# ent

id: cord-102246-2lmq9s4l
type: CELL_LINE
entity:
  Vero African green monkey kidney HeLa human epitheloid cervical carcinoma CHO Chinese hamster ovary HEp 2 human larynx epidermoid carcinoma PCK primary chicken kidney cell lines


id: cord-001572-ap4ro5me
type: PROTEIN
entity:
  AdvisoryCommittees CommitteesMeetingMaterials Blood VaccinesandOtherBiologics VaccinesandRelatedBiological


id: cord-001572-ap4ro5me
type: CELL_LINE
entity:
  human acute monocytic leukemia cell line THP 1 A human coronavirus responsible for the common cold massively kills dendritic cells


id: cord-001572-ap4ro5me
type: CHEMICAL
entity:
  rubella 3 4 5 Diploid cell lines have a finite lifespan and in these cell lines the chromosomes are paired Often these cells retain many characteristics of the cell types from which they originate The disadvantage of diploid cell lines lies


id: cord-002935-jq1xumrh
type: PROTEIN
entity:
  AlexaFluor 1 594 goat anti mouse IgG heavy light chains antibody Life Technologies Carlsbad CA at 37 C


id: cord-002935-jq1xumrh
type: PROTEIN
entity:
  horseradish peroxidase HRP conjugated goat anti mouse secondary antibody SeraCare Milford MA cat 074 1802


id: cord-002935-jq1xumrh
type: ORG
entity:
  A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays


id: cord-260107-gqbtkf0x
type: PROTEIN
entity:
  N D b ND ND ND ND ND ND ND ORF1a 12 354 ND ND ND ND ND ND ND ND ORF1ab 20 345 N D ND ND ND ND ND ND ND S 4 161 21448 A A A A C C C 272 K K K K T T T 21756 C T T T T T T


id: cord-266585-jfjrk9gy
type: DNA
entity:
  nucleotides 1 5751 lane 2 5752 8693 lane 3 8694 15 520 lane 4 15 521 20 900 lane 5 and 20 901 27 608 lane 6


id: cord-266585-jfjrk9gy
type: GGP
entity:
  proteins i e spike glycoprotein S envelope protein E membrane protein M and nucleocapsid protein N The 5 two third region of mRNA 1


id: cord-271638-0wsyl7vk
type: GGP
entity:
  myricetin 1 25 2 5 5 10 μM for 1 h at 37 C before infection then after adsorption the media containing myricetin


id: cord-271638-0wsyl7vk
type: GGP
entity:
  myricetin 2 5 5 10 20 μM treatment did not significantly influence the total expression levels of EGFR PI3K


id: cord-270683-982eqtog
type: CHEMICAL
entity:
  MgCl 2 2019 nCoV_N1 forward primer 5 0 GACCCCAAAATCAGCGAAAT 3 0 2019 nCoV_N3 reverse primer 5 0 TGTAGCACG


id: cord-010369-x9z8dg6a
type: PRODUCT
entity:
  Guidelines for Plaque Reduction Neutralization Testing of Human Antibodies to Dengue Viruses West Nile


id: cord-287488-h102xn29
type: PROTEIN
entity:
  LVCM COVID 19 working group members Bruna Larotonda Telezynski Camila Araujo Valério Fabyano Bruno Leal Ralyria Mello and Vanessa Nascimento Chalup and to the Brazilian Mail Company and the MCTIC of Brazil for the logistic support in virus distribution


id: cord-287488-h102xn29
type: ORG
entity:
  Department of Cell and Developmental Biology Institute of Biomedical Sciences University of São Paulo


id: cord-309934-kcyao9i9
type: PROTEIN
entity:
  Betaferon 0 8 and 200 IU mL for Alferon and 2 and 44 IU mL for Multiferon Betaferon Alferon Multiferon Wellferon


id: cord-309934-kcyao9i9
type: PROTEIN
entity:
  Betaferon 0 1 10 IU mL At 1 000 PFU this combination did not demonstrate observable synergistic inhibitory


id: cord-313596-kc8loqyj
type: DNA
entity:
  dsu029 sha 6525c7e5a16bc1ecd8fcdfda2cc6c82fe89c85c8 doc_id 313596 cord_uid kc8loqyj Continuous cell lines


id: cord-313596-kc8loqyj
type: CELL_LINE
entity:
  WHO Vero Cell Bank A collaborative study Evolutionary conservatism in arrangement of genetic material A comparative analysis of chromosome


id: cord-330772-i7cfmw9x
type: DISEASE
entity:
  Bacillus subtilis BC1212 Publisher s Note Springer Nature remains neutral with regard to jurisdictional claims


id: cord-305496-t8ykkekl
type: ORG
entity:
  the European 676 Society of Clinical Microbiology Clinical Characteristics of Coronavirus Disease 2019


id: cord-254916-y1rw9q11
type: PROTEIN
entity:
  pro bacterial expression product rabbit polyclonal 48 nsp4 FGQ4 transmembrane replicase protein synthetic peptide rabbit polyclonal 109 nsp5 DUE5 M pro bacterial expression product rabbit polyclonal 48 nsp6 GBZ7 transmembrane replicase protein synthetic peptide rabbit polyclonal 109 nsp8 DUK4 RNA polymerase


id: cord-263178-lvxxdvas
type: PROTEIN
entity:
  rHVR I II 7 10 0 10 10 10 6 10 4 10 0 10 0 10 0 10 3 10 5 10 7 10 10 10 7 10 10 10 10 10 10 10 10 10 rHVR I III 7 10 1 10 10 10 7 9 2 9 0 9 0 9 0 10 2 10 4 10 6 10 10 10 6 10 9 9 9 9 9 9 9 9 rHVR II III 8 10 2 10 10 10 6 8 1 8 0 8 0 8 0 10 1 10 3 10 6 10 10 10 7 10 8 8 8 8 8 8 8


id: cord-263178-lvxxdvas
type: PROTEIN
entity:
  rHVR I II III 7 10 0 10 10 10 6 10 5 10 0 10 0 10 0 10 2 10 5 10 7 10 10 10 8 10 10 10 10 10 10 10 10 10 Beaudette 9 10 1 10 10 10 4 9 4 9 1 9 0 9 0 10 2 10 4 10 6 10 10 10 6 10 9 9 9 9 9 9 9 9 ck CH LDL 091022 2 5 c 0 5 5 5 3 5 2 5 0 5 0 5 0 10 5 6 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 PBS 10 10 3 10 10 10 8 9 3 7 0 7 0 7 0 10 0 10 0 10 0 10 0 10 3 10 4 9 5 7 7 7 7 7 a


id: cord-297531-et1sli23
type: PROTEIN
entity:
  secondary antibody fluorescein isothiocyanate FITC conjugated goat anti mouse IgG Abcam Hangzhou China The


id: cord-298922-k568hlf4
type: PROTEIN
entity:
  polyclonal antibodies anti tubulin anti integrin 3 anti cystatin C anti protein S100 A2 anti apolipoprotein E4


id: cord-275863-qos9vu3r
type: DNA
entity:
  jir173 sha 3062cebd70447841d68c48fe83f0eb51b07a761d doc_id 275863 cord_uid qos9vu3r Dengue virus receptors


id: cord-275863-qos9vu3r
type: PROTEIN
entity:
  glycan antigen Lewis x A dendritic cell specific intercellular adhesion molecule 3 grabbing nonintegrin DC SIGN related protein


id: cord-332276-gs80celr
type: PROTEIN
entity:
  NF kB nuclear factor kappa B COX 2 cyclooxygenase 2 JNK c Jun N terminal kinase p38 MAPK p38 mitogen activated protein kinase ERK extracellular signal regulated kinase AP 1 activator protein 1


id: cord-332276-gs80celr
type: GGP
entity:
  kappa B COX 2 cyclooxygenase 2 JNK c Jun N terminal kinase p38 MAPK p38 mitogen activated protein kinase ERK extracellular


id: cord-331094-22366b81
type: PROTEIN
entity:
  EPI_ISL_450352 hCoV 19 Norway Trondheim E10 2020 EPI_ISL_450351 hCoV 19 Norway Trondheim E9 2020 EPI_ISL_450350 hCoV 19 Norway Trondheim S15 2020


id: cord-331094-22366b81
type: WORK_OF_ART
entity:
  the Treatment of Influenza Viral Infection Reviving Old Drugs to Fight Against a Long Lived Enemy Drug


id: cord-331094-22366b81
type: ORG
entity:
  High Throughput Screening and Identification of Potent Broad Spectrum Inhibitors of Coronaviruses Retrograde


id: cord-331094-22366b81
type: DISEASE
entity:
  herpes simplex virus 2 HSV 2 echovirus 1 EV1 human metapneumovirus HMPV RVFV and lymphocytic choriomeningitis virus LCMV


id: cord-288644-ywaefpe8
type: DISEASE
entity:
  Zsolt Daniel Noguera Julian Marc Paredes Roger Mateu Lourdes Quiñones Carles Erkizia Itziar Blanco Ignacio Valencia Alfonso Guallar Víctor Carrillo Jorge Blanco Julià Segalés


id: cord-288644-ywaefpe8
type: CHEMICAL
entity:
  plitidepsin cathepsin inhibitors nelfinavir mesylate hydrate interferon 2 alpha interferon gamma fenofibrate


id: cord-295559-yc8q62z8
type: DISEASE
entity:
  acute respiratory syndrome coronavirus Severe acute respiratory syndrome coronavirus SARS CoV infection


id: cord-289248-6mx4o0eb
type: DNA
entity:
  GeneArt High Order Genetic Assembly System The traditional method for assembly of an infectious cDNA


id: cord-289248-6mx4o0eb
type: DNA
entity:
  GeneArt Seamless Cloning and Assembly Kit Invitrogen Fig 1A creating plasmid p107109 MV The primer sequences


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


id: cord-312899-ot5pvtbl
type: CHEMICAL
entity:
  acyclovir ganciclovir cidofovir foscarnet interferon alpha 2a interferon alpha 2b amantadine zidovudine stavudine nevirapine abacavir


id: cord-329494-cdn52epy
type: CHEMICAL
entity:
  oligonucleotides Z F 5 GGATCCATGGGCAACTGCAACGGGGCATC 3 and Z trunc R 5 AAAAGCGGCCGCCTGGTGGTGGTGCTGTTGGCT 3 containing a BamHI


id: cord-283309-ovx5fzsg
type: DNA
entity:
  Spike S ORF3 Envelope E Membrane M Nucleocapsid N NS7a NS7b 3 SeACoV shared 95 nucleotide nt sequence


id: cord-323839-a4oejky0
type: DNA
entity:
  ATCAGACTCAGACTAATTCTCCTCGGCGGGCACATAGTGTAGCTAGTCAATCCATCATTGC Multiple nucleotide sequence alignment


id: cord-323839-a4oejky0
type: LAW
entity:
  ECDIPIGAGICASYQTQTNSPPRARSVASQSIIAYTMSLGAENSVAYSNNS ECDIPIGAGICASYQTQT SQSIIAYTMSLGAENSVAYSNNS SARS CoV


id: cord-316908-8ti75mru
type: PROTEIN
entity:
  dynamin II siDyn Sus scrofa 5 CAC CTC ATG ATC AAT AAC A 3 Chlorocebus sabaeus 5 CCT ACA TCA ACA CGA ACC A 3 clathrin heavy chain siCHC Sus scrofa 5 CCC ATA CCA TGA CTG ATG A 3 Chlorocebus sabaeus 5 GAT GAA CCT TAT GCA TGC A 3 EPS 15 siEPS15 Sus scrofa 5 CCT GTG GAT ATT CTT GGA A 3 Chlorocebus sabaeus 5


id: cord-316908-8ti75mru
type: GGP
entity:
  dynamin II siDyn Sus scrofa 5 CAC CTC ATG ATC AAT AAC A 3 Chlorocebus sabaeus 5 CCT ACA TCA ACA CGA ACC A 3 clathrin


# bib

id: cord-001572-ap4ro5me
title: Hematopoietic Cancer Cell Lines Can Support Replication of Sabin Poliovirus Type 1
author: Oosterhoff, Dinja
date: 2015-02-28
txt: ./txt/cord-001572-ap4ro5me.txt
cache: ./cache/cord-001572-ap4ro5me.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4359862/
doi: 10.1155/2015/358462
summary:
  To determine replication kinetics, the susceptible tumor cell lines were infected with Sabin poliovirus type 1 from the parental virus or virus that was passaged for 5 times on the hematopoietic cell lines at MOI 1 or MOI 0.01, and samples of the supernatant and cellular lysates were harvested at different time points. To determine whether hematopoietic cell lines can support replication of Sabin poliovirus type 1, cells were infected with an MOI of 1 and cells together with supernatant were harvested at day 3 (for all virus passages in Vero cells and for passages 3-5 on U937 cells) or day 6 after infection. In the supernatant of all cell lines tested, at day 4, a high virus titer, comparable to Sabin poliovirus type 1 replicated in Vero cells, was observed in the culture medium, indicating that virus replication was efficient during multiple rounds of replication.

abstract:
  Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus.


id: cord-002935-jq1xumrh
title: Testing therapeutics in cell-based assays: Factors that influence the apparent potency of drugs
author: Postnikova, Elena
date: 2018-03-22
txt: ./txt/cord-002935-jq1xumrh.txt
cache: ./cache/cord-002935-jq1xumrh.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5864066/
doi: 10.1371/journal.pone.0194880
summary:
  Here, we describe variable conditions tested during the development of our cell-based drug screen assays designed to identify compounds with anti-Ebola virus activity using established cell lines and human primary cells. The effect of multiple assay readouts and variable assay conditions, including virus input, time of infection, and the cell passage number, were compared, and the impact on the effective concentration for 50% and/ or 90% inhibition (EC(50), EC(90)) was evaluated using the FDA-approved compound, toremifene citrate. The CELIA was compared to the fluorescent assay (detected by regular plate reader or the HCI system) by testing the efficacy of toremifene citrate against EBOV infection in Huh 7 cells with a range of MOIs (0.1, 0.3, 1, and 3 ) at three different time points (24, 48 and 72 hpi) . The established CELIA was used to compare anti-EBOV activity of toremifene citrate in 3 different cell types, Vero E6, Huh 7 and MDMs using an MOI of 0.5 and a time point of 48 h (Fig 7) .

abstract:
  Identifying effective antivirals for treating Ebola virus disease (EVD) and minimizing transmission of such disease is critical. A variety of cell-based assays have been developed for evaluating compounds for activity against Ebola virus. However, very few reports discuss the variable assay conditions that can affect the results obtained from these drug screens. Here, we describe variable conditions tested during the development of our cell-based drug screen assays designed to identify compounds with anti-Ebola virus activity using established cell lines and human primary cells. The effect of multiple assay readouts and variable assay conditions, including virus input, time of infection, and the cell passage number, were compared, and the impact on the effective concentration for 50% and/ or 90% inhibition (EC(50), EC(90)) was evaluated using the FDA-approved compound, toremifene citrate. In these studies, we show that altering cell-based assay conditions can have an impact on apparent drug potency as measured by the EC(50). These results further support the importance of developing standard operating procedures for generating reliable and reproducible in vitro data sets for potential antivirals.


id: cord-003284-hjx2d5rq
author: Márquez-Jurado, Silvia
date: 2018-10-07
txt: ./txt/cord-003284-hjx2d5rq.txt
cache: ./cache/cord-003284-hjx2d5rq.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6212934/
doi: 10.3390/v10100547
summary:
  Furthermore, using this infectious clone we have generated a mutant ZIKV containing a single amino acid substitution (A175V) in the NS2A protein that presented reduced viral RNA synthesis in cell cultures, was highly attenuated in vivo and induced fully protection against a lethal challenge with ZIKV wild-type. To analyze the genetic stability of the recombinant ZIKV harboring the point mutation A175V in the coding region of the NS2A protein (rZIKV-RGN-mNS2A), total RNA was purified from Vero cells infected with viruses from passage 1 (P1) to passage 5 (P5) using the RNeasy minikit (Qiagen), according to the manufacturer''s specifications. To investigate whether the reduced RNA synthesis of rZIKV-RGN-mNS2A in Vero cells could result in viral attenuation in vivo, the ability of the mutant virus to induce pathogenesis was analyzed in A129 mice and compared with that of the parental rZIKV-RGN ( Figure 6 ).

title:
  An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo

abstract:
  The recent outbreaks of Zika virus (ZIKV), its association with Guillain–Barré syndrome and fetal abnormalities, and the lack of approved vaccines and antivirals, highlight the importance of developing countermeasures to combat ZIKV disease. In this respect, infectious clones constitute excellent tools to accomplish these goals. However, flavivirus infectious clones are often difficult to work with due to the toxicity of some flavivirus sequences in bacteria. To bypass this problem, several alternative approaches have been applied for the generation of ZIKV clones including, among others, in vitro ligation, insertions of introns and using infectious subgenomic amplicons. Here, we report a simple and novel DNA-launched approach based on the use of a bacterial artificial chromosome (BAC) to generate a cDNA clone of Rio Grande do Norte Natal ZIKV strain. The sequence was identified from the brain tissue of an aborted fetus with microcephaly. The BAC clone was fully stable in bacteria and the infectious virus was efficiently recovered in Vero cells through direct delivery of the cDNA clone. The rescued virus yielded high titers in Vero cells and was pathogenic in a validated mouse model (A129 mice) of ZIKV infection. Furthermore, using this infectious clone we have generated a mutant ZIKV containing a single amino acid substitution (A175V) in the NS2A protein that presented reduced viral RNA synthesis in cell cultures, was highly attenuated in vivo and induced fully protection against a lethal challenge with ZIKV wild-type. This BAC approach provides a stable and reliable reverse genetic system for ZIKV that will help to identify viral determinants of virulence and facilitate the development of vaccine and therapeutic strategies.


id: cord-004825-cdvnqfjz
title: The entry of Junin virus into Vero cells
author: Castilla, V.
date: 1994
txt: ./txt/cord-004825-cdvnqfjz.txt
cache: ./cache/cord-004825-cdvnqfjz.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7087180/
doi: 10.1007/bf01321064
summary:
  The entry mechanism of Junin virus (JV) into Vero cells was studied analyzing the effect of lysosomotropic compounds and acid pH on JV infection. Vero cells grown in coverslips were infected with JV (moi 1) and 15 mM ammonium chloride was added to the culture medium after adsorption. To reinforce that the membrane fusion activity of Junin virus is expressed at low pH, the formation of JV-induced syncytia in infected Vero cells incubated in medium at pH 5.5 or 7.5 was quantitated. In fact, a bypass of the ammonium chloride block of JV infection was achieved when the extracellular medium was at a pH below 6.1 (Fig. 5) , suggesting that the acidic conditions would probably trigger direct fusion of virus envelope with the cell membrane.

abstract:
  The entry mechanism of Junin virus (JV) into Vero cells was studied analyzing the effect of lysosomotropic compounds and acid pH on JV infection. Ammonium chloride, amantadine, chlorpheniramine and procaine inhibited JV production. The action of ammonium chloride was exerted at early times of infection. Virus internalization was inhibited and viral protein expression was not detected. When the extracellular medium was buffered at low pH, the ammonium chloride induced block on JV infection was overcome. Furthermore, JV was able to induce fusion of infected cells at pH 5.5 leading to polykaryoctye formation. Taken together, these results demonstrate that JV entry occurs through an endocytic mechanism requiring a low pH dependent membrane fusion.


id: cord-010369-x9z8dg6a
author: Saito, Kyoko
date: 2020-04-24
txt: ./txt/cord-010369-x9z8dg6a.txt
cache: ./cache/cord-010369-x9z8dg6a.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7182267/
doi: 10.1371/journal.pone.0232274
summary:
  Quantification of cellular and extracellular viral RNA revealed that high JEV production in Huh7.5.1–8 cells can be attributed to rapid viral replication kinetics and efficient virus release early in infection. Huh7.5.1-8 and Vero cells under high and low confluency were infected with JEV at MOI 0.1, and then the virus titer in culture supernatant was monitored from 1 to 4 d pi (Fig 2) . The culture supernatant of Huh7.5.1-8 cells exhibited a higher relative virus titer than that of Vero cells, particularly during the early infection times. These results suggested that the Huh7.5.1-8 cell line, compared with the Vero cell line, has a higher virus productivity and susceptibility to virus-induced cell death upon flavivirus infection. First, Huh7.5.1-8 cells produced higher amounts of infectious JEV and YFV than Vero cells early in infection (Figs 2 and 8A-8D ).

title:
  Comparative characterization of flavivirus production in two cell lines: Human hepatoma-derived Huh7.5.1-8 and African green monkey kidney-derived Vero

abstract:
  The Flaviviridae is a family of enveloped viruses with a positive-sense single-stranded RNA genome. It contains many viruses that threaten human health, such as Japanese encephalitis virus (JEV) and yellow fever virus (YFV) of the genus Flavivirus as well as hepatitis C virus of the genus Hepacivirus. Cell culture systems highly permissive for the Flaviviridae viruses are very useful for their isolation, propagation, and diagnosis, an understanding of their biology, and the development of vaccines and antiviral agents. Previously, we isolated a human hepatoma HuH-7-derived cell clone, Huh7.5.1–8, which is highly permissive to hepatitis C virus infection. Here, we have characterized flavivirus infection in the Huh7.5.1–8 cell line by comparing with that in the African green monkey kidney-derived Vero cell line, which is permissive for a wide spectrum of viruses. Upon infection with JEV, Huh7.5.1–8 cells produced a higher amount of virus particles early in infection and were more susceptible to virus-induced cell death than Vero cells. Similar outcomes were obtained when the cells were infected with another flavivirus, YFV (17D-204 strain). Quantification of cellular and extracellular viral RNA revealed that high JEV production in Huh7.5.1–8 cells can be attributed to rapid viral replication kinetics and efficient virus release early in infection. In a plaque assay, Huh7.5.1–8 cells developed JEV plaques more rapidly than Vero cells. Although this was not the case with YFV plaques, Huh7.5.1–8 cells developed higher numbers of YFV plaques than Vero cells. Sequence analysis of cDNA encoding an antiviral RNA helicase, RIG-I, showed that Huh7.5.1–8 cells expressed not only a full-length RIG-I mRNA with a known dominant-negative missense mutation but also variants without the mutation. However, the latter mRNAs lacked exon 5/6−12, indicating functional loss of RIG-I in the cells. These characteristics of the Huh7.5.1–8 cell line are helpful for flavivirus detection, titration, and propagation.


id: cord-023871-9vi0m378
title: Signaling Pathways of SARS-CoV In Vitro and In Vivo
author: Mizutani, Tetsuya
date: 2009-07-22
txt: ./txt/cord-023871-9vi0m378.txt
cache: ./cache/cord-023871-9vi0m378.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176218/
doi: 10.1007/978-3-642-03683-5_19
summary:
  AKT and JNK (Jun NH(2)-terminal kinase) signaling pathways are important to establish persistent infection of SARS-CoV in Vero E6 cells. SARS-CoV S protein expression induces release of interleukin-8 (IL-8) via ERK and p38 MAPK signaling pathways including activator protein 1 (AP-1) in A549 cells ). SARS-CoV infection induces apoptotic cell death in Vero E6 cells, via dephosphorylation of STAT3 by p38 MAPK activation, and inactivation of Akt, as previously described. Overexpression of SARS-CoV 3a protein in Vero E6 cells induces apoptosis, mediated through a caspase-8-dependent pathway or p38 MAPK Waye et al. Overexpression of severe acute respiratory syndrome coronavirus 3b protein induces both apoptosis and necrosis in Vero E6 cells The 3a protein of severe acute respiratory syndrome-associated coronavirus induces apoptosis in Vero E6 cells Microarray and real-time RT-PCR analyses of differential human gene expression patterns induced by severe acute respiratory syndrome (SARS) coronavirus infection of Vero cells

abstract:
  Severe acute respiratory syndrome (SARS) is a respiratory illness with variable symptoms that was recognized as the first near-pandemic infectious disease of the twenty-first century. A novel human coronavirus, named SARS coronavirus (SARS-CoV), derived from SARS patients was reported as the etiologic agent of SARS. Studying the signaling pathways of SARS-infected cells is key to understanding the molecular mechanism of SARS viral infection. Cell death is observed in cultured Vero E6 cells after SARS-CoV infection. From SARS-CoV infection to cell death, p38 mitogen-activated protein kinase (MAPK) is a key participant in the determination of cell death and survival. Two signaling pathways comprising signal transducer and activator of transcription 3 (STAT3) and p90 ribosomal S6 kinase (p90RSK) are downstream of p38 MAPK. AKT and JNK (Jun NH(2)-terminal kinase) signaling pathways are important to establish persistent infection of SARS-CoV in Vero E6 cells. Expression studies of SARS-CoV proteins indicate that the viral proteins are able to activate signaling pathways of host cells. The study of signaling pathways in SARS-CoV patients is difficult to perform compared with in vitro studies due to the effects of the human immune system. This review highlights recent progress in characterizing signal transduction pathways in SARS-CoV-infected cells in vitro and in vivo.


id: cord-102246-2lmq9s4l
author: Salvadori, Marcia R.
date: 2001-04-01
txt: ./txt/cord-102246-2lmq9s4l.txt
cache: ./cache/cord-102246-2lmq9s4l.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0378109701000908
doi: 10.1016/s0378-1097(01)00090-8
summary:
  title: Morphological and intracellular alterations induced by cytotoxin VT2y produced by Escherichia coli isolated from chickens with swollen head syndrome Abstract Recently, a novel verocytotoxin named VT2y was described which belongs to the STx family and is produced by Escherichia coli isolated from domestic poultry with swollen head syndrome (SHS). The VT2y toxin induced apoptosis in Vero, HeLa, CHO, CEF (primary chicken embryo fibroblast) and PCK (primary chicken kidney) cell lines. The aim of this study is to illustrate the morphological and intracellular alterations induced in vitro by cytotoxin VT2y in Vero, HeLa, CHO, HEp-2, PCK and CEF cell lines. The cytotoxin VT2y caused morphological alterations in Vero cells, such as cytoplasm vacuolization, blebbing of the plasma membrane, chromatin condensation within the nucleus, nuclear shrinkage and apoptotic bodies within 15 min. Escherichia coli strains isolated from pigs with edema disease produce a variant of Shiga-like toxin II Cytotoxin produced by Escherichia coli isolated from chickens with swollen head syndrome (SHS)

title:
  Morphological and intracellular alterations induced by cytotoxin VT2y produced by Escherichia coli isolated from chickens with swollen head syndrome

abstract:
  Abstract Recently, a novel verocytotoxin named VT2y was described which belongs to the STx family and is produced by Escherichia coli isolated from domestic poultry with swollen head syndrome (SHS). The VT2y toxin induced apoptosis in Vero, HeLa, CHO, CEF (primary chicken embryo fibroblast) and PCK (primary chicken kidney) cell lines. Morphological evidence (nuclear shrinkage, chromatin condensation and blebbing of the plasma membrane) of apoptosis could be distinguished in 15 min and was maximal at 1 h after treatment with VT2y. This was confirmed by the terminal dUTP nick-end-labeling (TUNEL) method.


id: cord-253616-7jyui5ca
title: Harringtonine Inhibits Zika Virus Infection through Multiple Mechanisms
author: Lai, Zheng-Zong
date: 2020-09-07
txt: ./txt/cord-253616-7jyui5ca.txt
cache: ./cache/cord-253616-7jyui5ca.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32906689/
doi: 10.3390/molecules25184082
summary:
  To investigate the anti-ZIKV activity of harringtonine, we assessed the inhibition of virus infection in Vero cells with MOI = 0.01 under different concentrations of harringtonine for 48 h. Intracellular viral RNA levels, protein expression levels and virus progeny in supernatants were respectively determined by RT-qPCR, western blotting and fluorescent focus assay (FFA). The dose-dependent anti-ZIKV activities of harringtonine were observed to decrease viral RNA/protein production and progeny yield ( Figure 1B-D) , indicating that virus propagation was suppressed. Harringtonine (625 nM) was administered at different stages of ZIKV infection with MOI = 0.1, and then the levels of ZIKV RNA in cells, as well as virus titers in supernatants, were determined after 24 h treatment. Harringtonine (625 nM) was administered at different stages of ZIKV infection with MOI = 0.1, and then the levels of ZIKV RNA in cells, as well as virus titers in supernatants, were determined after 24 h treatment.

abstract:
  Mosquito-borne Zika virus (ZIKV) is a Flavivirus that came under intense study from 2014 to 2016 for its well-known ability to cause congenital microcephaly in fetuses and neurological Guillain–Barré disease in adults. Substantial research on screening antiviral agents against ZIKV and preventing ZIKV infection are globally underway, but Food and Drug Administration (FDA)-approved treatments are not available yet. Compounds from Chinese medicinal herbs may offer an opportunity for potential therapies for anti-ZIKV infection. In this study, we evaluated the antiviral efficacy of harringtonine against ZIKV. Harringtonine possessed anti-ZIKV properties against the binding, entry, replication, and release stage through the virus life cycle. In addition, harringtonine have strong virucidal effects in ZIKV and exhibited prophylaxis antiviral ability prior ZIKV infection. The antiviral activity also observed in the treatment against Japanese encephalitis reporter virus (RP9-GFP strain). Overall, this study demonstrated that harringtonine would be a favorable potential candidate for the development of anti-ZIKV infection therapies.


id: cord-254317-n2knqj4z
title: The enhanced replication of an S-intact PEDV during coinfection with an S1 NTD-del PEDV in piglets
author: Su, Yunfang
date: 2018-11-27
txt: ./txt/cord-254317-n2knqj4z.txt
cache: ./cache/cord-254317-n2knqj4z.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/30593369/
doi: 10.1016/j.vetmic.2018.11.025
summary:
  Porcine epidemic diarrhea virus (PEDV) variants having a large deletion in the N-terminal domain of the S1 subunit of spike (S) protein were designated as S1 NTD-del PEDVs. They replicate well in experimentally infected pigs. Effect of mucin, bile and bile acids on the infection of PEDV icPC22A and icPC22A-S1Δ197 in Vero and IPEC-DQ cells Viruses (icPC22A or icPC22A-S1Δ197) were mixed with different concentrations of BM (0, 0.1, 0.3, 0.5 mg/mL) or PGM (0, 0.5, 1.0, 2.5, 5.0 mg/mL). Compared with the peak fecal PEDV N gene shedding titer (11.6 ± 0.2 log 10 copies/mL) of piglets in the icPC22A group (1 dpi), pigs in the coinfection group had a significantly higher peak titer (13.6 ± 0.7 log 10 copies/mL) ( Fig. 1B and Table 2 ) at a delayed time point (1.5 dpi). S1 NTD-del PEDV replicated to a lower peak titer in coinfection than that in single virus infection in both Vero cells and IPEC-DQ cells.

abstract:
  Porcine epidemic diarrhea virus (PEDV) variants having a large deletion in the N-terminal domain of the S1 subunit of spike (S) protein were designated as S1 NTD-del PEDVs. They replicate well in experimentally infected pigs. However, on farms they often co-infect pigs with the PEDV containing an intact S protein (S-intact PEDV). We aimed to characterize viral replication and pathogenesis in neonatal gnotobiotic pigs infected simultaneously with the two types of PEDV using two recombinant PEDVs: icPC22A and its S1 NTD-del form icPC22A-S1Δ197. Additionally, viral replication was compared in Vero and IPEC-DQ cells at the presence of bovine mucin (BM), porcine gastric mucin (PGM), swine bile and bile acids during inoculation. In the pigs coinfected with icPC22A and icPC22A-S1Δ197, icPC22A replicated to a higher peak titer than its infection of pigs without the presence of icPC22A-S1Δ197. The severity of diarrhea and intestinal atrophy were similar between icPC22A and the coinfection groups, but were significantly higher than icPC22A-S1Δ197 group. In Vero and IPEC-DQ cells, certain concentrations of BM, PGM, bile and bile acids increased significantly the infectivity of icPC22A but had no or negative effects on icPC22A-S1Δ197. These results indicated that the replication of the S-intact PEDV was enhanced during coinfection in piglets. This observation may be explained partially by the fact that mucin, bile and bile acids in gastrointestinal tract had facilitating effects on the infection of S-intact PEDV, but no/inhibition effects on S1 NTD-del PEDV.


id: cord-254916-y1rw9q11
author: Ogando, Natacha S.
date: 2020-06-22
txt: ./txt/cord-254916-y1rw9q11.txt
cache: ./cache/cord-254916-y1rw9q11.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32568027/
doi: 10.1099/jgv.0.001453
summary:
  The overall level of amino acid sequence identity of viral proteins ranges from about 65 % in the least conserved parts of the S protein to about 95 % in the most conserved replicative enzyme domains, prompting the coronavirus study group of the International Committee on the Taxonomy of Viruses to classify the new agent within the species Severe acute respiratory syndrome-related coronavirus, which also includes the 2003 SARS-CoV [1] . In this report, we describe a comparative study of the basic replication features of SARS-CoV and SARS-CoV-2 in Vero E6 cells, including growth kinetics, virus titres, plaque phenotype and an analysis of intracellular viral RNA and protein synthesis. One of them is the rapid evolution -during virus passaging in Vero cells -of a specific region of the SARS-CoV-2 S protein that contains the so-called furin-like cleavage site.

title:
  SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology

abstract:
  The sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread highlight the importance of understanding the full molecular details of coronavirus infection and pathogenesis. Here, we compared a variety of replication features of SARS-CoV-2 and SARS-CoV and analysed the cytopathology caused by the two closely related viruses in the commonly used Vero E6 cell line. Compared to SARS-CoV, SARS-CoV-2 generated higher levels of intracellular viral RNA, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium. Immunofluorescence microscopy of SARS-CoV-2-infected cells established extensive cross-reactivity of antisera previously raised against a variety of non-structural proteins, membrane and nucleocapsid protein of SARS-CoV. Electron microscopy revealed that the ultrastructural changes induced by the two SARS viruses are very similar and occur within comparable time frames after infection. Furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (remdesivir, alisporivir and chloroquine) is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha. An important difference between the two viruses is the fact that – upon passaging in Vero E6 cells – SARS-CoV-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene. These mutations change or delete a putative furin-like cleavage site in the region connecting the S1 and S2 domains and result in a very prominent phenotypic change in plaque assays.


id: cord-256370-cz88t29n
title: Isolation of a Novel Fusogenic Orthoreovirus from Eucampsipoda africana Bat Flies in South Africa
author: Jansen van Vuren, Petrus
date: 2016-02-29
txt: ./txt/cord-256370-cz88t29n.txt
cache: ./cache/cord-256370-cz88t29n.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.3390/v8030065
doi: 10.3390/v8030065
summary:
  This is the first report on isolation of an orthoreovirus from an arthropod host associated with bats, and phylogenetic and sequence data suggests that MAHLV constitutes a new species within the Orthoreovirus genus. Maximum Likelihood trees were prepared using amino acid sequences of all open reading frames from all segments, showing the placement of Mahlapitsi virus (MAHLV) in the Orthoreovirus genus relative to other viruses in this genus for which sequence is available on Genbank. A Maximum Likelihood tree, constructed with nucleic acid sequence data for the RNA-dependent RNA polymerase (RdRp) encoding segments of representative viruses from the different genera within Reoviridae (Figure 7) shows the placement of both isolates amongst other orthoreoviruses in the family. Maximum Likelihood trees were prepared using the deduced amino acid sequences from the open reading frames (ORF''s) of all the virus'' segments and those of other viruses in the Orthoreovirus genus (Figures 8-10) .

abstract:
  We report on the isolation of a novel fusogenic orthoreovirus from bat flies (Eucampsipoda africana) associated with Egyptian fruit bats (Rousettus aegyptiacus) collected in South Africa. Complete sequences of the ten dsRNA genome segments of the virus, tentatively named Mahlapitsi virus (MAHLV), were determined. Phylogenetic analysis places this virus into a distinct clade with Baboon orthoreovirus, Bush viper reovirus and the bat-associated Broome virus. All genome segments of MAHLV contain a 5' terminal sequence (5'-GGUCA) that is unique to all currently described viruses of the genus. The smallest genome segment is bicistronic encoding for a 14 kDa protein similar to p14 membrane fusion protein of Bush viper reovirus and an 18 kDa protein similar to p16 non-structural protein of Baboon orthoreovirus. This is the first report on isolation of an orthoreovirus from an arthropod host associated with bats, and phylogenetic and sequence data suggests that MAHLV constitutes a new species within the Orthoreovirus genus.


id: cord-260107-gqbtkf0x
title: Isolation and characterization of a Korean porcine epidemic diarrhea virus strain KNU-141112
author: Lee, Sunhee
date: 2015-10-02
txt: ./txt/cord-260107-gqbtkf0x.txt
cache: ./cache/cord-260107-gqbtkf0x.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0168170215300149
doi: 10.1016/j.virusres.2015.07.010
summary:
  In the present study, one Korean PEDV strain, KOR/KNU-141112/2014, was successfully isolated and serially propagated in Vero cells for over 30 passages. Our genomic analyses indicated that the Korean isolate KNU-141112 is genetically stable during the first 30 passages in cell culture and is grouped within subgroup G2b together with the recent re-emergent Korean strains. Our data indicated that KNU-141112 isolate is relatively stable during the first 30 passages in cell culture and is classified into subgroup G2b that includes PEDV strains responsible for recent severe outbreaks in Korea and the US. Although virus isolation in cell culture from clinical samples of naturally or experimentally infected pigs is fastidious, recent studies reported the successful isolation and propagation of several US original PEDV strains using Vero cells (Chen et al., 2014; Oka et al., 2014) . Cell culture isolation and sequence analysis of genetically diverse US porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene

abstract:
  Severe outbreaks of porcine epidemic diarrhea virus (PEDV) have re-emerged in Korea and rapidly swept across the country, causing tremendous economic losses to producers and customers. Despite the availability of PEDV vaccines in the domestic market, the disease continues to plague the Korean pork industry, raising issues regarding their protective efficacy and new vaccine development. Therefore, PEDV isolation in cell culture is urgently needed to develop efficacious vaccines and diagnostic assays and to conduct further studies on the virus biology. In the present study, one Korean PEDV strain, KOR/KNU-141112/2014, was successfully isolated and serially propagated in Vero cells for over 30 passages. The in vitro and in vivo characteristics of the Korean PEDV isolate were investigated. Virus production in cell culture was confirmed by cytopathology, immunofluorescence, and real-time RT-PCR. The infectious virus titers of the viruses during the first 30 passages ranged from 10(5.1) to 10(8.2) TCID(50) per ml. The inactivated KNU-141112 virus was found to mediate potent neutralizing antibody responses in immunized guinea pigs. Animal studies showed that KNU-141112 virus causes severe diarrhea and vomiting, fecal shedding, and acute atrophic enteritis, indicating that strain KNU-141112 is highly enteropathogenic in the natural host. In addition, the entire genomes or complete S genes of KNU-141112 viruses at selected cell culture passages were sequenced to assess the genetic stability and relatedness. Our genomic analyses indicated that the Korean isolate KNU-141112 is genetically stable during the first 30 passages in cell culture and is grouped within subgroup G2b together with the recent re-emergent Korean strains.


id: cord-263178-lvxxdvas
author: Shan, Dan
date: 2018-05-02
txt: ./txt/cord-263178-lvxxdvas.txt
cache: ./cache/cord-263178-lvxxdvas.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0168170217309103
doi: 10.1016/j.virusres.2018.04.013
summary:
  To study the roles of hypervariable regions (HVRs) in receptor-binding subunit S1 of the spike protein, we manipulated the genome of the IBV Beaudette strain using a reverse genetics system to construct seven recombinant strains by separately or simultaneously replacing the three HVRs of the Beaudette strain with the corresponding fragments from a QX-like nephropathogenic isolate ck/CH/LDL/091022 from China. We could not detect the replication with ck/CH/ LDL/091022 in Vero cells, so the neutralization tests were performed in 9-day-old SPF embryonated eggs to confirm whether the serotypes of the recombinant IBVs belonged to ck/CH/LDL/091022. Viral antigen was observed in Vero cells infected with the Beaudette strain and the seven recombinant IBVs (Fig. 1b) . S1 gene sequencing results confirmed that the heterogenous HVRs were stably maintained in the recombinant IBVs (Sup Fig. 1) , and no additional mutations were detected in the S protein after three passages in cells or eggs.

title:
  Effects of hypervariable regions in spike protein on pathogenicity, tropism, and serotypes of infectious bronchitis virus

abstract:
  To study the roles of hypervariable regions (HVRs) in receptor-binding subunit S1 of the spike protein, we manipulated the genome of the IBV Beaudette strain using a reverse genetics system to construct seven recombinant strains by separately or simultaneously replacing the three HVRs of the Beaudette strain with the corresponding fragments from a QX-like nephropathogenic isolate ck/CH/LDL/091022 from China. We characterized the growth properties of these recombinant IBVs in Vero cells and embryonated eggs, and their pathogenicity, tropism, and serotypes in specific pathogen-free (SPF) chickens. All seven recombinant IBVs proliferated in Vero cells, but the heterogenous HVRs could reduce their capacity for adsorption during in vitro infection. The recombinant IBVs did not significantly increase the pathogenicity compared with the Beaudette strain in SPF chickens, and they still shared the same serotype as the Beaudette strain, but the antigenic relatedness values between the recombinant strain and Beaudette strain generally decreased with the increase in the number of the HVRs exchanged. The results of this study demonstrate the functions of HVRs and they may help to develop a vaccine candidate, as well as providing insights into the prevention and control of IBV.


id: cord-263439-oquk4t96
title: Clathrin- and serine proteases-dependent uptake of porcine epidemic diarrhea virus into Vero cells
author: Park, Jung-Eun
date: 2014-10-13
txt: ./txt/cord-263439-oquk4t96.txt
cache: ./cache/cord-263439-oquk4t96.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0168170214003001
doi: 10.1016/j.virusres.2014.07.022
summary:
  Similar to other coronaviruses, PEDV spike protein mediates its cell entry by binding to cellular receptors and inducing membrane fusion between viral envelopes and cellular membranes. Taken together, our findings reveal that PEDV enters Vero cells via clathrin-mediated endocytosis and requires serine proteolysis during infection. Based on these observations, we concluded that an exogenous protease, like trypsin, was necessary to induce cell-cell fusion in PEDV-infected Vero cells but not essentially required for virus-cell entry. So, we hypothesized that PEDV entry into Vero cells under the trypsin-free condition most likely occurred inside endosomal compartments where cellular proteases might operate similar to trypsin, facilitating S-mediated fusion of PEDV with the endosomal membrane. The infection inhibition assay using various substrates that interfere with endocytosis or lysosomotropic agents revealed that PEDV enters Vero cells via clathrin-mediated endocytic uptake and delivery of virus to an acidic intracellular compartment.

abstract:
  Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus, is a causative agent of porcine enteric disease characterized by acute watery diarrhea and dehydration in sucking piglet. Similar to other coronaviruses, PEDV spike protein mediates its cell entry by binding to cellular receptors and inducing membrane fusion between viral envelopes and cellular membranes. However, the entry mechanism of PEDV is not studied. Here, we determined the entry mechanism of PEDV into Vero cells. Our data confirmed that PEDV entry followed clathrin-mediated endocytosis independence of caveolae-coated pit assembly. The internalized PEDV was co-localized with the clathrin-mediated endocytic marker, but not with the caveolae-mediated endocytic marker. In addition, cells treated with lysosomotropic agents and serine protease inhibitors were resistant to PEDV. Our data revealed that PEDV entry followed clathrin-mediated endocytosis and was dependent on a low pH and serine proteolysis for successful entry into cells.


id: cord-265263-r9e6bop3
title: Vaginal Lactobacillusgasseri CMUL57 can inhibit herpes simplex type 2 but not Coxsackievirus B4E2
author: Kassaa, Imad Al
date: 2015-03-10
txt: ./txt/cord-265263-r9e6bop3.txt
cache: ./cache/cord-265263-r9e6bop3.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/25752765/
doi: 10.1007/s00203-015-1101-8
summary:
  Tested lactobacilli displayed anti-HSV-2 activity when they were co-incubated with the virus prior to inoculating the mixture to Vero cell monolayers. This study shows that antiviral activity is due to the direct interaction between probiotic strains and enveloped virus. plantarum CMUL140 strain with HSV-2 on Vero cell monolayers has increased the cells viability by 65, 35 and 15 %, respectively, as compared to the control with 22 % viability, after exposure to the virus (Fig. 3a) . plantarum CMUL140-derived neutralized supernatants enhanced slightly the viability of Vero cell monolayer inoculated with 100 PFU HSV-2 with about 9.5, 5.4 and 2.4 %, respectively, due to reduction in viral infectious particles. gasseri CMUL57 at 10 8 CFU/ml or Dulbecco''s Modified Eagle''s Medium (DMEM, negative control) were incubated for 2 h at 37 °C in CO 2 atmosphere in the presence of HSV-2 (10 4 PFU/ml) or CVB4E2 (10 3 TCID 50 /ml).

abstract:
  This study aimed at demonstrating the antiviral activity of Lactobacillus gasseri CMUL57 (L. gasseri CMUL57), L. acidophilus CMUL67 and L. plantarum CMUL140 against herpes simplex type 2 (HSV-2) and Coxsackievirus B4E2 (CVB4E2), which are enveloped and naked viruses, respectively. These lactobacilli were non-cytotoxic and were able to reduce the cytopathic effect induced by HSV-2 in Vero cell monolayers. However, lactobacilli were not active against CVB4E2. Tested lactobacilli displayed anti-HSV-2 activity when they were co-incubated with the virus prior to inoculating the mixture to Vero cell monolayers. The detection of HSV-2 DNA by PCR in pellets of bacteria/virus mixtures let us to hypothesize that anti-HSV-2 activity of lactobacilli resulted from the viruses’ entrapment. This study showed the capabilities of vaginal lactobacilli to inhibit enveloped viruses such as HSV-2.


id: cord-266585-jfjrk9gy
author: Fang, Shouguo
date: 2007-02-05
txt: ./txt/cord-266585-jfjrk9gy.txt
cache: ./cache/cord-266585-jfjrk9gy.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0042682206005745
doi: 10.1016/j.virol.2006.08.020
summary:
  During construction of an infectious clone from a Vero cell-adapted coronavirus infectious bronchitis virus (IBV), we found that a G–C point mutation at nucleotide position 15526, causing Arg-to-Pro mutation at amino acid position 132 of the helicase protein, is lethal to the infectivity of IBV on Vero cells. Further characterization of the in vitro-synthesized full-length transcripts containing the G15526C mutation demonstrated that this mutation blocks the transcription of subgenomic RNAs. Substitution mutation of the Arg132 residue to a positively charged amino acid (Lys) affected neither the infectivity of the in vitro-synthesized transcripts nor the growth properties of the rescued virus. To further demonstrate that the failure to rescue infectious virus from the G15526C mutant transcripts is due to a defect in subgenomic RNA transcription, the full-length clones with and without the G15526C mutation were used to generate recombinant IBV expressing the enhanced green fluorescent protein (EGFP) by replacing the 5a gene with EGFP.

title:
  An arginine-to-proline mutation in a domain with undefined functions within the helicase protein (Nsp13) is lethal to the coronavirus infectious bronchitis virus in cultured cells

abstract:
  Genetic manipulation of the RNA genomes by reverse genetics is a powerful tool to study the molecular biology and pathogenesis of RNA viruses. During construction of an infectious clone from a Vero cell-adapted coronavirus infectious bronchitis virus (IBV), we found that a G–C point mutation at nucleotide position 15526, causing Arg-to-Pro mutation at amino acid position 132 of the helicase protein, is lethal to the infectivity of IBV on Vero cells. When the in vitro-synthesized full-length transcripts containing this mutation were introduced into Vero cells, no infectious virus was rescued. Upon correction of the mutation, infectious virus was recovered. Further characterization of the in vitro-synthesized full-length transcripts containing the G15526C mutation demonstrated that this mutation may block the transcription of subgenomic RNAs. Substitution mutation of the Arg132 residue to a positively charged amino acid Lys affected neither the infectivity of the in vitro-synthesized transcripts nor the growth properties of the rescued virus. However, mutation of the Arg132 residue to Leu, a conserved residue in other coronaviruses at the same position, reduced the recovery rate of the in vitro-synthesized transcripts. The recovered mutant virus showed much smaller-sized plaques. On the contrary, a G–C and a G–A point mutations at nucleotide positions 4330 and 9230, respectively, causing Glu–Gln and Gly–Glu mutations in or near the catalytic centers of the papain-like (Nsp3) and 3C-like (Nsp5) proteinases, did not show detectable detrimental effect on the rescue of infectious viruses and the infectivity of the rescued viruses.


id: cord-267446-rpv19oy6
author: Park, Jung-Eun
date: 2011-06-12
txt: ./txt/cord-267446-rpv19oy6.txt
cache: ./cache/cord-267446-rpv19oy6.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/21667287/
doi: 10.1007/s00705-011-1044-6
summary:
  The addition of trypsin was shown to induce fusion of the infected Vero cells, resulting in the formation of multiple syncytia, and produced a significant increase in virus titer after several passages. For example, infection by severe acute respiratory syndrome coronavirus (SARS-CoV) and murine hepatitis virus strain 2 (MHV-2) requires proteolytic cleavage in their target cells, which is mediated by trypsin-like proteases [24, 29, 32] . To investigate the effects of proteolytic cleavage of the surface protein of Vero cells and free virions by trypsin, Vero cells or KPEDV-9 were pre-treated with trypsin prior to infection. These results are consistent with the suggestion that trypsin is not absolutely essential for Vero-cell-adapted PEDV infection, as reported for other group 1 coronaviruses, but the titer increases during infection with trypsin-treated virus.

title:
  Receptor-bound porcine epidemic diarrhea virus spike protein cleaved by trypsin induces membrane fusion

abstract:
  Porcine epidemic diarrhea virus (PEDV) infection in Vero cells is facilitated by trypsin through an undefined mechanism. The present study describes the mode of action of trypsin in enhancing PEDV infection in Vero cells during different stage of the virus life cycle. During the viral entry stage, trypsin increased the penetration of Vero-cell-attached PEDV by approximately twofold. However, trypsin treatment of viruses before receptor binding did not enhance infectivity, indicating that receptor binding is essentially required for trypsin-mediated entry upon PEDV infection. Trypsin treatment during the budding stage of virus infection induces an obvious cytopathic effect in infected cells. Furthermore, we also show that the PEDV spike (S) glycoprotein is cleaved by trypsin in virions that are bound to the receptor, but not in free virions. These findings indicate that trypsin affects only cell-attached PEDV and increases infectivity and syncytium formation in PEDV-infected Vero cells by cleavage of the PEDV S protein. These findings strongly suggest that the PEDV S protein may undergo a conformational change after receptor binding and cleavage by exogenous trypsin, which induces membrane fusion.


id: cord-267613-hsc2x36j
title: Drug repurposing screens reveal FDA approved drugs active against SARS-Cov-2
author: Dittmar, Mark
date: 2020-06-19
txt: ./txt/cord-267613-hsc2x36j.txt
cache: ./cache/cord-267613-hsc2x36j.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.06.19.161042
doi: 10.1101/2020.06.19.161042
summary:
  Moreover, we found 9 drugs are antiviral in lung cells, 7 of which have been tested in humans, and 3 are FDA approved including Cyclosporine which we found is targeting Cyclophilin rather than Calcineurin for its antiviral activity. Previous studies found that the antiviral drug remdesivir, which was developed against the RNA-dependent RNA polymerase of Ebola virus, was also active against SARS-CoV-2 in vitro, with promising results in clinical trials (5) (6) (7) . Both cepharanthine and tetrandrine were previously shown to have antiviral activity against the human coronavirus OC43 and in recent studies on SARS-CoV-2 in Vero cell screens (13, 62, 63) . Strikingly, the activities of all of these drugs is similar in the two cell lines suggesting the same target and mechanism-of-action and that Cyclosporine would block SARS-CoV-2 in diverse infected tissues in vivo.

abstract:
  There are an urgent need for antivirals to treat the newly emerged SARS-CoV-2. To identify new candidates we screened a repurposing library of ~3,000 drugs. Screening in Vero cells found few antivirals, while screening in human Huh7.5 cells validated 23 diverse antiviral drugs. Extending our studies to lung epithelial cells, we found that there are major differences in drug sensitivity and entry pathways used by SARS-CoV-2 in these cells. Entry in lung epithelial Calu-3 cells is pH-independent and requires TMPRSS2, while entry in Vero and Huh7.5 cells requires low pH and triggering by acid-dependent endosomal proteases. Moreover, we found 9 drugs are antiviral in lung cells, 7 of which have been tested in humans, and 3 are FDA approved including Cyclosporine which we found is targeting Cyclophilin rather than Calcineurin for its antiviral activity. These antivirals reveal essential host targets and have the potential for rapid clinical implementation.


id: cord-270683-982eqtog
title: Isolation and characterization of severe acute respiratory syndrome coronavirus 2 in Turkey
author: Pavel, Shaikh Terkis Islam
date: 2020-09-16
txt: ./txt/cord-270683-982eqtog.txt
cache: ./cache/cord-270683-982eqtog.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1371/journal.pone.0238614
doi: 10.1371/journal.pone.0238614
summary:
  We determined that the Vero E6 and MA-104 cell lines are suitable for supporting SARS-CoV-2 that supports viral replication, development of cytopathic effect (CPE) and subsequent cell death. Phylogenetic analyses of the whole genome sequences showed that the hCoV-19/Turkey/ERAGEM-001/2020 strain clustered with the strains primarily from Australia, Canada, England, Iran and Kuwait and that the cases in the nearby clusters were reported to have travel history to Iran and to share the common unique nucleotide substitutions. For whole genome sequencing of hCoV-19/Turkey/ERAGEM-001/2020, Vero E6 cells infected with the virus were used for RNA extraction. The growth kinetics study showed that SARS-CoV-2 replicated rapidly and efficiently and could be detected within 6 h post-infection in Vero E6 and MA-104 cells (Fig 6A and 6B ). Immunoblotting analysis also confirmed that only Vero E6 and MA-104 cell lines infected with SARS-CoV-2 showed the expression of the virus specific proteins expression (Fig 5) .

abstract:
  Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and associated with severe respiratory illness emerged in Wuhan, China, in late 2019. The virus has been able to spread promptly across all continents in the world. The current pandemic has posed a great threat to public health concern and safety. Currently, there are no specific treatments or licensed vaccines available for COVID-19. We isolated SARS-CoV-2 from the nasopharyngeal sample of a patient in Turkey with confirmed COVID-19. We determined that the Vero E6 and MA-104 cell lines are suitable for supporting SARS-CoV-2 that supports viral replication, development of cytopathic effect (CPE) and subsequent cell death. Phylogenetic analyses of the whole genome sequences showed that the hCoV-19/Turkey/ERAGEM-001/2020 strain clustered with the strains primarily from Australia, Canada, England, Iran and Kuwait and that the cases in the nearby clusters were reported to have travel history to Iran and to share the common unique nucleotide substitutions.


id: cord-271638-0wsyl7vk
author: Li, Wenmiao
date: 2020-03-09
txt: ./txt/cord-271638-0wsyl7vk.txt
cache: ./cache/cord-271638-0wsyl7vk.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166354219305674
doi: 10.1016/j.antiviral.2020.104714
summary:
  Therefore, the natural dietary flavonoid myricetin has potential to be developed into a novel anti-HSV agent targeting both virus gD protein and cellular EGFR/PI3K/Akt pathway. As shown in Fig. 2C and E, myricetin treatment (20 μM) during adsorption significantly decreased the fluorescence of ICP5 protein on cell surface, compared to that in non-treated virus control cells, suggesting that myricetin may block virus adsorption process of HSV. As shown in Fig. 3A , in HSV-2 (MOI = 3.0) infected Vero cells, obvious syncytia with multinuclear cells were observed in non-treated virus control group (HSV-2) at 7 h p.i. However, treatment with myricetin (30, 15 μM) during 5-7 h p.i. markedly blocked syncytium formation only with a limited number of small syncytia, suggesting that myricetin may inhibit HSV-induced cell fusion. However, myricetin (2.5, 5, 10, 20 μM) treatment did not significantly influence the total expression levels of EGFR, PI3K and Akt proteins in HSV-2 infected Vero cells (Fig. 4G and H) .

title:
  Inhibition of herpes simplex virus by myricetin through targeting viral gD protein and cellular EGFR/PI3K/Akt pathway

abstract:
  Myricetin, a common dietary flavonoid, was reported to possess many different biological activities such as anti-oxidant, anti-inflammatory, and antiviral effects. In this study, we explored the anti-HSV effects and mechanisms of myricetin both in vitro and in vivo. The results showed that myricetin possessed anti-HSV-1 and HSV-2 activities with very low toxicity, superior to the effects of acyclovir. Myricetin may block HSV infection through direct interaction with virus gD protein to interfere with virus adsorption and membrane fusion, which was different from the nucleoside analogues such as acyclovir. Myricetin also down-regulate the cellular EGFR/PI3K/Akt signaling pathway to further inhibit HSV infection and its subsequent replication. Most importantly, intraperitoneal therapy of myricetin markedly improved mice survival and reduced virus titers in both lungs and spinal cord. Therefore, the natural dietary flavonoid myricetin has potential to be developed into a novel anti-HSV agent targeting both virus gD protein and cellular EGFR/PI3K/Akt pathway.


id: cord-272729-nbgdmavr
title: Ribavirin efficiently suppresses porcine nidovirus replication
author: Kim, Youngnam
date: 2012-10-27
txt: ./txt/cord-272729-nbgdmavr.txt
cache: ./cache/cord-272729-nbgdmavr.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0168170212004078
doi: 10.1016/j.virusres.2012.10.018
summary:
  Investigations into the mechanism of action of ribavirin against PRRSV and PEDV revealed that the addition of guanosine to the ribavirin treatment significantly reversed the antiviral effects, suggesting that depletion of the intracellular GTP pool by inhibiting IMP dehydrogenase may be essential for ribavirin activity. Further experiments revealed that suppression of ribavirin affects post-entry steps of the replication cycle of PRRSV and PEDV, including viral genomic and sg RNA synthesis, viral protein expression, and virus production. Several mechanisms of action for the antiviral activity of ribavirin have been suggested, including a reduction in cellular GTP pools via inosine monophosphate dehydrogenase (IMPDH) inhibition and increased mutation frequency on the virus genome leading to error catastrophe (Graci and Cameron, 2006) . Treatment of cells with ribavirin resulted in significant attenuation of postentry steps during the replication of porcine nidovirus, as determined by lower progeny production, diminished viral protein expression, and decreased synthesis of genomic RNA and sg mRNA.

abstract:
  Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) are porcine nidoviruses that represent emerging viral pathogens causing heavy economic impacts on the swine industry. Although ribavirin is a well-known antiviral drug against a broad range of both DNA and RNA viruses in vitro, its inhibitory effect and mechanism of action on porcine nidovirus replication remains to be elucidated. Therefore, the present study was conducted to determine whether ribavirin suppresses porcine nidovirus infection. Our results demonstrated that ribavirin treatment dose-dependently inhibited the replication of both nidoviruses. The antiviral activity of ribavirin on porcine nidovirus replication was found to be primarily exerted at early times post-infection. Treatment with ribavirin resulted in marked reduction of viral genomic and subgenomic RNA synthesis, viral protein expression, and progeny virus production in a dose-dependent manner. Investigations into the mechanism of action of ribavirin against PRRSV and PEDV revealed that the addition of guanosine to the ribavirin treatment significantly reversed the antiviral effects, suggesting that depletion of the intracellular GTP pool by inhibiting IMP dehydrogenase may be essential for ribavirin activity. Further sequencing analysis showed that the mutation frequency in ribavirin-treated cells was similar to that in untreated cells, indicating that ribavirin did not induce error-prone replication. Taken together, our data indicate that ribavirin might not only be a good therapeutic agent against porcine nidovirus, but also a potential candidate to be evaluated against other human and animal coronaviruses.


id: cord-273745-mwjh5se7
author: Meng, Fandan
date: 2014-03-25
txt: ./txt/cord-273745-mwjh5se7.txt
cache: ./cache/cord-273745-mwjh5se7.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0042682214000130
doi: 10.1016/j.virol.2014.01.010
summary:
  Three phage-displayed peptides designated H, S and F that recognize porcine aminopeptidase N (pAPN), the cellular receptor of porcine transmissible gastroenteritis virus (TGEV) were able to inhibit cell infection by TGEV. When Vero cells were pre-treated with peptide (cell pretreatment assay) prior to virus infection (Fig. 1B) , little changes in virus titers were observed between the control and peptide treatment groups; some small effects were observed with the rabbit anti-PEDV neutralizing antibodies. Finally, in the virus pretreatment assays where PEDV was incubated with peptides prior to cell infection (Fig. 1C) , the results indicated that both peptides H and S inhibited PEDV infectivity where EC 50 values were approximately 1 μg/ml and 62.5 μg/ml, respectively. Results clearly show that peptide H had no demonstrable effects on TGEV or PrV even at very high peptide concentrations (1 mM/ml) (Fig. 3) suggesting that a non-specific reactivity with virus envelopes is unlikely to be the cause for attenuating PEDV infectivity.

title:
  A phage-displayed peptide recognizing porcine aminopeptidase N is a potent small molecule inhibitor of PEDV entry

abstract:
  Three phage-displayed peptides designated H, S and F that recognize porcine aminopeptidase N (pAPN), the cellular receptor of porcine transmissible gastroenteritis virus (TGEV) were able to inhibit cell infection by TGEV. These same peptides had no inhibitory effects on infection of Vero cells by porcine epidemic diarrhea virus (PEDV). However, when PEDV, TGEV and porcine pseudorabies virus were incubated with peptide H (HVTTTFAPPPPR), only infection of Vero cells by PEDV was inhibited. Immunofluoresence assays indicated that inhibition of PEDV infection by peptide H was independent of pAPN. Western blots demonstrated that peptide H interacted with PEDV spike protein and that pre-treatment of PEDV with peptide H led to a higher inhibition than synchronous incubation with cells. These results indicate direct interaction with the virus is necessary to inhibit infectivity. Temperature shift assays demonstrated that peptide H inhibited pre-attachment of the virus to the cells.


id: cord-274110-nyyunoha
title: An inactivated West Nile Virus vaccine derived from a chemically synthesized cDNA system
author: Orlinger, Klaus K.
date: 2010-04-26
txt: ./txt/cord-274110-nyyunoha.txt
cache: ./cache/cord-274110-nyyunoha.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0264410X10002860
doi: 10.1016/j.vaccine.2010.02.092
summary:
  A cDNA comprising the complete genome of West Nile Virus (WNV) was generated by chemical synthesis using published sequence data, independent of any preformed viral components. The synthetic WNV, produced by transfection of in vitro transcribed RNA into cell culture, exhibited undistinguishable biological properties compared to the corresponding animal-derived wild-type virus. Taking advantage of the rapid progression of gene synthesis technology (for review [24] ), we intended to adopt such a synthetic approach to produce a flavivirus cDNA system for the generation of a synthetic WNV seed virus for use in vaccine development. The production and characterization of the resulting West Nile Virus, which fully matched the sequence of the in silico designed viral genome, confirms the feasibility and accuracy of the synthetic flavivirus reverse genetic system. Cover slips with fixed cells were dried, rehydrated with phosphate-buffered saline and treated with a polyclonal mouse anti-WNV serum (1:50 dilution) obtained after immunization of mice with a formalin-inactivated whole virus vaccine preparation.

abstract:
  A cDNA comprising the complete genome of West Nile Virus (WNV) was generated by chemical synthesis using published sequence data, independent of any preformed viral components. The synthetic WNV, produced by transfection of in vitro transcribed RNA into cell culture, exhibited undistinguishable biological properties compared to the corresponding animal-derived wild-type virus. No differences were found concerning viral growth in mammalian and insect cell lines and concerning expression of viral proteins in cells. There were also no significant differences in virulence in mice following intranasal challenge. After immunizations of mice with experimental vaccines derived from the synthetic and wild-type viruses, protection from lethal challenge was achieved with similar amounts of antigen. Both vaccine preparations also induced comparable levels of neutralizing antibodies in mice. In addition, the synthetic approach turned out to be very accurate, since the rescued WNV genome contained no undesired mutations. Thus, the first flavivirus based on chemical gene synthesis was indistinguishable from the parent virus. This demonstrates that virus isolates from animal sources are dispensable to derive seed viruses for vaccine production or research.


id: cord-275863-qos9vu3r
title: Lectin Switching During Dengue Virus Infection
author: Dejnirattisai, Wanwisa
date: 2011-06-15
txt: ./txt/cord-275863-qos9vu3r.txt
cache: ./cache/cord-275863-qos9vu3r.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/21606536/
doi: 10.1093/infdis/jir173
summary:
  In this report we have studied the interaction of dengue viruses produced in insect cells, tumor cell lines, and primary human dendritic cells (DCs) with DC-SIGN and L-SIGN. To formally prove that the loss of infection of DCs was a result of the loss of affinity of DC-produced virus for DC-SIGN, we went on to test infection on 3T3 cells expressing DC-SIGN and included in these assays the related C-type lectin L-SIGN ( Figure 3A ), which has also been reported to be a receptor for dengue virus. C6/36-and DC-derived viruses were incubated with increasing levels of pooled convalescent dengue immune serum and subsequently used to infect U937, a monocyte cell line that expresses the Fc receptor and which shows relatively low infectivity without the presence of enhancing antibodies. Viruses produced in both DCs and insect cells were susceptible to enhancement, over the same range of antibody concentrations, showing that DC-produced virus could exploit ADE to replicate in individuals undergoing a secondary dengue infection ( Figure 6A ).

abstract:
  Dengue virus receptors are relatively poorly characterized, but there has been recent interest in 2 C-type lectin molecules, dendritic cell–specific intercellular adhesion molecule 3 (ICAM-3)–grabbing nonintegrin (DC-SIGN) and its close homologue liver/lymph node–specific ICAM-3–grabbing integrin (L-SIGN), which can both bind dengue and promote infection. In this report we have studied the interaction of dengue viruses produced in insect cells, tumor cell lines, and primary human dendritic cells (DCs) with DC-SIGN and L-SIGN. Virus produced in primary DCs is unable to interact with DC-SIGN but remains infectious for L-SIGN–expressing cells. Skin-resident DCs may thus be a site of initial infection by insect-produced virus, but DCs will likely not participate in large-scale virus replication during dengue infection. These results reveal that differential glycosylation of dengue virus envelope protein is highly dependent on cell state and suggest that studies of virus tropism using virus prepared in insect cells or tumor cell lines should be interpreted with caution.


id: cord-276361-77cylm1o
title: HIV protease inhibitor nelfinavir inhibits replication of SARS-associated coronavirus
author: Yamamoto, Norio
date: 2004-06-04
txt: ./txt/cord-276361-77cylm1o.txt
cache: ./cache/cord-276361-77cylm1o.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0006291X04008150
doi: 10.1016/j.bbrc.2004.04.083
summary:
  title: HIV protease inhibitor nelfinavir inhibits replication of SARS-associated coronavirus Here we report that the HIV-1 protease inhibitor, nelfinavir, strongly inhibited replication of the SARS coronavirus (SARS-CoV). Experiments with various timings of drug addition revealed that nelfinavir exerted its effect not at the entry step, but at the post-entry step of SARS-CoV infection. We found that nelfinavir, a widely used HIV-1 protease inhibitor, could inhibit SARS-CoV replication efficiently. We screened our chemical library and found that nelfinavir could inhibit SARS-CoV replication in Vero E6 cells. Nelfinavir clearly inhibited the cytopathic effect (CPE) induced by infection with SARS-CoV (Fig. 1A) . Nelfinavir significantly inhibited SARS-CoV replication when used before infection (Figs. The other protease inhibitors including ritonavir had no effect on replication of SARS-CoV CC 50 , cytotoxic concentration of the compound that reduced cell viability to 50%. Our studies have clearly shown that nelfinavir can strongly inhibit the replication of SARS-CoV in Vero E6 cells.

abstract:
  A novel coronavirus has been identified as an etiological agent of severe acute respiratory syndrome (SARS). To rapidly identify anti-SARS drugs available for clinical use, we screened a set of compounds that included antiviral drugs already in wide use. Here we report that the HIV-1 protease inhibitor, nelfinavir, strongly inhibited replication of the SARS coronavirus (SARS-CoV). Nelfinavir inhibited the cytopathic effect induced by SARS-CoV infection. Expression of viral antigens was much lower in infected cells treated with nelfinavir than in untreated infected cells. Quantitative RT-PCR analysis showed that nelfinavir could decrease the production of virions from Vero cells. Experiments with various timings of drug addition revealed that nelfinavir exerted its effect not at the entry step, but at the post-entry step of SARS-CoV infection. Our results suggest that nelfinavir should be examined clinically for the treatment of SARS and has potential as a good lead compound for designing anti-SARS drugs.


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-279316-xz7aawem
title: Signal Transduction in SARS‐CoV‐Infected Cells
author: MIZUTANI, T.
date: 2007-04-23
txt: ./txt/cord-279316-xz7aawem.txt
cache: ./cache/cord-279316-xz7aawem.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/17470913/
doi: 10.1196/annals.1408.006
summary:
  Recent studies regarding SARS and SARS‐CoV have clarified that activation of mitogen‐activated protein kinases (MAPKs) plays important roles in upregulation of cytokine expression and apoptosis both in vitro and in vivo. For example, mitogen-activated protein kinases (MAPKs) are well-known signal transducers that respond to extracellular stimulation by cytokines, growth factors, viral infection, and stress, and in turn regulate cell differentiation, proliferation, survival, and apoptosis. Extracellular signal-regulated kinase (ERK) 1/2 was phosphorylated in SARS-CoV-infected Vero E6 cells, 27 whereas ERK1/2 was downregulated in N protein-expressing COS-1 cells as described below. Activation of the p38 MAPK signaling pathway and dephosphorylation of STAT3 via p38 MAPK induced by SARS-CoV infection have partially proapoptotic roles in Vero E6 cells. Importance of Akt signaling pathway for apoptosis in SARS-CoV-infected Vero E6 cells

abstract:
  abstract: Severe acute respiratory syndrome (SARS) is a newly found infectious disease that is caused by a novel human coronavirus, SARS coronavirus (SARS‐CoV). Because the mortality rate of SARS patients is very high, understanding the pathological mechanisms of SARS not only in vivo but in vitro is important for the prevention of SARS. Activation of signaling pathways caused by SARS‐CoV infection leads to the phosphorylation and activation of downstream molecules. Two conflicting cellular programs, apoptosis to eliminate virus‐infected cells and survival to delay apoptosis by producing antiviral cytokines, occur in SARS patients. Recent studies regarding SARS and SARS‐CoV have clarified that activation of mitogen‐activated protein kinases (MAPKs) plays important roles in upregulation of cytokine expression and apoptosis both in vitro and in vivo. Both Akt and p38 MAPK are keys for determination of cell survival or death in SARS‐CoV‐infected cells in vitro. Agents being developed to target these signaling cascades may be important for the design of anti‐SARS‐CoV drugs. This review highlights recent progress regarding SARS‐CoV biology, especially signal transduction in SARS‐CoV‐infected cells.


id: cord-279975-542qbbgp
author: Shibata, Isao
date: 2000-03-15
txt: ./txt/cord-279975-542qbbgp.txt
cache: ./cache/cord-279975-542qbbgp.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0378113599001996
doi: 10.1016/s0378-1135(99)00199-6
summary:
  title: Isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages This paper describes the isolation of porcine epidemic diarrhea (PED) virus in Vero and porcine cell cultures, and the influence of age on disease in experimental infection. PED virus was isolated from the small intestine of piglets inoculated with PED samples and cultured in Vero, porcine bladder and kidney cells propagated in collagen-coated tissue culture plates in maintenance medium (MM) containing trypsin. In porcine bladder and kidney cell cultures inoculated with isolated PED virus, cytopathic effects (CPE) including cell fusion were detected. Isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages Upon experimental infection, 2-and 7-day old pigs inoculated with PED virus developed severe diarrhea and died.

title:
  Isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages

abstract:
  This paper describes the isolation of porcine epidemic diarrhea (PED) virus in Vero and porcine cell cultures, and the influence of age on disease in experimental infection. PED virus was isolated from the small intestine of piglets inoculated with PED samples and cultured in Vero, porcine bladder and kidney cells propagated in collagen-coated tissue culture plates in maintenance medium (MM) containing trypsin. In porcine bladder and kidney cell cultures inoculated with isolated PED virus, cytopathic effects (CPE) including cell fusion were detected. Specific brilliant fluorescence was observed in the cytoplasm of these cells. Two- and 7-day old, and 2-, 4-, 8- and 12-week old specific pathogen-free (SPF) pigs were orally inoculated with PED virus isolated from an outbreak. All 2- and 7-day old pigs inoculated developed severe watery diarrhea from post-inoculation day (PID) 1 and died between PID 3 and 4. Although three of five 2-week old pigs developed diarrhea on PID 1–4, they eventually recovered. In the 4-week old group, three of five pigs had mild diarrhea for 1–2 days. None of the 8- and 12-week old pigs showed any clinical signs. Antibodies against PED virus were detected in all surviving pigs by virus neutralization (VN) test and immunofluorescence assay (IFA). Therefore, there is an age-dependent resistance to pathogenic PED virus infection in pigs.


id: cord-283309-ovx5fzsg
author: Yang, Yong-Le
date: 2019-08-09
txt: ./txt/cord-283309-ovx5fzsg.txt
cache: ./cache/cord-283309-ovx5fzsg.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0042682219302193
doi: 10.1016/j.virol.2019.08.006
summary:
  Six subgenomic mRNAs containing the leader-body junction sites, including a bicistronic mRNA encoding the accessory NS7a and NS7b genes, were experimentally identified in SeACoV-infected cells. Anti-Ac (Nsp3) staining also resulted in detection of perinuclear foci at four time points, indicating localization to the viral replication-transcription complexes (Fig. 1C) , which was similar to the pattern of Nsp3 antibody observed in SARS-CoV-infected Vero cells (Prentice et al., 2004) . DMVs are membrane structures where viral genomic RNA is recognized by the host cell machinery and translated into non-structural proteins (ORF1ab), assembling into viral replication-transcription complexes (Gosert et al., 2002) , whereas LVCVs are large circular organelles that are thought to originate from Golgi compartments expanding to accommodate numerous precursor virions Positions of forward (LF) and reverse primers (S1-R, sgORF3-R, sgE-R, sgM-R, sgN-R and NS7a-R/NS7-R) used for PCR amplification of distinct subgenomic mRNAs (sgRNAs) are indicated by arrows under the genome.

title:
  Characterization of a novel bat-HKU2-like swine enteric alphacoronavirus (SeACoV) infection in cultured cells and development of a SeACoV infectious clone

abstract:
  Swine enteric alphacoronavirus (SeACoV), also known as swine acute diarrhea syndrome coronavirus (SADS-CoV), belongs to the species Rhinolophus bat coronavirus HKU2. Herein, we report on the primary characterization of SeACoV in vitro. Four antibodies against the SeACoV spike, membrane, nucleocapsid and nonstructural protein 3 capable of reacting with viral antigens in SeACoV-infected Vero cells were generated. We established a DNA-launched SeACoV infectious clone based on the cell adapted passage-10 virus and rescued the recombinant virus with a unique genetic marker in cultured cells. Six subgenomic mRNAs containing the leader-body junction sites, including a bicistronic mRNA encoding the accessory NS7a and NS7b genes, were experimentally identified in SeACoV-infected cells. Cellular ultrastructural changes induced by SeACoV infection were visualized by electron microscopy. The availability of the SeACoV infectious clone and a panel of antibodies against different viral proteins will facilitate further studies on understanding the molecular mechanisms of SeACoV replication and pathogenesis.


id: cord-284322-synuzaxm
author: Borel, Nicole
date: 2010-07-27
txt: ./txt/cord-284322-synuzaxm.txt
cache: ./cache/cord-284322-synuzaxm.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/1471-2180-10-201
doi: 10.1186/1471-2180-10-201
summary:
  To confirm these initial observations, we established a cell culture model of mixed infections with Chlamydia and a cell culture-adapted porcine epidemic diarrhea virus (ca-PEDV) and hypothesized that this would result in the generation of persistent chlamydial forms. In contrast, dual infections with ca-PEDV and Chlamydia pecorum resulted in the exclusive production of aberrant inclusions containing between 2-50 ABs. Chlamydial inclusions in viral syncytia grew even larger than in non-viral infected Vero cells. The changes of chlamydial inclusion size by subsequent virus addition to Chlamydia abortus are different to those we observed in the Chlamydia pecorum dual infection experiments. TEM examinations of mixed infections (ca-PEDV and Chlamydia abortus or Chlamydia pecorum) revealed aberrant chlamydial inclusions containing fewer bacteria than typical inclusions and were located in viral syncytia or single cells without viral infection. Enlarged chlamydial inclusions were described in that study in the ca-PEDV co-infection model with Chlamydia abortus and Chlamydia pecorum but no further ultrastructural analysis has been subsequently performed.

title:
  Mixed infections with Chlamydia and porcine epidemic diarrhea virus - a new in vitro model of chlamydial persistence

abstract:
  BACKGROUND: Chlamydiae induce persistent infections, which have been associated with a wide range of chronic diseases in humans and animals. Mixed infections with Chlamydia and porcine epidemic diarrhea virus (PEDV) may result in generation of persistent chlamydial infections. To test this hypothesis, an in vitro model of dual infection with cell culture-adapted PEDV and Chlamydia abortus or Chlamydia pecorum in Vero cells was established. RESULTS: Infected cultures were investigated by immunofluorescence (IF), transmission electron microscopy (TEM) and re-infection experiments. By IF, Chlamydia-infected cells showed normal inclusions after 39 hpi. Dual infections with Chlamydia abortus revealed a heterogenous mix of inclusion types including small inclusions consisting of aberrant bodies (ABs), medium-sized inclusions consisting of ABs and reticulate bodies and normal inclusions. Only aberrant inclusions were observable in dual infection experiments with Chlamydia pecorum and PEDV. TEM examinations of mixed infections with Chlamydia abortus and Chlamydia pecorum revealed aberrant chlamydial inclusions containing reticulate-like, pleomorphic ABs, which were up to 2 μm in diameter. No re-differentiation into elementary bodies (EBs) was detected. In re-infection experiments, co-infected cells produced fewer EBs than monoinfected cells. CONCLUSIONS: In the present study we confirm that PEDV co-infection alters the developmental cycle of member species of the family Chlamydiaceae, in a similar manner to other well-described persistence induction methods. Interestingly, this effect appears to be partially species-specific as Chlamydia pecorum appears more sensitive to PEDV co-infection than Chlamydia abortus, as evidenced by TEM and IF observations of a homogenous population of aberrant inclusions in PEDV - Chlamydia pecorum co-infections.


id: cord-287488-h102xn29
author: Araujo, Danielle Bastos
date: 2020-10-23
txt: ./txt/cord-287488-h102xn29.txt
cache: ./cache/cord-287488-h102xn29.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/33111751/
doi: 10.1590/0074-02760200342
summary:
  BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was confirmed in Brazil in February 2020, the first cases were followed by an increase in the number of cases throughout the country, resulting in an important public health crisis that requires fast and coordinated responses. METHODS: After diagnosis in patients that returned from Italy to the São Paulo city in late February by RT-PCR, SARS-CoV-2 isolates were obtained in cell cultures and characterised by full genome sequencing, electron microscopy and in vitro replication properties. FINDINGS: The virus isolate was recovered from nasopharyngeal specimen, propagated in Vero cells (E6, CCL-81 and hSLAM), with clear cytopathic effects, and characterised by full genome sequencing, electron microscopy and in vitro replication properties. Virus stocks viable (titre 2.11 × 10(6) TCID50/mL, titre 1.5 × 10(6) PFUs/mL) and inactivated from isolate SARS.CoV2/SP02.2020.HIAE.Br were prepared and set available to the public health authorities and the scientific community in Brazil and abroad.

title:
  SARS-CoV-2 isolation from the first reported patients in Brazil and establishment of a coordinated task network

abstract:
  BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was confirmed in Brazil in February 2020, the first cases were followed by an increase in the number of cases throughout the country, resulting in an important public health crisis that requires fast and coordinated responses. OBJECTIVES: The objective of this work is to describe the isolation and propagation properties of SARS-CoV-2 isolates from the first confirmed cases of coronavirus disease 2019 (COVID-19) in Brazil. METHODS: After diagnosis in patients that returned from Italy to the São Paulo city in late February by RT-PCR, SARS-CoV-2 isolates were obtained in cell cultures and characterised by full genome sequencing, electron microscopy and in vitro replication properties. FINDINGS: The virus isolate was recovered from nasopharyngeal specimen, propagated in Vero cells (E6, CCL-81 and hSLAM), with clear cytopathic effects, and characterised by full genome sequencing, electron microscopy and in vitro replication properties. Virus stocks - viable (titre 2.11 × 10(6) TCID50/mL, titre 1.5 × 10(6) PFUs/mL) and inactivated from isolate SARS.CoV2/SP02.2020.HIAE.Br were prepared and set available to the public health authorities and the scientific community in Brazil and abroad. MAIN CONCLUSION: We believe that the protocols for virus growth and studies here described and the distribution initiative may constitute a viable model for other developing countries, not only to help a rapid effective pandemic response, but also to facilitate and support basic scientific research.


id: cord-288644-ywaefpe8
author: Rodon, Jordi
date: 2020-10-20
txt: ./txt/cord-288644-ywaefpe8.txt
cache: ./cache/cord-288644-ywaefpe8.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.04.23.055756
doi: 10.1101/2020.04.23.055756
summary:
  We have tested the antiviral activity of different clinically available compounds and their combinations by assessing their ability to inhibit viral induced cytopathic effect in vitro. Drug selection criteria first focused on compounds already being tested in clinical trials, along with well-known human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) protease inhibitors, as well as other compounds suggested to have potential activity against SARS-CoV-2 in molecular docking analysis or in vitro assays. Additional Food and Drug Administration (FDA)-approved compounds previously used to abrogate viral entry via clathrin-mediated endocytosis were also tested in this SARS-CoV-2-induced cytotoxicity assay (Supp . Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of plitidepsin and its combinations with hydroxychloroquine and remdesivir.

title:
  Pre-clinical search of SARS-CoV-2 inhibitors and their combinations in approved drugs to tackle COVID-19 pandemic

abstract:
  There is an urgent need to identify novel drugs against the new coronavirus. Although different antivirals are given for the clinical management of SARS-CoV-2 infection, their efficacy is still under evaluation. Here, we have screened existing drugs approved for human use in a variety of diseases, to compare how they counteract SARS-CoV-2-induced cytopathic effect and viral replication in vitro. Among the potential 72 antivirals tested herein that were previously proposed to inhibit SARS-CoV-2 infection, only 18% had in vitro antiviral activity. Moreover, only eight families had an IC50 below 25 µM or 102 IU/mL. These include chloroquine derivatives and remdesivir, along with plitidepsin, cathepsin inhibitors, nelfinavir mesylate hydrate, interferon 2-alpha, interferon-gamma, fenofibrate and camostat. Plitidepsin was the only clinically approved drug displaying nanomolar efficacy. Four of these families, including novel cathepsin inhibitors, blocked viral entry in a cell-type specific manner. Since the most effective antivirals usually combine therapies that tackle the virus at different steps of infection, we also assessed several drug combinations. Although no particular synergy was found, inhibitory combinations did not reduce their antiviral activity. Thus, these combinations could decrease the potential emergence of resistant viruses. Antivirals prioritized herein identify novel compounds and their mode of action, while independently replicating the activity of a reduced proportion of drugs which are mostly approved for clinical use. Combinations of these drugs should be tested in animal models to inform the design of fast track clinical trials.


id: cord-289248-6mx4o0eb
author: Wang, Yilong
date: 2018-05-01
txt: ./txt/cord-289248-6mx4o0eb.txt
cache: ./cache/cord-289248-6mx4o0eb.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.virol.2018.02.022
doi: 10.1016/j.virol.2018.02.022
summary:
  These two mutants grew to high titer in Vero cells, were genetically stable, and were significantly more attenuated in vitro and in vivo compared to the parental rMV-Hu191 vaccine strain. We next compared the replication kinetics of the rMV-Hu191 mutants and the parental virus in Vero cells in the time course of 120 h after infection (Fig. 5 ). These data suggest that rMVs carrying mutations in the SAM binding site were more attenuated in Vero cells than the parental MV vaccine strain. In this study, we successfully generated two recombinant measles viruses with amino acid substitutions in the SAM binding site of L protein and examined the effects of these mutations on viral replication, safety, and immunogenicity. We generated two recombinant MV-Hu191 carrying mutations in the SAM binding site, which not only grew to high titer in Vero cells and were genetically stable but also were significantly more attenuated and immunogenic compared to the currently used Chinese MV vaccine strain.

title:
  Enhancement of safety and immunogenicity of the Chinese Hu191 measles virus vaccine by alteration of the S-adenosylmethionine (SAM) binding site in the large polymerase protein

abstract:
  The live-attenuated measles virus (MV) vaccine based on the Hu191 strain has played a significant role in controlling measles in China. However, it has considerable adverse effects that may cause public health burden. We hypothesize that the safety and efficacy of MV vaccine can be improved by altering the S-adeno- sylmethionine (SAM) binding site in the conserved region VI of the large polymerase protein. To test this hypothesis, we established an efficient reverse genetics system for the rMV-Hu191 strain and generated two recombinant MV-Hu191 carrying mutations in the SAM binding site. These two mutants grew to high titer in Vero cells, were genetically stable, and were significantly more attenuated in vitro and in vivo compared to the parental rMV-Hu191 vaccine strain. Importantly, both MV-Hu191 mutants triggered a higher neutralizing antibody than rMV-Hu191 vaccine and provided complete protection against MV challenge. These results demonstrate its potential for an improved MV vaccine candidate.


id: cord-295559-yc8q62z8
author: Qian, Zhaohui
date: 2013-10-03
txt: ./txt/cord-295559-yc8q62z8.txt
cache: ./cache/cord-295559-yc8q62z8.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1371/journal.pone.0076469
doi: 10.1371/journal.pone.0076469
summary:
  Coronavirus S proteins are Class I viral fusion proteins like the HIV envelope (env), influenza hemagglutinin (HA) and paramyxovirus fusion (F) glycoproteins [17] , which typically require protease cleavage between the S1 and S2 domains ( Figure 1A ) to permit conformational changes in S2, activated by receptor binding and/or low pH, that mediate membrane fusion leading to virus entry and syncytia formation [3, 17, 18] . In addition to entry by endocytosis, we showed that, like SARS-CoV [21, 22] , MERS pseudovirions could enter susceptible Vero E6 cells at the plasma membrane if virions were first bound to cell surface receptors at 4°C at neutral pH in the presence of NH 4 Cl to inhibit acidification of endosomes, and also treated briefly at room temperature with trypsin to cleave the viral S protein.

title:
  Role of the Spike Glycoprotein of Human Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in Virus Entry and Syncytia Formation

abstract:
  Little is known about the biology of the emerging human group c betacoronavirus, Middle East Respiratory Syndrome coronavirus (MERS-CoV). Because coronavirus spike glycoproteins (S) mediate virus entry, affect viral host range, and elicit neutralizing antibodies, analyzing the functions of MERS-CoV S protein is a high research priority. MERS-CoV S on lentivirus pseudovirions mediated entry into a variety of cell types including embryo cells from New World Eptesicus fuscus bats. Surprisingly, a polyclonal antibody to the S protein of MHV, a group a murine betacoronavirus, cross-reacted in immunoblots with the S2 domain of group c MERS-CoV spike protein. MERS pseudovirions released from 293T cells contained only uncleaved S, and pseudovirus entry was blocked by lysosomotropic reagents NH(4)Cl and bafilomycin and inhibitors of cathepsin L. However, when MERS pseudovirions with uncleaved S protein were adsorbed at 4°C to Vero E6 cells, brief trypsin treatment at neutral pH triggered virus entry at the plasma membrane and syncytia formation. When 293T cells producing MERS pseudotypes co-expressed serine proteases TMPRSS-2 or -4, large syncytia formed at neutral pH, and the pseudovirions produced were non-infectious and deficient in S protein. These experiments show that if S protein on MERS pseudovirions is uncleaved, then viruses enter by endocytosis in a cathepsin L-dependent manner, but if MERS-CoV S is cleaved, either during virus maturation by serine proteases or on pseudovirions by trypsin in extracellular fluids, then viruses enter at the plasma membrane at neutral pH and cause massive syncytia formation even in cells that express little or no MERS-CoV receptor. Thus, whether MERS-CoV enters cells within endosomes or at the plasma membrane depends upon the host cell type and tissue, and is determined by the location of host proteases that cleave the viral spike glycoprotein and activate membrane fusion.


id: cord-297531-et1sli23
title: A novel glycoprotein D-specific monoclonal antibody neutralizes herpes simplex virus
author: Du, Ruikun
date: 2017-10-20
txt: ./txt/cord-297531-et1sli23.txt
cache: ./cache/cord-297531-et1sli23.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166354217304710
doi: 10.1016/j.antiviral.2017.10.013
summary:
  Our current investigation found a mAb, m27f that recognizes a new continuous epitope (residues 292 to 297) within the pro-fusion domain of HSV and possesses a high level of virus-neutralizing activity. Briefly, virusantibody mixture was incubated for 1 h at 4°C before inoculating over the Vero cells (pre-attachment), while prechilled (4°C for 15 min) Vero cells were infected with HSV-1 or HSV-2 (100 pfu/well) at 4°C for 1 h to allow virus adsorption before serial dilutions of antibodies were added (post-attachment). After 2 h of adsorption at 37°C, the virus inoculum was removed and cells were washed with PBS twice then incubated in DMEM containing 2% FBS in the presence of antibodies, m27f (500 μg/ml) and 21C11 (500 μg/ml), mouse IgG (500 μg/ml), or medium alone as a control. As shown in Fig. 4A , m27f completely inhibited cell-to-cell spread of both HSV-1 and HSV-2, as evidenced by observing the limited fluorescence caused by virus infection.

abstract:
  The worldwide prevalence of herpes simplex virus (HSV) and the shortage of efficient vaccines and novel therapeutic strategies against HSV are widely global concerns. The abundance on the virion and the major stimulus for the virus-neutralizing antibodies makes gD a predominant candidate for cure of HSV infection. In this study, we generated a monoclonal antibody (mAb), termed m27f, targeting to glycoprotein D (gD) of HSV-2, which also has cross-reactivity against HSV-1 gD. It has a high level of neutralizing activity against both HSV-1 and HSV-2, and binds to a highly conserved region (residues 292–297) within the pro-fusion domain of gD. It can effectively block HSV cell-to-cell spread in vitro. The pre- or post-attachment neutralization assay and syncytium formation inhibition assay revealed that m27f neutralizes HSV at the post-binding stage. Moreover, therapeutic administration of m27f completely prevented infection-related mortality of mice challenged with a lethal dose of HSV-2. Our newly identified epitope for the neutralizing antibody would facilitate studies of gD-based HSV entry or vaccine design, and m27f itself demonstrated a high potential for adaptation as a protective or therapeutic drug against HSV.


id: cord-298922-k568hlf4
author: Sun, Dongbo
date: 2015-06-15
txt: ./txt/cord-298922-k568hlf4.txt
cache: ./cache/cord-298922-k568hlf4.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093415000695
doi: 10.1016/j.jviromet.2015.03.002
summary:
  Among the disease-related functions, certain anti-viral pathways and proteins, such as the RIG-I-like receptor, Rap1, autophagy, mitogen-activated protein kinase, PI3K-Akt and Jak-STAT signaling pathways, and integrin β2/β3 and cystatin-C proteins, represented potential factors in PEDV infection. In our current study, we used a quantitative proteomics approach based on an iTRAQ tandem mass spectrometry (MS/MS) technique to identify proteins differentially expressed between PEDV-infected and mock-infected Vero E6 cells. To verify the differential expression of the selected DEPs, equivalent volumes of the cell lysate replicates from the PEDV-infected (V1-V3) and mock-infected (C1-C3) Vero E6 cells were pooled into the V and C samples, respectively, and western blotting was performed as described above, with the following exceptions: a 1:1000 dilution of the polyclonal antibodies anti-␤ tubulin, anti-integrin-␤3, anti-cystatin-C, anti-protein S100-A2, anti-apolipoprotein E4, and anti-centrin from rabbit (Beijing Biosynthesis Biotechnology, Beijing, China) was used as the primary antibody, and a 1:5000 dilution of the HRP-conjugated goat anti-rabbit IgG (Sigma-Aldrich, St. Louis, USA) was used as the secondary antibody.

title:
  Analysis of protein expression changes of the Vero E6 cells infected with classic PEDV strain CV777 by using quantitative proteomic technique

abstract:
  Recent outbreaks of porcine epidemic diarrhea virus (PEDV) have caused widespread concern. The identification of proteins associated with PEDV infection might provide insight into PEDV pathogenesis and facilitate the development of novel antiviral strategies. We analyzed the differential protein profile of PEDV-infected Vero E6 cells using mass spectrometry and an isobaric tag for relative and absolute quantification. A total of 126 proteins were identified that were differentially expressed between the PEDV-infected and mock-infected groups (P < 0.05, quantitative ratio ≥1.2), among which the expression of 58 proteins was up-regulated and that of 68 proteins was down-regulated in the PEDV-infected Vero E6 cells, involving in integrin β2/β3, cystatin-C. The Gene Ontology analysis indicated that the molecular function of the differentially expressed proteins (DEPs) was primarily related to binding and catalytic activity, and that the biological functions in which the DEPs are involved included metabolism, organismal systems, cellular processes, genetic information processing, environmental information processing, and diseases. Among the disease-related functions, certain anti-viral pathways and proteins, such as the RIG-I-like receptor, Rap1, autophagy, mitogen-activated protein kinase, PI3K-Akt and Jak-STAT signaling pathways, and integrin β2/β3 and cystatin-C proteins, represented potential factors in PEDV infection. Our findings provide valuable insight into PEDV-Vero E6 cell interactions.


id: cord-299509-7xjdryoq
author: Scholte, Florine E. M.
date: 2013-08-01
txt: ./txt/cord-299509-7xjdryoq.txt
cache: ./cache/cord-299509-7xjdryoq.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/23936484/
doi: 10.1371/journal.pone.0071047
summary:
  Infectious cDNA clones of viruses have become invaluable tools that allow reverse genetics studies to elucidate the contribution of specific amino acids or RNA structures to viraemia, virulence, antigenicity, replication kinetics, interactions with host factors, adaptation to new vectors, and many other aspects of the viral life cycle. To obtain a virus that -in terms of virulence, sensitivity to antiviral compounds, and CHIKV-host interactions -is expected to have the general characteristics of the E1-226V CHIKV strains that were circulating during the 2005-2009 outbreaks, we have constructed a completely synthetic CHIKV cDNA clone based on the consensus sequence of the aligned genomes of these recent isolates. Indirect immunofluorescence analysis of Vero E6 cells infected with CHIKV LS3, LS3-GFP, or ITA07-RA1 at various time points showed that the localization and expression kinetics of E2 and dsRNA were similar for the natural isolate and the synthetic viruses (Fig. 6) .

title:
  Characterization of Synthetic Chikungunya Viruses Based on the Consensus Sequence of Recent E1-226V Isolates

abstract:
  Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that re-emerged in 2004 and has caused massive outbreaks in recent years. The lack of a licensed vaccine or treatment options emphasize the need to obtain more insight into the viral life cycle and CHIKV-host interactions. Infectious cDNA clones are important tools for such studies, and for mechanism of action studies on antiviral compounds. Existing CHIKV cDNA clones are based on a single genome from an individual clinical isolate, which is expected to have evolved specific characteristics in response to the host environment, and possibly also during subsequent cell culture passaging. To obtain a virus expected to have the general characteristics of the recent E1-226V CHIKV isolates, we have constructed a new CHIKV full-length cDNA clone, CHIKV LS3, based on the consensus sequence of their aligned genomes. Here we report the characterization of this synthetic virus and a green fluorescent protein-expressing variant (CHIKV LS3-GFP). Their characteristics were compared to those of natural strain ITA07-RA1, which was isolated during the 2007 outbreak in Italy. In cell culture the synthetic viruses displayed phenotypes comparable to the natural isolate, and in a mouse model they caused lethal infections that were indistinguishable from infections with a natural strain. Compared to ITA07-RA1 and clinical isolate NL10/152, the synthetic viruses displayed similar sensitivities to several antiviral compounds. 3-deaza-adenosine was identified as a new inhibitor of CHIKV replication. Cyclosporin A had no effect on CHIKV replication, suggesting that cyclophilins -opposite to what was found for other +RNA viruses- do not play an essential role in CHIKV replication. The characterization of the consensus sequence-based synthetic viruses and their comparison to natural isolates demonstrated that CHIKV LS3 and LS3-GFP are suitable and representative tools to study CHIKV-host interactions, screen for antiviral compounds and unravel their mode of action.


id: cord-300379-db79kb5c
title: Potent Inhibition of Zika Virus Replication by Aurintricarboxylic Acid
author: Park, Jun-Gyu
date: 2019-04-12
txt: ./txt/cord-300379-db79kb5c.txt
cache: ./cache/cord-300379-db79kb5c.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/31031722/
doi: 10.3389/fmicb.2019.00718
summary:
  To quantify the ability of ATA to prevent ZIKV-induced apoptosis, tissue culture supernatants from ZIKV-infected Vero and A549 cells were harvested at 24, 48, and 72 h p.i. to measure the level of apoptotic signal as determined by caspase 3 and 7 activities ( Figure 5B) . ZIKV-infected cells showed FIGURE 4 | Aurintricarboxylic acid inhibition of ZIKV replication: Vero (A) and A549 (B) cells (24-well plate format, 2.5 × 10 5 cells/well, triplicates) were infected (MOI 0.1) with Paraiba/2015. In this study, we demonstrated that ATA (Figure 1 ) has limited toxicity (Figure 2) and an effective and dose-dependent antiviral activity against ZIKV infection (Figures 3, 4) in both monkey kidney epithelial Vero and human alveolar A549 cells. Notably, ATA can prevent ZIKV-induced CPE and apoptosis in both cell lines ( Figure 5 ) and has broad anti-viral activity against representative ZIKV strains from the African (Uganda/1947 and Nigeria/1968) and the Asian/American (Puerto Rico/2015 and French Polynesia/2013) lineages (Figure 6) .

abstract:
  Zika virus (ZIKV) is one of the recently emerging vector-borne viruses in humans and is responsible for severe congenital abnormalities such as microcephaly in the Western Hemisphere. Currently, only a few vaccine candidates and therapeutic drugs are being developed for the treatment of ZIKV infections, and as of yet none are commercially available. The polyanionic aromatic compound aurintricarboxylic acid (ATA) has been shown to have a broad-spectrum antimicrobial and antiviral activity. In this study, we evaluated ATA as a potential antiviral drug against ZIKV replication. The antiviral activity of ATA against ZIKV replication in vitro showed median inhibitory concentrations (IC(50)) of 13.87 ± 1.09 μM and 33.33 ± 1.13 μM in Vero and A549 cells, respectively; without showing any cytotoxic effect in both cell lines (median cytotoxic concentration (CC(50)) > 1,000 μM). Moreover, ATA protected both cell types from ZIKV-induced cytopathic effect (CPE) and apoptosis in a time- and concentration-dependent manner. In addition, pre-treatment of Vero cells with ATA for up to 72 h also resulted in effective suppression of ZIKV replication with similar IC(50). Importantly, the inhibitory effect of ATA on ZIKV infection was effective against strains of the African and Asian/American lineages, indicating that this inhibitory effect was not strain dependent. Overall, these results demonstrate that ATA has potent inhibitory activity against ZIKV replication and may be considered as a potential anti-ZIKV therapy for future clinical evaluation.


id: cord-305496-t8ykkekl
author: Stone, E. Taylor
date: 2020-08-21
txt: ./txt/cord-305496-t8ykkekl.txt
cache: ./cache/cord-305496-t8ykkekl.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.08.20.259838
doi: 10.1101/2020.08.20.259838
summary:
  One such tool for evaluating neutralizing antibody response is a 88 plaque/focus neutralization reduction test (PRNT/FRNT), which evaluates the ability of polyclonal 89 sera samples to prevent or reduce infection of a cell monolayer in vitro. We examined the impact of cell density on foci formation for both Vero WHO and Vero E6 cells 144 by plating identical dilutions of SARS-CoV-2 virus stocks on 96-well plates seeded with differing 145 numbers of WHO or E6 cells (3 × 104, 1.5 × 104 or 3 × 104 cells/well) one day prior to infection 146 of the cell monolayer. To determine the optimal time frame for infection of SARS-CoV-2 on a Vero WHO cell 172 monolayer to form individual foci, we tested a variety of incubation times. The FFA relies on an immunostaining protocol of an infected cell monolayer in order to 197 quantify infectious virus titer and is therefore dependent upon SARS-CoV-2-specific antibody 198

title:
  Characterization of cells susceptible to SARS-COV-2 and methods for detection of neutralizing antibody by focus forming assay

abstract:
  The SARS-CoV-2 outbreak and subsequent COVID-19 pandemic have highlighted the urgent need to determine what cells are susceptible to infection and for assays to detect and quantify SARS-CoV-2. Furthermore, the ongoing efforts for vaccine development have necessitated the development of rapid, high-throughput methods of quantifying infectious SARS-CoV-2, as well as the ability to screen human polyclonal sera samples for neutralizing antibodies against SARS-CoV-2. To this end, our lab has adapted focus forming assays for SARS-CoV-2 using Vero CCL-81 cells, referred to in this text as Vero WHO. Using the focus forming assay as the basis for screening cell susceptibility and to develop a focus reduction neutralization test. We have shown that this assay is a sensitive tool for determining SARS-CoV-2 neutralizing antibody titer in human, non-human primate, and mouse polyclonal sera following SARS-CoV-2 exposure. Additionally, we describe the viral growth kinetics of SARS-CoV-2 in a variety of different immortalized cell lines and demonstrate via human ACE2 and viral spike protein expression that these cell lines can support viral entry and replication.


id: cord-309469-2naxn580
author: An, Hongliu
date: 2019-01-05
txt: ./txt/cord-309469-2naxn580.txt
cache: ./cache/cord-309469-2naxn580.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.virol.2018.12.019
doi: 10.1016/j.virol.2018.12.019
summary:
  For example, polyadenylated nuclear (PAN) RNA encoded by Kaposi''s sarcoma-associated herpesvirus (Sun et al., 1996; Zhong and Ganem, 1997) , the~2-kb latency-associated transcript (LAT) expressed by Herpes simplex virus (Bloom, 2004 ), a con-served~5-kb intron expressed by Human cytomegalovirus (hCMV) (Kulesza and Shenk, 2004) , and a 7.2-kb RNA expressed by mouse CMV (Kulesza and Shenk, 2006) , U-rich RNAs (HSURs) produced by Herpesvirus saimiri (HVS), (Albrecht and Fleckenstein, 1992; Ensser and Fleckenstein, 2005) ; (3) Subgenomic ncRNAs from single-stranded RNA viruses by incomplete degradation of genomic RNA by the cellular 5-3′ exonuclease XRN1. The results confirmed previous report that IBV can synthesize sgRNA via template switch mediated by a noncanonical core sequence (Bentley et al., 2013) Notably, the mutant virus carrying four mutations (A27100U/A27111U/G27113C/G27114C) was also unable to produce ncRNA (Fig. 4) , suggesting these nucleotides are required for ncRNA production.

title:
  Identification and formation mechanism of a novel noncoding RNA produced by avian infectious bronchitis virus

abstract:
  Viral noncoding (nc) RNAs have been shown to play important roles in viral life cycle. Many viruses employ different mechanism to produce ncRNAs. Here, we report that coronavirus infectious bronchitis virus (IBV) produces a novel ncRNA in virus-infected cells. This ncRNA consists of 563 nucleotides excluding a poly(A) tail, is mainly derived from the 3′-untranslated region of IBV genome, and contains a 63-nt-long of terminal leader sequence derived from the 5′ end of the viral genome. Using mutagenesis and reverse genetics, we reveal that this ncRNA is a subgenomic RNA generated by discontinuous transcription mechanism.


id: cord-309934-kcyao9i9
title: Inhibition of SARS Coronavirus Infection In Vitro with Clinically Approved Antiviral Drugs
author: Tan, Emily L.C.
date: 2004-04-17
txt: ./txt/cord-309934-kcyao9i9.txt
cache: ./cache/cord-309934-kcyao9i9.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/15200845/
doi: 10.3201/eid1004.030458
summary:
  Here we report that certain interferon subtypes exhibit in vitro inhibitory activity against SARS-CoV and are candidates for follow-up studies in animal models and patients to determine their efficacy in vivo. A collection of 19 antiviral drugs was tested in the SARS-CoV CPE inhibition assay ( Table 2) . Because the criteria for ascertaining anti-SARS-CoV activity in this screen were set at 100% inhibition of CPE, and as high doses of interferons may result in severe clinical side effects, we chose to conduct further evaluations only in the interferons that showed complete inhibition from initial screen, namely, Wellferon, Multiferon, Betaferon, and Alferon. Betaferon, Alferon, Multiferon, Wellferon, and ribavirin inhibited CPE in SARS-CoV-infected Vero E6 cells, in decreasing order of potency. Ribavirin, a drug widely used in initial efforts to manage SARS infections, inhibited CPE completely at 500-5,000 µg/mL at virus loads of 100-10,000 PFU per well.

abstract:
  Severe acute respiratory syndrome (SARS) is an infectious disease caused by a newly identified human coronavirus (SARS-CoV). Currently, no effective drug exists to treat SARS-CoV infection. In this study, we investigated whether a panel of commercially available antiviral drugs exhibit in vitro anti–SARS-CoV activity. A drug-screening assay that scores for virus-induced cytopathic effects on cultured cells was used. Tested were 19 clinically approved compounds from several major antiviral pharmacologic classes: nucleoside analogs, interferons, protease inhibitors, reverse transcriptase inhibitors, and neuraminidase inhibitors. Complete inhibition of cytopathic effects of SARS-CoV in culture was observed for interferon subtypes, β-1b, α-n1, α-n3, and human leukocyte interferon α. These findings support clinical testing of approved interferons for the treatment of SARS.


id: cord-312899-ot5pvtbl
title: In vitro susceptibility of 10 clinical isolates of SARS coronavirus to selected antiviral compounds
author: Chen, F
date: 2004-09-30
txt: ./txt/cord-312899-ot5pvtbl.txt
cache: ./cache/cord-312899-ot5pvtbl.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S1386653204000551
doi: 10.1016/j.jcv.2004.03.003
summary:
  Commercial antiviral agents and pure chemical compounds extracted from traditional Chinese medicinal herbs were screened against 10 clinical isolates of SARS coronavirus by neutralisation tests with confirmation by plaque reduction assays. Interferon-beta-1a, leukocytic interferon-alpha, ribavirin, lopinavir, rimantadine, baicalin and glycyrrhizin showed antiviral activity. We report in this study on the in vitro antiviral susceptibility of 10 isolates of SARS coronavirus to commercially available antiviral agents and pure chemical compounds including baicalin, glycyrrhizin, and chlorogenic acid extracted from traditional Chinese herbs. Further testing by neutralization tests Table 3 Comparison of antiviral activity of 10 compounds against 10 strains of SARS-CoV in fRhK4 cell line, against the prototype strains (39849) with the other 9 isolates of SARS coronavirus against the active compounds confirmed detectable inhibitory activities for leukocytic interferon-alpha, interferon-beta-1a, ribavirin, lopinavir, rimantadine, and baicalin.

abstract:
  Abstract Effective antiviral agents are urgently needed to combat the possible return of severe acute respiratory syndrome (SARS). Commercial antiviral agents and pure chemical compounds extracted from traditional Chinese medicinal herbs were screened against 10 clinical isolates of SARS coronavirus by neutralisation tests with confirmation by plaque reduction assays. Interferon-beta-1a, leukocytic interferon-alpha, ribavirin, lopinavir, rimantadine, baicalin and glycyrrhizin showed antiviral activity. The two interferons were only active if the cell lines were pre-incubated with the drugs 16h before viral inoculation. Results were confirmed by plaque reduction assays. Antiviral activity varied with the use of different cell lines. Checkerboard assays for synergy were performed showing combinations of interferon beta-1a or leukocytic interferon-alpha with ribavirin are synergistic. Since the clinical and toxicity profiles of these agents are well known, they should be considered either singly or in combination for prophylaxis or treatment of SARS in randomised placebo controlled trials in future epidemics.


id: cord-313596-kc8loqyj
title: The Genome Landscape of the African Green Monkey Kidney-Derived Vero Cell Line
author: Osada, Naoki
date: 2014-09-28
txt: ./txt/cord-313596-kc8loqyj.txt
cache: ./cache/cord-313596-kc8loqyj.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/25267831/
doi: 10.1093/dnares/dsu029
summary:
  Detection of genomic rearrangements in the Vero JCRB0111 cell line Copy number variants were detected using the Control-FREEC software 32 with a 100-kb window size and 20-kb step size. To characterize SNVs in the Vero cell line nuclear genome, we mapped our paired-end reads to the reference genome of the rhesus macaque (M. Analysis of collected SRV-related short reads from all paired-end short reads of the Vero JCRB0111 cell line, followed by analyses of gene assignment and long terminal repeat (LTR) finding, identified the 8,367 bp complete SRV genome sequence. SRV variant sequences lacking the U3 and R regions of 3 0 LTR were instead detected in Vero ATCC CCL81-associated SRV, 47 while the results of this study suggested that some SRV copies in the Vero JCRB0111 cell genome were defective in the env-3 0 LTR region (Fig. 4) .

abstract:
  Continuous cell lines that originate from mammalian tissues serve as not only invaluable tools for life sciences, but also important animal cell substrates for the production of various types of biological pharmaceuticals. Vero cells are susceptible to various types of microbes and toxins and have widely contributed to not only microbiology, but also the production of vaccines for human use. We here showed the genome landscape of a Vero cell line, in which 25,877 putative protein-coding genes were identified in the 2.97-Gb genome sequence. A homozygous ∼9-Mb deletion on chromosome 12 caused the loss of the type I interferon gene cluster and cyclin-dependent kinase inhibitor genes in Vero cells. In addition, an ∼59-Mb loss of heterozygosity around this deleted region suggested that the homozygosity of the deletion was established by a large-scale conversion. Moreover, a genomic analysis of Vero cells revealed a female Chlorocebus sabaeus origin and proviral variations of the endogenous simian type D retrovirus. These results revealed the genomic basis for the non-tumourigenic permanent Vero cell lineage susceptible to various pathogens and will be useful for generating new sub-lines and developing new tools in the quality control of Vero cells.


id: cord-314546-fbddxbhd
author: Ko, Meehyun
date: 2020-08-16
txt: ./txt/cord-314546-fbddxbhd.txt
cache: ./cache/cord-314546-fbddxbhd.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32767684/
doi: 10.1002/jmv.26397
summary:
  Although nafamostat mesylate and camostat mesylate were not selected as potent antiviral drug candidates in our earlier study, we compared the antiviral efficacy of these drugs at this time in between Vero and Calu-3 cells following the discovery that TMPRSS2, a host protease necessary for priming viral spike glycoprotein, could be a target for COVID-19 antiviral development. 11 The discrepancy in IC 50 was specifically remarkable with nafamostat mesylate; the IC 50 decreased by approximately 6000 folds when the drug was used in the SARS-CoV-2-infected Calu-3 cells perhaps due to the dominant role of TMPRSS2-dependent viral entry in the Calu-3 human lung epithelial cells. In summary, we compared antiviral efficacy of the potential antiviral drug candidates against SARS-CoV-2 in between Vero and Calu-3 cells and found that nafamostat mesylate is the most potent antiviral drug candidate in vitro. Comparative analysis of antiviral efficacy of FDA-approved drugs against SARS-CoV-2 in human lung cells

title:
  Comparative analysis of antiviral efficacy of FDA‐approved drugs against SARS‐CoV‐2 in human lung cells

abstract:
  Drug repositioning represents an effective way to control the current COVID‐19 pandemic. Previously, we identified 24 FDA‐approved drugs which exhibited substantial antiviral effect against severe acute respiratory syndrome coronavirus 2 in Vero cells. Since antiviral efficacy could be altered in different cell lines, we developed an antiviral screening assay with human lung cells, which is more appropriate than Vero cell. The comparative analysis of antiviral activities revealed that nafamostat is the most potent drug in human lung cells (IC(50) = 0.0022 µM).


id: cord-316908-8ti75mru
author: Wei, Xiaona
date: 2020-02-10
txt: ./txt/cord-316908-8ti75mru.txt
cache: ./cache/cord-316908-8ti75mru.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32041637/
doi: 10.1186/s13567-020-0739-7
summary:
  Given that the differences in gene sequences and pathogenicity between classical and mutant strains of PEDV may lead to diverse invasion mechanisms, this study focused on the cellular entry pathways and cellular transport of the PEDV GI and GII subtype strains in Vero cells and IPEC-J2 cells. To clarify the specific endocytic pathways, systematic research methods were used and showed that PEDV enters cells via the clathrinand caveolae-mediated endocytosis pathways, in which dynamin II, clathrin heavy chain, Eps15, cholesterol, and caveolin-1 were indispensably involved. Our results showed that two the subtypes of PEDV utilized clathrin-, caveolae-, and lipid raft-mediated endocytosis to enter the Vero and IPEC-J2 cells, but the utilization efficiency of each endocytic pathway varied depending on the different genotypes and types of cells. We demonstrated that PEDV GI subtype GDS09 and GII subtype GDS01 strains could enter Vero and IPEC-J2 cells via the clathrin-, caveolae-, and lipid raft-mediated endocytosis pathways.

title:
  PEDV enters cells through clathrin-, caveolae-, and lipid raft-mediated endocytosis and traffics via the endo-/lysosome pathway

abstract:
  With the emergence of highly pathogenic variant strains, porcine epidemic diarrhea virus (PEDV) has led to significant economic loss in the global swine industry. Many studies have described how coronaviruses enter cells, but information on PEDV invasion strategies remains insufficient. Given that the differences in gene sequences and pathogenicity between classical and mutant strains of PEDV may lead to diverse invasion mechanisms, this study focused on the cellular entry pathways and cellular transport of the PEDV GI and GII subtype strains in Vero cells and IPEC-J2 cells. We first characterized the kinetics of PEDV entry into cells and found that the highest invasion rate of PEDV was approximately 33% in the IPEC-J2 cells and approximately 100% in the Vero cells. To clarify the specific endocytic pathways, systematic research methods were used and showed that PEDV enters cells via the clathrin- and caveolae-mediated endocytosis pathways, in which dynamin II, clathrin heavy chain, Eps15, cholesterol, and caveolin-1 were indispensably involved. In addition, lipid raft extraction assay showed that PEDV can also enter cells through lipid raft-mediated endocytosis. To investigate the trafficking of internalized PEDV, we found that PEDV entry into cells relied on low pH and internalized virions reached lysosomes through the early endosome–late endosome–lysosome pathway. The results concretely revealed the entry mechanisms of PEDV and provided an insightful theoretical basis for the further understanding of PEDV pathogenesis and guidance for new targets of antiviral drugs.


id: cord-323839-a4oejky0
author: Sasaki, Michihito
date: 2020-08-28
txt: ./txt/cord-323839-a4oejky0.txt
cache: ./cache/cord-323839-a4oejky0.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.08.28.271163
doi: 10.1101/2020.08.28.271163
summary:
  These results indicated that S gene mutants are resistant to the 1 5 9 treatment with TMPRRSS2 inhibitors, but are sensitive to antivirals that target post entry In an effort to understand the selection mechanisms underlying the generation of these 1 6 4 mutant variants, we estimated the frequency of S gene mutants in virus population of 1 6 5 SARS-CoV-2 that had undergone serial passage in cultured cells. In contrast, nucleotide sequence 1 7 2 deletions around the S1/S2 cleavage site corresponding to del1 and del2 mutants were 1 7 3 observed in all three biological replicates of SARS-CoV-2 populations passaged in Vero 1 7 4 cells (Fig. 5a) . Moreover, we must be very objective when interpreting the results 2 3 0 from studies using Vero-passaged virus, especially those focused on S protein cleavage, Cells were infected with either WT or S mutants of SARS-CoV-2 at an MOI of 1.

title:
  SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are generated in vitro during propagation in TMPRSS2-deficient cells

abstract:
  The spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains.


id: cord-329494-cdn52epy
title: Inhibition of Junín virus replication by small interfering RNAs
author: Artuso, María C.
date: 2009-07-08
txt: ./txt/cord-329494-cdn52epy.txt
cache: ./cache/cord-329494-cdn52epy.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.antiviral.2009.07.001
doi: 10.1016/j.antiviral.2009.07.001
summary:
  The efficacy of this Z2-siRNA against JUNV was also demonstrated in virus-infected cells, by testing infectious virus plaque formation (92.8% JUNV yield reduction), viral RNA level or antigen expression, as well as in cells transfected with Z-specific reporter plasmids (91% reduction in expression of Z-EGFP fusion protein). (E) Expression of viral antigens in Vero cells transfected with Z2-siRNA or X-siRNA and infected with JUNV was detected by immunofluorescence assay using a rabbit anti-JUNV polyclonal serum. The efficacy of this agent against JUNV was also demonstrated in virus-infected cells by testing infectious virus plaque formation, viral RNA or antigen expression, as well as in cells transfected with Z-specific reporter plasmids. The present study represents the first report of virus inhibition mediated by RNA interference for a New World arenavirus, a group in the family including four agents of severe viral hemorrhagic fevers in South America (JUNV, Machupo, Sabiá and Guanarito viruses).

abstract:
  Junín virus (JUNV), the etiological agent of the Argentine hemorrhagic fever, has a single-stranded RNA genome with ambisense expression which encodes for five proteins. In previous works we have demonstrated that the Z arenavirus matrix protein represents an attractive target for antiviral therapy. With the aim of studying a new alternative therapeutic mechanism, four Z-specific siRNAs (Z1- to Z4-siRNAs) were tested showing variable efficacy. The most effective inhibitor was Z2-siRNA targeted at the region encompassed by nt 179–197 of Z gene. The efficacy of this Z2-siRNA against JUNV was also demonstrated in virus-infected cells, by testing infectious virus plaque formation (92.8% JUNV yield reduction), viral RNA level or antigen expression, as well as in cells transfected with Z-specific reporter plasmids (91% reduction in expression of Z-EGFP fusion protein). Furthermore, the lack of effect of this Z-siRNA on the expression of other JUNV proteins, such as N and GPC, confirmed the specificity of action exerted by Z2-siRNA on Z transcript. Thus, the present study represents the first report of virus inhibition mediated by RNA interference for a New World arenavirus.


id: cord-330772-i7cfmw9x
author: Peng, Ju-Yi
date: 2019-12-03
txt: ./txt/cord-330772-i7cfmw9x.txt
cache: ./cache/cord-330772-i7cfmw9x.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/31797149/
doi: 10.1186/s13568-019-0916-0
summary:
  The in vitro toxicity and antiviral ability of the surfactin-like peptide in the BLFP crude extract against PEDV were evaluated using the Vero cells. To study the antiviral activity of BLFP crude extract against PEDV, the biosurfactants were added at different time points during the viral infection. No statically significant difference in the average daily gain was noted among all groups each week BLFP crude extract with PEDV-infected cells during the whole study. Similarly, extracellular viral RNA levels in PEDV-infected cells cultured with biosurfactants were significantly lower than those without BLFP crude extract 24 and 48 HPI (Fig. 8b) . b Extracellular viral RNA levels in the supernatants of PEDV-infected Vero cells treated with or without BLFP crude extract were determined by real-time reverse transcription (RT)-PCR. d Intracellular viral RNA levels in the supernatants of PEDV-infected Vero cells treated with or without BLFP crude extract were determined by real-time RT-PCR.

title:
  Evaluation of antiviral activity of Bacillus licheniformis-fermented products against porcine epidemic diarrhea virus

abstract:
  Bacillus licheniformis (B. licheniformis) is commonly used as probiotic and its secondary metabolites are attractive anti-microbial candidate. In the present study, we aimed to evaluate the antiviral activity of crude extracts from B. licheniformis against porcine epidemic diarrhea virus (PEDV), a highly contagious enveloped porcine virus that has caused great economic loss in pigs. In vivo, PEDV-infected piglets supplemented with air-dried solid state fermentative cultivate containing B. licheniformis-fermented products (BLFP) showed milder clinical symptoms and decreased viral shedding. Importantly, no significant systemic pathological lesions and no reduction in average daily gain were noted in pigs supplemented with the BLFP, which suggests that it is safe for use in pigs. In vitro experiments revealed that while B. licheniformis crude extracts exhibited no toxicity in Vero cells, co-cultivation of B. licheniformis crude extracts with PEDV significantly reduced viral infection and replication. Summarized current results suggest that the B. licheniformis-fermented products could be a novel candidate food additive for reducing the impact of PED on the swine industry.


id: cord-331094-22366b81
title: Potential Antiviral Options against SARS-CoV-2 Infection
author: Ianevski, Aleksandr
date: 2020-06-13
txt: ./txt/cord-331094-22366b81.txt
cache: ./cache/cord-331094-22366b81.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32545799/
doi: 10.3390/v12060642
summary:
  We also screened 136 safe-in-man broad-spectrum antivirals against the SARS-CoV-2 infection in Vero-E6 cells and identified nelfinavir, salinomycin, amodiaquine, obatoclax, emetine and homoharringtonine. After the initial screening, we identified apilimod, emetine, amodiaquine, obatoclax, homoharringtonine, salinomycin, arbidol, posaconazole and nelfinavir as compounds that rescued virus-infected cells from death (AUC from 285 to 585; Table S1 ). We next profiled transcriptional responses to nelfinavir, amodiaquine or both drugs in virus-or mock-infected Vero-E6 cells at 24 h. Anti-SARS-CoV-2 activity of safe-in man broad-spectrum antivirals in Vero-E6 cells. Here, we found that combinations of nelfinavir with salinomycin, amodiaquine, obatoclax, emetine or homoharringtonine were synergistic against SARS-CoV-2 in Vero-E6 cells. Thus, the amodiaquine and nelfinavir combination could result in better efficacy and decreased toxicity for the treatment of SARS-CoV-2 and perhaps other viral infections. Transcriptomic analysis of mock-and SARS-CoV-2-infected Vero-E6 cells treated with nelfinavir, amodiaquine or both drugs.

abstract:
  As of June 2020, the number of people infected with severe acute respiratory coronavirus 2 (SARS-CoV-2) continues to skyrocket, with more than 6.7 million cases worldwide. Both the World Health Organization (WHO) and United Nations (UN) has highlighted the need for better control of SARS-CoV-2 infections. However, developing novel virus-specific vaccines, monoclonal antibodies and antiviral drugs against SARS-CoV-2 can be time-consuming and costly. Convalescent sera and safe-in-man broad-spectrum antivirals (BSAAs) are readily available treatment options. Here, we developed a neutralization assay using SARS-CoV-2 strain and Vero-E6 cells. We identified the most potent sera from recovered patients for the treatment of SARS-CoV-2-infected patients. We also screened 136 safe-in-man broad-spectrum antivirals against the SARS-CoV-2 infection in Vero-E6 cells and identified nelfinavir, salinomycin, amodiaquine, obatoclax, emetine and homoharringtonine. We found that a combination of orally available virus-directed nelfinavir and host-directed amodiaquine exhibited the highest synergy. Finally, we developed a website to disseminate the knowledge on available and emerging treatments of COVID-19.


id: cord-331680-qlzhtxs0
title: Potential Opportunity of Antisense Therapy of COVID-19 on an in Vitro Model
author: Goryachev, A.N.
date: 2020-11-03
txt: ./txt/cord-331680-qlzhtxs0.txt
cache: ./cache/cord-331680-qlzhtxs0.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.11.02.363598
doi: 10.1101/2020.11.02.363598
summary:
  In accordance with the purpose of the study, the following tasks were set: -to select the nucleotide sequence of the virus that is supposed to be inhibited, -to carry out the synthesis of oligonucleotide, -to determine cytotoxicity and antiviral activity in an in vitro experiment on cell culture. The assessment of antiviral activity of the drug in addition to cytopathic action was also taken into account by reducing the infectious titer of the virus in the culture of Vero cells E6 according to PCR RNA SARS-CoV-2, determined by the threshold of the number of reaction cycles (cycle treshold, Ct) in various dilutions of the study drug. Determination of the antiviral efficacy of the antisense oligonucleotide according to the treatment scheme (administration of the drug 24 hours after infection) was taken into account by the decrease in the infectious titer of the virus in the culture of Vero E6 cells by the cytopathic effect.

abstract:
  Data on potential effectiveness and prospects of treatment of new coronavirus infection of COVID-19 caused by virus SARS-CoV-2 with the help of antisense oligonucleotides acting against RNA of virus on an in vitro model are given. The ability of antisense oligonucleotides to suppress viral replication in diseases caused by coronaviruses using the example of SARS and MERS is shown. The identity of the initial regulatory section of RNA of various coronaviruses was found within 50 - 100 nucleotides from the 5’-end, which allows using antisense suppression of this RNA fragment. A new RNA fragment of the virus present in all samples of coronovirus SARS-CoV-2 has been identified, the suppression of which with the help of an antisense oligonucleotide can be effective in the treatment of COVID-19. The study of the synthesized antisense oligonucleotide 5’ - AGCCGAGTGACAGCC ACACAG, complementary to the selected virus RNA sequence, was carried out. The low toxicity of the preparations of this group in the cell culture study and the ability to reduce viral load at high doses according to real time-PCR data are shown. The cytopathogenic dose exceeds 2 mg / ml. At a dosage of 1 mg / ml, viral replication is reduced by 5-13 times. Conclusions are made about the prospects of this direction and the feasibility of using the inhalation way of drug administration into the body.


id: cord-332276-gs80celr
author: Tan, Yee‐Joo
date: 2007-08-20
txt: ./txt/cord-332276-gs80celr.txt
cache: ./cache/cord-332276-gs80celr.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/17714515/
doi: 10.1111/j.1462-5822.2007.01034.x
summary:
  In two independent studies, it was demonstrated that the inhibition of apoptosis, either by caspase inhibitors or by overexpression of the Bcl-2 protein, did not affect SARS-CoV replication in Vero cells (Ren et al., 2005; Bordi et al., 2006) , suggesting that apoptosis does not play a role in facilitating viral release. The mechanisms for induction of apoptosis by these SARS-CoV proteins are unclear, although in some cases, it could be related to their abilities to interfere with cellular functions, such as blocking cell cycle progression, altering membrane permeability, activating signal transduction pathways, upregulating transcription factors and other regulatory genes (Table 1 ). (2007) The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) gene 7 products contribute to virus-induced apoptosis. Over-expression of 7a, a protein specifically encoded by the severe acute respiratory syndrome (SARS) -coronavirus, induces apoptosis via a caspase-dependent pathway

title:
  Regulation of cell death during infection by the severe acute respiratory syndrome coronavirus and other coronaviruses

abstract:
  Both apoptosis and necrosis have been observed in cells infected by various coronaviruses, suggesting that the regulation of cell death is important for viral replication and/or pathogenesis. Expeditious research on the severe acute respiratory syndrome (SARS) coronavirus, one of the latest discovered coronaviruses that infect humans, has provided valuable insights into the molecular aspects of cell‐death regulation during infection. Apoptosis was observed in vitro, while both apoptosis and necrosis were observed in tissues obtained from SARS patients. Viral proteins that can regulate apoptosis have been identified, and many of these also have the abilities to interfere with cellular functions. Occurrence of cell death in host cells during infection by other coronaviruses, such as the mouse hepatitis virus and transmissible porcine gastroenteritis virus, has also being extensively studied. The diverse cellular responses to infection revealed the complex manner by which coronaviruses affect cellular homeostasis and modulate cell death. As a result of the complex interplay between virus and host, infection of different cell types by the same virus does not necessarily activate the same cell‐death pathway. Continuing research will lead to a better understanding of the regulation of cell death during viral infection and the identification of novel antiviral targets.


id: cord-333208-tibtngy8
title: Antiviral Role of IFITM Proteins in African Swine Fever Virus Infection
author: Muñoz-Moreno, Raquel
date: 2016-04-26
txt: ./txt/cord-333208-tibtngy8.txt
cache: ./cache/cord-333208-tibtngy8.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1371/journal.pone.0154366
doi: 10.1371/journal.pone.0154366
summary:
  The interferon-induced transmembrane (IFITM) protein family is a group of antiviral restriction factors that impair flexibility and inhibit membrane fusion at the plasma or the endosomal membrane, restricting viral progression at entry. The role of IFITM2 in the inhibition of ASFV in Vero cells could be related to impaired endocytosis-mediated viral entry and alterations in the cholesterol efflux, suggesting that IFITM2 is acting at the late endosome, preventing the decapsidation stage of ASFV. Thus, our goal in the current work was to test whether the IFITM family of proteins affected early entry steps of ASFV infection in Vero cell cultures using the cell-adapted Ba71V isolate. Confocal microscopy experiments revealed that, IFITM1 was mainly distributed at the plasma membrane and to a lesser extent in perinuclear compartments, resembling endosomal structures (Fig 3C, lower left panel) , while endogenous IFITM1 was barely detected in Vero cells containing the empty vector (Fig 3C, upper left panel) .

abstract:
  The interferon-induced transmembrane (IFITM) protein family is a group of antiviral restriction factors that impair flexibility and inhibit membrane fusion at the plasma or the endosomal membrane, restricting viral progression at entry. While IFITMs are widely known to inhibit several single-stranded RNA viruses, there are limited reports available regarding their effect in double-stranded DNA viruses. In this work, we have analyzed a possible antiviral function of IFITMs against a double stranded DNA virus, the African swine fever virus (ASFV). Infection with cell-adapted ASFV isolate Ba71V is IFN sensitive and it induces IFITMs expression. Interestingly, high levels of IFITMs caused a collapse of the endosomal pathway to the perinuclear area. Given that ASFV entry is strongly dependent on endocytosis, we investigated whether IFITM expression could impair viral infection. Expression of IFITM1, 2 and 3 reduced virus infectivity in Vero cells, with IFITM2 and IFITM3 having an impact on viral entry/uncoating. The role of IFITM2 in the inhibition of ASFV in Vero cells could be related to impaired endocytosis-mediated viral entry and alterations in the cholesterol efflux, suggesting that IFITM2 is acting at the late endosome, preventing the decapsidation stage of ASFV.


id: cord-339012-4juhmjaj
author: Hou, Wei
date: 2020-07-28
txt: ./txt/cord-339012-4juhmjaj.txt
cache: ./cache/cord-339012-4juhmjaj.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.07.28.224576
doi: 10.1101/2020.07.28.224576
summary:
  Instead, PEDV down-regulated the expression of a set of zinc finger proteins with putative antiviral activity and enhanced the expression of the transmembrane serine protease gene TMPRSS13 (alias MSPL) to support its own infection by virus-cell membrane fusion (Shi et al, 2017, Viruses, 9(5):114). Furthermore, by comprehensive datamining in biological and chemical databases and consulting related literature we identified sets of PEDV-response genes with potential to influence i) the metabolism of biogenic amines (e.g. histamine), ii) the formation of cilia and "synaptic clefts" between cells, iii) epithelial mucus production, iv) platelets activation, and v) physiological processes in the body regulated by androgenic hormones (like blood pressure, salt/water balance and energy homeostasis). The detected sets of differential expressed genes (DEGs) for PEDV and MRV were analyzed by gene set enrichment analysis (GSEA) using functional bioinformatic programs to retrieve biological processes (pathways and Gene Ontology terms [GO-term]) and associations with chemical compounds, including drugs.

title:
  Rapid host response to an infection with Coronavirus. Study of transcriptional responses with Porcine Epidemic Diarrhea Virus

abstract:
  The transcriptional response in Vero cells (ATCC® CCL-81) infected with the coronavirus Porcine Epidemic Diarrhea Virus (PEDV) was measured by RNAseq analysis 4 and 6 hours after infection. Differential expressed genes (DEGs) in PEDV infected cells were compared to DEGs responding in Vero cells infected with Mammalian Orthoreovirus (MRV). Functional analysis of MRV and PEDV DEGs showed that MRV increased the expression level of several cytokines and chemokines (e.g. IL6, CXCL10, IL1A, CXCL8 [alias IL8]) and antiviral genes (e.g. IFI44, IFIT1, MX1, OASL), whereas for PEDV no enhanced expression was observed for these “hallmark” antiviral and immune effector genes. Pathway and Gene Ontology “enrichment analysis” revealed that PEDV infection did not stimulate expression of genes able to activate an acquired immune response, whereas MRV did so within 6h. Instead, PEDV down-regulated the expression of a set of zinc finger proteins with putative antiviral activity and enhanced the expression of the transmembrane serine protease gene TMPRSS13 (alias MSPL) to support its own infection by virus-cell membrane fusion (Shi et al, 2017, Viruses, 9(5):114). PEDV also down-regulated expression of Ectodysplasin A, a cytokine of the TNF-family able to activate the canonical NFKB-pathway responsible for transcription of inflammatory genes like IL1B, TNF, CXCL8 and PTGS2. The only 2 cytokine genes found up-regulated by PEDV were Cardiotrophin-1, an IL6-type cytokine with pleiotropic functions on different tissues and types of cells, and Endothelin 2, a neuroactive peptide with vasoconstrictive properties. Furthermore, by comprehensive datamining in biological and chemical databases and consulting related literature we identified sets of PEDV-response genes with potential to influence i) the metabolism of biogenic amines (e.g. histamine), ii) the formation of cilia and “synaptic clefts” between cells, iii) epithelial mucus production, iv) platelets activation, and v) physiological processes in the body regulated by androgenic hormones (like blood pressure, salt/water balance and energy homeostasis). The information in this study describing a “very early” response of epithelial cells to an infection with a coronavirus may provide pharmacologists, immunological and medical specialists additional insights in the underlying mechanisms of coronavirus associated severe clinical symptoms including those induced by SARS-CoV-2. This may help them to fine-tune therapeutic treatments and apply specific approved drugs to treat COVID-19 patients.


id: cord-343132-qqhivgkq
author: Chang-Liao, Wan-Ping
date: 2020-07-15
txt: ./txt/cord-343132-qqhivgkq.txt
cache: ./cache/cord-343132-qqhivgkq.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32760370/
doi: 10.3389/fmicb.2020.01578
summary:
  The results showed that the intracellular extracts of Ln. mesenteroides YPK30 possessed in vitro prophylactic, therapeutic, and direct-inhibitory effects against PEDV in the Vero cell model. As shown in Figure 3 , the metabolic activity of Vero cells pretreated with the intracellular extracts of Ln. mesenteroides, regardless of which strain, were similar to those pretreated with IFN-α2b (p > 0.05) but were significantly higher than the un-pretreated cells (p < 0.05), indicating that all the Ln. mesenteroides strains isolated from kefir grains possessed in vitro prophylactic effects against PEDV. durans, Lb. kefiri, Lc. lactis, and Ln. mesenteroides , were isolated from kefir grains, and the in vitro prophylactic effects of the intracellular extracts of these four species against PEDV infection in Vero cells were compared. Therefore, the in vitro prophylactic and therapeutic effects of the intracellular extracts of Ln. mesenteroides YPK30 against PEDV in Vero cells seem not be attributed to the direct interaction of bacterial components or metabolites with virus.

title:
  Isolation of a Leuconostoc mesenteroides Strain With Anti-Porcine Epidemic Diarrhea Virus Activities From Kefir Grains

abstract:
  Swine grown under commercial conditions are vulnerable to environmental exposure to several viruses, which may cause infectious diseases and spread easily and rapidly, resulting in significant economic losses in animal husbandry. Previous studies have suggested that probiotics seem to be a new and promising alternative to vaccinations to protect animals against potential viral infections. In this study, we used the Vero cell culture model of infection to study porcine epidemic diarrhea virus (PEDV). We screened lactic acid bacteria (LAB) with anti-PEDV potential from kefir grains, which are starter cultures used to ferment milk into kefir. Twenty-nine LAB strains were isolated and identified as Enterococcus durans, Lactobacillus kefiri, Lactococcus lactis, and Leuconostoc mesenteroides, according to 16S ribosomal RNA (rRNA) and rpoA gene sequence analyses. The anti-PEDV activities of the LAB intracellular extracts were compared, and the intracellular extracts of Ln. mesenteroides showed higher anti-PEDV activities than that of the other species. Among the Ln. mesenteroides strains, a strain designated YPK30 showed a higher growth rate than that of the other strains and was further evaluated for its anti-PEDV activity. The results showed that the intracellular extracts of Ln. mesenteroides YPK30 possessed in vitro prophylactic, therapeutic, and direct-inhibitory effects against PEDV in the Vero cell model. The expression levels of Type 1 interferon (IFN)-dependent genes, including Myxovirus resistance 1 (MX1) and interferon-stimulated gene 15 (ISG15), were significantly increased after treatment with intracellular extracts of Ln. mesenteroides YPK30 for 24 h. Such expression suggests that the anti-PEDV activity of Ln. mesenteroides YPK30 could be attributed to its up-regulatory effect on the expression of MX1 and ISG15 genes. These results suggested that Ln. mesenteroides YPK30 has the potential to provide some levels of host protection against PEDV infections.


id: cord-343515-fad1yyqx
title: Inhibition of SARS–CoV-2 by type I and type III interferons
author: Felgenhauer, Ulrike
date: 2020-10-09
txt: ./txt/cord-343515-fad1yyqx.txt
cache: ./cache/cord-343515-fad1yyqx.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1074/jbc.ac120.013788
doi: 10.1074/jbc.ac120.013788
summary:
  For SARS-CoV-2 (dark gray bars), statistically significant negative correlation coefficients (CC) were obtained for both cell lines, indicating that viral replication is increasingly inhibited by IFN-a. Observations were similar when the input MOI was reduced to 0.001 (Fig. S1 ), except that titers of SARS-CoV-1 in Calu-3 cells were already very low in the absence of any IFN-a, resulting in a nonsignificant effect of additional IFN. Our data thus indicate that (i) if anything, ruxolitinib is an enhancer rather than an inhibitor of SARS-CoV-2 multiplication, and (ii) the boosting effect is most likely due to inhibition of the antiviral JAK/ STAT signaling pathway, because it is not present in the IFN induction-deficient Vero E6 cells. For the statistical testing of the dose-response effect of IFN (type I and III) against SARS-coronaviruses, the typical regression procedures were not applicable because of several values below the detection limit and some ties in the data.

abstract:
  The recently emerged severe acute respiratory syndrome coronavirus-2 (SARS–CoV-2) is the causative agent of the devastating COVID-19 lung disease pandemic. Here, we tested the inhibitory activities of the antiviral interferons of type I (IFN-α) and type III (IFN-λ) against SARS–CoV-2 and compared them with those against SARS–CoV-1, which emerged in 2003. Using two mammalian epithelial cell lines (human Calu-3 and simian Vero E6), we found that both IFNs dose-dependently inhibit SARS–CoV-2. In contrast, SARS–CoV-1 was restricted only by IFN-α in these cell lines. SARS–CoV-2 generally exhibited a broader IFN sensitivity than SARS–CoV-1. Moreover, ruxolitinib, an inhibitor of IFN-triggered Janus kinase/signal transducer and activator of transcription signaling, boosted SARS–CoV-2 replication in the IFN-competent Calu-3 cells. We conclude that SARS–CoV-2 is sensitive to exogenously added IFNs. This finding suggests that type I and especially the less adverse effect–prone type III IFN are good candidates for the management of COVID-19.


id: cord-345689-5ns1onkw
author: Kusters, Inca C.
date: 2009-01-30
txt: ./txt/cord-345689-5ns1onkw.txt
cache: ./cache/cord-345689-5ns1onkw.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/B9780123694089000111
doi: 10.1016/b978-0-12-369408-9.00011-1
summary:
  Taking into account all the uncertainties and anticipating the worst-case scenario, many laboratories and vaccine manufacturers started working on a vaccine approach against SARS infection, largely based on what was known from animal CoVs. In this chapter, we will discuss the necessity for international cooperation and the importance of discretionary funding for rapidly developing a prototype vaccine candidate. When the laboratory work on the SARS-CoV vaccine development started, no data were available on the inactivation characteristics of the virus. The results from the experiments performed to evaluate the viral loss of the SARS-CoV due to drying on glass surface were also surprising: 35 -42 days were necessary to inactivate the virus to the detection limit of the technique. Based on our experience to date, the inactivated, adjuvanted SARS-CoV prototype vaccine seems to be a good candidate for further evaluation in Phase 1 studies.

title:
  Manufacturing Vaccines for an Emerging Viral Infection–Specific Issues Associated with the Development of a Prototype SARS Vaccine

abstract:
  Abstract The world was struck by surprise when a Severe Acute Respiratory Syndrome (SARS) epidemic started in 2003 in China. This disease had never been observed in man before; the SARS-Coronavirus causing the disease was unknown. With the uncertainty about the future impact of this epidemic, an important international collaboration started spontaneously sharing scientific knowledge and reagents. Resources became quickly available, and public and private efforts were undertaken to develop rapidly a vaccine. We will discuss here the importance of the international collaboration and the availability of funding. Moreover, we will review the most important and challenging steps during the industrial development of the SARS vaccine highlighting the difficulties in terms of safety working with such a highly pathogenic, unknown virus. We will emphasize the industrial perspectives on inactivation and decontamination experiments, the selection of the most promising vaccine candidate, the production process and the choice and use of animal models in such a pressing and difficult situation. Finally, we will briefly review the unique regulatory environment created during this period for the development of a SARS vaccine.


id: cord-349689-njb6619x
author: Khan, Mohsin
date: 2010-03-24
txt: ./txt/cord-349689-njb6619x.txt
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url: https://www.ncbi.nlm.nih.gov/pubmed/20336760/
doi: 10.1002/jmv.21663
summary:
  The goal of the current study was to evaluate the doseand time-dependent effects of chloroquine on CHIKV replication, and to elucidate the mechanism of viral inhibition in Vero cells. Anti-viral activity was assessed by performing cell viability assays on cells that had been infected with CHIKV in the presence of various concentrations of ammonium chloride, ribavirin, or chloroquine. The mechanism of inhibition of CHIKV activity by chloroquine was assessed by comparing the effects of chloroquine to those of a known lysomotropic agent (ammonium chloride) that interferes with early stages of infection, and a standard anti-viral compound (ribavirin) that inhibits virus replication during late stages of infection. To determine the anti-CHIKV activity of chloroquine, we analyzed virus yield in Vero cells treated with drug as compared to untreated infected control cells. We demonstrated that chloroquine is an effective anti-viral agent against CHIKV infection in Vero cells in culture, thus, demonstrating the in vitro prophylactic and therapeutic potential of chloroquine in inhibiting CHIVK infection.

title:
  Assessment of in vitro prophylactic and therapeutic efficacy of chloroquine against chikungunya virus in vero cells

abstract:
  The resurgence of Chikungunya virus (CHIKV) in the form of unprecedented and explosive epidemics in India and the Indian Ocean islands after a gap of 32 years is a major public health concern. Currently, there is no specific therapy available to treat CHIKV infection. In the present study, the in vitro prophylactic and therapeutic effects of chloroquine on CHIKV replication in Vero cells were investigated. Inhibitory effects were observed when chloroquine was administered pre‐infection, post‐infection, and concurrent with infection, suggesting that chloroquine has prophylactic and therapeutic potential. The inhibitory effects were confirmed by performing a plaque reduction neutralization test (PRNT), real‐time reverse transcriptase (RT)‐PCR analysis of viral RNA levels, and cell viability assays. Chloroquine diminished CHIKV infection in a dose‐dependent manner, with an effective concentration range of 5–20 µM. Concurrent addition of drug with virus, or treatment of cells prior to infection drastically reduced virus infectivity and viral genome copy number by ≥99.99%. The maximum inhibitory effect of chloroquine was observed within 1–3 hr post‐infection (hpi), and treatment was ineffective once the virus successfully passed through the early stages of infection. The mechanism of inhibition of virus activity by chloroquine involved impaired endosomal‐mediated virus entry during early stages of virus replication, most likely through the prevention of endocytosis and/or endosomal acidification, based on a comparative evaluation using ammonium chloride, a known lysosomotropic agent. J. Med. Virol. 82: 817–824, 2010. © 2010 Wiley‐Liss, Inc.


id: cord-351377-xorj8tnz
author: Kao, Chi-Fei
date: 2018-10-04
txt: ./txt/cord-351377-xorj8tnz.txt
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url: https://www.ncbi.nlm.nih.gov/pubmed/30287770/
doi: 10.3390/v10100543
summary:
  Moreover, inoculation with iPEDVPT-P96 elicited comparable levels of anti-PEDV specific plasma IgG and fecal/salivary IgA, neutralizing antibody titers, and similar but less effective immunoprotection against the virulent PEDVPT-P5 challenge compared to the parental PEDVPT-P96. In the present study, an infectious cDNA clone of an attenuated G2b PEDV strain was successfully generated for the first time, and the in vitro and in vivo data indicate that iPEDVPT-P96 is further attenuated but remains immunogenic compared to its parental PEDVPT-P96 viral stock. While one piglet in the PEDVPT-P96 group showed intermittent loose diarrhea (score = 1) and viral shedding at 6 to 11 days post-inoculation (DPI) with the peak viral titer of 1.45 ± 3.24 log 10 RNA copies/mL at 8 DPI (Figure 4) , no evidence of PEDV-associated clinical signs and fecal viral shedding were demonstrated in both iPEDVPT-P96 and mock groups.

title:
  The Characterization of Immunoprotection Induced by a cDNA Clone Derived from the Attenuated Taiwan Porcine Epidemic Diarrhea Virus Pintung 52 Strain

abstract:
  The porcine epidemic diarrhea virus (PEDV) poses a great threat to the global swine industries and the unreliable protection induced by the currently available vaccines remains a major challenge. We previously generated a genogroup 2b (G2b) PEDV Taiwan Pintung 52 (PEDVPT) strain, PEDVPT-P96, and determined its promising host immune response against the virulent PEDVPT-P5 strain. To study the attenuation determinants of PEDVPT-P96 and establish a PEDVPT-P96-based recombinant vector as a vaccine platform for further antigenicity modification, iPEDVPT-P96, a full-length cDNA clone of PEDVPT-P96, was established. Comparing to the parental PEDVPT-P96 virus, the iPEDVPT-P96 virus showed efficient replication kinetics with a delayed decline of viral load and similar but much more uniform plaque sizes in Vero cells. In the 5-week-old piglet model, fecal viral shedding was observed in the PEDVPT-P96-inoculated piglets, whereas those inoculated with iPEDVPT-P96 showed neither detectable fecal viral shedding nor PEDV-associated clinical signs. Moreover, inoculation with iPEDVPT-P96 elicited comparable levels of anti-PEDV specific plasma IgG and fecal/salivary IgA, neutralizing antibody titers, and similar but less effective immunoprotection against the virulent PEDVPT-P5 challenge compared to the parental PEDVPT-P96. In the present study, an infectious cDNA clone of an attenuated G2b PEDV strain was successfully generated for the first time, and the in vitro and in vivo data indicate that iPEDVPT-P96 is further attenuated but remains immunogenic compared to its parental PEDVPT-P96 viral stock. The successful development of the iPEDVPT-P96 cDNA clone could allow for the manipulation of the viral genome to study viral pathogenesis and facilitate the rapid development of effective vaccines.


id: cord-351525-306syrrn
author: Yang, Yong-Le
date: 2019-11-26
txt: ./txt/cord-351525-306syrrn.txt
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url: https://doi.org/10.1128/jvi.01448-19
doi: 10.1128/jvi.01448-19
summary:
  title: Broad Cross-Species Infection of Cultured Cells by Bat HKU2-Related Swine Acute Diarrhea Syndrome Coronavirus and Identification of Its Replication in Murine Dendritic Cells In Vivo Highlight Its Potential for Diverse Interspecies Transmission We first demonstrated that SADS-CoV possesses a broad species tropism and is able to infect cell lines from diverse species, including bats, mice, rats, gerbils, hamsters, pigs, chickens, nonhuman primates, and humans. As a brief summary of the results, 21 of the 24 cell lines showed significant susceptibility to SADS-CoV infection, defined by efficient viral replication, antigen expression, and the appearance of cytopathic effect (CPE). As some cells did not display CPE after SADS-CoV infection, all cell lines were subsequently tested for viral M protein expression by immunofluorescence assay (IFA) Fig. 1) , revealing the same range as seen by CPE in the different cell lines (data not shown).

title:
  Broad Cross-Species Infection of Cultured Cells by Bat HKU2-Related Swine Acute Diarrhea Syndrome Coronavirus and Identification of Its Replication in Murine Dendritic Cells In Vivo Highlight Its Potential for Diverse Interspecies Transmission

abstract:
  Outbreaks of severe diarrhea in neonatal piglets in Guangdong, China, in 2017 resulted in the isolation and discovery of a novel swine enteric alphacoronavirus (SeACoV) derived from the species Rhinolophus bat coronavirus HKU2 (Y. Pan, X. Tian, P. Qin, B. Wang, et al., Vet Microbiol 211:15–21, 2017). SeACoV was later referred to as swine acute diarrhea syndrome CoV (SADS-CoV) by another group (P. Zhou, H. Fan, T. Lan, X.-L. Yang, et al., Nature 556:255–258, 2018). The present study was set up to investigate the potential species barriers of SADS-CoV in vitro and in vivo. We first demonstrated that SADS-CoV possesses a broad species tropism and is able to infect cell lines from diverse species, including bats, mice, rats, gerbils, hamsters, pigs, chickens, nonhuman primates, and humans. Trypsin contributes to but is not essential for SADS-CoV propagation in vitro. Furthermore, C57BL/6J mice were inoculated with the virus via oral or intraperitoneal routes. Although the mice exhibited only subclinical infection, they supported viral replication and prolonged infection in the spleen. SADS-CoV nonstructural proteins and double-stranded RNA were detected in splenocytes of the marginal zone on the edge of lymphatic follicles, indicating active replication of SADS-CoV in the mouse model. We identified that splenic dendritic cells (DCs) are the major targets of virus infection by immunofluorescence and flow cytometry approaches. Finally, we demonstrated that SADS-CoV does not utilize known CoV receptors for cellular entry. The ability of SADS-CoV to replicate in various cells lines from a broad range of species and the unexpected tropism for murine DCs provide important insights into the biology of this bat-origin CoV, highlighting its possible ability to cross interspecies barriers. IMPORTANCE Infections with bat-origin coronaviruses (CoVs) (severe acute respiratory syndrome CoV [SARS-CoV] and Middle East respiratory syndrome CoV [MERS-CoV]) have caused severe illness in humans after “host jump” events. Recently, a novel bat-HKU2-like CoV named swine acute diarrhea syndrome CoV (SADS-CoV) has emerged in southern China, causing lethal diarrhea in newborn piglets. It is important to assess the species barriers of SADS-CoV infection since the animal hosts (other than pigs and bats) and zoonotic potential are still unknown. An in vitro susceptibility study revealed a broad species tropism of SADS-CoV, including various rodent and human cell lines. We established a mouse model of SADS-CoV infection, identifying its active replication in splenic dendritic cells, which suggests that SADS-CoV has the potential to infect rodents. These findings highlight the potential cross-species transmissibility of SADS-CoV, although further surveillance in other animal populations is needed to fully understand the ecology of this bat-HKU2-origin CoV.


id: cord-352511-gkm7i62s
author: Yamada, Yoshiyuki
date: 2009-07-02
txt: ./txt/cord-352511-gkm7i62s.txt
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url: https://www.ncbi.nlm.nih.gov/pubmed/19572016/
doi: 10.1371/journal.pone.0006130
summary:
  title: Acquisition of Cell–Cell Fusion Activity by Amino Acid Substitutions in Spike Protein Determines the Infectivity of a Coronavirus in Cultured Cells Here we report that acquisition of the cell–cell fusion activity by amino acid mutations in the S protein determines the infectivity of IBV in cultured cells. This study demonstrates that acquisition of the cell–cell fusion activity in S protein determines the selection and/or adaptation of a coronavirus from chicken embryo to cultured cells of human and animal origins. In this study, we report that acquisition of the cell-cell fusion activity by point mutations in the spike (S) protein of avian coronavirus infectious bronchitis virus (IBV) plays a critical role in adaptation and/or selection of a variant that infects cultured cells. Sequence comparison of two S protein constructs, S(EP3) and S(CK), cloned from EP3 and CK-adapted IBV strains, respectively, showed amino acid substitutions at 31 positions (Fig. 1a) .

title:
  Acquisition of Cell–Cell Fusion Activity by Amino Acid Substitutions in Spike Protein Determines the Infectivity of a Coronavirus in Cultured Cells

abstract:
  Coronavirus host and cell specificities are determined by specific interactions between the viral spike (S) protein and host cell receptor(s). Avian coronavirus infectious bronchitis (IBV) has been adapted to embryonated chicken eggs, primary chicken kidney (CK) cells, monkey kidney cell line Vero, and other human and animal cells. Here we report that acquisition of the cell–cell fusion activity by amino acid mutations in the S protein determines the infectivity of IBV in cultured cells. Expression of S protein derived from Vero- and CK-adapted strains showed efficient induction of membrane fusion. However, expression of S protein cloned from the third passage of IBV in chicken embryo (EP3) did not show apparent syncytia formation. By construction of chimeric S constructs and site-directed mutagenesis, a point mutation (L857-F) at amino acid position 857 in the heptad repeat 1 region of S protein was shown to be responsible for its acquisition of the cell–cell fusion activity. Furthermore, a G405-D point mutation in the S1 domain, which was acquired during further propagation of Vero-adapted IBV in Vero cells, could enhance the cell–cell fusion activity of the protein. Re-introduction of L857 back to the S gene of Vero-adapted IBV allowed recovery of variants that contain the introduced L857. However, compensatory mutations in S1 and some distant regions of S2 were required for restoration of the cell–cell fusion activity of S protein carrying L857 and for the infectivity of the recovered variants in cultured cells. This study demonstrates that acquisition of the cell–cell fusion activity in S protein determines the selection and/or adaptation of a coronavirus from chicken embryo to cultured cells of human and animal origins.


id: cord-355440-20yq6zj0
author: Klingström, Jonas
date: 2006-09-06
txt: ./txt/cord-355440-20yq6zj0.txt
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extension: .txt
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url: https://www.ncbi.nlm.nih.gov/pubmed/16955520/
doi: 10.1002/eji.200535587
summary:
  In this study, we compared the effects of NO and peroxynitrite on hantavirus replication and free mature virions in vitro, and of inducible nitric oxide synthase (iNOS) in hantavirus‐infected suckling mice. In the present study, we investigated the effect of NO and peroxynitrite on hantavirus replication in Vero E6 cells and on free mature virions by using S-nitroso-Nacetylpenicillamine (SNAP; an NO-donor), 3-morpholinosydnonimine hydrochloride (SIN-1; a peroxynitrite donor), and by stimulating endogenous NO production by iNOS with cytokines. From cells treated with 50 lM SNAP, 85% less viable virus was obtained, whereas lower concentrations of SNAP had no detectable effect on the virus replication, as compared to NAP-treated or medium controls ( Fig. 2A , and data not shown). However, the finding that iNOS -/suckling mice had higher levels of replicating virus than controls is in line with the finding that cytokine-stimulated NO production inhibited hantavirus replication in Vero E6 cells.

title:
  Nitric oxide and peroxynitrite have different antiviral effects against hantavirus replication and free mature virions

abstract:
  Reactive nitrogen intermediates (RNI), like nitric oxide (NO) and peroxynitrite, have antiviral effects against certain viruses. Hantaviruses, like other members of the Bunyaviridae family, have previously not been shown to be sensitive to RNI. In this study, we compared the effects of NO and peroxynitrite on hantavirus replication and free mature virions in vitro, and of inducible nitric oxide synthase (iNOS) in hantavirus‐infected suckling mice. The NO‐generating compound S‐nitroso‐N‐acetylpenicillamine (SNAP), as well as cytokine‐induced NO, strongly inhibited hantavirus replication in Vero E6 cells, while pretreatment of free virions with SNAP only had a limited effect on their viability. In contrast, 3‐morpholinosydnonimine hydrochloride (SIN‐1), a peroxynitrite donor, inhibited virus replication only to a very low extent in vitro, but pretreatment of virus with SIN‐1 led to a considerably lowered viability of the virions. Infections of various human cell types per se did not induce NO production. The viral titers in iNOS(–/–) mice were higher compared to the controls, suggesting that NO inhibits hantavirus replication in vivo. In conclusion, we show that NO has strong antiviral effects on hantavirus replication, and peryoxynitrite on mature free virions, suggesting that different RNI can have different effects on various parts of the replication cycle for the same virus.


