key: cord-288701-nx9fg4yn
authors: Mari, Viviana; Losurdo, Michele; Lucente, Maria Stella; Lorusso, Eleonora; Elia, Gabriella; Martella, Vito; Patruno, Giovanni; Buonavoglia, Domenico; Decaro, Nicola
title: Multiplex real-time RT-PCR assay for bovine viral diarrhea virus type 1, type 2 and HoBi-like pestivirus
date: 2015-12-17
journal: J Virol Methods
DOI: 10.1016/j.jviromet.2015.12.003
sha: 
doc_id: 288701
cord_uid: nx9fg4yn

HoBi-like pestiviruses are emerging pestiviruses that infect cattle causing clinical forms overlapping to those induced by bovine viral diarrhea virus (BVDV) 1 and 2. As a consequence of their widespread distribution reported in recent years, molecular tools for rapid discrimination among pestiviruses infecting cattle are needed. The aim of the present study was to develop a multiplex real-time RT-PCR assay, based on the TaqMan technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging HoBi-like strains. The assay was found to be sensitive, specific and repeatable, ensuring detection of as few as 10(0)–10(1) viral RNA copies. No cross-reactions between different pestiviral species were observed even in samples artificially contaminated with more than one pestivirus. Analysis of field samples tested positive for BVDV-1, BVDV-2 or HoBi-like virus by a nested PCR protocol revealed that the developed TaqMan assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time RT-PCR assay previously developed for HoBi-like pestivirus detection showed cross-reactivity with few high-titre BVDV-2 samples.

The genus Pestivirus belongs to the Flaviviridae family and includes 4 recognised species: Bovine viral diarrhea virus 1 and 2 (BVDV-1 and BVDV-2), Classical swine fever virus (CSFV), and Border disease virus (BDV) (Simmonds et al., 2011) .

Pestiviruses have a positive single-stranded RNA genome, approximately 12.3 kb in length, composed by a single open reading frame (ORF), preceded and followed by 2 untranslated regions (5 and 3 UTR). The single ORF encodes for a polyprotein that gives origin to 12 smaller proteins by viral cleavage: N pro , C, E rns , E1, E2, p7, NS2/NS3, NS4A, NS4B, NS5A, NS5B (Simmonds et al., 2011) . Among all the genomic regions, the 5 UTR, N pro and E2 are widely used for comparison and phylogenetic analysis (Bauermann et al., 2013) . On the basis of the capacity to cause a cytopathic effect (cpe) in cell cultures, two BVDV biotypes are known, cytopathogenic (cp) and non-cytopathogenic (ncp), both involved in the pathogenesis of mucosal disease (MD), a fatal outcome of BVDV infection in persistently infected (PI) calves (Brownlie et al., 1984; Bolin et al., 1985) .

Pestivirus infection leads to significant economic losses worldwide showing a wide range of clinical signs, including mild upper respiratory signs, a transient decrease in circulating white blood cells, and a low-grade, short-term fever. However, there are virulent strains that cause severe respiratory disease, gastroenteric disorders, hemorrhagic syndrome, and pneumonia (Baker, 1995; Brownlie, 1990; Corapi et al., 1989) . Reproductive disorders represent one of the most important consequences of the BVDV infection (Houe et al., 2006) . Infection of pregnant cows during the first trimester of gestation with ncp BVDV strains, leads to failure of fertilization, return to estrus, abortion, congenital malformations, stillbirths, or the birth of PI animals which may appear normal or sometimes smaller and with congenital malformations. They are constantly viremic and BVDV seronegative, representing the main source of BVDV infection in the herd through their body fluids, so that eradication programs rely on the detection and slaughtering of these animals (Bauermann et al., 2013) .

Because of their RNA nature, pestivirus genomes accumulate several mutations during replication that may lead to the emergence of new strains, lineages or species (Kirkland et al., 2007;  Schirrmeier et al., 2004; Vilcek et al., 2005) . Recently, additional four pestivirus species have been proposed within the genus Pestivirus: Pestivirus of giraffe, Pronghorn virus, Bungowannah virus, and HoBi-like pestivirus (Bauermann et al., 2013) . The prototype strain (HoBi D32/00) of this emerging group of pestiviruses, also known as BVDV-3 or atypical pestiviruses (Larska et al., 2012; Liu et al., 2009) , was detected as contaminant of a batch of fetal bovine serum (FBS) imported from Brazil (Schirrmeier et al., 2004) . Natural infections by HoBi-like pestiviruses have been reported in South America (Cortez et al., 2006; Weber et al., 2014) , Asia (Kampa et al., 2010; Haider et al., 2014; Mishra et al., 2014) and Italy (Decaro et al., 2011 (Decaro et al., , 2012a (Decaro et al., , 2012c (Decaro et al., , 2013a (Decaro et al., , 2013b . The virus has been associated to respiratory disease (Decaro et al., 2011 (Decaro et al., , 2012c (Decaro et al., , 2013a and abortions (Decaro et al., 2012a) . Several molecular methods are available for the detection and characterisation of pestiviruses. Most of them either do not detect HoBi-like pestiviruses at all or detect them with low efficiency (Schirrmeier et al., 2004; Ståhl et al., 2007 Ståhl et al., , 2010 . A recently developed real-time RT-PCR assay is able to detect HoBi-like pestiviruses without providing any simultaneous detection of BVDV-1 and BVDV-2 (Liu et al., 2008) . However, the oligonucleotides used in this assay cross-react with high-titre BVDV-2 samples (Decaro et al., 2012b (Decaro et al., , 2013b . On the other hand, the nested PCR approach established by Sullivan and Akkina (1995) misclassifies HoBi-like viruses as BVDV-2 (Decaro et al., 2012b) . In order to detect all pestiviruses infecting cattle using a single tool, a novel real-time RT-PCR assay has been recently developed (Losurdo et al., 2015) . In addition, a nested PCR (nPCR) protocol has been set up for pestivirus typing (Decaro et al., 2012b) . This assay has been proven to be specific as no cross-reactions between BVDV-1, BVDV-2 and HoBi-like pestiviruses were observed. However, the test is time consuming and labor intensive and can be exposed to a high risk of crosscontamination due to the post-PCR manipulations. To overcome these limitations, we have developed a multiplex real-time RT-PCR assay for simultaneous detection of the different species of bovine pestiviruses, including the emerging HoBi-like group, allowing a rapid, sensitive and specific diagnosis of pestivirus infection and characterisation of the viral species.

The full-length genome of reference strains of BVDV-1, BVDV-2, HoBi-like pestivirus, BDV and CSFV were obtained from the GenBank database (http://www.ncbi.nlm.nih.gov/Genbank/index. html) and aligned using the BioEdit software package (Hall, 1999) .

Forward and reverse primers were designed using Primer3 software, version 4.0 (http://frodo.wi.mit.edu/primer3/) to amplify a 164-nt fragment of the 5 UTR conserved region of all aligned pestiviruses. Three specific probes to detect BVDV-1, BVDV-2 and HoBi-like pestivirus were designed using Beacon Designer Software, version 2.06 (Premier Biosoft International, Palo Alto, CA, USA) (Fig. 1) . Primers and probes were synthesised by Eurofins Genomics and are reported in Table 1 .

Pestivirus strains BVDV-1 NADL (courtesy of Dr Ferrari, Istituto Zooprofilattico Sperimentale di Lombardia ed Emilia Romagna, Brescia, Italy), BVDV-2 232/02 (Decaro et al., 2004) , HoBi-like strain 1/10-1 Italy (Decaro et al., 2011) , BDV BD91 (Buonavoglia et al., 1994) , and an RNA extract of the CSFV lapinised Chinese vaccine (courtesy of Istituto Zooprofilattico Sperimentale di Lombardia ed Emilia Romagna, Brescia, Italy) were used to evaluate the specificity of the test. To rule out any cross-reactivity between bovine pestiviruses and other bovine viral pathogens, isolates of the following viruses were also tested: bovine coronavirus (Decaro et al., 2008) , bovine rotaviruses (Pratelli et al., 1999) , bovine respiratory syncytial virus (vaccine strain BRSV/375, CattleMaster 4, Zoetis Italia srl), bovine parainfluenza virus (vaccine strain TS RLB 103, CattleMaster 4, Zoetis Italia srl), and bovine herpesvirus types 1 (Thiry et al., 2006) and 4 (Tempesta et al., 1996) .

A total of 159 pestivirus positive field samples were analysed. Ninety-eight samples were recruited from a previous study and had been already characterised by the nPCR assay (Decaro et al., 2012b) , whereas additional 61 specimens were detected more recently during an epidemiological survey for HoBi-like pestivirus (unpublished data). The analysed samples included 28 tissue samples from aborted fetuses, 23 respiratory specimens from calves with respiratory disease, 19 fecal samples from calves with enteritis and 89 EDTA-blood samples from PI animals.

RNA was extracted from all samples using QIAamp ® cador ® Pathogen Mini Kit (Qiagen S.p.A., Milan, Italy), according to manufacturer's instructions.

RNA standards for BVDV-1, BVDV-2 and HoBi-like pestivirus were obtained amplifying a fragment of the 5 UTR region of reference strains BVDV-1 NADL, BVDV-2 232/06 and HoBi-like pestivirus Italy-1/10-1, using the common forward primer 324 (Vilcek et al., 1994) and three different pestivirus specific reverse primers (BVD1-690R: 5 -TCTATGCACACATAAATGTGGTA-3 , BVD2-657R: 5 -ACTACCGGTCACTCTGCCAACTCTCCTA-3 , BVD3-692R: 5 -TCGGTACACACATACATGTGATA-3 ), designed using Primer3 software, version 0.4.0.

The RT-PCR products were cloned into TOPO ® XL PCR Cloning vector (Invitrogen Srl, Milan, Italy) and transcribed with RiboMAX TM Large Scale RNA Production System-T7 (Promega Italia, Milan, Italy) from the T7 promoter, according to the manufacturer's guidelines. After DNAse treatment, the transcripts were purified using QIAamp ® RNA Easy kit (Qiagen S.p.A., Milan, Italy) and quantified by spectrophotometric analysis. Ten-fold dilutions of the RNA transcripts, representing 10 0 to 10 9 copies RNA l −1 of template, were carried out in a mixed fecal/nasal swab suspension from a calf that tested PCR negative for pestivirus RNA (Sullivan and Akkina, 1995; Decaro et al., 2012b) . Aliquots of each dilution were frozen at −70 • C and used only once.

Reverse transcription of 1 l of duplicates of the standard dilutions and RNA extracts was carried out using GeneAmp ® RNA PCR kit (Life Technologies Italia Applera Italia, Monza, Italy) in a 20l reaction volume containing PCR buffer 1× (KCl 50 mM, Tris-HCl 10 mM, pH 8.3), MgCl 2 5 mM, 1 mM of each deoxynucleotide (dATP, dCTP, dGTP, dTTP), RNase Inhibitor 1 U, MuLV reverse transcriptase 2.5 U, random hexamers 2.5 U. Reverse-transcription was carried out at 42 • C for 30 min, followed by a denaturation step at 99 • C for 5 min.

The triplex real-time PCR targeting the 5 UTR gene of BVDV-1, BVDV-2 and HoBi-like pestivirus was performed on a CFX96 TM Real-Time System (Bio-Rad Laboratories Srl, Milan, Italy) in a 25-l reaction mixture containing 12.5 l of iTaq TM Universal Probes Supermix (Bio-Rad Laboratories Srl, Milan, Italy), 600 nM of primers Pesti-qF and Pesti-qR, 200 nM of probes BVD1-Pb and BVD3-Pb and 400 nM of probe BVD2-Pb, and 10 l of c-DNA. The thermal protocol consisted of activation of iTaq DNA polymerase at 95 • C for 10 min, followed by 45 cycles of denaturation at 95 • C for 15 s and annealing/extension at 60 • C for 1 min. The detection of the Table 1 Primers and TaqMan probes used in the multiplex real-time RT-PCR assay for discrimination of bovine pestiviruses and other oligonucleotides used in the study. increasing fluorescent signal was carried out during the extension step of the reaction and the data was analysed with the appropriate sequence detector software (Bio-Rad CFX Manager v. 3.1, Bio-Rad Laboratories Srl). In order to verify the absence of RNA losses during the extraction step and the presence of RT-PCR inhibitors in the RNA templates, an internal control (IC), consisting of an RNA synthetic transcript containing the M gene of canine coronavirus (CCoV) type II (Decaro et al., 2005) , was added to the lysis buffer (AVL buffer, QIAGEN S.p.A.) at a concentration of 10,000 RNA copies ml −1 of buffer prior to nucleic acid extraction. The fixed amount of the IC added to each sample had been calculated to give a mean C T value in a genotypespecific real-time RT-PCR assay (Decaro et al., 2005) of 34.18 with a S.D. of 0.65 as calculated by 50 separate runs. Samples in which the C T value for the IC was >35.48 (average plus 2 S.D.) were excluded from the analysis.

Specificity of the assay was evaluated by testing pestivirus reference strains and other bovine viruses including bovine respiratory syncytial virus, bovine coronavirus, bovine rotavirus, bovine herpesvirus 1 and 4 and bovine parainfluenza virus. Serial ten-fold dilutions of the BVDV-1, BVDV-2 and HoBi-like pestivirus standards containing from 10 0 to 10 9 copies of RNA transcripts and the correlate C T values were used to set the standard curves for respective absolute quantifications.

Bovine nasal and faecal swabs and EDTA-blood samples that had tested negative for pestivirus and distilled water were used as negative controls and blank, respectively.

The sensitivity of the multiplex real-time RT-PCR assay was evaluated using 10-fold dilutions of EDTA-blood samples containing about 10 7 , 10 6 and 10 7 copies of BVDV-1, BVDV-2 and HoBi-like pestivirus RNA, respectively, made in a EDTA-blood sample from a calf tested negative for BVDV. The same sample dilutions were submitted to nPCR (Decaro et al., 2012b) and to the HoBi-like TaqMan assay (Liu et al., 2008) for a comparison.

Intra-assay repeatability was evaluated testing 10 times the same samples in one experiment, and the inter-assay repeatability was verified repeating the experiment 10 times. Clinical samples containing virus amounts spanning the whole sensitivity limits of the multiplex real-time RT-PCR assay were selected for repeatability evaluation. Coefficients of variation (CVs) were calculated by dividing the standard deviation of each tested sample by its mean and multiplying that result by 100.

The detection of pestivirus RNA in clinical samples and RNA transcript dilutions was carried out using a nPCR protocol previously developed for the characterisation of bovine pestiviruses (Decaro et al., 2012b) . First-and second-step amplifications were carried out using SuperScript TM One-Step RT-PCR for Long Templates (Life Technologies Italia) and AmpliTaq Gold (Life Technologies Italia), as previously described (Decaro et al., 2012b) . Oligonucleotides are reported in Table 1 .

To compare the performance of the developed triplex assay with the only existing real-time RT-PCR assay claimed to detect specifically this group of viruses, all clinical samples were tested by means of the Liu's assay (Liu et al., 2008) . Reverse transcription and realtime PCR were carried out as described for the triplex assay using oligonucleotides listed in Table 1 .

No fluorescence signal was detected from either negative controls or distilled water and all of the other bovine pathogens, including the related pestiviruses BDV and CSFV, were not detected by the developed multiplex real-time RT-PCR. The assay was proven to be species specific since BVDV-1, BVDV-2 and HoBi-like pestivirus were correctly detected by the specific probes and no cross-reaction between the three pestiviruses was observed. The standard curves generated for each pestivirus using ten-fold dilutions of standard RNA covered a linear range of at least nine orders of magnitude (from 10 0 /10 1 to 10 9 copies of standard RNA) and linearity was observed over the entire quantification range (slopes of −3.294, −2.977 and −3.412 for BVDV-1, BVDV-2 and HoBi-like pestivirus, respectively). Coefficients of regression (R 2 ) were 0.996, 0.994 and 0.997 for BVDV-1, BVDV-2 and HoBi-like pestivirus, respectively.

The sensitivity of the assay was set at 10 0 RNA copies for BVDV-1 and at 10 1 RNA copies for BVDV-2 and HoBi-like pestivirus. Nested PCR had the same sensitivity in the case of BVDV-2 and HoBi-like pestivirus, but it was 1-log less sensitive than the TaqMan assay when BVDV-1 was processed. In addition, the specific TaqMan assay by Liu et al. (2008) was able to detect as few as 10 1 HoBi-like pestivirus RNA copies.

The repeatability was evaluated by calculating the intra-and interassay CVs. BVDV-1 CVs ranged from 9.48% (samples containing 4 × 10 2 copies RNA l −1 ) to 53.13% (1.8 × 10 3 copies RNA l −1 ); BVDV-2 CVs varied from 23.66% (1 × 10 5 copies RNA l −1 ) to 50.38% (4 × 10 3 copies RNA l −1 ); HoBi-like pestivirus CVs were between 9.01% (8 × 10 2 copies RNA l −1 ) and 21.50% (6 × 10 4 copies RNA l −1 ). Interassay CVs ranges were comprised between 24.89% (5 × 10 2 copies RNA l −1 ) and 47.65% (2 × 10 4 copies RNA l −1 ) for BVDV-1, between 16.63% 1 × 10 5 copies RNA l −1 ) and 49.68% (2 × 10 2 copies RNA l −1 ) for BVDV-2; between 13.18% (8 × 10 2 copies RNA l −1 ) and 16.73% (6 × 10 4 copies RNA l −1 ) for HoBilike pestivirus.

The IC was detected in all the examined samples, with C T values below the threshold value of 34.76, thus confirming the absence of RNA losses during nucleic acid extraction or DNA polymerase inhibition during real-time PCR.

In order to rule out any interference between the speciesspecific TaqMan probes contained in the same mix, a pestivirus negative EDTA-blood sample was also spiked with low (10 3 copies) and high (10 8 copies) concentrations of standard RNA of BVDV-1, BVDV-2 and HoBi-like pestivirus and the viral loads in the spiked samples were quantified using the developed assay. The RNA titres of the pestivirus species were calculated correctly, showing that no interference occurred during detection and quantitation of the different viruses contained in the same sample (data not shown).

By analysis of 159 field samples tested positive by nPCR (Decaro et al., 2012b) , there was a perfect agreement between gel-based and real-time RT-PCR assays. BVDV-1 and BVDV-2 were detected in 103 and 15 samples, respectively, whereas 41 specimens that had been collected from two different cattle herds in southern Italy tested positive for HoBi-like pestivirus.

The amount of RNA detected in the samples covered a wide range of copies per microlitre of template, ranging from 4.02 × 10 1 to 7.26 × 10 7 (BVDV-1), from 5.78 × 10 1 to 8.45 × 10 6 (BVDV-2) and from 3.72 × 10 1 to 2.75 × 10 7 (HoBi-like pestivirus).

The Liu assay (Liu et al., 2008) was able to type correctly all HoBi-like strains and did not recognise any of the BVDV-1 positive samples, but 3 out of 15 BVDV-2 strains were mistyped as HoBi-like viruses. The BVDV-2 titres of the mistyped samples were above 10 6 RNA copies l −1 of template.

Several PCR-based methods have been developed for sensitive and rapid detection of BVDV in clinical samples (Bhudevi and Weinstock, 2003; Young et al., 2006; La Rocca and Sandvik, 2009; Yan et al., 2011; Fan et al., 2012; Zhang et al., 2014; Losurdo et al., 2015) , but only few methods are currently available for unambiguous discrimination between BVDV-1 and BVDV-2 (Sullivan and Akkina, 1995; Letellier and Kerkhofs, 2003; Baxi et al., 2006; LeBlanc et al., 2010) . The emergence of HoBi-like pestivirus that causes clinical pictures overlapping those induced by BVDV-1 and BVDV-2 (Bauermann et al., 2012 (Bauermann et al., , 2013 (Bauermann et al., , 2014 , posed several concerns as for the ability of existing diagnostic methods to efficiently detect this emerging group of pestiviruses (Schirrmeier et al., 2004) . The panpestivirus RT-PCR developed by Vilcek et al. (1994) , which is commonly used for BVDV molecular screening, fails to detect or detects with low efficiency HoBi-like strains due to the presence of a mismatch at the 3 end of primer 324 that prevents the correct primer annealing. Other conventional and real-time RT-PCR protocols are able to detect the novel pestivirus but do not provide any virus characterisation, which is helpful to assess virus epidemiology (Elvander et al., 1998; Letellier et al., 1999; Gaede et al., 2005; Hoffmann et al., 2006; Losurdo et al., 2015) . A Taq-Man assay that was claimed to be specific for HoBi-like pestivirus was recently developed (Liu et al., 2008) , but this assay could not discriminate simultaneously BVDV-1 and BVDV-2 and showed partial cross-reaction with high-titre BVDV-2 samples (Decaro et al., 2012b (Decaro et al., , 2013b . This cross-reaction was confirmed by the present study since 3 out 15 BVDV-2 positive samples reacted as HoBi-like strains when tested by the Liu's assay.

Recently, a nPCR assay was established for simultaneous discrimination of all pestiviruses infecting cattle, including HoBi-like pestivirus (Decaro et al., 2012b) . This method was specific and reliable, but also cumbersome as it requires two separate steps, an RT-PCR followed by the nested amplification; in addition, it presents a certain risk of cross-contamination between positive and negative samples.

To overcome the limitations of existing methods, we have developed a triplex real-time RT-PCR assay, based on the Taq-Man chemistry, which was able to differentiate efficiently BVDV-1, BVDV-2 and HoBi-like pestivirus. The assay was proven to be repeatable and linear over a range of at least 9 orders of magnitude, from 10 0 /10 1 to 10 9 RNA copies, thus ensuring an accurate measurement of pestivirus RNA loads in clinical samples. In comparison with nPCR, the real-time RT-PCR assay was equally or slightly more sensitive and less time-consuming. Labeling the speciesspecific probes with different fluorophores enables a correct characterisation of the pestiviral strains contained in clinical samples. In addition, the 96-well format of the real-time PCR plates ensures a high throughput, with the chance to test simultaneously several samples, which is useful for large-scale epidemiological surveys. The developed assay is a closed system in which the tube is never opened post-amplification, and this reduces the possibility of cross-contamination of new samples with previously amplified products. A certain carryover may occur due to the separation between RT and fluorogenic PCR, but we preferred a two-step assay, since one-tube methods are less sensitive than a two-step RT-PCR procedure (Nakamura et al., 1993; Bustin, 2000) and the risk of RNA degradation is increased if analyses are performed over a long period of time (Postollec et al., 2011) . In order to further reduce the risk of contamination, we have strictly separated the different working steps and carried out pipetting in different laminar flow hoods. In addition, the two-step assay requires more work, thus reducing the laboratory capacity. Another limitation of the study is that the analysed pestiviral strains were geographically homogeneous, so that theoretically less common subtypes or divergent viruses circulating in different countries could not be correctly characterised. However, sequence alignment of the oligonucleotide binding regions showed that they are conserved within the same viral species for BVDV-1 and BVDV-2. As for HoBi-like viruses, even the more divergent strains recently identified in Asia (Mishra et al., 2014; Haider et al., 2014) displayed only few mismatches, which should be tolerated by the oligonucleotides, including the TaqMan probe (Fig. 1) . Anyway, the assay needs to be validated with those divergent strains that seem to be widespread in the Asian continent. The developed assay does not recognise BDV, which was recently detected in cattle (Strong et al., 2010; Braun et al., 2013) . However, at the moment the epidemiological situation does not require including BDV in the diagnostic algorithm for pestivirus detection in cattle.

In conclusion, the triplex TaqMan assay can be used in extensive epidemiological surveys, thus helping assess the best prophylactic measures against pestiviruses infecting cattle and facilitating eradication programmes against these viruses, which are responsible for marked economic losses in cattle herds worldwide.

The clinical manifestations of bovine viral diarrhea infection

Antigenic relationships between Bovine viral diarrhea virus 1 and 2 and HoBi virus: possible impacts on diagnosis and control

Hobi-like viruses: an emerging group of pestiviruses

Generation of calves persistently infected with HoBi-like pestivirus and comparison of methods for detection of these persistent infections

A one-step multiplex real-time RT-PCR for detection and typing of bovine viral diarrhea viruses

Detection of bovine viral diarrhea virus in formalin fixed paraffin embedded tissue sections by real time RT-PCR (Taqman)

Severe clinical disease induced in cattle persistently infected with noncytopathic bovine viral diarrhea virus by superinfection with cytopathic bovine viral diarrhea virus

Infection of cattle with Border disease virus by sheep on communal alpine pastures

Experimental production of fatal mucosal disease in cattle

The pathogenesis of bovine virus diarrhoea virus infections

Persistent pestivirus infection in sheep in Apulia (southern Italy)

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

Severe thrombocytopenia in young calves experimentally infected with noncytopathic bovine viral diarrhea virus

Genetic characterization of Brazilian bovine viral diarrhea virus isolates by partial nucleotide sequencing of the 5 -UTR region

Malattia delle mucose da BVDV tipo 2: descrizione di un focolaio in Puglia

Genotypespecific fluorogenic RT PCR assays for the detection and quantitation of canine coronavirus type I and type II RNA in faecal samples of dogs

Respiratory disease associated with bovine coronavirus infection in cattle herds in Southern Italy

Atypical pestivirus and severe respiratory disease in calves

Hobi'-like pestivirus in aborted bovine fetuses

A nested PCR approach for unambiguous typing of pestiviruses infecting cattle

Hobi-like pestivirus: both biotypes isolated from a diseased animal

Persistent infection caused by Hobi-like pestivirus

Detection of a Hobi-like virus in archival samples suggests circulation of this emerging pestivirus species in Europe prior to

Mucosal diseaselike syndrome in a calf persistently infected by Hobi-like pestivirus

An experimental study of a concurrent primary infection with bovine respiratory syncytial virus (BRSV) and bovine viral diarrhoea virus (BVDV) in calves

A reverse transcription loop-mediated isothermal amplification method for rapid detection of bovine viral diarrhea virus

Detection and species specific differentiation of pestiviruses using real-time RT-PCR

Identification and epidemiology of a rare HoBi-like pestivirus strain in Bangladesh

BioEdit: a user-friendly biological sequence alignment and analysis program for Windows 95/98/NT

A universal heterologous internal control system for duplex real-time RT-PCR assays used in a detection system for pestiviruses

Test strategies in bovine viral diarrhea virus control and eradication campaigns in Europe

Bovine herpesvirus type 1 (BHV-1) and bovine viral diarrhoea virus (BVDV) infections in dairy herds: self clearance and the detection of seroconversions against a new atypical pestivirus

Identification of a novel virus in pigs-Bungowannah virus: a possible new species of pestivirus

A short target real-time RT-PCR assay for detection of pestiviruses infecting cattle

Kinetics of single and dual infection of calves with an Asian atypical bovine pestivirus and a highly virulent strain of bovine viral diarrhoea virus 1

A novel combination of TaqMan RT-PCR and a suspension microarray assay for the detection and species identification of pestiviruses

Detection and genotyping of bovine diarrhea virus by reverse transcription-polymerase chain amplification of the 50 untranslated region

Real-time PCR for simultaneous detection and genotyping of bovine viral diarrhea virus

A TaqMan real-time RT-PCR assay for selective detection of atypical bovine pestiviruses in clinical samples and biological products

Phylogeny, classification and evolutionary insights into pestiviruses

Development of a TaqMan assay for sensitive detection of all pestiviruses infecting cattle, including the emerging HoBi-like strains

Identification and molecular characterization of novel and divergent HoBi-like pestiviruses from naturally infected cattle in India

Amplification and detection of a single molecule of human immunodeficiency virus RNA

Recent advances in quantitative PCR (qPCR) applications in food microbiology

Characterization by polymerase chain reaction of ruminant rotaviruses isolated in Italy

Genetic and antigenic characterization of an atypical pestivirus isolate, a putative member of a novel pestivirus species

Family Flaviviridae

Natural infection of cattle with an atypical 'HoBi'-like pestivirus-implications for BVD control and for the safety of biological products

Atypical 'HoBi'-like pestiviruses-Recent findings and implications

Antigenic and genetic characterisation of border disease viruses isolated from UK cattle

A nested polymerase chain reaction assay to differentiate pestiviruses

Evaluation of the pathogenicity of a bovine herpesvirus-4 (BHV-4) strain in pregnant rabbits

A live attenuated glycoprotein E negative bovine herpesvirus 1 vaccine induces a partial cross-protection against caprine herpesvirus 1 infection in goats

Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis

Characterization of a novel pestivirus originating from a pronghorn antelope

Clinical presentation resembling mucosal disease associated with 'HoBi'-like pestivirus in a field outbreak

Combination of reverse transcription real-time polymerase chain reaction and antigen capture enzymelinked immunosorbent assay for the detection of animals persistently infected with Bovine viral diarrhea virus

Real-time RT-PCR detection of Bovine Viral Diarrhoea virus in whole blood using an external RNA reference

Comparison of conventional RT-PCR, reverse-transcription loop-mediated isothermal amplification, and SYBR green I-based real-time RT-PCR in the rapid detection of bovine viral diarrhea virus nucleotide in contaminated commercial bovine sera batches

This work was supported by grants from Italian Ministry of University, project PON01 02589/1 "MicroMap -Sviluppo di una piattaforma tecnologica multiplex per diagnostica molecolare, portatile ed automatizzata, basata sulla logica strumentale del Labon-chip, in grado di consentire applicazioni multiparametriche in campo infettivologico".