases. All 15 HPyV6-positive patients were coinfected with other respiratory viruses, of which influenza virus A (IFVA) (8/15, 53.3 %) and respiratory syncytial virus (7/15, 46.7 %) were most common. All 15 HPyV6-positive patients were diagnosed with lower respiratory tract infections, and their viral loads ranged from 1.38 to 182.42 copies/μl nasopharyngeal aspirate specimen. The most common symptoms were cough (100 %) and fever (86.7 %). The complete 4926-bp genome (BJ376 strain, GenBank accession number KM387421) was amplified and showed 100 % identity to HPyV6 strain 607a. CONCLUSIONS: The prevalence of HPyV6 was 1.7 % in nasopharyngeal aspirate specimens from hospitalized children with respiratory tract infections, as analyzed by real-time PCR. Because the coinfection rate was high and the viral load low, it was not possible to establish a correlation between HPyV6 and respiratory diseases. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0390-5) contains supplementary material, which is available to authorized users.


id: cord-292581-6ipzvryb
title: Elevated levels of vitamin D and deficiency of mannose binding lectin in dengue hemorrhagic fever
author: Alagarasu, Kalichamy
date: 2012-05-04
txt: ./txt/cord-292581-6ipzvryb.txt
cache: ./cache/cord-292581-6ipzvryb.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/22559908/
doi: 10.1186/1743-422x-9-86
summary:
  BACKGROUND: Altered plasma concentrations of vitamin D and mannose binding lectin (MBL), components of innate immunity, have been shown to be associated with the pathogenesis of viral infections. The objective of the present study was to find out whether plasma concentrations of MBL and vitamin D are different in patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). Therefore, we investigated the levels of plasma vitamin D and MBL in dengue infected patients in the context of disease severity and immune status. When the patients were grouped based on immune status and disease severity, secondary DHF cases had significantly higher concentrations of vitamin D as compared to secondary DF cases (P < 0.050). Analysis of circulating concentrations of MBL in dengue cases and healthy controls revealed no significant difference between the two groups suggesting that the MBL mediated pathway of complement activation might be inhibited or may not be induced during DENV infection.

abstract:
  BACKGROUND: Altered plasma concentrations of vitamin D and mannose binding lectin (MBL), components of innate immunity, have been shown to be associated with the pathogenesis of viral infections. The objective of the present study was to find out whether plasma concentrations of MBL and vitamin D are different in patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). THE RESULTS: The plasma concentrations of vitamin D and MBL were assessed in 48 DF cases, 45 DHF cases and 20 apparently healthy controls using ELISA based methods. Vitamin D concentrations were found to be higher among both DF and DHF cases as compared to healthy controls (P < 0.005 and P < 0.001). Vitamin D concentrations were not different between DF and DHF cases. When the dengue cases were classified into primary and secondary infections, secondary DHF cases had significantly higher concentrations of vitamin D as compared to secondary DF cases (P < 0.050). MBL concentrations were not significantly different between healthy controls and dengue cases. MBL concentrations were observed to be lower in DHF cases as compared to DF cases (P < 0.050). Although MBL levels were not different DF and DHF cases based on immune status, the percentage of primary DHF cases (50%) having MBL levels lower than 500 ng/ml were less compared to primary DF cases (P = 0.038). CONCLUSIONS: The present study suggests that higher concentrations of vitamin D might be associated with secondary DHF while deficiency of MBL may be associated with primary DHF.


id: cord-292794-okh6i4l1
author: Wang, Bin
date: 2012-06-27
txt: ./txt/cord-292794-okh6i4l1.txt
cache: ./cache/cord-292794-okh6i4l1.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/22738661/
doi: 10.1186/1743-422x-9-127
summary:
  Results demonstrated that there was no detectable virus load in all the vaccine-immunized mice, while empty vector control group showed high lung viral titers ( Figure 4 ). Results indicated that all the mice that had been vaccinated with MHa had a detectable virus level, although showed a reduction in mean viral titers in both challenge groups compared with vector control, the reduction did not reach significance (p = 0.06 for rPan09 group and p = 0.67 for G11 group, Figure 4 ). The present study lays the foundation for universal swine influenza vaccine development, and call for further investigations in which the heterologous immune response should be further enhanced, such as the addition of molecular adjuvants [31, 32] and/or more copies of conserved viral protein encoding genes [33] , and the usage of DNA-prime protein/virus-boost immunization schedule [34, 35] .

title:
  Protective efficacy of a broadly cross-reactive swine influenza DNA vaccine encoding M2e, cytotoxic T lymphocyte epitope and consensus H3 hemagglutinin

abstract:
  BACKGROUND: Pigs have been implicated as mixing reservoir for the generation of new pandemic influenza strains, control of swine influenza has both veterinary and public health significance. Unlike human influenza vaccines, strains used for commercially available swine influenza vaccines are not regularly replaced, making the vaccines provide limited protection against antigenically diverse viruses. It is therefore necessary to develop broadly protective swine influenza vaccines that are efficacious to both homologous and heterologous virus infections. In this study, two forms of DNA vaccines were constructed, one was made by fusing M2e to consensus H3HA (MHa), which represents the majority of the HA sequences of H3N2 swine influenza viruses. Another was made by fusing M2e and a conserved CTL epitope (NP147-155) to consensus H3HA (MNHa). Their protective efficacies against homologous and heterologous challenges were tested. RESULTS: BALB/c mice were immunized twice by particle-mediated epidermal delivery (gene gun) with the two DNA vaccines. It was shown that the two vaccines elicited substantial antibody responses, and MNHa induced more significant T cell-mediated immune response than MHa did. Then two H3N2 strains representative of different evolutional and antigenic clusters were used to challenge the vaccine-immunized mice (homosubtypic challenge). Results indicated that both of the DNA vaccines prevented homosubtypic virus infections completely. The vaccines’ heterologous protective efficacies were further tested by challenging with a H1N1 swine influenza virus and a reassortant 2009 pandemic strain. It was found that MNHa reduced the lung viral titers significantly in both challenge groups, histopathological observation showed obvious reduction of lung pathogenesis as compared to MHa and control groups. CONCLUSIONS: The combined utility of the consensus HA and the conserved M2e and CTL epitope can confer complete and partial protection against homologous and heterologous challenges, respectively, in mouse model. This may provide a basis for the development of universal swine influenza vaccines.


id: cord-293938-40zyv1h8
title: Coronaviruses and the human airway: a universal system for virus-host interaction studies
author: Jonsdottir, Hulda R.
date: 2016-02-06
url: https://doi.org/10.1186/s12985-016-0479-5
doi: 10.1186/s12985-016-0479-5
abstract:
  Human coronaviruses (HCoVs) are large RNA viruses that infect the human respiratory tract. The emergence of both Severe Acute Respiratory Syndrome and Middle East Respiratory syndrome CoVs as well as the yearly circulation of four common CoVs highlights the importance of elucidating the different mechanisms employed by these viruses to evade the host immune response, determine their tropism and identify antiviral compounds. Various animal models have been established to investigate HCoV infection, including mice and non-human primates. To establish a link between the research conducted in animal models and humans, an organotypic human airway culture system, that recapitulates the human airway epithelium, has been developed. Currently, different cell culture systems are available to recapitulate the human airways, including the Air-Liquid Interface (ALI) human airway epithelium (HAE) model. Tracheobronchial HAE cultures recapitulate the primary entry point of human respiratory viruses while the alveolar model allows for elucidation of mechanisms involved in viral infection and pathogenesis in the alveoli. These organotypic human airway cultures represent a universal platform to study respiratory virus-host interaction by offering more detailed insights compared to cell lines. Additionally, the epidemic potential of this virus family highlights the need for both vaccines and antivirals. No commercial vaccine is available but various effective antivirals have been identified, some with potential for human treatment. These morphological airway cultures are also well suited for the identification of antivirals, evaluation of compound toxicity and viral inhibition.


id: cord-295207-0p6x4lwx
title: Peptide inhibition of human cytomegalovirus infection
author: Melnik, Lilia I
date: 2011-02-22
txt: ./txt/cord-295207-0p6x4lwx.txt
cache: ./cache/cord-295207-0p6x4lwx.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/1743-422x-8-76
doi: 10.1186/1743-422x-8-76
summary:
  The aim of this study was to develop therapeutic peptides targeting glycoprotein B (gB), a major glycoprotein of HCMV that is highly conserved across the Herpesviridae family, that specifically inhibit fusion of the viral envelope with the host cell membrane preventing HCMV entry and infection. Previous studies have suggested that synthetic peptides corresponding to or overlapping with sequences in viral fusion proteins that have positive WWIHS scores can sometimes serve as viral entry inhibitors [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] . For example, Enfurvitide (Fuzeon) is a 36-amino acid peptide that overlaps with a WWIHS score-positive sequence in the transmembrane protein (TM) of HIV-1, and prevents viral fusion and entry of the virus. These results suggest that the HCMV inhibitory peptides identified here may serve as Figure 2 Determination of regions within gB that display a high propensity to interact with the lipid surface of cell membranes by using Wimley-White Interfacial Hydrophobicity Scale (WWIHS).

abstract:
  BACKGROUND: Human cytomegalovirus (HCMV) is the most prevalent congenital viral infection in the United States and Europe causing significant morbidity and mortality to both mother and child. HCMV is also an opportunistic pathogen in immunocompromised individuals, including human immunodeficiency virus (HIV)- infected patients with AIDS, and solid organ and allogeneic stem cell transplantation recipients. Current treatments for HCMV-associated diseases are insufficient due to the emergence of drug-induced resistance and cytotoxicity, necessitating novel approaches to limit HCMV infection. The aim of this study was to develop therapeutic peptides targeting glycoprotein B (gB), a major glycoprotein of HCMV that is highly conserved across the Herpesviridae family, that specifically inhibit fusion of the viral envelope with the host cell membrane preventing HCMV entry and infection. RESULTS: Using the Wimley-White Interfacial Hydrophobicity Scale (WWIHS), several regions within gB were identified that display a high potential to interact with lipid bilayers of cell membranes and hydrophobic surfaces within proteins. The ability of synthetic peptides analogous to WWIHS-positive sequences of HCMV gB to inhibit viral infectivity was evaluated. Human foreskin fibroblasts (HFF) were infected with the Towne-GFP strain of HCMV (0.5 MOI), preincubated with peptides at a range of concentrations (78 nm to 100 μM), and GFP-positive cells were visualized 48 hours post-infection by fluorescence microscopy and analyzed quantitatively by flow cytometry. Peptides that inhibited HCMV infection demonstrated different inhibitory concentration curves indicating that each peptide possesses distinct biophysical properties. Peptide 174-200 showed 80% inhibition of viral infection at a concentration of 100 μM, and 51% and 62% inhibition at concentrations of 5 μM and 2.5 μM, respectively. Peptide 233-263 inhibited infection by 97% and 92% at concentrations of 100 μM and 50 μM, respectively, and 60% at a concentration of 2.5 μM. While peptides 264-291 and 297-315, individually failed to inhibit viral infection, when combined, they showed 67% inhibition of HCMV infection at a concentration of 0.125 μM each. CONCLUSIONS: Peptides designed to target putative fusogenic domains of gB provide a basis for the development of novel therapeutics that prevent HCMV infection.


id: cord-300434-obvm2en0
author: Ivancic-Jelecki, Jelena
date: 2018-07-18
txt: ./txt/cord-300434-obvm2en0.txt
cache: ./cache/cord-300434-obvm2en0.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/s12985-018-1020-9
doi: 10.1186/s12985-018-1020-9
summary:
  BACKGROUND: Small hydrophobic (SH) gene is one of the mostly diverse genomic regions of human respiratory syncytial virus (HRSV). When analysis was restricted to strains with identical HVR2 nucleotide sequences and differing SH protein sequences, 75% of differences observed in the SH ectodomain were located within region coding for amino acids 49–51. In molecular epidemiology studies, HRSV surveillance and genotyping are based on sequences of the second, C-terminal hypervariable region of the G gene (HVR2). Following our previous analysis of HVR2 sequences of strains detected within a limited geographical area (the Zagreb region) and limited time frame (March 2011 to March 2014) [16] , the goal of this study was to investigate whether virus variability and inter-genotype differences observed for HVR2 are also present in the SH gene.

title:
  Variability analysis and inter-genotype comparison of human respiratory syncytial virus small hydrophobic gene

abstract:
  BACKGROUND: Small hydrophobic (SH) gene is one of the mostly diverse genomic regions of human respiratory syncytial virus (HRSV). Its coding region constitutes less than 50% of the complete gene length, enabling SH gene to be highly variable and the SH protein highly conserved. In standard HRSV molecular epidemiology studies, solely sequences of the second hypervariable region of the glycoprotein gene (HVR2) are analyzed. To what extent do the strains identical in HVR2 differ elsewhere in genomes is rarely investigated. Our goal was to investigate whether diversity and inter-genotype differences observed for HVR2 are also present in the SH gene. METHODS: We sequenced 198 clinical samples collected within a limited area and time frame. In this HRSV collection, rapid and significant changes in HVR2 occurred. RESULTS: Over 20% of strains from this pool (containing HRSV genotypes NA1, ON1, GA5, BA9 and BA10) would be incorrectly assumed to be identical to another strain if only the HVR2 region was analysed. The majority of differences found in SH gene were located in the 5′ untranslated region (UTR). Seven indels were detected, one was genotype GA5 specific. An in-frame deletion of 9 nucleotides (coding for amino acids 49–51) was observed in one of group A strains. Fifteen different SH protein sequences were detected; 68% of strains possessed the consensus sequence and most of others differed from the consensus in only one amino acid (only 4 strains differed in 2 amino acids). The majority of differing amino acids in group A viruses had the same identity as the corresponding amino acids in group B strains. When analysis was restricted to strains with identical HVR2 nucleotide sequences and differing SH protein sequences, 75% of differences observed in the SH ectodomain were located within region coding for amino acids 49–51. CONCLUSIONS: Basing HRSV molecular epidemiology studies solely on HVR2 largely underestimates the complexity of circulating virus populations. In strain identification, broadening of the genomic target sequence to SH gene would provide a more comprehensive insight into viral pool versatility and its evolutionary processes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12985-018-1020-9) contains supplementary material, which is available to authorized users.


id: cord-300837-d0a8y5qh
title: A Functional Henipavirus Envelope Glycoprotein Pseudotyped Lentivirus Assay System
author: Khetawat, Dimple
date: 2010-11-12
url: https://www.ncbi.nlm.nih.gov/pubmed/21073718/
doi: 10.1186/1743-422x-7-312
abstract:
  BACKGROUND: Hendra virus (HeV) and Nipah virus (NiV) are newly emerged zoonotic paramyxoviruses discovered during outbreaks in Queensland, Australia in 1994 and peninsular Malaysia in 1998/9 respectively and classified within the new Henipavirus genus. Both viruses can infect a broad range of mammalian species causing severe and often-lethal disease in humans and animals, and repeated outbreaks continue to occur. Extensive laboratory studies on the host cell infection stage of HeV and NiV and the roles of their envelope glycoproteins have been hampered by their highly pathogenic nature and restriction to biosafety level-4 (BSL-4) containment. To circumvent this problem, we have developed a henipavirus envelope glycoprotein pseudotyped lentivirus assay system using either a luciferase gene or green fluorescent protein (GFP) gene encoding human immunodeficiency virus type-1 (HIV-1) genome in conjunction with the HeV and NiV fusion (F) and attachment (G) glycoproteins. RESULTS: Functional retrovirus particles pseudotyped with henipavirus F and G glycoproteins displayed proper target cell tropism and entry and infection was dependent on the presence of the HeV and NiV receptors ephrinB2 or B3 on target cells. The functional specificity of the assay was confirmed by the lack of reporter-gene signals when particles bearing either only the F or only G glycoprotein were prepared and assayed. Virus entry could be specifically blocked when infection was carried out in the presence of a fusion inhibiting C-terminal heptad (HR-2) peptide, a well-characterized, cross-reactive, neutralizing human mAb specific for the henipavirus G glycoprotein, and soluble ephrinB2 and B3 receptors. In addition, the utility of the assay was also demonstrated by an examination of the influence of the cytoplasmic tail of F in its fusion activity and incorporation into pseudotyped virus particles by generating and testing a panel of truncation mutants of NiV and HeV F. CONCLUSIONS: Together, these results demonstrate that a specific henipavirus entry assay has been developed using NiV or HeV F and G glycoprotein pseudotyped reporter-gene encoding retrovirus particles. This assay can be conducted safely under BSL-2 conditions and will be a useful tool for measuring henipavirus entry and studying F and G glycoprotein function in the context of virus entry, as well as in assaying and characterizing neutralizing antibodies and virus entry inhibitors.


id: cord-300908-i80tuhqk
author: Yu, Fuxun
date: 2015-08-04
txt: ./txt/cord-300908-i80tuhqk.txt
cache: ./cache/cord-300908-i80tuhqk.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/26239826/
doi: 10.1186/s12985-015-0350-0
summary:
  title: Application of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for the detection of SFTSV-specific human IgG and IgM antibodies by indirect ELISA rSFTSV-N protein based indirect IgG and IgM enzyme linked immunosorbent assay (ELISA) systems were established to detect specific human IgG and IgM antibodies, respectively. Application of the rSFTSV-N protein based indirect IgG ELISA to the 115 serum samples showed results that perfectly matched those of the total antibody sandwich ELISA with a sensitivity and specificity of 100 %. To determine the appropriate dilutions of serum samples for the indirect IgG and IgM ELISAs using the rSFTSV-N protein mentioned above, samples from healthy volunteers and SFTS confirmed patients were diluted two-fold from 1:100 up to 1:1000. To evaluate the usefulness for diagnosis of the rSFTSV-N protein we developed, we established an indirect IgG and IgM ELISA for the laboratory diagnosis of SFTSV infection in humans with the serum samples as clinical specimens.

title:
  Application of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for the detection of SFTSV-specific human IgG and IgM antibodies by indirect ELISA

abstract:
  BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease that was first reported in China in 2011. It is caused by SFTS virus (SFTSV) which is a member of the Phlebovirus genus in the Bunyaviridae family. SFTSV has been classified as a BSL3 pathogen. There is a need to develop safe and affordable serodiagnostic methods for proper clinical management of infected patients. METHODS: The full length nucleocapsid (N) gene of SFTSV Yamaguchi strain was amplified by RT-PCR and cloned to an expression vector pQE30. The recombinant (r) SFTSV-N protein was expressed by using Escherichia coli (E. coli) expression system and purified under native conditions. rSFTSV-N protein based indirect IgG and IgM enzyme linked immunosorbent assay (ELISA) systems were established to detect specific human IgG and IgM antibodies, respectively. One hundred fifteen serum samples from clinically suspected-SFTS patients were used to evaluate the newly established systems and the results were compared with the total antibody detecting sandwich ELISA system. RESULTS: The native form of recombinant (r) SFTSV-N protein was expressed and purified. Application of the rSFTSV-N protein based indirect IgG ELISA to the 115 serum samples showed results that perfectly matched those of the total antibody sandwich ELISA with a sensitivity and specificity of 100 %. The rSFTSV-N protein based indirect IgM ELISA missed 8 positive samples that were detected by the total antibody sandwich ELISA. The sensitivity and specificity of rSFTSV-N-IgM capture ELISA were 90.59 and 100 %, respectively. CONCLUSIONS: The rSFTSV-N protein is highly immunoreactive and a good target for use as an assay antigen in laboratory diagnosis. Its preparation is simpler in comparison with that used for the total antibody sandwich system. Our rSFTSV-N protein-based IgG and IgM ELISA systems have the advantage of distinguishing two types of antibodies and require small volume of serum sample only. They are safe to use for diagnosis of SFTS virus infection and especially fit in large-scale epidemiological investigations.


id: cord-302170-k2apj469
author: Fang, Peng
date: 2015-10-24
url: https://doi.org/10.1186/s12985-015-0405-2
doi: 10.1186/s12985-015-0405-2
title:
  Assessment of reference gene stability in Rice stripe virus and Rice black streaked dwarf virus infection rice by quantitative Real-time PCR

abstract:
  BACKGROUND: Stably expressed reference gene(s) normalization is important for the understanding of gene expression patterns by quantitative Real-time PCR (RT-qPCR), particularly for Rice stripe virus (RSV) and Rice black streaked dwarf virus (RBSDV) that caused seriously damage on rice plants in China and Southeast Asia. METHODS: The expression of fourteen common used reference genes of Oryza sativa L. were evaluated by RT-qPCR in RSV and RBSDV infected rice plants. Suitable normalization reference gene(s) were identified by geNorm and NormFinder algorithms. RESULTS: UBQ 10 + GAPDH and UBC + Actin1 were identified as suitable reference genes for RT-qPCR normalization under RSV and RBSDV infection, respectively. When using multiple reference genes, the expression patterns of OsPRIb and OsWRKY, two virus resistance genes, were approximately similar with that reported previously. Comparatively, by using single reference gene (TIP41-Like), a weaker inducible response was observed. CONCLUSIONS: We proposed that the combination of two reference genes could obtain more accurate and reliable normalization of RT-qPCR results in RSV- and RBSDV-infected plants. This work therefore sheds light on establishing a standardized RT-qPCR procedure in RSV- and RBSDV-infected rice plants, and might serve as an important point for discovering complex regulatory networks and identifying genes relevant to biological processes or implicated in virus. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0405-2) contains supplementary material, which is available to authorized users.


id: cord-303313-b8lesr2g
title: Virus detection and identification using random multiplex (RT)-PCR with 3'-locked random primers
author: Clem, Amy L
date: 2007-06-28
url: https://www.ncbi.nlm.nih.gov/pubmed/17598900/
doi: 10.1186/1743-422x-4-65
abstract:
  BACKGROUND: PCR-based detection and identification of viruses assumes a known, relatively stable genome. Unfortunately, high mutation rates may lead to extensive changes in viral nucleic acid sequences making dedicated PCR primer use problematic. Furthermore, in bioterrorism, viral consensus sequences can be genetically modified as a countermeasure to RT-PCR and DNA chip detection. Accordingly, there is a great need for the development of rapid and universal virus detection and identification technologies. RESULTS: We report herein that viral genomic DNA or RNA can be separated from host nucleic acids in plasma by filtration and nuclease digestion, and randomly amplified in a single PCR using a mixture of primers designed to be resistant to primer-dimer amplification (5'-VVVVVVVVAA-3', V = A, G or C; 3(8 )or 6561 primers). We have termed this novel PCR method Random Multiplex (RT)-PCR since hundreds of overlapping PCR amplifications occur simultaneously. Using this method, we have successfullydetected and partially sequenced 3 separate viruses in human plasma without using virus-specific reagents (i.e., Adenovirus Type 17, Coxsackievirus A7, and Respiratory Syncytial Virus B). The method is sensitive to ~1000 genome equivalents/ml and may represent the fastest means of detection of unknown viruses. CONCLUSION: These studies suggest that the further development of random multiplex (RT)-PCR may lead to a diagnostic assay that can universally detect viruses in donated blood products as well as in patients suffering with idiopathic disease states of possible viral etiology.


id: cord-303494-tofch4j7
title: Identification of VP1 peptides diagnostic of encephalomyocarditis virus from swine
author: Bai, Juan
date: 2014-12-30
url: https://doi.org/10.1186/s12985-014-0226-8
doi: 10.1186/s12985-014-0226-8
abstract:
  BACKGROUND: Encephalomyocarditis virus (EMCV) can cause myocarditis, respiratory failure, reproductive failure, and sudden death in pre-weaned piglets, which has been isolated in China. EMCV VP1 protein was one of the most important structural proteins and played an important role in the protective immunity. In this study, 10 monoclonal antibodies (McAbs) against EMCV VP1 were screened and identified. RESULTS: Epitope mapping results indicated that McAbs (6E11, 7A7, 7C9) specifically recognized the linear epitopes V(2)ENAEK(7), McAbs (1D1, 2A2, 5A1, 5A11, 5G1) recognized the epitope F(19)VAQPVY(25), and McAbs 1G8 and 3A9 recognized P(42)IGAFTVK(49). Protein sequence alignment of VP1 with 16 EMCV isolates indicated that the epitope F(19)VAQPVY(25) was conserved in all the reference strains. The epitopes P(42)IGAFTVK(49) and V(2)ENAEK(7) only had 1 or 2 variable amino acid among the reference strains. The 3D model analysis results showed that these epitopes presented as spheres were shown within the context of the complete particle. CONCLUSIONS: In this study, ten McAbs against EMCV VP1 were developed and three B-cells epitopes (2-7aa, 19-25aa and 42-49aa) were defined in VP1. All the results herein will promote the future investigations into the function of VP1 of EMCV and development of diagnostic methods of EMCV.


id: cord-304058-i8cywew0
author: Pfefferle, Susanne
date: 2009-08-24
url: https://www.ncbi.nlm.nih.gov/pubmed/19698190/
doi: 10.1186/1743-422x-6-131
title:
  Reverse genetic characterization of the natural genomic deletion in SARS-Coronavirus strain Frankfurt-1 open reading frame 7b reveals an attenuating function of the 7b protein in-vitro and in-vivo

abstract:
  During the outbreak of SARS in 2002/3, a prototype virus was isolated from a patient in Frankfurt/Germany (strain Frankfurt-1). As opposed to all other SARS-Coronavirus strains, Frankfurt-1 has a 45-nucleotide deletion in the transmembrane domain of its ORF 7b protein. When over-expressed in HEK 293 cells, the full-length protein but not the variant with the deletion caused interferon beta induction and cleavage of procaspase 3. To study the role of ORF 7b in the context of virus replication, we cloned a full genome cDNA copy of Frankfurt-1 in a bacterial artificial chromosome downstream of a T7 RNA polymerase promoter. Transfection of capped RNA transcribed from this construct yielded infectious virus that was indistinguishable from the original virus isolate. The presumed Frankfurt-1 ancestor with an intact ORF 7b was reconstructed. In CaCo-2 and HUH7 cells, but not in Vero cells, the variant carrying the ORF 7b deletion had a replicative advantage against the parental virus (4- and 6-fold increase of virus RNA in supernatant, respectively). This effect was neither associated with changes in the induction or secretion of type I interferon, nor with altered induction of apoptosis in cell culture. However, pretreatment of cells with interferon beta caused the deleted virus to replicate to higher titers than the parental strain (3.4-fold in Vero cells, 7.9-fold in CaCo-2 cells). In Syrian Golden Hamsters inoculated intranasally with 10e4 plaque forming units of either virus, mean titers of infectious virus and viral RNA in the lungs after 24 h were increased 23- and 94.8-fold, respectively, with the deleted virus. This difference could explain earlier observations of enhanced virulence of Frankfurt-1 in Hamsters as compared to other SARS-Coronavirus reference strains and identifies the SARS-CoV 7b protein as an attenuating factor with the SARS-Coronavirus genome. Because attenuation was focused on the early phase of infection in-vivo, ORF 7b might have contributed to the delayed accumulation of virus in patients that was suggested to have limited the spread of the SARS epidemic.


id: cord-305315-0qt7eth0
author: Cao, Liyan
date: 2015-08-18
txt: ./txt/cord-305315-0qt7eth0.txt
cache: ./cache/cord-305315-0qt7eth0.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/s12985-015-0345-x
doi: 10.1186/s12985-015-0345-x
summary:
  title: Porcine epidemic diarrhea virus inhibits dsRNA-induced interferon-β production in porcine intestinal epithelial cells by blockade of the RIG-I-mediated pathway In this study, porcine small intestinal epithelial cells (IECs), the target cells of PEDV, were used as the infection model in vitro to identify the possible molecular mechanisms of PEDV-inhibition IFN-β production. CONCLUSION: Taken together, our data demonstrated for the first time that PEDV infection of its target cell line, IECs, inhibited dsRNA-mediated IFN-β production by blocking the activation of IPS-1 in RIG-I-mediated pathway. PEDV failed to induce IFN-β expression and inhibited poly (I:C)-mediated IFN-β production in IECs Type I IFNs (IFN-α/β) are critical to the host antiviral innate immune response. In summary, the findings of the present study suggested that PEDV-infection in IECs inhibits dsRNA-induced IFN-β induction by interfering with IRF-3 activity associated with RIG-I-mediated signaling pathway.

title:
  Porcine epidemic diarrhea virus inhibits dsRNA-induced interferon-β production in porcine intestinal epithelial cells by blockade of the RIG-I-mediated pathway

abstract:
  BACKGROUND: The lack of optimal porcine cell lines has severely impeded the study and progress in elucidation of porcine epidemic diarrhea virus (PEDV) pathogenesis. Vero cell, an African green monkey kidney cell line, was often used to isolate and propagate PEDV. Nonetheless, the target cells of PEDV in vivo are intestinal epithelial cells, during infection, intestinal epithelia would be damaged and resulted in digestive disorders. The immune functions of porcine epithelial cells and interactions with other immune cell populations display a number of differences compared to other species. Type I interferon (IFN) plays an important role in antiviral immune response. Limited reports showed that PEDV could inhibit type I interferon production. In this study, porcine small intestinal epithelial cells (IECs), the target cells of PEDV, were used as the infection model in vitro to identify the possible molecular mechanisms of PEDV-inhibition IFN-β production. RESULTS: PEDV not only failed to induce IFN-β expression, but also inhibited dsRNA-mediated IFN-β production in IECs. As the key IFN-β transcription factors, we found that dsRNA-induced activation of IFN regulatory factor 3 (IRF-3) was inhibited after PEDV infection, but not nuclear factor-kappaB (NF-κB). To identify the mechanism of PEDV intervention with dsRNA-mediated IFN-β expression more accurately, the role of individual molecules of RIG-I signaling pathway were investigated. In the upstream of IRF-3, TANK-binding kinase 1 (TBK1)-or inhibitor of κB kinase-ε (IKKε)-mediated IFN-β production was not blocked by PEDV, while RIG-I-and its adapter molecule IFN-β promoter stimulator 1 (IPS-1)-mediated IFN-β production were completely inhibited after PEDV infection. CONCLUSION: Taken together, our data demonstrated for the first time that PEDV infection of its target cell line, IECs, inhibited dsRNA-mediated IFN-β production by blocking the activation of IPS-1 in RIG-I-mediated pathway. Our studies offered new visions in understanding of the interaction between PEDV and host innate immune system.


id: cord-307364-j86t65qu
author: Uccellini, Lorenzo
date: 2014-08-08
txt: ./txt/cord-307364-j86t65qu.txt
cache: ./cache/cord-307364-j86t65qu.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/25106433/
doi: 10.1186/1743-422x-11-144
summary:
  In situ hybridization confirmed the presence of intracellular, intracytoplasmic viral nucleic acids in the lungs of infected snakes. Phylogenetic analysis based on a 1,136 amino acid segment of the polyprotein suggests that this virus may represent a new species in the subfamily Torovirinae. CONCLUSIONS: This report of a novel nidovirus in ball pythons may provide insight into the pathogenesis of respiratory disease in this species and enhances our knowledge of the diversity of nidoviruses. Here we report the discovery of a novel nidovirus in a collection of ball pythons (Python regius) in upstate New York with pneumonia, tracheitis and esophagitis. Based on the phylogenetic position and the genetic distances between In situ hybridization to a 934 nt fragment of the genomic polyprotein 1ab region was used to assess viral infection and distribution in the lung tissue. Identification of a novel nidovirus in an outbreak of fatal respiratory disease in ball pythons (Python regius)

title:
  Identification of a novel nidovirus in an outbreak of fatal respiratory disease in ball pythons (Python regius)

abstract:
  BACKGROUND: Respiratory infections are important causes of morbidity and mortality in reptiles; however, the causative agents are only infrequently identified. FINDINGS: Pneumonia, tracheitis and esophagitis were reported in a collection of ball pythons (Python regius). Eight of 12 snakes had evidence of bacterial pneumonia. High-throughput sequencing of total extracted nucleic acids from lung, esophagus and spleen revealed a novel nidovirus. PCR indicated the presence of viral RNA in lung, trachea, esophagus, liver, and spleen. In situ hybridization confirmed the presence of intracellular, intracytoplasmic viral nucleic acids in the lungs of infected snakes. Phylogenetic analysis based on a 1,136 amino acid segment of the polyprotein suggests that this virus may represent a new species in the subfamily Torovirinae. CONCLUSIONS: This report of a novel nidovirus in ball pythons may provide insight into the pathogenesis of respiratory disease in this species and enhances our knowledge of the diversity of nidoviruses.


id: cord-308397-kqftn75t
author: Sun, Jia-zeng
date: 2014-12-03
url: https://www.ncbi.nlm.nih.gov/pubmed/25465595/
doi: 10.1186/s12985-014-0210-3
title:
  MicroRNA miR-320a and miR-140 inhibit mink enteritis virus infection by repression of its receptor, feline transferrin receptor

abstract:
  Mink enteritis virus (MEV) is one of the most important pathogens in the mink industry. Recent studies have shed light into the role of microRNAs (miRNAs), small noncoding RNAs of length ranging from 18–23 nucleotides (nt), as critical modulators in the host-pathogen interaction networks. We previously showed that miRNA miR-181b can inhibit MEV replication by repression of viral non-structural protein 1 expression. Here, we report that two other miRNAs (miR-320a and miR-140) inhibit MEV entry into feline kidney (F81) cells by downregulating its receptor, transferrin receptor (TfR), by targeting the 3′ untranslated region (UTR) of TfR mRNA, while being themselves upregulated.


id: cord-310372-qc6941pm
author: Ji, Jun
date: 2011-04-22
url: https://doi.org/10.1186/1743-422x-8-184
doi: 10.1186/1743-422x-8-184
title:
  Phylogenetic distribution and predominant genotype of the avian infectious bronchitis virus in China during 2008-2009

abstract:
  BACKGROUND: The nephropathogenic avian infectious bronchitis (IB) caused unprecedented economic losses to the commercial chicken industry of China in 2008-2009. To investigate the prevalence of nephropathogenic IB in China, eighty IBV isolates from different provinces during 2008-2009 were identified by dwarf embryo test and RT-PCR. RESULTS: The strains were mostly isolated in winter and spring with a wide age range of IB outbreaks, from 4 to 69 days. By the virus recovery trials, 70/80 of the strains resulted in the deaths or distresses of birds from nephritis. To learn more about the molecular evolutionary characteristics of the circulating field strains, the coding region of major spike 1 (S1) protein gene of these strains was RT-PCR amplified and sequenced. Compared to the published representative strains, nucleotides and amino acids sequence analysis indicated that the S1 genes of these strains and the reference strains displayed homologies ranging from 75.1% to 99.8% and from 73.1% to 99.8% respectively. S1 protein of the major pandemic strains contained 540 or 542 amino acids with the cleavage site of HRRRR or RRFRR. Phylogenetic analysis revealed that recent field isolates of IBV in China were mostly belonged to A2-branch (QXIBV-branch) and HN08-branch, only one isolate was belonged to Gray-branch and M41-branch respectively. Most of the 80 strains showed evolutionarily distant from vaccine strains. CONCLUSIONS: The results of this study suggested that nephropathogenic IBVs were mainly A2-like strains in China during 2008-2009.


id: cord-310536-u30cufg7
author: Finger, Paula Fonseca
date: 2018-12-12
url: https://doi.org/10.1186/s12985-018-1096-2
doi: 10.1186/s12985-018-1096-2
title:
  Combined use of ELISA and Western blot with recombinant N protein is a powerful tool for the immunodiagnosis of avian infectious bronchitis

abstract:
  BACKGROUND: The avian infectious bronchitis virus (IBV) remains a significant source of loss in the poultry industry and early diagnosis is required to prevent the disease from spreading. This study examined the combined use of an ELISA and Western blot (WB) to detect antibodies against the nucleocapsid protein (N) of IBV. The coding sequence for N was amplified by RT-PCR and expressed in Escherichia coli. A soluble recombinant N protein (rN) of approximately 50 kDa was obtained. A total of 389 sera were tested against the rN in ELISA and the results were compared with those of the commercial IDEXX IBV Ab test. ELISA-rN achieved a 90.34% sensitivity and 90.16% specificity. WB confirmed all false negative sera in ELISA-rN or IDEXX test as truly positive. The current study indicate that the combined use of rN in ELISA and WB is a powerful tool for the immunodiagnosis of avian infectious bronchitis. METHODS: Constructed recombinant pAE/n expression vectors were used to transform E. coli BL21(DE3) Star competent cells (Invitrogen). The rN of infectious bronchitis virus was purified by affinity chromatography using HisTrap HP 1 mL columns pre-packed with pre-charged Ni Sepharose in the ÄKTAprime Automated Liquid Chromatography system (GE Healthcare). A total of 389 serum samples from chickens were used to develop and evaluate the ELISA-rN test. To standardize the indirect ELISA development, serum dilutions (1:100, 1:200 and 1:400) and different concentrations of purified rN antigen (50, 100 and 200 ng/well) were tested. Positive and negative sera for IBV were used as controls. The results were compared with those obtained from a commercial kit. Serum samples scored as negative with the commercial kit but as positive with the ELISA-rN were further analysed by Western blot analyses using the rN protein as an antigen. The results of the ELISA-rN were compared to the commercial kit results using receiver-operating characteristics curves, area under the curve, and confidence intervals with the software GraphPad Prism version 6.0 for Windows (GraphPad Software, USA). RESULTS: The expected cDNA fragment of approximately 1240 bp was successfully amplified by PCR using primers designed to select for the coding region of the N protein. The rN was expressed as a soluble protein to avoid the refolding steps and, after purification a yield of 10 mg/L of rN was obtained. The SDS-PAGE results demonstrated the presence of two distinct bands that had a molecular mass of approximately 45 and 50 KDa. Out of 244 sera that scored positive in the commercial ELISA IDEXX IBV Ab Test, 220 were also positive in the ELISA-rN, yielding an ELISA-rN test sensitivity of 90.16%. Out of 145 sera that scored negative in the IDEXX IBV Ab Test, 131 also scored negative in the ELISA-rN, indicating a specificity of 90.34%. Sera that tested negative in the ELISA-rN and positive in the commercial test also reacted with the rN protein in Western blot. CONCLUSIONS: The association between the ELISA and Western blot techniques developed in this study with a subunit of IBV (rN) were able to detect antibodies that the commercial ELISA did not detect suggesting that the ELISA-rN has greater sensitivity.


id: cord-313598-2t40ss6h
title: Throat and nasal swabs for molecular detection of respiratory viruses in acute pharyngitis
author: Ali, Mohsin
date: 2015-10-29
url: https://doi.org/10.1186/s12985-015-0408-z
doi: 10.1186/s12985-015-0408-z
abstract:
  BACKGROUND: Detection of specific respiratory viruses is important for surveillance programs, where nasopharyngeal or nasal swabs have traditionally been used. Our objective was to determine whether sampling with a throat swab provides incremental benefit—when used in conjunction with a nasal swab—to detect respiratory viruses among patients with acute pharyngitis in the outpatient setting. FINDINGS: Among 83 university students with acute pharyngitis, we detected respiratory viruses with molecular assays on two samples collected per student: with a flocked nasal mid-turbinate swab and a rayon throat swab. Forty-eight (58 %) patients had virus-positive samples, with 49 virus positives detected by either swab (one patient had a dual viral co-infection). The most common viruses were rhinovirus, coronavirus, and influenza A virus. Specifically, 29 virus positives were detected by both swabs, 14 exclusively by the nasal swab, and six exclusively by the throat swab. The additional six virus positives detected by the throat swab corresponded to an absolute increase in viral detection of 7.1 % (95 % CI: 1.2–12.9 %); the specific viruses detected were four rhinoviruses and two coronaviruses. CONCLUSIONS: The flocked nasal swab samples respiratory viruses well, even among patients whose primary complaint is a sore throat. The rayon throat swab has modest incremental value over and above using the flocked nasal mid-turbinate swab alone, which suggests that while throat swabs alone would not be adequate for respiratory viral surveillance, they may have value as a supplementary test.


id: cord-314201-6njwigco
title: Universal primers that amplify RNA from all three flavivirus subgroups
author: Maher-Sturgess, Sheryl L
date: 2008-01-24
url: https://doi.org/10.1186/1743-422x-5-16
doi: 10.1186/1743-422x-5-16
abstract:
  BACKGROUND: Species within the Flavivirus genus pose public health problems around the world. Increasing cases of Dengue and Japanese encephalitis virus in Asia, frequent outbreaks of Yellow fever virus in Africa and South America, and the ongoing spread of West Nile virus throughout the Americas, show the geographical burden of flavivirus diseases. Flavivirus infections are often indistinct from and confused with other febrile illnesses. Here we review the specificity of published primers, and describe a new universal primer pair that can detect a wide range of flaviviruses, including viruses from each of the recognised subgroups. RESULTS: Bioinformatic analysis of 257 published full-length Flavivirus genomes revealed conserved regions not previously targeted by primers. Two degenerate primers, Flav100F and Flav200R were designed from these regions and used to generate an 800 base pair cDNA product. The region amplified encoded part of the methyltransferase and most of the RNA-dependent-RNA-polymerase (NS5) coding sequence. One-step RT-PCR testing was successful using standard conditions with RNA from over 60 different flavivirus strains representing about 50 species. The cDNA from each virus isolate was sequenced then used in phylogenetic analyses and database searches to confirm the identity of the template RNA. CONCLUSION: Comprehensive testing has revealed the broad specificity of these primers. We briefly discuss the advantages and uses of these universal primers.


id: cord-314954-otc0pc09
author: Liu, Xiaoli
date: 2010-09-13
txt: ./txt/cord-314954-otc0pc09.txt
cache: ./cache/cord-314954-otc0pc09.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/1743-422x-7-223
doi: 10.1186/1743-422x-7-223
summary:
  In this study, the C-terminus of DEV UL26 protein (designated UL26c), which contains the whole of UL26.5, was expressed, and the recombinant UL26c protein was used to immunize BALB/c mice to generate monoclonal antibodies (mAb). Both western blotting and ELISA showed that 1C8 could react specifically with both chicken embryonic fibroblasts (CEF) infected with DEV ( Figure 1B and Figure 1C ) and the recombinant protein UL26c ( Figure 1D and Figure 1C ). The results of both western blotting and ELISA showed that this peptide defined by mAb 1C8 could react with murine anti-DEV antibody ( Figure 4 ), which demonstrated that the epitope had good reactivity. A series of 17 fragments that spanned the UL26c protein were expressed with a GST tag in this study, and used to screen for the minimal epitope recognized by mAb 1C8 using western blotting and ELISA. The specificity and reactivity of the mAb 1C8 were also determined by western blotting using recombinant UL26c protein and CEF infected by DEV, respectively.

title:
  Identification of a novel linear B-cell epitope in the UL26 and UL26.5 proteins of Duck Enteritis Virus

abstract:
  BACKGROUND: The Unique Long 26 (UL26) and UL26.5 proteins of herpes simplex virus are known to function during the assembly of the viruses. However, for duck enteritis virus (DEV), which is an unassigned member of the family Herpesviridae, little information is available about the function of the two proteins. In this study, the C-terminus of DEV UL26 protein (designated UL26c), which contains the whole of UL26.5, was expressed, and the recombinant UL26c protein was used to immunize BALB/c mice to generate monoclonal antibodies (mAb). The mAb 1C8 was generated against DEV UL26 and UL26.5 proteins and used subsequently to map the epitope in this region. Both the mAb and its defined epitope will provide potential tools for further study of DEV. RESULTS: A mAb (designated 1C8) was generated against the DEV UL26c protein, and a series of 17 partially overlapping fragments that spanned the DEV UL26c were expressed with GST tags. These peptides were subjected to enzyme-linked immunosorbent assay (ELISA) and western blotting analysis using mAb 1C8 to identify the epitope. A linear motif, (520)IYYPGE(525), which was located at the C-terminus of the DEV UL26 and UL26.5 proteins, was identified by mAb 1C8. The result of the ELISA showed that this epitope could be recognized by DEV-positive serum from mice. The (520)IYYPGE(525 )motif was the minimal requirement for reactivity, as demonstrated by analysis of the reactivity of 1C8 with several truncated peptides derived from the motif. Alignment and comparison of the 1C8-defined epitope sequence with those of other alphaherpesviruses indicated that the motif (521)YYPGE(525 )in the epitope sequence was conserved among the alphaherpesviruses. CONCLUSION: A mAb, 1C8, was generated against DEV UL26c and the epitope-defined minimal sequence obtained using mAb 1C8 was (520)IYYPGE(525). The mAb and the identified epitope may be useful for further study of the design of diagnostic reagents for DEV.


id: cord-315688-ba5dus2j
author: He, Lei
date: 2012-08-28
txt: ./txt/cord-315688-ba5dus2j.txt
cache: ./cache/cord-315688-ba5dus2j.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/22929207/
doi: 10.1186/1743-422x-9-176
summary:
  title: In vitro inhibition of transmissible gastroenteritis coronavirus replication in swine testicular cells by short hairpin RNAs targeting the ORF 7 gene In the reporter assays, three of four shRNA expression plasmids were able to inhibit significantly the expression of ORF 7 gene and replication of TGEV, as shown by real-time quantitative RT-PCR analysis of viral ORF 7 and N genes and detection of virus titers (TCID(50)/ml). Here, we demonstrate that RNAi targeting of the ORF 7 gene of TGEV, introduced by short hairpin RNAs (shRNAs), is capable of inhibiting virus replication and protecting swine testicular (ST) cells from the destruction induced by TGEV, which may be not only a new anti-TGEV strategy, but also a new approach to the study of its pathogenesis. The present study demonstrated the use of RNAi against TGEV via shRNA-expressing plasmid vector pGPU6-GFP, which significantly reduced viral genomic RNA replication and protected ST cells from TGEV destruction, by targeting ORF 7 gene.

title:
  In vitro inhibition of transmissible gastroenteritis coronavirus replication in swine testicular cells by short hairpin RNAs targeting the ORF 7 gene

abstract:
  BACKGROUND: Transmissible gastroenteritis (TGE) is a highly contagious viral disease of swine, characterized by severe vomiting, diarrhea, and high mortality. Currently, the vaccines for it are only partially effective and no specific drug is available for treatment of TGE virus (TGEV) infection. RNA interference has been confirmed as a new approach for controlling viral infections. In this study, the inhibitory effect of short hairpin RNAs (shRNAs) targeting the ORF 7 gene of TGEV on virus replication was examined. RESULTS: Four theoretically effective sequences of TGEV ORF 7 gene were designed and selected for construction of shRNA expression plasmids. In the reporter assays, three of four shRNA expression plasmids were able to inhibit significantly the expression of ORF 7 gene and replication of TGEV, as shown by real-time quantitative RT-PCR analysis of viral ORF 7 and N genes and detection of virus titers (TCID(50)/ml). Stable swine testicular (ST) cells expressing the shRNAs were established. Observation of the cytopathic effect and apoptosis, as well as a cell proliferation assay demonstrated that the three shRNAs were capable of protecting ST cells against TGEV destruction, with high specificity and efficiency. CONCLUSIONS: Our results indicated that plasmid-transcribed shRNAs targeting the ORF 7 gene in the TGEV genome effectively inhibited expression of the viral target gene and viral replication in vitro. These findings provide evidence that the shRNAs have potential therapeutic application for treatment of TGE.


id: cord-316142-wg1qwmj1
author: Weiss, Sabrina
date: 2017-09-18
url: https://doi.org/10.1186/s12985-017-0843-0
doi: 10.1186/s12985-017-0843-0
title:
  A novel Coltivirus-related virus isolated from free-tailed bats from Côte d’Ivoire is able to infect human cells in vitro

abstract:
  BACKGROUND: Zoonotic transmission events play a major role in the emergence of novel diseases. While such events are virtually impossible to predict, wildlife screening for potential emerging pathogens can be a first step. Driven by recent disease epidemics like severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and Ebola, bats have gained special interest as reservoirs of emerging viruses. METHODS: As part of a bigger study investigating pathogens in African bats we screened animals for the presence of known and unknown viruses. RESULTS: We isolated and characterised a novel reovirus from blood of free-tailed bats (Chaereophon aloysiisabaudiae) captured in 2006 in Côte d’Ivoire. The virus showed closest relationship with two human pathogenic viruses, Colorado tick fever virus and Eyach virus, and was able to infect various human cell lines in vitro. CONCLUSION: The study shows the presence of a coltivirus-related virus in bats from Sub-Sahara Africa. Serological studies could help to assess its impact on humans or wildlife health.


id: cord-316234-vtjsfi2c
title: New oligonucleotide microarray for rapid diagnosis of avian viral diseases
author: Sultankulova, Kulyaisan T.
date: 2017-04-05
url: https://doi.org/10.1186/s12985-017-0738-0
doi: 10.1186/s12985-017-0738-0
abstract:
  BACKGROUND: We developed a new oligonucleotide microarray comprising 16 identical subarrays for simultaneous rapid detection of avian viruses: avian influenza virus (AIV), Newcastle disease virus (NDV), infection bronchitis virus (IBV), and infectious bursal disease virus (IBDV) in single- and mixed-virus infections. The objective of the study was to develop an oligonucleotide microarray for rapid diagnosis of avian diseases that would be used in the course of mass analysis for routine epidemiological surveillance owing to its ability to test one specimen for several infections. METHODS AND RESULTS: The paper describes the technique for rapid and simultaneous diagnosis of avian diseases such as avian influenza, Newcastle disease, infectious bronchitis and infectious bursal disease with use of oligonucleotide microarray, conditions for hybridization of fluorescent-labelled viral cDNA on the microarray and its specificity tested with use of AIV, NDV, IBV, IBDV strains as well as biomaterials from poultry. Sensitivity and specificity of the developed microarray was evaluated with use of 122 specimens of biological material: 44 cloacal swabs from sick birds and 78 tissue specimens from dead wild and domestic birds, as well as with use of 15 AIV, NDV, IBV and IBDV strains, different in their origin, epidemiological and biological characteristics (RIBSP Microbial Collection). This microarray demonstrates high diagnostic sensitivity (99.16% within 95% CI limits 97.36–100%) and specificity (100%). Specificity of the developed technique was confirmed by direct sequencing of NP and M (AIV), VP2 (IBDV), S1 (IBV), NP (NDV) gene fragments. CONCLUSION: Diagnostic effectiveness of the developed DNA microarray is 99.18% and therefore it can be used in mass survey for specific detection of AIV, NDV, IBV and IBDV circulating in the region in the course of epidemiological surveillance. Rather simple method for rapid diagnosis of avian viral diseases that several times shortens duration of assay versus classical diagnostic methods is proposed.


id: cord-317773-jdq1d98i
title: Enhanced inhibition of Avian leukosis virus subgroup J replication by multi-target miRNAs
author: Meng, Qing-Wen
date: 2011-12-22
url: https://doi.org/10.1186/1743-422x-8-556
doi: 10.1186/1743-422x-8-556
abstract:
  BACKGROUND: Avian leukosis virus (ALV) is a major infectious disease that impacts the poultry industry worldwide. Despite intensive efforts, no effective vaccine has been developed against ALV because of mutations that lead to resistant forms. Therefore, there is a dire need to develop antiviral agents for the treatment of ALV infections and RNA interference (RNAi) is considered an effective antiviral strategy. RESULTS: In this study, the avian leukosis virus subgroup J (ALV-J) proviral genome, including the gag genes, were treated as targets for RNAi. Four pairs of miRNA sequences were designed and synthesized that targeted different regions of the gag gene. The screened target (i.e., the gag genes) was shown to effectively suppress the replication of ALV-J by 19.0-77.3%. To avoid the generation of escape variants during virus infection, expression vectors of multi-target miRNAs were constructed using the multi-target serial strategy (against different regions of the gag, pol, and env genes). Multi-target miRNAs were shown to play a synergistic role in the inhibition of ALV-J replication, with an inhibition efficiency of viral replication ranging from 85.0-91.2%. CONCLUSION: The strategy of multi-target miRNAs might be an effective method for inhibiting ALV replication and the acquisition of resistant mutations.


id: cord-318686-we6pveus
author: Ehlen, Lukas
date: 2016-05-04
url: https://www.ncbi.nlm.nih.gov/pubmed/27142375/
doi: 10.1186/s12985-016-0531-5
title:
  Epithelial cell lines of the cotton rat (Sigmodon hispidus) are highly susceptible in vitro models to zoonotic Bunya-, Rhabdo-, and Flaviviruses

abstract:
  BACKGROUND: Small mammals such as bats and rodents have been increasingly recognized as reservoirs of novel potentially zoonotic pathogens. However, few in vitro model systems to date allow assessment of zoonotic viruses in a relevant host context. The cotton rat (Sigmodon hispidus) is a New World rodent species that has a long-standing history as an experimental animal model due to its unique susceptibility to human viruses. Furthermore, wild cotton rats are associated with a large variety of known or potentially zoonotic pathogens. METHODS: A method for the isolation and culture of airway epithelial cell lines recently developed for bats was applied for the generation of rodent airway and renal epithelial cell lines from the cotton rat. Continuous cell lines were characterized for their epithelial properties as well as for their interferon competence. Susceptibility to members of zoonotic Bunya-, Rhabdo-, and Flaviviridae, in particular Rift Valley fever virus (RVFV), vesicular stomatitis virus (VSV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV) was tested. Furthermore, novel arthropod-derived viruses belonging to the families Bunya-, Rhabdo-, and Mesoniviridae were tested. RESULTS: We successfully established airway and kidney epithelial cell lines from the cotton rat, and characterized their epithelial properties. Cells were shown to be interferon-competent. Viral infection assays showed high-titre viral replication of RVFV, VSV, WNV, and TBEV, as well as production of infectious virus particles. No viral replication was observed for novel arthropod-derived members of the Bunya-, Rhabdo-, and Mesoniviridae families in these cell lines. CONCLUSION: In the current study, we showed that newly established cell lines from the cotton rat can serve as host-specific in vitro models for viral infection experiments. These cell lines may also serve as novel tools for virus isolation, as well as for the investigation of virus-host interactions in a relevant host species.


id: cord-318853-mxyxwkhx
author: Sallie, Richard
date: 2005-08-22
txt: ./txt/cord-318853-mxyxwkhx.txt
cache: ./cache/cord-318853-mxyxwkhx.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/16115320/
doi: 10.1186/1743-422x-2-70
summary:
  Perhaps more importantly, ineluctable generation of broad phenotypic diversity after viral RNA is translated to protein quasispecies suggests a mechanism of disease that specifically targets, and functionally disrupts, the host cell surface molecules – including hormone, lipid, cell signalling or neurotransmitter receptors – that viruses co-opt for cell entry. Hence, as relative concentrations of wild-type and variant viral proteins vary, alteration of both processivity and fidelity of RNA pol results, permitting viruses to adaptively respond to environmental changes, including immune recognition and reaction to evolving cell receptors. As clear evidence exists for viral disruption of leptin function [106] and virus-associated weight gain in humans [107] and monkeys [108] , is it possible the global epidemics of type II diabetes mellitus, insulin resistance, hyperlipidaemia and obesity now prevalent [109] [110] [111] [112] [113] [114] [115] [116] , are just that; epidemics fundamentally caused by viruses that co-opt insulin or leptin or other associated receptors for cell access and generate protein quasispecies that disrupt receptor function?

title:
  Replicative homeostasis II: Influence of polymerase fidelity on RNA virus quasispecies biology: Implications for immune recognition, viral autoimmunity and other "virus receptor" diseases

abstract:
  Much of the worlds' population is in active or imminent danger from established infectious pathogens, while sporadic and pandemic infections by these and emerging agents threaten everyone. RNA polymerases (RNA(pol)) generate enormous genetic and consequent antigenic heterogeneity permitting both viruses and cellular pathogens to evade host defences. Thus, RNA(pol )causes more morbidity and premature mortality than any other molecule. The extraordinary genetic heterogeneity defining viral quasispecies results from RNA(pol )infidelity causing rapid cumulative genomic RNA mutation a process that, if uncontrolled, would cause catastrophic loss of sequence integrity and inexorable quasispecies extinction. Selective replication and replicative homeostasis, an epicyclical regulatory mechanism dynamically linking RNApol fidelity and processivity with quasispecies phenotypic diversity, modulating polymerase fidelity and, hence, controlling quasispecies behaviour, prevents this happening and also mediates immune escape. Perhaps more importantly, ineluctable generation of broad phenotypic diversity after viral RNA is translated to protein quasispecies suggests a mechanism of disease that specifically targets, and functionally disrupts, the host cell surface molecules – including hormone, lipid, cell signalling or neurotransmitter receptors – that viruses co-opt for cell entry. This mechanism – "Viral Receptor Disease (VRD)" – may explain so-called "viral autoimmunity", some classical autoimmune disorders and other diseases, including type II diabetes mellitus, and some forms of obesity. Viral receptor disease is a unifying hypothesis that may also explain some diseases with well-established, but multi-factorial and apparently unrelated aetiologies – like coronary artery and other vascular diseases – in addition to diseases like schizophrenia that are poorly understood and lack plausible, coherent, pathogenic explanations.


id: cord-319954-lh32rhhe
author: Loo, Liat Hui
date: 2013-09-25
txt: ./txt/cord-319954-lh32rhhe.txt
cache: ./cache/cord-319954-lh32rhhe.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/1743-422x-10-294
doi: 10.1186/1743-422x-10-294
summary:
  Immunoblotting of lysates prepared from pCAGGS/M transfected cells with MAbM3F8 (anti-M) revealed the presence of a 28 kDa protein, the expected size of the HMPV M protein ( Figure 1C ). Only pCAGGS/F-cmyc transfected cells immunoprecipitated and immunoblotted with anti-cmyc showed the presence of F proteins species corresponding in size to the monomeric and oligomeric F protein ( Figure 1D ). Similarly lysates immunoprecipitated with anti-FLAG and immunoblotted using anti-cmyc revealed increasing amounts of F145 with increasing DSP concentration, together with a cmyc-tagged protein species >300 kDa ( Figure 2F ) whose molecular mass was consistent with higher oligomeric forms of the F protein. We failed to detect the presence of the M protein in detergent extracts prepared from pCAGGS/M transfected cells that were immunoprecipitated with influenza virus anti-NP (i.e. a non-specific antibody) and immunblotted with anti-M ( Figure 4C ); confirming the specificity of the M protein immunoprecipitation.

title:
  Evidence for the interaction of the human metapneumovirus G and F proteins during virus-like particle formation

abstract:
  BACKGROUND: Human metapneumovirus (HMPV) is now a major cause of lower respiratory infection in children. Although primary isolation of HMPV has been achieved in several different cell lines, the low level of virus replication and the subsequent recovery of low levels of infectious HMPV have hampered biochemical studies on the virus. These experimental methodologies usually require higher levels of biological material that can be achieved following HMPV infection. In this study we demonstrate that expression of the HMPV F, G and M proteins in mammalian cells leads to HMPV virus-like particles (VLP) formation. This experimental strategy will serve as a model system to allow the process of HMPV virus assembly to be examined. METHODS: The HMPV F, G and M proteins were expressed in mammalian cell lines. Protein cross-linking studies, sucrose gradient centrifugation and in situ imaging was used to examine interactions between the virus proteins. VLP formation was examined using sucrose density gradient centrifugation and electron microscopy analysis. RESULTS: Analysis of cells co-expressing the F, G and M proteins demonstrated that these proteins interacted. Furthermore, in cells co-expression the three HMPV proteins the formation VLPs was observed. Image analysis revealed the VLPs had a similar morphology to the filamentous virus morphology that we observed on HMPV-infected cells. The capacity of each protein to initiate VLP formation was examined using a VLP formation assay. Individual expression of each virus protein showed that the G protein was able to form VLPs in the absence of the other virus proteins. Furthermore, co-expression of the G protein with either the M or F proteins facilitated their incorporation into the VLP fraction. CONCLUSION: Co-expression of the F, G and M proteins leads to the formation of VLPs, and that incorporation of the F and M proteins into VLPs is facilitated by their interaction with the G protein. Our data suggests that the G protein plays a central role in VLP formation, and further suggests that the G protein may also play a role in the recruitment of the F and M proteins to sites of virus particle formation during HMPV infection.


id: cord-320617-ucm7wx8b
author: B’Krong, Nguyen Thi Thuy Chinh
date: 2018-04-12
url: https://www.ncbi.nlm.nih.gov/pubmed/29650033/
doi: 10.1186/s12985-018-0980-0
title:
  Enterovirus serotypes in patients with central nervous system and respiratory infections in Viet Nam 1997–2010

abstract:
  BACKGROUND: Enteroviruses are the most common causative agents of human illness. Enteroviruses have been associated with regional and global epidemics, recently, including with severe disease (Enterovirus A71 and D68), and are of interest as emerging viruses. Here, we typed Enterovirus A-D (EV) from central nervous system (CNS) and respiratory infections in Viet Nam. METHODS: Data and specimens from prospective observational clinical studies conducted between 1997 and 2010 were used. Species and serotypes were determined using type-specific RT-PCR and viral protein 1 or 4 (VP1, VP4) sequencing. RESULTS: Samples from patients with CNS infection (51 children – 10 CSF and 41 respiratory/rectal swabs) and 28 adults (28 CSF) and respiratory infection (124 children – 124 respiratory swabs) were analysed. Twenty-six different serotypes of the four Enterovirus species (A-D) were identified, including EV-A71 and EV-D68. Enterovirus B was associated with viral meningitis in children and adults. Hand, foot and mouth disease associated Enteroviruses A (EV-A71 and Coxsackievirus [CV] A10) were detected in children with encephalitis. Diverse serotypes of all four Enterovirus species were found in respiratory samples, including 2 polio-vaccine viruses, but also 8 CV-A24 and 8 EV-D68. With the exception of EV-D68, the relevance of these viruses in respiratory infection remains unknown. CONCLUSION: We describe the diverse spectrum of enteroviruses from patients with CNS and respiratory infections in Viet Nam between 1997 and 2010. These data confirm the global circulation of Enterovirus genera and their associations and are important for clinical diagnostics, patient management, and outbreak response. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12985-018-0980-0) contains supplementary material, which is available to authorized users.


id: cord-320693-de1lmzl1
title: Antiviral activity of Piscidin 1 against pseudorabies virus both in vitro and in vivo
author: Hu, Han
date: 2019-07-31
url: https://doi.org/10.1186/s12985-019-1199-4
doi: 10.1186/s12985-019-1199-4
abstract:
  BACKGROUND: Swine-origin virus infection spreading widely could cause significant economic loss to porcine industry. Novel antiviral agents need to be developed to control this situation. METHODS: In this study, we evaluated the activities of five broad-spectrum antimicrobial peptides (AMPs) against several important swine-origin pathogenic viruses by TCID(50) assay. Plaque reduction assay and cell apoptosis assay were also used to test the activity of the peptides. Protection effect of piscidin against pseudorabies virus (PRV) was also examined in mouse model. RESULTS: Piscidin (piscidin 1), caerin (caerin 1.1) and maculatin (maculatin 1.1) could inhibit PRV by direct interaction with the virus particles in a dose-dependent manner and they could also protect the cells from PRV-induced apoptosis. Among the peptides tested, piscidin showed the strongest activity against PRV. Moreover, in vivo assay showed that piscidin can reduce the mortality of mice infected with PRV. CONCLUSION: In vitro and in vivo experiments indicate that piscidin has antiviral activity against PRV.


id: cord-321756-a7eh4dkb
title: Respiratory viruses in children hospitalized for acute lower respiratory tract infection in Ghana
author: Kwofie, Theophilus B
date: 2012-04-10
url: https://doi.org/10.1186/1743-422x-9-78
doi: 10.1186/1743-422x-9-78
abstract:
  BACKGROUND: Acute respiratory tract infections are one of the major causes of morbidity and mortality among young children in developing countries. Information on the viral aetiology of acute respiratory infections in developing countries is very limited. The study was done to identify viruses associated with acute lower respiratory tract infection among children less than 5 years. METHOD: Nasopharyngeal samples and blood cultures were collected from children less than 5 years who have been hospitalized for acute lower respiratory tract infection. Viruses and bacteria were identified using Reverse Transcriptase Real-Time Polymerase Chain Reaction and conventional biochemical techniques. RESULTS: Out of 128 patients recruited, 33(25.88%%, 95%CI: 18.5% to 34.2%) were positive for one or more viruses. Respiratory Syncytial Virus (RSV) was detected in 18(14.1%, 95%CI: 8.5% to 21.3%) patients followed by Adenoviruses (AdV) in 13(10.2%, 95%CI: 5.5% to 16.7%), Parainfluenza (PIV type: 1, 2, 3) in 4(3.1%, 95%CI: 0.9% to 7.8%) and influenza B viruses in 1(0.8%, 95%CI: 0.0 to 4.3). Concomitant viral and bacterial co-infection occurred in two patients. There were no detectable significant differences in the clinical signs, symptoms and severity for the various pathogens isolated. A total of 61.1% (22/36) of positive viruses were detected during the rainy season and Respiratory Syncytial Virus was the most predominant. CONCLUSION: The study has demonstrated an important burden of respiratory viruses as major causes of childhood acute respiratory infection in a tertiary health institution in Ghana. The data addresses a need for more studies on viral associated respiratory tract infection.


id: cord-321814-vt6yio6x
title: Isolation and characterization of a variant porcine epidemic diarrhea virus in China
author: Pan, Yongfei
date: 2012-09-12
url: https://doi.org/10.1186/1743-422x-9-195
doi: 10.1186/1743-422x-9-195
abstract:
  An outbreak of diarrhea in pigs started in Guangdong, South China in January 2011. Cases were characterized by watery diarrhea, dehydration and vomiting, with 80–100% morbidity and 50–90% mortality in suckling piglets. The causative agent of the diarrhea was ultimately identified as porcine epidemic diarrhea virus (PEDV). In this study, we isolated a PEDV strain designated CHGD-01 from piglet intestines using Vero cell cultures, and its specific cytopathic effects were confirmed in susceptible cells by direct immunofluorescence testing and electron microscopy. The complete genome of CHGD-01 was shown to be 28,035 nucleotides in length, with a similar structure to that of PEDV reference strains. Phylogenetic analyses based on the whole genome revealed that CHGD-01 shared nucleotide sequence identities of 98.2–98.4% with two other Chinese isolates reported in the same year, thus constituting a new cluster. Amino acid sequence analysis based on individual virus genes indicated a close relationship between the spike protein gene of CHGD-01 and the field strain KNU0802 in Korea. Its ORF3 and nucleoprotein genes, however, were divergent from all other sequenced PEDV isolate clusters and therefore formed a new group, suggesting a new variant PEDV isolate in China. Further studies will be required to determine the immunogenicity and pathogenicity of this new variant.


id: cord-322024-yrqpq9cf
author: Jevšnik, Monika
date: 2013-02-05
url: https://www.ncbi.nlm.nih.gov/pubmed/23379823/
doi: 10.1186/1743-422x-10-46
title:
  Detection of human coronaviruses in simultaneously collected stool samples and nasopharyngeal swabs from hospitalized children with acute gastroenteritis

abstract:
  BACKGROUND: Human coronaviruses (HCoVs) are a well-known cause of respiratory infections but their role in gastrointestinal infections is unclear. The objective of our study was to assess the significance of HCoVs in the etiology of acute gastroenteritis (AGE) in children <6 years of age. METHODS: Stool samples and nasopharyngeal (NP) swabs collected from 260 children hospitalized for AGE (160 also had respiratory symptoms) and 157 otherwise healthy control children admitted for elective surgery were tested for the presence of four HCoVs using real time RT-PCR. Registered at ClinicalTrials.gov (reg. NCT00987519). RESULTS: HCoVs were more frequent in patients with AGE than in controls (23/260, 8.8% versus 4/151, 2.6%; odds ratio, OR 3.3; 95% confidence interval, CI 1.3–10.0; P = 0.01). Three of four HCoV-positive members in the control group, asymptomatic when sampled, recalled gastrointestinal or respiratory symptoms within the previous 14 days. In patients with AGE, HCoVs were present in NP samples more often than in stools (22/256, 8.6%, versus 6/260, 2.3%; P = 0.0004). In 5/6 children with HCoVs detected in stools, the viruses were also detected in NP swabs. Patients had a significantly higher probability of HCoV detection in stool (OR 4; 95% CI 1.4–15.3; P = 0.006) and also in stool and/or NP (OR 3.3, 95% CI 1.3–10.0; P = 0.01) than healthy controls. All four HCoVs species were detected in stool and NP samples. CONCLUSIONS: Although HCoVs were more frequently detected in patients with AGE than in the control group, high prevalence of HCoVs in NP swabs compounded by their low occurrence in stool samples and detection of other viruses in stool samples, indicate that HCoVs probably play only a minor role in causing gastrointestinal illness in children <6 years old.


id: cord-322238-8iwljdoi
author: Chen, Qin
date: 2010-08-29
txt: ./txt/cord-322238-8iwljdoi.txt
cache: ./cache/cord-322238-8iwljdoi.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/20799985/
doi: 10.1186/1743-422x-7-206
summary:
  Loop-mediated isothermal amplification (LAMP) was developed to detect the TGEV by incubation at 60°C for 1 h and the product specificity was confirmed by HphI digestion. By using serial sample dilutions as templates, the detection limit of LAMP was about 10 pg RNA, 10 times more sensitive than that of PCR and could be comparable to the nest-PCR. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method, which amplifies DNA/RNA with high specificity, sensitivity and rapidity under isothermal condition [5] . As a kind of nucleic acid amplification method, LAMP could not only qualitatively detect the TGEV, but also quantitatively analyze the virus. In conclusion, this study demonstrates that the LAMP method established could detect only the TGEV and no cross-reaction with other viruses, the detection limit was about 10 pg RNA, which was 10 times more sensitive than that of PCR and could be comparable to the nest-PCR.

title:
  Detection of swine transmissible gastroenteritis coronavirus using loop-mediated isothermal amplification

abstract:
  A conserved nucleic acid fragment of the nucleocapsid gene of Swine Transmissible Gastroenteritis Coronavirus (TGEV) was chosen as the target, six special primers were designed successfully. Loop-mediated isothermal amplification (LAMP) was developed to detect the TGEV by incubation at 60°C for 1 h and the product specificity was confirmed by HphI digestion. Standard curves with high accuracy for TGEV quantization was constructed by adding 1 × SYBR greenI in the LAMP reaction. The assay established in this study was found to detect only the TGEV and no cross-reaction with other viruses, demonstrating its high specificity. By using serial sample dilutions as templates, the detection limit of LAMP was about 10 pg RNA, 10 times more sensitive than that of PCR and could be comparable to the nest-PCR.


id: cord-322738-nvyknvc7
title: Viral aetiology influenza like illnesses in Santa Cruz, Bolivia (2010–2012)
author: Delangue, Julie
date: 2014-02-24
url: https://doi.org/10.1186/1743-422x-11-35
doi: 10.1186/1743-422x-11-35
abstract:
  BACKGROUND: Acute respiratory infections represent a serious public health issue worldwide but virological aetiologies of Influenza Like Illnesses (ILIs) remain largely unknown in developing countries. This study represents the first attempt to characterise viral aetiologies of ILIs in Bolivia. METHODS: It was performed in Santa Cruz city from January 2010 to September 2012, based on 564 naso-pharyngeal swabs collected in a National Reference Laboratory and real-time PCR techniques, viral cultures and phylogenetic analyses. RESULTS: 50.2% of samples were positive for at least one virus with influenza viruses (Flu A: ~15%; Flu B: ~9%), rhinoviruses (~8%), coronaviruses (~5%) and hRSV (~4%) being the most frequently identified. The pattern of viral infections varied according to age groups. The elucidation rate was the highest (>60%) amongst patients under 10 yo and the lowest (<40%) amongst patients ≥60 yo. Nearly 3% of samples showed dual viral infections. Epidemiological peaks were associated with a predominant virus but generally included 30-50% of infections by different viruses. Unexpectedly, the frequency of influenza in the 0–4 yo population was very low and a complete hRSV eclipse occurred in 2011. Genetic analyses indicated that distinct evolutionary lineages of Flu A(H1N1)pdm2009, Flu A/H3N2 and Flu B have co-circulated in Bolivia in the study period, originating from Central and North America, Europe, Asia and Australia. CONCLUSION: Our results emphasise the requirement for a reinforced epidemiological and genetic follow-up of influenza and other ILIs in Bolivia to further inform the preparation of vaccines used in the region, guide vaccination campaigns and improve the medical management of patients.


id: cord-322937-lakdi3x8
author: Kang, Xiao-ping
date: 2010-06-02
url: https://www.ncbi.nlm.nih.gov/pubmed/20515509/
doi: 10.1186/1743-422x-7-113
title:
  A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus

abstract:
  A duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was improved for simultaneous detection of highly pathogenic H5N1 avian influenza virus and pandemic H1N1 (2009) influenza virus, which is suitable for early diagnosis of influenza-like patients and for epidemiological surveillance. The sensitivity of this duplex real-time RT-PCR assay was 0.02 TCID(50 )(50% tissue culture infective dose) for H5N1 and 0.2 TCID(50 )for the pandemic H1N1, which was the same as that of each single-target RT-PCR for pandemic H1N1 and even more sensitive for H5N1 with the same primers and probes. No cross reactivity of detecting other subtype influenza viruses or respiratory tract viruses was observed. Two hundred and thirty-six clinical specimens were tested by comparing with single real-time RT-PCR and result from the duplex assay was 100% consistent with the results of single real-time RT-PCR and sequence analysis.


id: cord-323009-frej2qmb
author: Nakouné, Emmanuel
date: 2013-02-08
url: https://www.ncbi.nlm.nih.gov/pubmed/23391188/
doi: 10.1186/1743-422x-10-49
title:
  First introduction of pandemic influenza A/H1N1 and detection of respiratory viruses in pediatric patients in Central African Republic

abstract:
  BACKGROUND: Acute viral respiratory illnesses in children in sub-Saharan Africa have received relatively little attention, although they are much more frequent causes of morbidity and mortality than in developed countries. Active surveillance is essential to identify the causative agents and to improve clinical management, especially in the context of possible circulation of pandemic viruses. FINDINGS: A prospective study was conducted in the Central African Republic (CAR) between January and December 2010 among infants and children aged 0–15 years attending sentinel sites for influenza-like illness or acute respiratory illness. Nasopharyngeal swabs were collected, and one-step real-time and multiplex reverse transcription-polymerase chain reaction were used to detect respiratory viruses. Respiratory viruses were detected in 49 of the 329 (14.9%) nasopharyngeal samples: 29 (8.8%) contained influenza viruses (5 (1.5%) had pandemic influenza A/H1N1 virus and 24 (7.3%) had influenza B viruses), 11 (3.3%) contained parainfluenza viruses types 1 and 3 and 9 (2.7%) contained human respiratory syncytial virus. Most cases were detected during the rainy season in the CAR. Analysis of the amplicon sequences confirmed the identity of each detected virus. CONCLUSIONS: The influenza surveillance system in the CAR has provided valuable data on the seasonality of influenza and the circulation of other respiratory viruses. Our network could therefore play a valuable role in the prevention and control of influenza epidemics in the CAR.


id: cord-325172-a8ntxnmm
title: Antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus
author: Yip, Ming Shum
date: 2014-05-06
url: https://doi.org/10.1186/1743-422x-11-82
doi: 10.1186/1743-422x-11-82
abstract:
  BACKGROUND: Public health risks associated to infection by human coronaviruses remain considerable and vaccination is a key option for preventing the resurgence of severe acute respiratory syndrome coronavirus (SARS-CoV). We have previously reported that antibodies elicited by a SARS-CoV vaccine candidate based on recombinant, full-length SARS-CoV Spike-protein trimers, trigger infection of immune cell lines. These observations prompted us to investigate the molecular mechanisms and responses to antibody-mediated infection in human macrophages. METHODS: We have used primary human immune cells to evaluate their susceptibility to infection by SARS-CoV in the presence of anti-Spike antibodies. Fluorescence microscopy and real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) were utilized to assess occurrence and consequences of infection. To gain insight into the underlying molecular mechanism, we performed mutational analysis with a series of truncated and chimeric constructs of fragment crystallizable γ receptors (FcγR), which bind antibody-coated pathogens. RESULTS: We show here that anti-Spike immune serum increased infection of human monocyte-derived macrophages by replication-competent SARS-CoV as well as Spike-pseudotyped lentiviral particles (SARS-CoVpp). Macrophages infected with SARS-CoV, however, did not support productive replication of the virus. Purified anti-viral IgGs, but not other soluble factor(s) from heat-inactivated mouse immune serum, were sufficient to enhance infection. Antibody-mediated infection was dependent on signaling-competent members of the human FcγRII family, which were shown to confer susceptibility to otherwise naïve ST486 cells, as binding of immune complexes to cell surface FcγRII was necessary but not sufficient to trigger antibody-dependent enhancement (ADE) of infection. Furthermore, only FcγRII with intact cytoplasmic signaling domains were competent to sustain ADE of SARS-CoVpp infection, thus providing additional information on the role of downstream signaling by FcγRII. CONCLUSIONS: These results demonstrate that human macrophages can be infected by SARS-CoV as a result of IgG-mediated ADE and indicate that this infection route requires signaling pathways activated downstream of binding to FcγRII receptors.


id: cord-325555-be78qely
author: Lyoo, Hey Rhyoung
date: 2015-02-26
url: https://www.ncbi.nlm.nih.gov/pubmed/25888736/
doi: 10.1186/s12985-015-0269-5
title:
  Constant up-regulation of BiP/GRP78 expression prevents virus-induced apoptosis in BHK-21 cells with Japanese encephalitis virus persistent infection

abstract:
  BACKGROUND: Persistent infection of the Japanese Encephalitis Virus (JEV) has been reported in clinical cases, experimental animals, and various cell culture systems. We previously reported the establishment of spontaneous JEV persistent infection, assisted by defective interfering particle accumulation and/or attenuated helper viruses, in BHK-21 cells devoid of virus-induced apoptosis, cBS6-2 and cBS6-3. However, cell-specific factors may play important roles in controlling JEV replication and have never been assessed for this specific phenomenon. Recent evidence suggests that viruses have evolved various mechanisms to cope with endoplasmic reticulum stress signaling pathways for their efficient amplification and transmission, including the unfolded protein response (UPR). RESULTS: To identify the host cell factors that affect JEV persistence, we investigated the expression of essential UPR factors in cBS6-2 and cBS6-3 cells. Of the selected UPR factors tested, the most noticeable deviations from those of the normal BHK-21 cells with JEV acute infection were as follows: the suppression of C/EBP homologous binding protein (CHOP) and the constant up-regulation of immunoglobulin binding protein (BiP) expression in cBS6-2 and cBS6-3 cells. In JEV acute infection on normal BHK-21 cells, silencing CHOP expression through specific siRNA blocked cell death almost completely. Meanwhile, depletion of BiP by specific siRNA unlocked CHOP expression in cBS6-2 and cBS6-3 cells, resulting in massive cell death. Fulminant apoptotic cell death for both cell clones on tunicamycin treatment revealed that the JEV persistently infected cells still contained functional arms for cell fate decisions. CONCLUSIONS: BHK-21 cells with JEV persistent infection strive against virus-induced apoptosis through constant up-regulation of BiP expression, resulting in the complete depletion of CHOP even with apparent virus amplification in the cells. Accordingly, the attenuation of virus replication as well as the modifications to cell metabolism could be additional factors contributing to the development of JEV persistent infection in mammalian cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0269-5) contains supplementary material, which is available to authorized users.


id: cord-327013-gc6o8ou3
author: Kim, Heui Man
date: 2020-07-06
url: https://www.ncbi.nlm.nih.gov/pubmed/32631440/
doi: 10.1186/s12985-020-01375-1
title:
  Characterization of neuraminidase inhibitor-resistant influenza virus isolates from immunocompromised patients in the Republic of Korea

abstract:
  BACKGROUND: The emergence of influenza viruses resistant to anti-influenza drugs is a threat to global public health. The Korea Centers for Disease Control and Prevention operates the Korea Influenza and Respiratory Viruses Surveillance System (KINRESS) to monitor epidemics of influenza and Severe Acute Respiratory Infection (SARI) to identify mutated influenza viruses affecting drug resistance, pathogenesis, and transmission. METHODS: Oropharyngeal swab samples were collected from KINRESS and SARI during the 2018–2019 season. The specimens confirmed influenza virus using real-time RT-PCR on inoculated MDCK cells. HA and NA sequences of the influenza viruses were analyzed for phylogeny and mutations. Neuraminidase inhibition and hemagglutination inhibition assays were utilized to characterize the isolates. RESULTS: Two A(H1N1)pdm09 isolates harboring an H275Y substitution in the neuraminidase sequence were detected in patients with acute hematologic cancer. They had prolonged respiratory symptoms, with the virus present in the respiratory tract despite oseltamivir and peramivir treatment. Through the neuraminidase inhibition assay, both viruses were found to be resistant to oseltamivir and peramivir, but not to zanamivir. Although hemagglutinin and neuraminidase phylogenetic analyses suggested that the 2 A(H1N1)pdm09 isolates were not identical, their antigenicity was similar to that of the 2018–19 influenza vaccine virus. CONCLUSIONS: Our data indicate the utility of monitoring influenza-infected immunocompromised patients in general hospitals for the early detection of emerging neuraminidase inhibitor-resistant viruses and maintaining continuous laboratory surveillance of patients with influenza-like illness in sentinel clinics to monitor the spread of such new variants. Finally, characterization of the virus can inform the risk assessment for future epidemics and pandemics caused by drug-resistant influenza viruses.


id: cord-329517-3yn80r9h
author: Yang, Jin-Long
date: 2009-09-15
url: https://doi.org/10.1186/1743-422x-6-142
doi: 10.1186/1743-422x-6-142
title:
  Development of a fluorescent quantitative real-time polymerase chain reaction assay for the detection of Goose parvovirus in vivo

abstract:
  BACKGROUND: Goose parvovirus (GPV) is a Dependovirus associated with latent infection and mortality in geese. Currently, it severely affects geese production worldwide. The objective of this study was to develop a fluorescent quantitative real-time polymerase chain reaction (PCR) (FQ-PCR) assay for fast and accurate quantification of GPV DNA in infected goslings, which can aid in the understanding of the regular distribution pattern and the nosogenesis of GPV in vivo. RESULTS: The detection limit of the assay was 2.8 × 10(1 )standard DNA copies, with a sensitivity of 3 logs higher than that of the conventional gel-based PCR assay targeting the same gene. The real-time PCR was reproducible, as shown by satisfactory low intraassay and interassay coefficients of variation. CONCLUSION: The high sensitivity, specificity, simplicity, and reproducibility of the GPV fluorogenic PCR assay, combined with a high throughput, make this method suitable for a broad spectrum of GPV etiology-related applications.


id: cord-329520-dt0jku7v
author: Hon, Kam LE
date: 2010-07-21
url: https://doi.org/10.1186/1743-422x-7-169
doi: 10.1186/1743-422x-7-169
title:
  Influenza or not influenza: Analysis of a case of high fever that happened 2000 years ago in Biblical time

abstract:
  The Bible describes the case of a woman with high fever cured by our Lord Jesus Christ. Based on the information provided by the gospels of Mark, Matthew and Luke, the diagnosis and the possible etiology of the febrile illness is discussed. Infectious diseases continue to be a threat to humanity, and influenza has been with us since the dawn of human history. If the postulation is indeed correct, the woman with fever in the Bible is among one of the very early description of human influenza disease. Infectious diseases continue to be a threat to humanity, and influenza has been with us since the dawn of human history. We analysed a case of high fever that happened 2000 years ago in Biblical time and discussed possible etiologies.


id: cord-329680-ekxsv91t
author: Yu, Yunjia
date: 2019-12-23
url: https://www.ncbi.nlm.nih.gov/pubmed/31870450/
doi: 10.1186/s12985-019-1266-x
title:
  Inhibition effects of patchouli alcohol against influenza a virus through targeting cellular PI3K/Akt and ERK/MAPK signaling pathways

abstract:
  BACKGROUND: Patchouli alcohol (PA) is a tricyclic sesquiterpene extracted from Pogostemonis Herba, which is a traditional Chinese medicine used for therapy of inflammatory diseases. Recent studies have shown that PA has various pharmacological activities, including anti-bacterial and anti-viral effects. METHODS: In this study, the anti-influenza virus (IAV) activities and mechanisms were investigated both in vitro and in vivo. The inhibitory effects of PA against IAV in vitro were evaluated by plaque assay and immunofluorescence assay. The neuraminidase inhibition assay, hemagglutination inhibition (HI) assay, and western blot assay were used to explore the anti-viral mechanisms. The anti-IAV activities in vivo were determined by mice pneumonia model and HE staining. RESULTS: The results showed that PA significantly inhibited different IAV strains multiplication in vitro, and may block IAV infection through inactivating virus particles directly and interfering with some early stages after virus adsorption. Cellular PI3K/Akt and ERK/MAPK signaling pathways may be involved in the anti-IAV actions of PA. Intranasal administration of PA markedly improved mice survival and attenuated pneumonia symptoms in IAV infected mice, comparable to the effects of Oseltamivir. CONCLUSIONS: Therefore, Patchouli alcohol has the potential to be developed into a novel anti-IAV agent in the future.


id: cord-330251-dwjijmwz
title: Nucleolar localization of influenza A NS1: striking differences between mammalian and avian cells
author: Volmer, Romain
date: 2010-03-17
url: https://www.ncbi.nlm.nih.gov/pubmed/20236536/
doi: 10.1186/1743-422x-7-63
abstract:
  In mammalian cells, nucleolar localization of influenza A NS1 requires the presence of a C-terminal nucleolar localization signal. This nucleolar localization signal is present only in certain strains of influenza A viruses. Therefore, only certain NS1 accumulate in the nucleolus of mammalian cells. In contrast, we show that all NS1 tested in this study accumulated in the nucleolus of avian cells even in the absence of the above described C-terminal nucleolar localization signal. Thus, nucleolar localization of NS1 in avian cells appears to rely on a different nucleolar localization signal that is more conserved among influenza virus strains.


id: cord-330777-xcwppaux
author: Esposito, Susanna
date: 2010-04-30
url: https://www.ncbi.nlm.nih.gov/pubmed/20433729/
doi: 10.1186/1743-422x-7-85
title:
  Collection by trained pediatricians or parents of mid-turbinate nasal flocked swabs for the detection of influenza viruses in childhood

abstract:
  This study evaluated the efficiency of pediatric mid-turbinate nasal flocked swabs used by parents in 203 children aged 6 months to 5 years with signs and symptoms of respiratory disease. Two nasal samples were collected from each child in a randomised sequence: one by a trained pediatrician and one by a parent. The real-time polymerase chain reaction influenza virus detection rates were similar in the samples collected using the two methods (Cohen's kappa = 0.86), as were the cycle threshold values. In comparison with the pediatrician-collected samples, the sensitivity and specificity of the parental collections were respectively 89.3% (95% confidence interval [CI]: 77.8-100%) and 97.7% (95% CI: 95.5-100%), and the positive and negative predictive values were respectively 86.2% (95% CI: 73.7-95.1%) and 98.2% (95% CI: 96.4-100%). The children were significantly more satisfied with the parental collections (median values ± standard deviation, 1.59 ± 0.55 vs 3.51 ± 0.36; p < 0.0001). These findings show that mid-turbinate nasal flocked swabs specifically designed for infants and children can be used by parents without reducing the influenza virus detection rate. Moreover, the direct involvement of parents significantly increases patient acceptance, thus simplifying collection and suggesting that this novel swab design should be considered for epidemiological surveys and vaccine efficacy studies.


id: cord-331542-wy068c6o
title: Identification of a novel B-cell epitope in the spike protein of porcine epidemic diarrhea virus
author: Kong, Ning
date: 2020-04-03
url: https://doi.org/10.1186/s12985-020-01305-1
doi: 10.1186/s12985-020-01305-1
abstract:
  BACKGROUND: Porcine epidemic diarrhea virus (PEDV) infection causes an acute enteric tract infectious disease characterized by vomiting, anorexia, dehydration, weight loss and high mortality in neonatal piglets. During PEDV infection, the spike protein (S) is a major virion structural protein interacting with receptors and inducing neutralizing antibodies. However, the neutralizing B-cell epitopes within PEDV S protein have not been well studied. METHODS: To accurately identify the important immunodominant region of S1, the purified truncated S1 proteins (SA, SB, SC, SD and SE) were used to immunize BALB/c mice to prepare polyclonal antibodies. The antisera titers were determined by indirect ELISA, western blot and IFA after four immunizations to find the important immunodominant region of S1, and then purified the immunodominant region of S1 protein and immunized mice to generate the special antibodies, and then used recombinant peptides to determine the B-cell epitopes of monoclonal antibodies. RESULTS: Five antisera of recombinant proteins of the spike protein region of PEDV were generated and we found that only the polyclonal antibody against part of the S1 region (signed as SE protein, residues 666–789) could recognize the native PEDV. Purified SE protein was used to immunize BALB/c mice and generate mAb 2E10. Pepscan of the SE protein demonstrated that SE16 ((722)SSTFNSTREL(731)) is the minimal linear epitope required for reactivity with the mAb 2E10. Further investigation indicated that the epitope SE16 was localized on the surface of PEDV S protein in the 3D structure. CONCLUSIONS: A mAb 2E10 that is specifically bound to PEDV was generated and identified a specific linear B-cell epitope (SE16, (722)SSTFNSTREL(731)) of the mAb. The epitope region of PEDV S1 localized in the different regions in comparison with the earlier identified epitopes. These findings enhance the understanding of the PEDV spike protein structure for vaccine design and provide a potential use for developing diagnostic methods to detect PEDV.


id: cord-333349-tsfxpaj6
author: Chai, Weidong
date: 2014-08-08
txt: ./txt/cord-333349-tsfxpaj6.txt
cache: ./cache/cord-333349-tsfxpaj6.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/25103309/
doi: 10.1186/1743-422x-11-140
summary:
  title: Elevated dietary zinc oxide levels do not have a substantial effect on porcine reproductive and respiratory syndrome virus (PPRSV) vaccination and infection The purpose of this study was to determine the influence of dietary zinc oxide supplementation on vaccination and challenge infection with PRRSV. CONCLUSIONS: Our results suggest that higher levels of dietary ZnO do not have the potential to stimulate or modulate systemic immune responses after vaccination and heterologous PRRSV infection to an extent that could improve the clinical and virological outcome. In contrast to Vanhee et al., we chose a single-vaccination approach and challenge-infected the animals with a heterologous type I PRRSV (95,38% sequence identity for the envelope glycoprotein GP5, which bears a major neutralizing epitope) in order test the influence of elevated Zn levels on an suboptimal antigen stimulus and on crossprotection. Elevated dietary zinc oxide levels do not have a substantial effect on porcine reproductive and respiratory syndrome virus (PPRSV) vaccination and infection

title:
  Elevated dietary zinc oxide levels do not have a substantial effect on porcine reproductive and respiratory syndrome virus (PPRSV) vaccination and infection

abstract:
  BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important infectious agents for the swine industry worldwide. Zinc (Zn) salts, which are widely used as a dietary supplement in swine nutrition, have shown antiviral effects in vitro as well as in vivo. The purpose of this study was to determine the influence of dietary zinc oxide supplementation on vaccination and challenge infection with PRRSV. FINDINGS: The clinical course of PRRS and the success of vaccination with an experimental inactivated vaccine were compared between animals receiving a conventional diet (50 ppm Zn, control group) and diets supplemented with Zn oxide (ZnO) at final Zn concentrations of 150 or 2,500 ppm. Pigs receiving higher dietary Zn levels showed a tendency towards higher neutralizing antibody levels after infection, while dietary Zn levels did not substantially influence the number of antiviral IFN-gamma secreting cells (IFN-gamma-SC) or percentages of blood immune cell subsets after infection. Finally, feeding higher dietary Zn levels reduced neither clinical symptoms nor viral loads. CONCLUSIONS: Our results suggest that higher levels of dietary ZnO do not have the potential to stimulate or modulate systemic immune responses after vaccination and heterologous PRRSV infection to an extent that could improve the clinical and virological outcome. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1743-422X-11-140) contains supplementary material, which is available to authorized users.


id: cord-333913-roftm446
title: Klassevirus 1, a previously undescribed member of the family Picornaviridae, is globally widespread
author: Holtz, Lori R
date: 2009-06-24
txt: ./txt/cord-333913-roftm446.txt
cache: ./cache/cord-333913-roftm446.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/19552824/
doi: 10.1186/1743-422x-6-86
summary:
  In an effort to identify novel viruses that may be causal agents of diarrhea, we used high throughput mass sequencing to analyze stool samples collected from patients with acute diarrhea. RESULTS: Sequences with limited similarity to known picornaviruses were detected in a stool sample collected in Australia from a child with acute diarrhea. We also detected klassevirus 1 by RT-PCR in a diarrhea specimen collected from a patient in St. Louis, United States as well as in untreated sewage collected in Barcelona, Spain. Extracted nucleic acid from a stool specimen collected in 1984 from a child with acute diarrhea was subjected to high throughput mass sequencing using 454 pyroseqencing technology. Phylogenetic analysis of the VP3/VP1, P2, and P3 regions of the genome demonstrated that this virus sequence is highly divergent from all previously described picornaviruses (Figure 2A -C).

abstract:
  BACKGROUND: Diarrhea is the third leading infectious cause of death worldwide and is estimated to be responsible for approximately 2 million deaths a year. While many infectious causes of diarrhea have been established, approximately 40% of all diarrhea cases are of unknown etiology. In an effort to identify novel viruses that may be causal agents of diarrhea, we used high throughput mass sequencing to analyze stool samples collected from patients with acute diarrhea. RESULTS: Sequences with limited similarity to known picornaviruses were detected in a stool sample collected in Australia from a child with acute diarrhea. Using a combination of mass sequencing, RT-PCR, 5' RACE and 3' RACE, a 6383 bp fragment of the viral genome was sequenced. Phylogenetic analysis demonstrated that this virus was highly divergent from, but most closely related to, members of the genus Kobuvirus. We have tentatively named this novel virus klassevirus 1. We also detected klassevirus 1 by RT-PCR in a diarrhea specimen collected from a patient in St. Louis, United States as well as in untreated sewage collected in Barcelona, Spain. CONCLUSION: Klassevirus 1 is a previously undescribed picornavirus that is globally widespread and present on at least three continents. Further investigations to determine whether klassevirus 1 is a human pathogen are needed.


id: cord-335865-pibq3iwh
author: Jumat, Muhammad Raihan
date: 2014-11-19
txt: ./txt/cord-335865-pibq3iwh.txt
cache: ./cache/cord-335865-pibq3iwh.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/25408253/
doi: 10.1186/s12985-014-0198-8
summary:
  title: Imaging analysis of human metapneumovirus-infected cells provides evidence for the involvement of F-actin and the raft-lipid microdomains in virus morphogenesis METHODS: HMPV-infected LLC-MK2 cells were stained with antibodies that recognised the HMPV fusion protein (F protein), attachment protein (G protein) and matrix protein (M protein), and fluorescent probes that detect GM1 within lipid-raft membranes (CTX-B-AF488) and F-actin (Phalloidin-FITC). Cells co-expressing recombinant HMPV G and F proteins formed virus-like particles and were co-stained with antibodies that recognise the recombinant G and F proteins and phalloidin-FITC and CTX-B-AF594, and the distribution of the G and F proteins, GM1 and F-actin determined. We also observed these co-stained filamentous structures spreading to the non-infected cells, suggesting that F-actin may play a role in virus transmission in the monolayer, in a similar manner to that proposed for RSV [17, 25, 30] . HMPV-infected cells were stained with anti-G and CTX-B-AF488, and examined using confocal microscopy at an optical plane that allowed the virus filaments to be visualized ( Figure 4D ).

title:
  Imaging analysis of human metapneumovirus-infected cells provides evidence for the involvement of F-actin and the raft-lipid microdomains in virus morphogenesis

abstract:
  BACKGOUND: Due to difficulties of culturing Human metapneumovirus (HMPV) much of the current understanding of HMPV replication can be inferred from other closely related viruses. The slow rates of virus replication prevent many biochemical analyses of HMPV particles. In this study imaging was used to examine the process of HMPV morphogenesis in individually infected LLC-MK2 cells, and to better characterise the sites of HMPV assembly. This strategy has circumvented the problems associated with slow replication rates and allowed us to characterise both the HMPV particles and the sites of HMPV morphogenesis. METHODS: HMPV-infected LLC-MK2 cells were stained with antibodies that recognised the HMPV fusion protein (F protein), attachment protein (G protein) and matrix protein (M protein), and fluorescent probes that detect GM1 within lipid-raft membranes (CTX-B-AF488) and F-actin (Phalloidin-FITC). The stained cells were examined by confocal microscopy, which allowed imaging of F-actin, GM1 and virus particles in HMPV-infected cells. Cells co-expressing recombinant HMPV G and F proteins formed virus-like particles and were co-stained with antibodies that recognise the recombinant G and F proteins and phalloidin-FITC and CTX-B-AF594, and the distribution of the G and F proteins, GM1 and F-actin determined. RESULTS: HMPV-infected cells stained with anti-F, anti-G or anti-M revealed a filamentous staining pattern, indicating that the HMPV particles have a filamentous morphology. Staining of HMPV-infected cells with anti-G and either phalloidin-FITC or CTX-B-AF488 exhibited extensive co-localisation of these cellular probes within the HMPV filaments. This suggested that lipid-raft membrane domains and F-actin structures are present at the site of the virus morphogenesis, and are subsequently incorporated into the HMPV filaments. Furthermore, the filamentous virus-like particles that form in cells expressing the G protein formed in cellular structures containing GM1 and F-actin, suggesting the G protein contains intrinsic targeting signals to the sites of virus assembly. CONCLUSIONS: These data suggest that HMPV matures as filamentous particles and that virus morphogenesis occurs within lipid-raft microdomains containing localized concentrations of F-actin. The similarity between HMPV morphogenesis and the closely related human respiratory syncytial virus suggests that involvement of F-actin and lipid-raft microdomains in virus morphogenesis may be a common feature of the Pneumovirinae. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-014-0198-8) contains supplementary material, which is available to authorized users.


id: cord-336510-qzm9wgde
title: Macrophages and cytokines in the early defence against herpes simplex virus
author: Ellermann-Eriksen, Svend
date: 2005-08-03
txt: ./txt/cord-336510-qzm9wgde.txt
cache: ./cache/cord-336510-qzm9wgde.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/16076403/
doi: 10.1186/1743-422x-2-59
summary:
  In a first wave of responses, cytokines, primarily type I interferons (IFN) and tumour necrosis factor are produced and exert a direct antiviral effect and activate the macrophages themselves. Generally the type I IFNs exhibit a huge range of biological effects, such as antiviral and antiproliferative effects, stimulation of immune cells such as T cells, natural killer (NK) cells, monocytes, macrophages, and dendritic cells, increased expression of MHC-I, activation of pro-apoptotic genes and inhibition of anti-apoptotic mechanisms, modulation of cellular differentiation, and inhibition of angiogenesis [171] . Effect of IL-4 and IL-13 on IFN-gamma-induced production of nitric oxide in mouse macrophages infected with herpes simplex virus type 2 Herpes Simplex virus type 1-induced interferon production and activation of natural killer cells in mice NF-kappaB activation is responsible for the synergistic effect of herpes simplex virus type 2 infection on interferon-gamma-induced nitric oxide production in macrophages

abstract:
  Herpes simplex virus (HSV) type 1 and 2 are old viruses, with a history of evolution shared with humans. Thus, it is generally well-adapted viruses, infecting many of us without doing much harm, and with the capacity to hide in our neurons for life. In rare situations, however, the primary infection becomes generalized or involves the brain. Normally, the primary HSV infection is asymptomatic, and a crucial element in the early restriction of virus replication and thus avoidance of symptoms from the infection is the concerted action of different arms of the innate immune response. An early and light struggle inhibiting some HSV replication will spare the host from the real war against huge amounts of virus later in infection. As far as such a war will jeopardize the life of the host, it will be in both interests, including the virus, to settle the conflict amicably. Some important weapons of the unspecific defence and the early strikes and beginning battle during the first days of a HSV infection are discussed in this review. Generally, macrophages are orchestrating a multitude of anti-herpetic actions during the first hours of the attack. In a first wave of responses, cytokines, primarily type I interferons (IFN) and tumour necrosis factor are produced and exert a direct antiviral effect and activate the macrophages themselves. In the next wave, interleukin (IL)-12 together with the above and other cytokines induce production of IFN-γ in mainly NK cells. Many positive feed-back mechanisms and synergistic interactions intensify these systems and give rise to heavy antiviral weapons such as reactive oxygen species and nitric oxide. This results in the generation of an alliance against the viral enemy. However, these heavy weapons have to be controlled to avoid too much harm to the host. By IL-4 and others, these reactions are hampered, but they are still allowed in foci of HSV replication, thus focusing the activity to only relevant sites. So, no hero does it alone. Rather, an alliance of cytokines, macrophages and other cells seems to play a central role. Implications of this for future treatment modalities are shortly considered.


id: cord-336870-nirg3269
title: The newly emerged COVID-19 disease: a systemic review
author: Abebe, Endeshaw Chekol
date: 2020-07-08
url: https://www.ncbi.nlm.nih.gov/pubmed/32641059/
doi: 10.1186/s12985-020-01363-5
abstract:
  Coronaviruses are large family-RNA viruses that belong to the order Nidovirales, family Coronaviridae, subfamily Coronavirinae. The novel COVID-19 infection, caused by a beta coronavirus called SARS-CoV-2, is a new outbreak that has been emerged in Wuhan, China in December 2019. The most common symptoms of COVID-19 are fever, cough, and dyspnea. As per the March 12, 2020, WHO report, more than 125,048 confirmed COVID-19 cases and over 4613 deaths have been identified in more than 117 countries. It is now regarded as a pandemic that seriously spread and attack the world. The primary means of transmission is person to person through droplets that occurred during coughing or sneezing, through personal contact (shaking hands), or by touching contaminated objects. So far, there is no effective therapy and vaccine available against this novel virus and therefore, only supportive care is used as the mainstay of management of patients with COVID-19. The mortality rate of COVID-19 is considerable. This work aimed to provide insight on the newly emerged COVID-19, in the hope to gain a better understanding on the general overview, epidemiology, transmission, clinical features, diagnosis, treatment, and clinical outcomes as well as the prevention and control of COVID-19.


id: cord-337003-7ygcfzii
author: Mehrbod, Parvaneh
date: 2017-11-09
url: https://www.ncbi.nlm.nih.gov/pubmed/29121968/
doi: 10.1186/s12985-017-0884-4
title:
  Association of IFITM3 rs12252 polymorphisms, BMI, diabetes, and hypercholesterolemia with mild flu in an Iranian population

abstract:
  BACKGROUND: IFITM3 has been suggested to be associated with infection in some ethnic groups. Diabetes and hypercholesterolemia are also important clinical conditions that can predispose individuals to infection. The aim of this study was to investigate the association of rs12252 C polymorphism, BMI, diabetes, and hypercholesterolemia with mild flu in an Iranian population. METHODS: We conducted a case-control study, including 79 mild flu and 125 flu-negative individuals attending primary care centers of three provinces of Iran (i.e, Markazi, Semnan, and Zanjan). Pharyngeal swab specimens were collected from all participants, and were subjected to RNA and DNA extractions for Real-time PCR and PCR tests. All PCR products were then sequenced to find T/C polymorphisms in the rs12252 region. Data on demographic, anthropometric, and clinical variables were collected from participants’ medical records available in the primary care centers. The data was analyzed using DNASIS (v. 2.5) and Stata (v.11) software. RESULTS: All participants were of Fars ethnic background. The allele frequency for rs12252-C was found to be 9.49% among cases and 2.40% among controls. Carriers of the rs12252 C allele (CT + CC genotypes) showed 5.92 folds increase in the risk of mild flu comparing to the T allele homozygotes (P value: 0.007). We also found a significant positive association between rs12252 C allele heterozygote and mild flu (OR: 7.62, P value: 0.008), but not in C allele homozygote group (OR: 2.71, P value: 0.406). Similarly, we did not find a significant association between mild flu and BMI (OR: 1.06, P value: 0.087), diabetes (OR: 0.61, P value: 0.392), and hypercholesterolemia (OR: 0.50, P value: 0.393) in multivariable logistic regression. CONCLUSIONS: This is the first study evaluating the association between rs12252 polymorphisms, diabetes, hypercholesterolemia, and BMI and susceptibility to mild flu in an Iranian population. Our results suggest a significant positive association between mild flu and rs12252 C allele heterozygous and carriage. Future replication of the strong association observed here between rs12252 C allele carriage and mild flu might candidate this polymorphism as a genetic marker for early screening of susceptibility to mild flu. Lack of significant association between C allele homozygous and mild flu, observed in this study, might be the result of small sample size in this group. TRIAL REGISTRATION: IR.PII.REC.1395.3. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12985-017-0884-4) contains supplementary material, which is available to authorized users.


id: cord-337128-yyz7z0xj
author: Abdel-Moneim, Ahmed S
date: 2009-02-05
txt: ./txt/cord-337128-yyz7z0xj.txt
cache: ./cache/cord-337128-yyz7z0xj.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/1743-422x-6-15
doi: 10.1186/1743-422x-6-15
summary:
  title: Immunohistochemistry for detection of avian infectious bronchitis virus strain M41 in the proventriculus and nervous system of experimentally infected chicken embryos Aim of this study was to investigate by immunohistochemistry (IHC) the tissue tropism of avian infectious bronchitis virus (IBV) strain M41 in experimentally infected chicken embryos. Using IHC, antigens of IBV were detected in nasal epithelium, trachea, lung, spleen, myocardial vasculature, liver, gastrointestinal tract, kidney, skin, sclera of the eye, spinal cord, as well as in brain neurons of the inoculated embryos. IBV antigens were detected in the nasal epithelium, trachea, lung, spleen, myocardium, liver, gizzard, proventriculus, kidney, skin, sclera of the eye, spinal cord, as well as in neurons of the central nervous system in infected embryos (Table 1, Figure 1 ).

title:
  Immunohistochemistry for detection of avian infectious bronchitis virus strain M41 in the proventriculus and nervous system of experimentally infected chicken embryos

abstract:
  BACKGROUND: Infectious bronchitis virus primarily induces a disease of the respiratory system, different IBV strains may show variable tissue tropisms and also affect the oviduct and the kidneys. Proventriculitis was also associated with some new IBV strains. Aim of this study was to investigate by immunohistochemistry (IHC) the tissue tropism of avian infectious bronchitis virus (IBV) strain M41 in experimentally infected chicken embryos. RESULTS: To this end chicken embryos were inoculated in the allantoic sac with 10(3 )EID(50 )of IBV M41 at 10 days of age. At 48, 72, and 120 h postinoculation (PI), embryos and chorioallantoic membranes (CAM) were sampled, fixed, and paraffin-wax embedded. Allantoic fluid was also collected and titrated in chicken embryo kidney cells (CEK). The sensitivity of IHC in detecting IBV antigens in the CAM of inoculated eggs matched the virus reisolation and detection in CEK. Using IHC, antigens of IBV were detected in nasal epithelium, trachea, lung, spleen, myocardial vasculature, liver, gastrointestinal tract, kidney, skin, sclera of the eye, spinal cord, as well as in brain neurons of the inoculated embryos. These results were consistent with virus isolation and denote the wide tissue tropism of IBV M41 in the chicken embryo. Most importantly, we found infection of vasculature and smooth muscle of the proventriculus which has not seen before with IBV strain M41. CONCLUSION: IHC can be an additional useful tool for diagnosis of IBV infection in chickens and allows further studies to foster a deeper understanding of the pathogenesis of infections with IBV strains of different virulence. Moreover, these results underline that embryonic tissues in addition to CAM could be also used as possible source to generate IBV antigens for diagnostic purposes.


id: cord-337274-1fw0xiin
author: Si, Wei
date: 2010-05-01
txt: ./txt/cord-337274-1fw0xiin.txt
cache: ./cache/cord-337274-1fw0xiin.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/20433759/
doi: 10.1186/1743-422x-7-86
summary:
  title: A multiplex reverse transcription-nested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus A multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR) method was developed for the detection and differentiation of wild-type and vaccine strains of canine distemper virus (CDV). The multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR) method could be used to effectively detect and differentiate between wild-type CDV-infected dogs from dogs which were vaccinated with CDV vaccine, which would make it useful in clinical diagnosis and epidemiological monitoring. In summary, the multiplex RT-nPCR developed in this study is a highly specific and sensitive assay for the rapid detection and differentiation of wild-type and vaccine strains of CDV. A multiplex reverse transcription-nested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus

title:
  A multiplex reverse transcription-nested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus

abstract:
  A multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR) method was developed for the detection and differentiation of wild-type and vaccine strains of canine distemper virus (CDV). A pair of primers (P1 and P4) specific for CDV corresponding to the highly conserved region of the CDV genome were used as a common primer pair in the first-round PCR of the nested PCR. Primers P2 specific for CDV wild-type strains, were used as the forward primer together with the common reverse primer P4 in the second round of nested PCR. Primers P3, P5 specific for CDV wild-type strain or vaccine strain, were used as the forward primer together with the common reverse primer P4+P6 in the second round of nested PCR. A fragment of 177 bp was amplified from vaccine strain genomic RNA, and a fragment of 247 bp from wild-type strain genomic RNA in the RT-nPCR, and two fragments of 247 bp and 177 bp were amplified from the mixed samples of vaccine and wild-type strains. No amplification was achieved for uninfected cells, or cells infected with Newcastle disease virus (NDV), canine parvovirus (CPV), canine coronavirus (CCV), rabies virus (RV), or canine adenovirus (CAV). The RT-nPCR method was used to detect 30 field samples suspected of canine distemper from Heilongjiang and Jilin Provinces, and 51 samples in Shandong province. As a result of 30 samples, were found to be wild-type-like, and 5 to be vaccine-strain-like. The RT-nPCR method can be used to effectively detect and differentiate wild-type CDV-infected dogs from dogs vaccinated with CDV vaccine, and thus can be used in clinical detection and epidemiological surveillance.


id: cord-337636-3yc0ribg
title: A novel two-step, direct-to-PCR method for virus detection off swabs using human coronavirus 229E
author: Morehouse, Zachary P.
date: 2020-08-25
txt: ./txt/cord-337636-3yc0ribg.txt
cache: ./cache/cord-337636-3yc0ribg.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/s12985-020-01405-y
doi: 10.1186/s12985-020-01405-y
summary:
  Herein, we have developed a method to detect virus off swabs using solely shaker-mill based mechanical lysis and the transfer of the viral lysate directly to a PCR assay for virus detection, bypassing the substantial reagent and time investments required for extraction and purification steps. Swabs were spiked in serial dilutions from 1.2 × 10(6) to 1.2 × 10(1) copies/mL and then placed in 2 mL tubes with viral transport media (VTM) to mimic the specimen collection procedures in the clinic prior to processing via shaker-mill homogenization. RESULTS: HCoV-229E in vitro spiked swabs were processed in a novel two-step, direct-to-PCR methodology for viral detection. CONCLUSION: We have proven that the shaker-mill homogenization-based two-step, direct-to-PCR procedures provides sufficient viral lysis off swabs, where the resulting lysate can be used directly in PCR for the detection of HCoV-229E. Using human coronavirus 229E (HCoV-229E) as our model organism, we developed a novel two-step methodology of optimized shaker-mill homogenization parameters that allowed for direct-to-PCR viral detection.

abstract:
  BACKGROUND: Currently, one of the most reliable methods for viral infection detection are polymerase chain reaction (PCR) based assays. This process is time and resource heavy, requiring multiple steps of lysis, extraction, purification, and amplification procedures. Herein, we have developed a method to detect virus off swabs using solely shaker-mill based mechanical lysis and the transfer of the viral lysate directly to a PCR assay for virus detection, bypassing the substantial reagent and time investments required for extraction and purification steps. METHODS: Using Human Coronavirus 229E (HCoV-229E) as a model system, we spiked swabs in vitro for proof-of-concept testing. Swabs were spiked in serial dilutions from 1.2 × 10(6) to 1.2 × 10(1) copies/mL and then placed in 2 mL tubes with viral transport media (VTM) to mimic the specimen collection procedures in the clinic prior to processing via shaker-mill homogenization. After homogenization, 1 μL of lysate was processed using RT-qPCR for amplification of the nucleocapsid (N) gene, qualifying viral detection. RESULTS: HCoV-229E in vitro spiked swabs were processed in a novel two-step, direct-to-PCR methodology for viral detection. After running 54 swabs, we confidently determined our limit of detection to be 1.2 × 10(3) viral copies/mL with 96.30% sensitivity. CONCLUSION: We have proven that the shaker-mill homogenization-based two-step, direct-to-PCR procedures provides sufficient viral lysis off swabs, where the resulting lysate can be used directly in PCR for the detection of HCoV-229E. This finding allows for reductions in the time and resources required for PCR based virus detection in comparison to the traditional extraction-to-PCR methodology.


id: cord-338372-xsg0j92t
title: Permissiveness of human hepatoma cell lines for HCV infection
author: Sainz, Bruno
date: 2012-01-24
txt: ./txt/cord-338372-xsg0j92t.txt
cache: ./cache/cord-338372-xsg0j92t.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/1743-422x-9-30
doi: 10.1186/1743-422x-9-30
summary:
  While varying luciferase activity after parallel HCVpp inoculation is a phenotype we have seen among Huh7 cell lines from different laboratories (Additional file 1: Figure S3 and [34] ), to functionally test whether these cells express suboptimal levels of any of the known HCV entry factors, we transiently transfected PLC, Hep3B and HepG2-CD81 cells with vectors expressing the four known human HCV receptors and reassessed their permissiveness for HCVpp and HCVcc infection 48 h post-transfection. Notably, we have previously shown that IFN-mediated activation of the innate cellular interferon response inhibits HCV RNA replication and subsequent production of de novo HCV virions ( [45] and data not shown), thus the reduced steady-state HCVcc infection levels observed in Hep3B and HepG2-CD81 cells and the decline of HCV infection observed in PLC cells, despite these cells exhibiting comparable infection initiation permissiveness, may be at least in part a consequence of HCV-induced innate immune signaling in these hepatoma cell lines.

abstract:
  BACKGROUND: Although primary and established human hepatoma cell lines have been evaluated for hepatitis C virus (HCV) infection in vitro, thus far only Huh7 cells have been found to be highly permissive for infectious HCV. Since our understanding of the HCV lifecycle would benefit from the identification of additional permissive cell lines, we assembled a panel of hepatic and non-hepatic cell lines and assessed their ability to support HCV infection. Here we show infection of the human hepatoma cell lines PLC/PRF/5 and Hep3B with cell culture-derived HCV (HCVcc), albeit to lower levels than that achieved in Huh7 cells. To better understand the reduced permissiveness of PLC and Hep3B cells for HCVcc infection, we performed studies to evaluate the ability of each cell line to support specific steps of the viral lifecycle (i.e. entry, replication, egress and spread). RESULTS: We found that while the early events in HCV infection (i.e. entry plus replication initiation) are cumulatively equivalent or only marginally reduced in PLC and Hep3B cells, later steps of the viral life cycle such as steady-state replication, de novo virus production and/or spread are impaired to different degrees in PLC and Hep3B cultures compared to Huh7 cell cultures. Interestingly, we also observed that interferon stimulated gene (i.e. ISG56) expression was significantly and differentially up-regulated in PLC and Hep3B cells following viral infection. CONCLUSIONS: We conclude that the restrictions observed later during HCV infection in these cell lines could in part be attributed to HCV-induced innate signaling. Nevertheless, the identification of two new cell lines capable of supporting authentic HCVcc infection, even at reduced levels, expands the current repertoire of cell lines amendable for the study of HCV in vitro and should aid in further elucidating HCV biology and the cellular determinants that modulate HCV infection.


id: cord-339209-oe8onyr9
title: Mesoniviruses are mosquito-specific viruses with extensive geographic distribution and host range
author: Vasilakis, Nikos
date: 2014-05-20
txt: ./txt/cord-339209-oe8onyr9.txt
cache: ./cache/cord-339209-oe8onyr9.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/1743-422x-11-97
doi: 10.1186/1743-422x-11-97
summary:
  The organization of each genome was similar to that described previously for the mesoniviruses (NDiV, CavV, HanaV, NseV and MenoV), featuring a long 5''-untranslated region (5''-UTR) of 359 to 370 nt, six major long open reading frames (ORFs), and a long terminal region of 1780 to 1804 nt preceding the poly[A] tail ( Figure 2 ). To determine the phylogenetic relationships of the newly identified insect viruses, maximum likelihood (ML) phylogenetic trees were constructed based on the amino acid alignments of ORF2a (unprocessed S protein) and a concatenated region of the highly conserved domains within ORF1ab (3CL pro , RdRp and ZnHel1). A Clustal X alignment of the mesonivirus ORF3a proteins and individual structural analyses using SignalP and TMHMM and NetNGlyc (www.expasy.org) indicated that each is a class I transmembrane glycoprotein with a predicted N-termimal signal peptide, an ectodomain containing a conserved set of 6 cysteine residues and a single conserved N-glycosylation site, a transmembrane domain and a C-terminal cytoplasmic domain ( Figure 4A, 4D) .

abstract:
  BACKGROUND: The family Mesoniviridae (order Nidovirales) comprises of a group of positive-sense, single-stranded RNA ([+]ssRNA) viruses isolated from mosquitoes. FINDINGS: Thirteen novel insect-specific virus isolates were obtained from mosquitoes collected in Indonesia, Thailand and the USA. By electron microscopy, the virions appeared as spherical particles with a diameter of ~50 nm. Their 20,129 nt to 20,777 nt genomes consist of positive-sense, single-stranded RNA with a poly-A tail. Four isolates from Houston, Texas, and one isolate from Java, Indonesia, were identified as variants of the species Alphamesonivirus-1 which also includes Nam Dinh virus (NDiV) from Vietnam and Cavally virus (CavV) from Côte d’Ivoire. The eight other isolates were identified as variants of three new mesoniviruses, based on genome organization and pairwise evolutionary distances: Karang Sari virus (KSaV) from Java, Bontag Baru virus (BBaV) from Java and Kalimantan, and Kamphaeng Phet virus (KPhV) from Thailand. In comparison with NDiV, the three new mesoniviruses each contained a long insertion (180 – 588 nt) of unknown function in the 5’ region of ORF1a, which accounted for much of the difference in genome size. The insertions contained various short imperfect repeats and may have arisen by recombination or sequence duplication. CONCLUSIONS: In summary, based on their genome organizations and phylogenetic relationships, thirteen new viruses were identified as members of the family Mesoniviridae, order Nidovirales. Species demarcation criteria employed previously for mesoniviruses would place five of these isolates in the same species as NDiV and CavV (Alphamesonivirus-1) and the other eight isolates would represent three new mesonivirus species (Alphamesonivirus-5, Alphamesonivirus-6 and Alphamesonivirus-7). The observed spatiotemporal distribution over widespread geographic regions and broad species host range in mosquitoes suggests that mesoniviruses may be common in mosquito populations worldwide.


id: cord-339744-xrit0w5i
author: Feng, Bo
date: 2017-11-03
txt: ./txt/cord-339744-xrit0w5i.txt
cache: ./cache/cord-339744-xrit0w5i.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/29100522/
doi: 10.1186/s12985-017-0875-5
summary:
  Our previous microarray analysis showed that H9N2 virus infection and inactivated viral particle inoculation increased the expression of interferon-inducible transmembrane protein 1 (IFITM1) in human umbilical vein endothelial cells (HUVECs). In present study, we deeply investigated the expression patterns of IFITM1 and IFITM1-mediated antiviral response induced by H9N2 virus infection and inactivated viral particle inoculation in HUVECs. Epithelial cells that are considered target cells of the influenza virus were selected as a reference control. The results indicated that the cellular interaction between intracellular molecules and viral particles might be involved in the induction of IFITM1 expression in HUVECs. To determine the antiviral activity of IFITM1 protein induced by H9N2 virus infection, HUVECs or BEAS-2Bs were infected with H9N2 virus at MOI of 5 and incubated for 1 h, then cells were transfected with IFITM1 specific siRNA or control siRNA for 36 h.

title:
  Investigation of antiviral state mediated by interferon-inducible transmembrane protein 1 induced by H9N2 virus and inactivated viral particle in human endothelial cells

abstract:
  BACKGROUND: Endothelial cells are believed to play an important role in response to virus infection. Our previous microarray analysis showed that H9N2 virus infection and inactivated viral particle inoculation increased the expression of interferon-inducible transmembrane protein 1 (IFITM1) in human umbilical vein endothelial cells (HUVECs). In present study, we deeply investigated the expression patterns of IFITM1 and IFITM1-mediated antiviral response induced by H9N2 virus infection and inactivated viral particle inoculation in HUVECs. Epithelial cells that are considered target cells of the influenza virus were selected as a reference control. METHODS: First, we quantified the expression levels of IFITM1 in HUVECs induced by H9N2 virus infection or viral particle inoculation using quantitative real-time PCR and western blot. Second, we observed whether hemagglutinin or neuraminidase affected IFITM1 expression in HUVECs. Finally, we investigated the effect of induced-IFITM1 on the antiviral state in HUVECs by siRNA and activation plasmid transfection. RESULTS: Both H9N2 virus infection and viral particle inoculation increased the expression of IFITM1 without elevating the levels of interferon-ɑ/β in HUVECs. HA or NA protein binding alone is not sufficient to increase the levels of IFITM1 and interferon-ɑ/β in HUVECs. IFITM1 induced by viral particle inoculation significantly decreased the virus titers in culture supernatants of HUVECs. CONCLUSIONS: Our results showed that inactivated viral particle inoculation increased the expression of IFITM1 at mRNA and protein levels. Moreover, the induction of IFITM1 expression mediated the antiviral state in HUVECs.


id: cord-340046-kgbvld0y
title: Exhaled breath condensate sampling is not a new method for detection of respiratory viruses
author: Houspie, Lieselot
date: 2011-03-04
txt: ./txt/cord-340046-kgbvld0y.txt
cache: ./cache/cord-340046-kgbvld0y.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/21375748/
doi: 10.1186/1743-422x-8-98
summary:
  BACKGROUND: Exhaled breath condensate (EBC) sampling has been considered an inventive and novel method for the isolation of respiratory viruses. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms (defined in Table 1 ), using a commercially available condenser (RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA). Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract.

abstract:
  BACKGROUND: Exhaled breath condensate (EBC) sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections.


id: cord-340883-zf8jbhdl
author: He, Zhongping
date: 2007-03-27
txt: ./txt/cord-340883-zf8jbhdl.txt
cache: ./cache/cord-340883-zf8jbhdl.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/17386116/
doi: 10.1186/1743-422x-4-32
summary:
  title: Using patient-collected clinical samples and sera to detect and quantify the severe acute respiratory syndrome coronavirus (SARS-CoV) Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect and quantify SARS-CoV in 934 sera and self-collected throat washes and fecal samples from 271 patients with laboratory-confirmed SARS managed at a single institution. The highest SARS-CoV RT-PCR rates (70.4–86.3%) and viral loads (log(10 )4.5–6.1) were seen in fecal samples collected 2–4 weeks after the onset of clinical illness. The aim of this study was to detect and quantify SARS-CoV using RT-PCR in sera and throat washes and stools self-collected by 271 patients with laboratory confirmed SARS managed at a single institution. The use of patient self-collected throat washings may reduce risks to healthcare workers, although lower respiratory tract samples such as sputum, NPAs or bronchoalveolar lavage fluid are likely to have higher viral loads and offer increased likelihood of SARS-CoV detection by RT-PCR.

title:
  Using patient-collected clinical samples and sera to detect and quantify the severe acute respiratory syndrome coronavirus (SARS-CoV)

abstract:
  BACKGROUND: Severe acute respiratory syndrome (SARS) caused a large outbreak of pneumonia in Beijing, China, in 2003. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect and quantify SARS-CoV in 934 sera and self-collected throat washes and fecal samples from 271 patients with laboratory-confirmed SARS managed at a single institution. RESULTS: SARS-CoV detection rates in sera were highest in the first 9 days of illness, whereas detection was highest in throat washes 5–14 days after onset of symptoms. The highest SARS-CoV RT-PCR rates (70.4–86.3%) and viral loads (log(10 )4.5–6.1) were seen in fecal samples collected 2–4 weeks after the onset of clinical illness. Fecal samples were frequently SARS-CoV RT-PCR positive beyond 40 days, and occasional sera still had SARS-CoV detected after 3 weeks of illness. CONCLUSION: In the context of an extensive outbreak with major pressure on hospital resources, patient self-collected samples are an alternative to nasopharyngeal aspirates for laboratory confirmation of SARS-CoV infection.


id: cord-341168-3gd1w2kn
author: Beer, Christiane
date: 2006-04-02
txt: ./txt/cord-341168-3gd1w2kn.txt
cache: ./cache/cord-341168-3gd1w2kn.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/16579862/
doi: 10.1186/1743-422x-3-21
summary:
  We have previously shown that A-MLV entry is closely associated with cholesterol-rich microdomains like rafts and caveolae [1] and that A-MLV envelope protein is associated with rafts in infected cells suggesting a possible role of rafts in A-MLV assembly [2] . As rafts are suggested to be pre-caveolae [11] and a large fraction of the A-MLV receptor Pit2 was found associated with cholesterol-rich microdomains [1] , we have here investigated if rafts and caveolae are involved in the early steps of A-MLV binding. As GM1 is a general marker for cholesterol-rich microdomains, we investigated if these regions of preferred A-MLV binding were also enriched in caveolin-1 (cav-1), a major structural protein of caveolae. Using confocal microscopy we found that A-MLV binds preferentially to large GM1-positive membrane regions of NIH3T3 cells, which are most likely rafts. In addition, A-MLV binding to large rafts was independent of plasma membrane cholesterol indicating that the A-MLV receptor Pit2 or other virus interacting proteins were still present in these regions.

title:
  Amphotropic murine leukemia virus is preferentially attached to cholesterol-rich microdomains after binding to mouse fibroblasts

abstract:
  BACKGROUND: We have recently shown that amphotropic murine leukemia virus (A-MLV) can enter the mouse fibroblast cell line NIH3T3 via caveola-dependent endocytosis. But due to the size and omega-like shape of caveolae it is possible that A-MLV initially binds cells outside of caveolae. Rafts have been suggested to be pre-caveolae and we here investigate whether A-MLV initially binds to its receptor Pit2, a sodium-dependent phosphate transporter, in rafts or caveolae or outside these cholesterol-rich microdomains. RESULTS: Here, we show that a high amount of cell-bound A-MLV was attached to large rafts of NIH3T3 at the time of investigation. These large rafts were not enriched in caveolin-1, a major structural component of caveolae. In addition, they are rather of natural occurrence in NIH3T3 cells than a result of patching of smaller rafts by A-MLV. Thus cells incubated in parallel with vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped MLV particles showed the same pattern of large rafts as cells incubated with A-MLV, but VSV-G pseudotyped MLV particles did not show any preference to attach to these large microdomains. CONCLUSION: The high concentration of A-MLV particles bound to large rafts of NIH3T3 cells suggests a role of these microdomains in early A-MLV binding events.


id: cord-341321-paucodwz
title: Complete genome sequence of a highly divergent astrovirus isolated from a child with acute diarrhea
author: Finkbeiner, Stacy R
date: 2008-10-14
txt: ./txt/cord-341321-paucodwz.txt
cache: ./cache/cord-341321-paucodwz.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/1743-422x-5-117
doi: 10.1186/1743-422x-5-117
summary:
  We have previously described the identification of several sequence fragments with limited sequence identity to known astroviruses in a stool specimen obtained from a child with acute diarrhea, suggesting that a novel virus was present. Like other astroviruses, the AstV-MLB1 genome was predicted to encode three open reading frames (ORF1a, ORF1b, and ORF2) and contained both 5'' and 3'' non-translated regions (NTR), as well as a poly-A tail. In the less conserved regions II-IV, AstV-MLB1 shared only 5-27% amino acid identity to the known human astroviruses. Complete sequencing and genome analysis of Astrovirus MLB1 revealed that the virus has three open reading 1a 787 28 28 NA 29 29 NA NA 29 9 9 NA 10 22 24 1b 511 54 54 NA 54 54 NA NA 54 36 35 NA 36 47 44 2 756 24 24 24 23 23 24 24 24 15 16 16 11 18 19 frames sharing the same organization as other astroviruses.

abstract:
  BACKGROUND: Astroviruses infect a variety of mammals and birds and are causative agents of diarrhea in humans and other animal hosts. We have previously described the identification of several sequence fragments with limited sequence identity to known astroviruses in a stool specimen obtained from a child with acute diarrhea, suggesting that a novel virus was present. RESULTS: In this study, the complete genome of this novel virus isolate was sequenced and analyzed. The overall genome organization of this virus paralleled that of known astroviruses, with 3 open reading frames identified. Phylogenetic analysis of the ORFs indicated that this virus is highly divergent from all previously described animal and human astroviruses. Molecular features that are highly conserved in human serotypes 1–8, such as a 3'NTR stem-loop structure and conserved nucleotide motifs present in the 5'NTR and ORF1b/2 junction, were either absent or only partially conserved in this novel virus. CONCLUSION: Based on the analyses described herein, we propose that this newly discovered virus represents a novel species in the family Astroviridae. It has tentatively been named Astrovirus MLB1.


id: cord-342117-r2chpw7y
title: Inhibitory effect of small interfering RNA on dengue virus replication in mosquito cells
author: Wu, Xinwei
date: 2010-10-14
txt: ./txt/cord-342117-r2chpw7y.txt
cache: ./cache/cord-342117-r2chpw7y.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/20946645/
doi: 10.1186/1743-422x-7-270
summary:
  The antiviral effect of siRNA was evaluated by measurement of cell survival rate using the MTT method and viral RNA was quantitated with real-time RT-PCR. CONCLUSIONS: Our data showed that synthetic siRNA against the DEN-1 membrane glycoprotein precursor gene effectively inhibited DEN-1 viral RNA replication and increased C6/36 cell survival rate. Therefore, in this study, we designed and synthesized 21 nt siRNAs against the DEN-I viral PrM gene, and investigated their inhibition effects of dengue virus replication in transfected C6/36 cells. These data indicate that siRNA against DEN viral genome can effectively inhibit viral RNA replication in the C6/36 cells, protect host cell from viral attack, suggesting its potential role in prevention and treatment of dengue fever. Our data showed that synthetic siRNA against the DEN membrane glycoprotein precursor gene effectively inhibited DEN viral RNA replication and increased C6/36 cell survival rate. Inhibition of viral gene expression and replication in mosquito cells by dsRNA-triggered RNA interference

abstract:
  BACKGROUND: Dengue viruses (DENs) are the wildest transmitted mosquito-borne pathogens throughout tropical and sub-tropical regions worldwide. Infection with DENs can cause severe flu-like illness and potentially fatal hemorrhagic fever. Although RNA interference triggered by long-length dsRNA was considered a potent antiviral pathway in the mosquito, only limited studies of the value of small interfering RNA (siRNA) have been conducted. RESULTS: A 21 nt siRNA targeting the membrane glycoprotein precursor gene of DEN-1 was synthesized and transfected into mosquito C6/36 cells followed by challenge with DEN. The stability of the siRNA in cells was monitored by flow cytometry. The antiviral effect of siRNA was evaluated by measurement of cell survival rate using the MTT method and viral RNA was quantitated with real-time RT-PCR. The presence of cells containing siRNA at 0.25, 1, 3, 5, 7 days after transfection were 66.0%, 52.1%, 32.0%, 13.5% and 8.9%, respectively. After 7 days incubation with DEN, there was reduced cytopathic effect, increased cell survival rate (76.9 ± 4.5% vs 23.6 ± 14.6%) and reduced viral RNA copies (Ct value 19.91 ± 0.63 vs 14.56 ± 0.39) detected in transfected C6/36 cells. CONCLUSIONS: Our data showed that synthetic siRNA against the DEN-1 membrane glycoprotein precursor gene effectively inhibited DEN-1 viral RNA replication and increased C6/36 cell survival rate. siRNA may offer a potential new strategy for prevention and treatment of DEN infection.


id: cord-342157-qjyooq68
title: Comparative analysis of full genomic sequences among different genotypes of dengue virus type 3
author: King, Chwan-Chuen
date: 2008-05-21
txt: ./txt/cord-342157-qjyooq68.txt
cache: ./cache/cord-342157-qjyooq68.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/18495043/
doi: 10.1186/1743-422x-5-63
summary:
  In our study, complete genomic sequencing of DENV-3 strains collected from different geographical locations and isolation years were determined and the sequence diversity as well as selection pressure sites in the DENV genome other than within the E gene were also analyzed. RESULTS: Using maximum likelihood and Bayesian approaches, our phylogenetic analysis revealed that the Taiwan''s indigenous DENV-3 isolated from 1994 and 1998 dengue/DHF epidemics and one 1999 sporadic case were of the three different genotypes – I, II, and III, each associated with DENV-3 circulating in Indonesia, Thailand and Sri Lanka, respectively. Compared to the prototype strain H87, several unique amino acid substitutions that serve as unique signature sites for each genotype were found within the full genomic sequences of the selected DENV-3 isolates from Taiwan or other countries and are listed by the order of the gene in Table 3 .

abstract:
  BACKGROUND: Although the previous study demonstrated the envelope protein of dengue viruses is under purifying selection pressure, little is known about the genetic differences of full-length viral genomes of DENV-3. In our study, complete genomic sequencing of DENV-3 strains collected from different geographical locations and isolation years were determined and the sequence diversity as well as selection pressure sites in the DENV genome other than within the E gene were also analyzed. RESULTS: Using maximum likelihood and Bayesian approaches, our phylogenetic analysis revealed that the Taiwan's indigenous DENV-3 isolated from 1994 and 1998 dengue/DHF epidemics and one 1999 sporadic case were of the three different genotypes – I, II, and III, each associated with DENV-3 circulating in Indonesia, Thailand and Sri Lanka, respectively. Sequence diversity and selection pressure of different genomic regions among DENV-3 different genotypes was further examined to understand the global DENV-3 evolution. The highest nucleotide sequence diversity among the fully sequenced DENV-3 strains was found in the nonstructural protein 2A (mean ± SD: 5.84 ± 0.54) and envelope protein gene regions (mean ± SD: 5.04 ± 0.32). Further analysis found that positive selection pressure of DENV-3 may occur in the non-structural protein 1 gene region and the positive selection site was detected at position 178 of the NS1 gene. CONCLUSION: Our study confirmed that the envelope protein is under purifying selection pressure although it presented higher sequence diversity. The detection of positive selection pressure in the non-structural protein along genotype II indicated that DENV-3 originated from Southeast Asia needs to monitor the emergence of DENV strains with epidemic potential for better epidemic prevention and vaccine development.


id: cord-342906-51296y8d
title: Aerosolized avian influenza virus by laboratory manipulations
author: Li, Zhiping
date: 2012-08-06
url: https://doi.org/10.1186/1743-422x-9-146
doi: 10.1186/1743-422x-9-146
abstract:
  BACKGROUND: Avian H5N1 influenza viruses present a challenge in the laboratory environment, as they are difficult to collect from the air due to their small size and relatively low concentration. In an effort to generate effective methods of H5N1 air removal and ensure the safety of laboratory personnel, this study was designed to investigate the characteristics of aerosolized H5N1 produced by laboratory manipulations during research studies. RESULTS: Normal laboratory procedures used to process the influenza virus were carried out independently and the amount of virus polluting the on-site atmosphere was measured. In particular, zootomy, grinding, centrifugation, pipetting, magnetic stirring, egg inoculation, and experimental zoogenetic infection were performed. In addition, common accidents associated with each process were simulated, including breaking glass containers, syringe injection of influenza virus solution, and rupturing of centrifuge tubes. A micro-cluster sampling ambient air pollution collection device was used to collect air samples. The collected viruses were tested for activity by measuring their ability to induce hemagglutination with chicken red blood cells and to propagate in chicken embryos after direct inoculation, the latter being detected by reverse-transcription PCR and HA test. The results showed that the air samples from the normal centrifugal group and the negative-control group were negative, while all other groups were positive for H5N1. CONCLUSIONS: Our findings suggest that there are numerous sources of aerosols in laboratory operations involving H5N1. Thus, laboratory personnel should be aware of the exposure risk that accompanies routine procedures involved in H5N1 processing and take proactive measures to prevent accidental infection and decrease the risk of virus aerosol leakage beyond the laboratory.


id: cord-343893-sophqqne
title: Feline aminopeptidase N is not a functional receptor for avian infectious bronchitis virus
author: Chu, Victor C
date: 2007-02-26
txt: ./txt/cord-343893-sophqqne.txt
cache: ./cache/cord-343893-sophqqne.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/17324273/
doi: 10.1186/1743-422x-4-20
summary:
  Feline aminopeptidase N (fAPN) serves as a functional receptor for most group 1 coronaviruses including feline infectious peritonitis virus (FIPV), canine coronavirus, transmissible gastroenteritis virus (TGEV), and human coronavirus 229E (HCoV-229E). Group 1 CoVs -including human coronavirus-229E (HCoV-229E), feline infectious peritonitis virus (FIPV), transmissible gastroenteritis virus (TGEV) and canine coronavirus (CCV) -utilize human, feline, porcine, and canine aminopeptidase N (APN) as functional receptors during virus entry [10] [11] [12] [13] . We also cultured seven strains of IBV, including Arkansas 99, Arkansas_DPI, California 99, Connecticut 46, Holland 52, Iowa 97, and Massachusetts 41 (designated as Ark99, Ark_DPI, CA99, Conn46, H52, Iowa97, and Mass41) as candidates to test for fAPN utilization by group 3 avian CoVs. Surprisingly, expression of fAPN did not increase viral infection in any of the strains tested. To verify the functionality of fAPN as a coronavirus receptor, we first tested its ability to rescue FIPV-1146 and TGEV infection of non-permissive cells, as reported in previous studies [11] .

abstract:
  BACKGROUND: Coronaviruses are an important cause of infectious diseases in humans, including severe acute respiratory syndrome (SARS), and have the continued potential for emergence from animal species. A major factor in the host range of a coronavirus is its receptor utilization on host cells. In many cases, coronavirus-receptor interactions are well understood. However, a notable exception is the receptor utilization by group 3 coronaviruses, including avian infectious bronchitis virus (IBV). Feline aminopeptidase N (fAPN) serves as a functional receptor for most group 1 coronaviruses including feline infectious peritonitis virus (FIPV), canine coronavirus, transmissible gastroenteritis virus (TGEV), and human coronavirus 229E (HCoV-229E). A recent report has also suggested a role for fAPN during IBV entry (Miguel B, Pharr GT, Wang C: The role of feline aminopeptidase N as a receptor for infectious bronchitis virus. Brief review. Arch Virol 2002, 147:2047–2056. RESULTS: Here we show that, whereas both transient transfection and constitutive expression of fAPN on BHK-21 cells can rescue FIPV and TGEV infection in non-permissive BHK cells, fAPN expression does not rescue infection by the prototype IBV strain Mass41. To account for the previous suggestion that fAPN could serve as an IBV receptor, we show that feline cells can be infected with the prototype strain of IBV (Mass 41), but with low susceptibility compared to primary chick kidney cells. We also show that BHK-21 cells are slightly susceptible to certain IBV strains, including Ark99, Ark_DPI, CA99, and Iowa97 (<0.01% efficiency), but this level of infection is not increased by fAPN expression. CONCLUSION: We conclude that fAPN is not a functional receptor for IBV, the identity of which is currently under investigation.


id: cord-343908-f3v8wi9h
author: Ammayappan, Arun
date: 2008-12-22
txt: ./txt/cord-343908-f3v8wi9h.txt
cache: ./cache/cord-343908-f3v8wi9h.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/19102764/
doi: 10.1186/1743-422x-5-157
summary:
  title: Complete genomic sequence analysis of infectious bronchitis virus Ark DPI strain and its evolution by recombination An infectious bronchitis virus Arkansas DPI (Ark DPI) virulent strain was sequenced, analyzed and compared with many different IBV strains and coronaviruses. Comparative sequence analysis of Ark DPI with other IBV strains shows striking similarity to the Conn, Gray, JMK, and Ark 99, which were circulating during that time period. The complete genome sequence analysis of Ark DPI strain shows striking similarity to the Conn, Gray, JMK, and Ark 99 IBV strains, which were circulating during that time period [1, [19] [20] [21] . Phylogenetic tree analysis of complete Ark DPI genome sequence with other IBV strains Cloning and sequencing of genes encoding structural proteins of avian infectious bronchitis virus Molecular cloning and sequence comparison of the S1 glycoprotein of the Gray and JMK strains of avian infectious bronchitis virus

title:
  Complete genomic sequence analysis of infectious bronchitis virus Ark DPI strain and its evolution by recombination

abstract:
  An infectious bronchitis virus Arkansas DPI (Ark DPI) virulent strain was sequenced, analyzed and compared with many different IBV strains and coronaviruses. The genome of Ark DPI consists of 27,620 nucleotides, excluding poly (A) tail, and comprises ten open reading frames. Comparative sequence analysis of Ark DPI with other IBV strains shows striking similarity to the Conn, Gray, JMK, and Ark 99, which were circulating during that time period. Furthermore, comparison of the Ark genome with other coronaviruses demonstrates a close relationship to turkey coronavirus. Among non-structural genes, the 5'untranslated region (UTR), 3C-like proteinase (3CL(pro)) and the polymerase (RdRp) sequences are 100% identical to the Gray strain. Among structural genes, S1 has 97% identity with Ark 99; S2 has 100% identity with JMK and 96% to Conn; 3b 99%, and 3C to N is 100% identical to Conn strain. Possible recombination sites were found at the intergenic region of spike gene, 3'end of S1 and 3a gene. Independent recombination events may have occurred in the entire genome of Ark DPI, involving four different IBV strains, suggesting that genomic RNA recombination may occur in any part of the genome at number of sites. Hence, we speculate that the Ark DPI strain originated from the Conn strain, but diverged and evolved independently by point mutations and recombination between field strains.


id: cord-346574-u28y1ttw
author: Chen, Keyan
date: 2012-08-24
txt: ./txt/cord-346574-u28y1ttw.txt
cache: ./cache/cord-346574-u28y1ttw.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/1743-422x-9-172
doi: 10.1186/1743-422x-9-172
summary:
  At present, various laboratory methods are available for the detection and surveillance of PHE-CoV, including virus isolation [1] , hemagglutination/hemagglutination inhibition (HA/HI) tests [13] , immunohistochemistry (IHC) assays [10] , and molecular tools such as nestedpolymerase chain reaction (nested PCR) and reverse transcriptase-polymerase chain reaction (RT-PCR) that enable detection of specific CoV RNA sequences from infected tissues [14, 15] . (1) (2) (3) In this study, an immunochromatographic strip with high sensitivity and specificity was developed for the detection of PHE-CoV, combining monoclonal antibody (MAb) and colloidal gold immunochromatography (GICA), and the resulting product is suitable for the surveillance of PHE-CoV. Thus, a lot of brain tissue samples were collected from deceased piglets with suspected PHE-CoV infection, and using RT-PCR and ELISA as reference test, the relative specificity and sensitivity of the immunochromatographic strip were determined to be 100% and 97.78%, respectively.

title:
  Development and evaluation of an immunochromatographic strip for rapid detection of porcine hemagglutinating encephalomyelitis virus

abstract:
  BACKGROUND: The incidence of PHE among pigs in many countries is on the rise, and it has caused great economic losses to the pig industry. Therefore, the development of a sensitive, specific, and easily-performed assay is crucial for the rapid detection and surveillance of PHE-CoV infection and transmission. RESULTS: An immunochromatographic strip was developed for the detection of PHE-CoV. The colloidal gold-labeled MAb 4D4 was used as the detection reagent, and the MAb 1E2 and goat anti-mouse IgG coated the strip's test and control lines, respectively. The immunochromatographic strip was capable of specifically detecting PHE-CoV with a HA unit of 2 within 10 min. Storage of the strips at room temperature for 6 months or at 4°C for 12 months did not change their sensitivity or specificity. Using RT-PCR as a reference test, the relative specificity and sensitivity of the immunochromatographic strip were determined to be 100% and 97.78%, respectively. There was an excellent agreement between the results obtained by RT-PCR and the immunochromatographic strips (kappa = 0.976). Additionally, there was a strong agreement between the sandwich enzyme-linked immunosorbent assay (ELISA) and immunochromatographic strips (Kappa = 0.976). When the immunochromatographic strips were used for diagnosing PHE-CoV infection in the Jilin Province, the PHE-CoV-positive rate ranged from 61.54% in the Jilin district to 17.95% in the Songyuan district. CONCLUSIONS: Based on its high specificity, sensitivity, and stability, the immunochromatographic strip would be suitable for on-site detection of PHE-CoV for surveillance and epidemiological purposes.


id: cord-347319-lcuma3eh
title: Lysosomotropic agents as HCV entry inhibitors
author: Ashfaq, Usman A
date: 2011-04-12
txt: ./txt/cord-347319-lcuma3eh.txt
cache: ./cache/cord-347319-lcuma3eh.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/1743-422x-8-163
doi: 10.1186/1743-422x-8-163
summary:
  HCV has two envelop proteins named as E1 and E2 which play an important role in cell entry through two main pathways: direct fusion at the plasma membrane and receptor-mediated endocytosis. To investigate the antiviral effect of LA (Chloroquine and NH(4)Cl) on pH dependent endocytosis, HCV pseudoparticles (HCVpp) of 1a and 3a genotype were produced and used to infect liver cells. For antiviral screening of Chloroquine and NH(4)Cl, liver cells were infected with HCVpp of 3a and 1a genotype in the presence or absence of different concentrations of Chloroquine and NH4Cl and there luciferase activity was determined by using a luminometer. We therefore tested the infectivity of HCVpp after treatment of target cells with different concentrations of Chloroquine and NH 4 Cl. HCVpp of 1a and 3a genotype demonstrated dose-dependent inhibition in the presence of Chloroquine and NH 4 Cl. Chloroquine and NH 4 Cl showed greater than 50% reduction of virus infectivity at 50 μM and 10 mM concentration respectively, suggesting a pH-sensitive route of virus entry (Figure 2a and 2b ).

abstract:
  HCV has two envelop proteins named as E1 and E2 which play an important role in cell entry through two main pathways: direct fusion at the plasma membrane and receptor-mediated endocytosis. Fusion of the HCV envelope proteins is triggered by low pH within the endosome. Lysosomotropic agents (LA) such as Chloroquine and Ammonium chloride (NH(4)Cl) are the weak bases and penetrate in lysosome as protonated form and increase the intracellular pH. To investigate the antiviral effect of LA (Chloroquine and NH(4)Cl) on pH dependent endocytosis, HCV pseudoparticles (HCVpp) of 1a and 3a genotype were produced and used to infect liver cells. The toxicological effects of Chloroquine and NH(4)Cl were tested in liver cells through MTT cell proliferation assay. For antiviral screening of Chloroquine and NH(4)Cl, liver cells were infected with HCVpp of 3a and 1a genotype in the presence or absence of different concentrations of Chloroquine and NH4Cl and there luciferase activity was determined by using a luminometer. The results demonstrated that Chloroquine and NH(4)Cl showed more than 50% reduction of virus infectivity at 50 μM and 10 mM concentrations respectively. These results suggest that inhibition of HCV at fusion step by increasing the lysosomal pH will be better option to treat chronic HCV.


id: cord-347569-9fvbshz2
author: Balakrishnan, Krishnan Nair
date: 2020-10-27
txt: ./txt/cord-347569-9fvbshz2.txt
cache: ./cache/cord-347569-9fvbshz2.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/33109247/
doi: 10.1186/s12985-020-01436-5
summary:
  On the other hand, negative control siRNA and untreated groups showed higher viral titer than siRNA treated groups: 4 log 10 TCID50/ml at day 10 p.i and 8 log 10 TCID50/ml at day 18 p.i. Analysis of RCMV ALL-03 infected cells undergoing apoptosis/necrosis upon combination siRNAs treatment were studied using flow cytometry. Taking into consideration as positive treatment control group, commercial drug GCV exhibited better rate of CPE inhibition (Fig. 7) compared to other combination siR-NAs but lesser efficient than dpc + ie2b siRNAs. In order to understand more on the role of each combination siRNAs during RCMV ALL-03 infection, all the four combination siRNAs targeting different regions were analyzed individually with specific primers. During the search of effective treatment for CMV, idea of controlling the disease by means of inhibiting virus replication and gene expression by combinations of two and more siRNAs had been explored in this study.

title:
  Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03)

abstract:
  BACKGROUND: Cytomegalovirus (CMV) is an opportunistic pathogen that causes severe complications in congenitally infected newborns and non-immunocompetent individuals. Developing an effective vaccine is a major public health priority and current drugs are fronting resistance and side effects on recipients. In the present study, with the aim of exploring new strategies to counteract CMV replication, several anti-CMV siRNAs targeting IE2 and DNA polymerase gene regions were characterized and used as in combinations for antiviral therapy. METHODS: The rat embryo fibroblast (REF) cells were transfected with multi siRNA before infecting with CMV strain ALL-03. Viral growth inhibition was measured by tissue culture infectious dose (TCID50), cytopathic effect (CPE) and droplet digital PCR (ddPCR) while IE2 and DNA polymerase gene knockdown was determined by real-time PCR. Ganciclovir was deployed as a control to benchmark the efficacy of antiviral activities of respective individual siRNAs. RESULTS: There was no significant cytotoxicity encountered for all the combinations of siRNAs on REF cells analyzed by MTT colorimetric assay (P > 0.05). Cytopathic effects (CPE) in cells infected by RCMV ALL-03 had developed significantly less and at much slower rate compared to control group. The expression of targeted genes was downregulated successfully resulted in significant reduction (P < 0.05) of viral mRNA and DNA copies (dpb + dpc: 79%, 68%; dpb + ie2b: 68%, 60%; dpb + dpc + ie2b: 48%, 42%). Flow cytometry analysis showed a greater percentage of viable and early apoptosis of combined siRNAs-treated cells compared to control group. Notably, the siRNAs targeting gene regions were sequenced and mutations were not encountered, thereby avoiding the formation of mutant with potential resistant viruses. CONCLUSIONS: In conclusion. The study demonstrated a tremendous promise of innovative approach with the deployment of combined siRNAs targeting at several genes simultaneously with the aim to control CMV replication in host cells.


id: cord-348204-365z3qxz
author: Harun, Mohammad Syamsul Reza
date: 2013-11-09
url: https://doi.org/10.1186/1743-422x-10-329
doi: 10.1186/1743-422x-10-329
title:
  Transcriptional profiling of feline infectious peritonitis virus infection in CRFK cells and in PBMCs from FIP diagnosed cats

abstract:
  BACKGROUND: Feline Infectious Peritonitis (FIP) is a lethal systemic disease, caused by the FIP Virus (FIPV); a virulent mutant of Feline Enteric Coronavirus (FECV). Currently, the viruses virulence determinants and host gene expressions during FIPV infection are not fully understood. METHODS: RNA sequencing of Crandell Rees Feline Kidney (CRFK) cells, infected with FIPV strain 79–1146 at 3 hours post infection (h.p.i), were sequenced using the Illumina next generation sequencing approach. Bioinformatic’s analysis, based on Felis catus 2X annotated shotgun reference genome, using CLC bio Genome Workbench mapped both control and infected cell reads to 18899 genes out of 19046 annotated genes. Kal’s Z test statistical analysis was used to analyse the differentially expressed genes from the infected CRFK cells. Real time RT-qPCR was developed for further transcriptional profiling of three genes (PD-1, PD-L1 and A3H) in infected CRFK cells and Peripheral Blood Mononuclear Cells (PBMCs) from healthy and FIP-diseased cats. RESULTS: Based on Kal’s Z-test, with False Discovery Rate (FDR) <0.05 and >1.99 fold change on gene expressions, a total of 61 genes were differentially expressed by both samples, where 44 genes were up-regulated and the remainder were down-regulated. Most genes were closely clustered together, suggesting a homogeneous expression. The majority of the genes that were significantly regulated, were those associated with monocytes-macrophage and Th1 cell functions, and the regulation of apoptosis. Real time RT-qPCR developed focusing on 2 up-regulated genes (PD-L1 and A3H) together with an apoptosis associated gene PD-1 expressions in FIPV infected CRFK cells and in PBMCs from healthy and FIP diagnosed cats produced concordant results with transcriptome data. CONCLUSION: The possible roles of these genes, and their importance in feline coronaviruses infection, are discussed.


id: cord-348876-v55piprx
author: Zhao, Guangyu
date: 2010-01-18
txt: ./txt/cord-348876-v55piprx.txt
cache: ./cache/cord-348876-v55piprx.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/1743-422x-7-9
doi: 10.1186/1743-422x-7-9
summary:
  In the present study, we designed a tetra-branched multiple antigenic peptide (MAP)-based vaccine, designated M2e-MAP, which contains the sequence overlapping the highly conserved extracellular domain of matrix protein 2 (M2e) of a HPAI H5N1 virus, and investigated its immune responses and cross-protection against different clades of H5N1 viruses. In the present study, we designed and synthesized a tetra-branched multiple antigenic peptide (MAP) derived from the M2e sequence of H5N1 virus VN/1194 strain, denoted as M2e-MAP, with an aim to develop a M2e-based vaccine for induction of M2e-specific immune responses and cross-protection of the vaccinated animals against lethal challenge of divergent H5N1 virus strains. After receiving the lethal dose (10 LD 50 ) of two H5N1 virus strains, the M2e-MAP vaccinated mice were further evaluated in terms of cross-protective ability by daily observation of the clinical symptoms, including weight loss and survival rate for two weeks, and then histopathological examination following removal of lung tissues.

title:
  An M2e-based multiple antigenic peptide vaccine protects mice from lethal challenge with divergent H5N1 influenza viruses

abstract:
  BACKGROUND: A growing concern has raised regarding the pandemic potential of the highly pathogenic avian influenza (HPAI) H5N1 viruses. Consequently, there is an urgent need to develop an effective and safe vaccine against the divergent H5N1 influenza viruses. In the present study, we designed a tetra-branched multiple antigenic peptide (MAP)-based vaccine, designated M2e-MAP, which contains the sequence overlapping the highly conserved extracellular domain of matrix protein 2 (M2e) of a HPAI H5N1 virus, and investigated its immune responses and cross-protection against different clades of H5N1 viruses. RESULTS: Our results showed that M2e-MAP vaccine induced strong M2e-specific IgG antibody responses following 3-dose immunization of mice with M2e-MAP in the presence of Freunds' or aluminium (alum) adjuvant. M2e-MAP vaccination limited viral replication and attenuated histopathological damage in the challenged mouse lungs. The M2e-MAP-based vaccine protected immunized mice against both clade1: VN/1194 and clade2.3.4: SZ/406H H5N1 virus challenge, being able to counteract weight lost and elevate survival rate following lethal challenge of H5N1 viruses. CONCLUSIONS: These results suggest that M2e-MAP presenting M2e of H5N1 virus has a great potential to be developed into an effective subunit vaccine for the prevention of infection by a broad spectrum of HPAI H5N1 viruses.


id: cord-349287-mwj2qby4
title: MERS coronavirus: diagnostics, epidemiology and transmission
author: Mackay, Ian M.
date: 2015-12-22
txt: ./txt/cord-349287-mwj2qby4.txt
cache: ./cache/cord-349287-mwj2qby4.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/s12985-015-0439-5
doi: 10.1186/s12985-015-0439-5
summary:
  The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. First cases of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infections in France, investigations and implications for the prevention of human-tohuman transmission

abstract:
  The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users.


id: cord-349683-3qdnnwvd
author: Zhang, Xuefeng
date: 2018-07-24
txt: ./txt/cord-349683-3qdnnwvd.txt
cache: ./cache/cord-349683-3qdnnwvd.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/s12985-018-1026-3
doi: 10.1186/s12985-018-1026-3
summary:
  The levels of anti-Ad antibodies in the Ad5-null control group (Ad empty vector, 10 11 VP/animal) were higher than those detected for the animals that received the same-dose (high-dose 10 11 VP/ animal) of Ad-HBV group, which may have resulted from gene inserting of truncated Pol, Core and Env antigen. The neutralization activity of the highest-titers serum samples from every animal in the Ad5-null control group and the three Ad-HBV groups (low, mid, and high doses) were then measured by the Ad vector luciferase-expressing inhibition assay. As shown in Table 3 and Fig. 5 , administration with Ad-HBV significantly improved the IFN-γ and IL-2 expression levels of the livers in the mid-dose, high-dose, and Ad-null control group (p < 0.05), but not in the low-dose group.

title:
  Immunogenicity of adenovirus-vector vaccine targeting hepatitis B virus: non-clinical safety assessment in non-human primates

abstract:
  BACKGROUND: A new promising therapeutic approach has emerged for patients chronically infected by the hepatitis B virus (HBV) with the development of a non-replicative adenovirus vector vaccine candidate (Ad-HBV). The vaccine encodes a fusion protein composed of a truncated HBV core protein, mutated polymerase protein, and two envelope domains. In this study, we assessed the immunogenicity of Ad-HBV administered to cynomolgus monkeys during a non-clinical safety assessment. METHODS: The virus was subcutaneously administered at 1.0 × 10(9) viral particles (VP)/animal (low-dose group), 1.0 × 10(10) VP/animal (mid-dose group), and 1.0 × 10(11) VP/animal (high-dose group); the control groups were administered an Ad5-null virus (1.0 × 10(11) VP/animal) and saline only. RESULTS: Except for inflammatory cell infiltration under the skin at the injection sites and transient elevation of body temperature and serum albumin, no Ad-HBV-related toxic effects were noted in any treatment group. Moreover, interferon (IFN)-γ enzyme-linked immunospot assays showed that Ad-HBV induced the targeting of T cells to a broad spectrum of HBV-specific epitopes spanning all three of the selected HBV immunogens (core, polymerase, and envelope domains) in a dose-dependent manner. Although anti-Ad antibody was produced in all groups (except for the saline control), the antibody titers were significantly lower in the high-dose Ad-HBV group than in the group that received the same dose of the Ad-null empty vector. In addition, the IFN-γ and IL-2 expression levels in the liver were significantly improved for the mid-dose, high-dose, and Ad-null control group (p < 0.05), but not for the low-dose group. CONCLUSIONS: Taken together, this safety assessment indicates that the Ad-HBV candidate vaccine is a potent specific immunotherapeutic agent, supporting its further clinical development as an anti-HBV infection vaccine. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12985-018-1026-3) contains supplementary material, which is available to authorized users.


id: cord-351920-igmb2yfe
author: Oma, Veslemøy Sunniva
date: 2016-06-13
txt: ./txt/cord-351920-igmb2yfe.txt
cache: ./cache/cord-351920-igmb2yfe.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/s12985-016-0555-x
doi: 10.1186/s12985-016-0555-x
summary:
  The aims of the study were to investigate the duration and quantity of BCoV shedding in feces and nasal secretions related to clinical signs, the presence of virus in blood and tissues and to test the hypothesis that seropositive calves are not infectious to naïve in-contact calves three weeks after BCoV infection. In two experimental studies, infected calves were not protected against reinfection with a different BCoV strain three weeks after the first challenge, but did not develop clinical signs [19, 20] . The majority of experimental studies have used BCoV inoculation as challenge procedure, which may influence clinical signs and viral shedding, and thereby the transmission potential compared to natural infection. The present study showed that calves infected with BCoV shed viral RNA for five weeks, and harbored viral RNA in intestinal tissues and lymph nodes even longer.

title:
  Bovine coronavirus in naturally and experimentally exposed calves; viral shedding and the potential for transmission

abstract:
  BACKGROUND: Bovine coronavirus (BCoV) is a widely distributed pathogen, causing disease and economic losses in the cattle industry worldwide. Prevention of virus spread is impeded by a lack of basic knowledge concerning viral shedding and transmission potential in individual animals. The aims of the study were to investigate the duration and quantity of BCoV shedding in feces and nasal secretions related to clinical signs, the presence of virus in blood and tissues and to test the hypothesis that seropositive calves are not infectious to naïve in-contact calves three weeks after BCoV infection. METHODS: A live animal experiment was conducted, with direct contact between animal groups for 24 h as challenge procedure. Four naïve calves were commingled with a group of six naturally infected calves and sequentially euthanized. Two naïve sentinel calves were commingled with the experimentally exposed group three weeks after exposure. Nasal swabs, feces, blood and tissue samples were analyzed for viral RNA by RT-qPCR, and virus isolation was performed on nasal swabs. Serum was analyzed for BCoV antibodies. RESULTS: The calves showed mild general signs, and the most prominent signs were from the respiratory system. The overall clinical score corresponded well with the shedding of viral RNA the first three weeks after challenge. General depression and cough were the signs that correlated best with shedding of BCoV RNA, while peak respiratory rate and peak rectal temperature appeared more than a week later than the peak shedding. Nasal shedding preceded fecal shedding, and the calves had detectable amounts of viral RNA intermittently in feces through day 35 and in nasal secretions through day 28, however virus isolation was unsuccessful from day six and day 18 from the two calves investigated. Viral RNA was not detected in blood, but was found in lymphatic tissue through day 42 after challenge. Although the calves were shedding BCoV RNA 21 days after infection the sentinel animals were not infected. CONCLUSIONS: Prolonged shedding of BCoV RNA can occur, but detection of viral RNA does not necessarily indicate a transmission potential. The study provides valuable information with regard to producing scientifically based biosecurity advices. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-016-0555-x) contains supplementary material, which is available to authorized users.


id: cord-351942-u9zpyy29
author: Tan, Bing
date: 2016-11-25
txt: ./txt/cord-351942-u9zpyy29.txt
cache: ./cache/cord-351942-u9zpyy29.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/27884154/
doi: 10.1186/s12985-016-0648-6
summary:
  title: Isolation and characterization of adenoviruses infecting endangered golden snub-nosed monkeys (Rhinopithecus roxellana) FINDINGS: We conducted a surveillance of viral infection in endangered golden snub-nosed monkeys (Rhinopithecus roxellana) in the subfamily Colobinae in China, and found that 5.1% of sampled individuals were positive for adenovirus. Golden snub-nosed monkeys (Rhinopithecus roxellana) living in Shennongjia Nature Reserve (SNR) in Hubei, China, are an endangered species belonging to the subfamily Colobinae [14] . For investigating the antibody prevalence against the AdV infection, virus neutralization assay was performed using archived serum samples collected from 8 golden snub-nosed monkeys in SNR [23] . The present isolate from these monkeys is distantly related to known AdV types, and likely represents a novel species in the genus Mastadenovirus. AdVs: Adenoviruses; HAdV: Human mastadenovirus; OWMs: Old World monkeys; SAdV: Simian adenovirus or mastadenovirus; SNR: Shennongjia Nature Reserve; WIV: Wuhan Institute of Virology

title:
  Isolation and characterization of adenoviruses infecting endangered golden snub-nosed monkeys (Rhinopithecus roxellana)

abstract:
  BACKGROUND: Adenoviruses are important pathogens with the potential for interspecies transmission between humans and non-human primates. Although many adenoviruses have been identified in monkeys, the knowledge of these viruses from the Colobinae members is quite limited. FINDINGS: We conducted a surveillance of viral infection in endangered golden snub-nosed monkeys (Rhinopithecus roxellana) in the subfamily Colobinae in China, and found that 5.1% of sampled individuals were positive for adenovirus. One of the adenoviruses (SAdV-WIV19) was successfully isolated and its full-length genome was sequenced. The full-length genome of WIV19 is 33,562 bp in size, has a G + C content of 56.2%, and encodes 35 putative genes. Sequence analysis revealed that this virus represents a novel species in the genus Mastadenovirus. Diverse cell lines, including those of human origin, were susceptible to WIV19. CONCLUSION: We report the first time the isolation and full-length genomic characterization of an adenovirus from the subfamily Colobinae. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-016-0648-6) contains supplementary material, which is available to authorized users.


id: cord-352361-jh31omg2
author: Nobach, Daniel
date: 2020-01-30
txt: ./txt/cord-352361-jh31omg2.txt
cache: ./cache/cord-352361-jh31omg2.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32000801/
doi: 10.1186/s12985-020-1289-3
summary:
  Although several rodents and other small mammals are known as important reservoirs for many viruses, bats (order: Chiroptera) represent the vast majority of identified natural reservoirs of several virus families/species to date [1, 14] . In conclusion, due to the continuous detection of new viruses in bats, the unclear situation regarding additional potential BoDV-1-reservoirs and molecular evidence for co-evolution of bats and bornaviruses, this study was conducted to investigate the potential presence of the most common orthobornaviruses in bats from endemic and non-endemic areas in Germany. Although the bicolored white-toothed shrew has been identified as indigenous reservoir of BoDV-1, other potential reservoirs or animal carriers are still unknown so that further investigations of small mammals including bat species are urgently needed. Distribution of Borna disease virus antigen and RNA in tissues of naturally infected bicolored white-toothed shrews, Crocidura leucodon, supporting their role as reservoir host species

title:
  No evidence for European bats serving as reservoir for Borna disease virus 1 or other known mammalian orthobornaviruses

abstract:
  BACKGROUND: The majority of emerging infectious diseases are zoonotic in nature and originate from wildlife reservoirs. Borna disease, caused by Borna disease virus 1 (BoDV-1), is an infectious disease affecting mammals, but recently it has also been shown to cause fatal encephalitis in humans. The endemic character of Borna disease points towards a nature-bound reservoir, with only one shrew species identified as reservoir host to date. Bats have been identified as reservoirs of a variety of zoonotic infectious agents. Endogenous borna-like elements in the genome of certain bat species additionally point towards co-evolution of bats with bornaviruses and therefore raise the question whether bats could serve as a potential reservoir of orthobornaviruses. METHODS: Frozen brain samples (n = 257) of bats of seven different genera from Germany were investigated by orthobornaviral RT-PCR. Additionally, tissue slides of formalin-fixed paraffin-embedded material of a subset of these bats (n = 140) were investigated for orthobornaviral phosphoprotein by immunohistochemistry. RESULTS: The brain samples were tested by RT-PCR without any evidence of orthobornavirus specific amplicons. Immunohistochemistry revealed a faint immunoreaction in 3/140 bats but with an untypical staining pattern for viral antigen. CONCLUSIONS: RT-PCR-screening showed no evidence for orthobornaviral RNA in the investigated bats. However, immunohistochemistry results should be investigated further to elucidate whether the reaction might be associated with expressed endogenous bornaviral elements or other so far unknown bornaviruses.


id: cord-354138-ps3rzjve
author: Zhao, Fu-Rong
date: 2014-03-14
txt: ./txt/cord-354138-ps3rzjve.txt
cache: ./cache/cord-354138-ps3rzjve.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/1743-422x-11-49
doi: 10.1186/1743-422x-11-49
summary:
  However, prevalence of A (H1N1) pdm09 influenza virus infection in cats in northeastern China is unknown. However, prevalence of A (H1N1) pdm09 influenza virus infection in cats in northeastern China is unknown. Therefore, the prevalence of A (H1N1) pdm09 influenza virus infections was performed among cats in northeastern China in this study. Therefore, the prevalence of A (H1N1) pdm09 influenza virus infections was performed among cats in northeastern China in this study. Additionally, in order to have a timely data for pandemic (H1N1) 2009 prevalence in northeastern China, 115 blood samples were retrospectively analyzed from pet dogs and pet cats in Harbin in 2008. Perhaps cats were at a higher probability of infection in northeastern China, due to they exposures in dense populations of humans with high influenza A (H1N1) pdm09 attack rates. Serologic evidence of pandemic influenza virus H1N1 2009 infection in cats in China

title:
  Serological report of pandemic (H1N1) 2009 infection among cats in Northeastern China in 2012-02 and 2013-03

abstract:
  BACKGROUND: Influenza A virus has a wide range of hosts. It has not only infected human, but also been reported interspecies transmission from humans to other animals, such as pigs, poultry, dogs and cats. However, prevalence of A (H1N1) pdm09 influenza virus infections in cats in northeastern China is unknown. Therefore, the prevalence of A (H1N1) pdm09 influenza virus infections was performed among cats in northeastern China in this study. FINDINGS: Of all samples in this study, the overall seroprevalence of pandemic (H1N1) 2009 infection in cats was 21% (240/1140). It also showed a higher prevalence rate of pandemic(H1N1) 2009 infection in pet cats (30.6%) than roaming cats (11%) based on NT. In addition, the results also showed a trend of difference in term of species of cats and it was statistically significant. CONCLUSIONS: This is the first survey on the seroprevalence of pandemic (H1N1) 2009 infection among cats in northeastern China. This study has observed a relatively high seroprevalence of pandemic (H1N1) 2009 among different cat populations in northeastern China, similar seroprevalence studies should be conducted elsewhere.


id: cord-355119-sdg9zdc1
author: Lin, Huixing
date: 2016-04-22
txt: ./txt/cord-355119-sdg9zdc1.txt
cache: ./cache/cord-355119-sdg9zdc1.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/s12985-016-0529-z
doi: 10.1186/s12985-016-0529-z
summary:
  In the immune protective efficiency tests, the neutralizing antibody titers in sera, the colostrum and the milk on 7th day after farrowing of the inactivated YC2014 PEDV strain immunized group were significantly higher than the inactivated CV777 immunized group and the inactivated DR13 immunized group (P < 0.05). In the present study, a PEDV strain, YC2014, was isolated from intestinal samples of suckling piglets with acute diarrhea in 2014, the evolutionary characteristics and the immune protective efficiency of YC2014 were also determined. The neutralizing antibody titer in the colostrum and the milk on 7th day after farrowing of the YC2014 PEDV strain immunized group was also significantly higher than the other three groups (P < 0.05, Fig. 4c ). In this study, we successfully isolated the YC2014 PEDV strain from porcine intestinal samples in dead piglets during outbreaks of acute diarrhea. Sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (PEDV) strains in China

title:
  Epidemic strain YC2014 of porcine epidemic diarrhea virus could provide piglets against homologous challenge

abstract:
  BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is the main causative agent of porcine epidemic diarrhea (PED). Since December 2010, a large-scale outbreak of diarrhea has been observed in swine farms in China. Accumulated evidence indicates that this large-scale outbreak of diarrhea were caused by highly virulent PEDV variants. METHODS: A PEDV strain, YC2014, was isolated from intestinal samples of suckling piglets with acute diarrhea in 2014. The complete genomic sequence of YC2014 and the nucleotide sequence of S gene were aligned with sequences of published isolates using MEGA 5.1 software. The immune protective efficiency of YC2014 were determined by testing PEDV neutralizing antibodies in sera, the colostrum and the milk on 7th day after farrowing of the immunized sows. The diarrhea symptoms of piglets after challenge were also observed. RESULTS: Phylogenetic analysis of the complete genomic sequence of YC2014 and the nucleotide sequence of S gene demonstrated that the YC2014 PEDV strain was clustered with the PEDV epidemic strains, with >99 % nucleotide identity to these PEDV strains. The S gene sequence of YC2014 shared only 93.9 % ~ 94.4 % identities with classical CV777, DR13 and JS2008 strains, with 15 nucleotide insertion in three sites and three nucleotide deletion in one site. The amino acid (AA) sequence of S gene of YC2014 shared only 92.8 % ~ 93.4 % identities with classical CV777, DR13 and JS2008 strains, with 5 AA insertion in two sites and 1 AA deletion in one site. In the immune protective efficiency tests, the neutralizing antibody titers in sera, the colostrum and the milk on 7th day after farrowing of the inactivated YC2014 PEDV strain immunized group were significantly higher than the inactivated CV777 immunized group and the inactivated DR13 immunized group (P < 0.05). The traditional inactivated PEDV vaccines made from CV777 or DR13 could not protect piglets from YC2014 challenge, while inactivated YC2014 could provide piglets with 100 % protection against YC2014 challenge. CONCLUSIONS: The results showed that, great antigenicity variation had occurred to this YC2014 PEDV strain. The YC2014 PEDV strain could provide piglets against homologous challenge. It is critical for future pathogenic and antigenic studies, as well as for the development of effective preventive and control vaccines against PEDV.


id: cord-355489-tkvfneje
title: Phylogenetic history demonstrates two different lineages of dengue type 1 virus in Colombia
author: Mendez, Jairo A
date: 2010-09-14
url: https://doi.org/10.1186/1743-422x-7-226
doi: 10.1186/1743-422x-7-226
abstract:
  BACKGROUND: Dengue Fever is one of the most important viral re-emergent diseases affecting about 50 million people around the world especially in tropical and sub-tropical countries. In Colombia, the virus was first detected in the earliest 70's when the disease became a major public health concern. Since then, all four serotypes of the virus have been reported. Although most of the huge outbreaks reported in this country have involved dengue virus serotype 1 (DENV-1), there are not studies about its origin, genetic diversity and distribution. RESULTS: We used 224 bp corresponding to the carboxyl terminus of envelope (E) gene from 74 Colombian isolates in order to reconstruct phylogenetic relationships and to estimate time divergences. Analyzed DENV-1 Colombian isolates belonged to the formerly defined genotype V. Only one virus isolate was clasified in the genotype I, likely representing a sole introduction that did not spread. The oldest strains were closely related to those detected for the first time in America in 1977 from the Caribbean and were detected for two years until their disappearance about six years later. Around 1987, a split up generated 2 lineages that have been evolving separately, although not major aminoacid changes in the analyzed region were found. CONCLUSION: DENV-1 has been circulating since 1978 in Colombia. Yet, the phylogenetic relationships between strains isolated along the covered period of time suggests that viral strains detected in some years, although belonging to the same genotype V, have different recent origins corresponding to multiple re-introduction events of viral strains that were circulating in neighbor countries. Viral strains used in the present study did not form a monophyletic group, which is evidence of a polyphyletic origin. We report the rapid spread patterns and high evolution rate of the different DENV-1 lineages.


id: cord-355906-yeaw9nr8
title: Tackling dengue fever: Current status and challenges
author: Nedjadi, Taoufik
date: 2015-12-09
txt: ./txt/cord-355906-yeaw9nr8.txt
cache: ./cache/cord-355906-yeaw9nr8.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/26645066/
doi: 10.1186/s12985-015-0444-8
summary:
  Recent advances in molecular biology have revealed that the genetic makeup of the three elements of dengue infection (the virus, the vector, and the host) plays a primordial role in the pathogenesis of the disease and could potentially contribute to the DHF progression [19, 24, 35] . Dengue virus serotype-1 antigen was expressed in a vector based on pediatric live-attenuated Schwarz measles vaccine (MV) by using the envelope domain III (EDIII) fused with the ectodomain of the membrane protein (ectoM). The Centers for Disease Control and Prevention (USA) have also developed a live-attenuated vaccine named DENVax, which was found to be highly immunogenic in both children and adults and has currently entered phase I clinical trial in the United States [96, 97] .

abstract:
  According to recent statistics, 96 million apparent dengue infections were estimated worldwide in 2010. This figure is by far greater than the WHO prediction which indicates the rapid spread of this disease posing a growing threat to the economy and a major challenge to clinicians and health care services across the globe particularly in the affected areas. This article aims at bringing to light the current epidemiological and clinical status of the dengue fever. The relationship between genetic mutations, single nucleotide polymorphism (SNP) and the pathophysiology of disease progression will be put into perspective. It will also highlight the recent advances in dengue vaccine development. Thus far, a significant progress has been made in unraveling the risk factors and understanding the molecular pathogenesis associated with the disease. However, further insights in molecular features of the disease and the development of animal models will enormously help improving the therapeutic interventions and potentially contribute to finding new preventive measures for population at risk.


id: cord-356064-q56jnhss
author: Bartel, Sebastian
date: 2011-08-01
txt: ./txt/cord-356064-q56jnhss.txt
cache: ./cache/cord-356064-q56jnhss.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/1743-422x-8-380
doi: 10.1186/1743-422x-8-380
summary:
  title: Proteome analysis of vaccinia virus IHD-W-infected HEK 293 cells with 2-dimensional gel electrophoresis and MALDI-PSD-TOF MS of on solid phase support N-terminally sulfonated peptides In this report the proteome of HEK293 cells infected with Vaccinia Virus strain IHD-W was analyzed by 2-dimensional gel electrophoresis and MALDI-PSD-TOF MS in a bottom-up approach. Derivatization of peptides with 4-sulfophenyl isothiocyanate (SPITC) carried out on ZipTipμ-C18 columns enabled protein identification via the peptides'' primary sequence, providing improved s/n ratios as well as signal intensities of the PSD spectra. The E3L gene product has been identified in HEK 293 cells infected with active VACV IHD-W, which is in correlation to proteome analysis of VACV virions that indicate that the putative double-stranded RNA binding protein (D-1) is not present in the virion but is expressed in the early replication phase. Proteome analysis of vaccinia virus IHD-W-infected HEK 293 cells with 2-dimensional gel electrophoresis and MALDI-PSD-TOF MS of on solid phase support N-terminally sulfonated peptides

title:
  Proteome analysis of vaccinia virus IHD-W-infected HEK 293 cells with 2-dimensional gel electrophoresis and MALDI-PSD-TOF MS of on solid phase support N-terminally sulfonated peptides

abstract:
  BACKGROUND: Despite the successful eradication of smallpox by the WHO-led vaccination programme, pox virus infections remain a considerable health threat. The possible use of smallpox as a bioterrorism agent as well as the continuous occurrence of zoonotic pox virus infections document the relevance to deepen the understanding for virus host interactions. Since the permissiveness of pox infections is independent of hosts surface receptors, but correlates with the ability of the virus to infiltrate the antiviral host response, it directly depends on the hosts proteome set. In this report the proteome of HEK293 cells infected with Vaccinia Virus strain IHD-W was analyzed by 2-dimensional gel electrophoresis and MALDI-PSD-TOF MS in a bottom-up approach. RESULTS: The cellular and viral proteomes of VACV IHD-W infected HEK293 cells, UV-inactivated VACV IHD-W-treated as well as non-infected cells were compared. Derivatization of peptides with 4-sulfophenyl isothiocyanate (SPITC) carried out on ZipTipμ-C18 columns enabled protein identification via the peptides' primary sequence, providing improved s/n ratios as well as signal intensities of the PSD spectra. The expression of more than 24 human proteins was modulated by the viral infection. Effects of UV-inactivated and infectious viruses on the hosts' proteome concerning energy metabolism and proteins associated with gene expression and protein-biosynthesis were quite similar. These effects might therefore be attributed to virus entry and virion proteins. However, the modulation of proteins involved in apoptosis was clearly correlated to infectious viruses. CONCLUSIONS: The proteome analysis of infected cells provides insight into apoptosis modulation, regulation of cellular gene expression and the regulation of energy metabolism. The confidence of protein identifications was clearly improved by the peptides' derivatization with SPITC on a solid phase support. Some of the identified proteins have not been described in the context of poxvirus infections before and need to be further characterised to identify their meaning for apoptosis modulation and pathogenesis.


