key: cord-0839506-bne77cap
authors: Letellier, C; Kerkhofs, P; Wellemans, G; Vanopdenbosch, E
title: Detection and genotyping of bovine diarrhea virus by reverse transcription-polymerase chain amplification of the 5′ untranslated region
date: 1999-01-01
journal: Vet Microbiol
DOI: 10.1016/s0378-1135(98)00267-3
sha: 23c806983dd9270c82f7a51284fd361d55da7481
doc_id: 839506
cord_uid: bne77cap

A reverse-transcription polymerase chain reaction (RT-PCR) was developed to differentiate the bovine diarrhea virus (BVDV) from other pestiviruses, and to determine the genotype of the BVDV isolates. For this purpose, primer pairs were selected in the 5′ untranslated region (5′UTR). The primers B(E) and B(2) were located in highly conserved regions and were pestivirus-specific. Two primer pairs named B(3)B(4) and B(5)B(6) were specific of BVDV genotypes I and II, respectively. With this technique, an amplification product of the expected size was obtained with either the B(3)B(4) or the B(5)B(6) primer pairs for the 107 BVDV isolates tested but not for BDV or CSFV. For some isolates that were grouped in the genotype II, sequence analysis of the PCR fragments confirmed their classification into this genotype.

Bovine viral diarrhea is an important disease of cattle. Acute infection is usually mild and often subclinical, and is resolved with the appearance of neutralizing antibodies. Transplacental infection of the fetus with a noncytopathic BVDV strain during the first trimester of gestation may lead to persistently infected (p.i.), immunotolerant calves. When these animals are superinfected with an antigenically similar cytopathic strain they may suffer of the fatal mucosal disease (Brownlie, 1991) . Another fatal outcome of BVDV infection is associated with severe thrombocytopenia and hemorrhagic lesions. Several outbreaks of this syndrome have been reported, essentially in North America but Veterinary Microbiology 64 (1999) 155±167 cases have also been reported in Europe (Corapi et al., 1989; Broes et al., 1992; Pellerin et al., 1994) .

BVDV belongs to the pestivirus genus that also comprises border disease virus of sheep (BDV) and classical swine fever virus (CSFV). The classification of virus types was made according to the host-species that was infected. Pestiviruses are, however, able to cross the species barrier. BVDV can cross-infect cattle, sheep, goats and pigs (Carlsson, 1991; Paton et al., 1992) . BDV is an ovine pathogen that occasionally infects pigs (Roehe et al., 1992; Edwards et al., 1995; Vilcek and Belak, 1996) . Differentiation between CSFV and other pestiviruses can be accomplished by the use of CSFV-specific monoclonal antibodies (Mabs) but the search for ruminant pestivirus-specific Mabs has failed due to a great antigenic diversity (Edwards et al., 1991) .

RT-PCR has been proved to be a rapid and sensitive method to detect viral nucleic acids and this technique has been used by several investigators for the detection of pestiviruses using oligonucleotide primers located in conserved regions of the viral genome (Lopez et al., 1991; Katz et al., 1993; Ridpath et al., 1993; Wirz et al., 1993; Hofmann et al., 1994; Horner et al., 1995; Tajima et al., 1995; Canal et al., 1996) . Most of the PCR primers that were selected in the 5 0 UTR recognized the greatest number of pestivirus isolates but failed to differentiate BVDV from other pestiviruses Vilcek et al., 1994) . Specific detection of CSFV by RT-PCR has been described using primers located in the coding part of the genome or by the sequence analysis of the PCR product amplified using primers located in a highly conserved region of the 5 0 UTR Wirz et al., 1993; Hofmann et al., 1994) . The gp25, gp48, p54 and p80 regions did also serve as template for BVDV amplification. However, these RT-PCR tests failed to detect all BVDV isolates (Vilcek et al., 1994; Tajima et al., 1995) .

Based on the sequence comparison of 5 0 UTR, the BVDV isolates were subdivided in two genotypes. Sequence homology within each group was very high (>93%) while homology between group I and II droped near 74% (Pellerin et al., 1994; Ridpath et al., 1994) .

An accurate and fast differentiation between BVDV and another pestivirus is of great importance for the development of control measures, particularly when an outbreak of CSFV is suspected. Sensitive detection of BVDV contamination in FCS and cell culture would help diagnostic work and would improve the safety of veterinary and human vaccines.

The aim of this study was to develop a RT-PCR test that allowed the detection of BVDV and differentiation between both genotypes. Published 5 0 UTR sequences were aligned and two pairs of primers were selected. Belgium field BVDV isolates were tested by amplification with both sets of primers. Sequence analysis of the PCR products confirmed the appartenance of some field isolates to the genotype II.

The BVDV reference strain NADL was received from Dr. J.M. Aynaud (INRA, Thiverval, France), the C24V strain from Dr. Straver (CDI, Amsterdam), the 3620/Han89 isolate (Hewicker-Trautwein et al., 1995) from Dr. B. Liess and the New York and BDV Aveyron (Chappuis et al., 1986) strains were received from Prof. E. Thiry (University of Lie Áge). The viruses were propagated on bovine fetal kidney cells grown in minimum essential medium (MEM) supplemented with pestivirus free fetal bovine serum. CSFV was kindly provided by Dr. F. Koenen (CERVA).

Organs (lungs, intestines, brains) were obtained from 64 BVDV-positive cattle with respiratory, digestive or neurological symptoms. These were routine diagnostic samples obtained between 1991 and 1996 that were assessed as BVDV positive by a direct immunofluorescence test realized on frozen tissue sections. Homogenate extracts were then used for RNA isolation. Leucocytes pellets were prepared from 39 immunotolerant cattle which had previously been detected by an antigen capture ELISA kit (Rhone Me Ârieux).

Oligonucleotide primers were designed from the 5 0 UTR. The sequence of two pairs of primers, designated B 1 B 2 and B E B 2 , was located in a highly conserved region of BVDV, CSFV and BDV. These primers were tested for the amplification of pestivirus cDNAs. Their sequence, position according to the NADL genome sequence (Collett et al., 1988) and the expected size of the amplified products are presented in Table 1 .

Two pairs of primers were designed from the alignment of sequences published by others (Pellerin et al., 1994) . The sequences were conserved in a BVDV genotype but not between genotype I and II. The B 3 B 4 pair was tested for the amplification of BVDV type 1 sequences and the B 5 B 6 pair was used for BVDV type 2 amplification.

RNA was extracted using 1 ml TRIZOL (GIBCO BRL) according to the manufacturer's instructions. The extraction was accomplished using 200 ml of organ homogenate extracts or a pellet of leucocytes corresponding to 1.5 ml of heparinized and hemolyzed blood. The RNA was resuspended in 10 ml DEPC-treated water. The reverse transcription was carried out in a volume of 20 ml containing 50 mM Tris±HCl (pH 8.3), 75 mM KCl, 3 mM MgCl 2 , 10 mM DTT, 0.5 mM dNTP, 150 pmol of the reverse primer B 2 , 200 u M-MLV reverse transcriptase (GIBCO BRL) and 2 ml RNA. The cDNA was synthesized at 378C for 15 min and the enzyme was inactivated for 5 min at 958C.

The amplification of cDNA by PCR was carried out in a total volume of 50 ml containing 20 mM Tris±HCl (pH 8.4), 50 mM KCl, 3 mM MgCl 2 , 0.5 mM dNTP, 75 pmol of each primer, 2 ml cDNA and 2.5 u Taq DNA polymerase (GIBCO BRL). The reaction was heated in a thermocycle for 5 min at 958C and then submitted to 35 cycles of amplification. For the B 1 B 2 and B E B 2 primer pairs, the conditions of amplification were 1 min at 948C, 1 min at 578C, and 1 min at 728C. For the B 3 B 4 and B 5 B 6 primer pairs, the conditions were 1 min at 948C, 1 min at 518C, and 1 min at 728C. Amplified products were separated by electrophoresis in 1.5% agarose gel in Tris±borate EDTA buffer.

After electrophoresis of the B E B 2 amplification products, the agarose gel was soaked two times for 15 min in denaturation solution (1.5 M NaCl, 0.5 M NaOH), then in neutralization solution (0.5 M Tris±HCl (pH 7.5), 1.5 M NaCl). The DNA was then transfered to two positively charged nylon membranes (Boehringer) and the DNA was cross-linked by UV irradiation. The filter was incubated for 30 min at 448C in DIG Easy Hyb solution (Boehringer). Then 100 pmol of 3 0 DIG labeled B 4 or B 6 oligonucleotide (Eurogentec) was added and the incubation was continued overnight at 448C.

Filters were washed and the hybridized probes were detected using DIG luminescent detection kit (Boehringer) according to the manufacturer's instructions.

The PCR products obtained with the B 1 B 2 or B E B 2 primers were purified on Centricon 100 filters and were cloned in a PGEM-T vector (Promega). Plasmid DNA was purified using Qiagen tip 20 columns. A minimum of two clones were sequenced with the sequencing primer M13±21, dye terminator cycle sequencing kit (Perkin-Elmer) and ABI PRISM 310 automated cycle sequencer according to the manufacturer's recommendations.

Alignment of the sequences was realized using the PILEUP program included in the GCG software package (Devereux et al., 1984) . Distances were calculated by the Kimura 2-parameter method (Kimura, 1980) and used to construct trees using the GROWTREE program of the GCG. Sequence analysis was achieved using the Belgian EMBnet Node facility.

Total RNA extracted from infected cell cultures was submitted to RT-PCR by using the B E B 2 pestivirus-specific primers. A PCR product of the expected size (287 bp) was amplified from BVDV reference strains NADL, New York, C24V, 3620/Han89, BDV Aveyron and CSFV, and from the BVDV field isolate UVR420.

The results obtained for BVDV NADL, New York, UVR420, BDV Aveyron and CSFV are shown in Fig. 1(A) . In Fig. 1(B) , the same cDNAs were amplified with BVDV type I and type II-specific primer pairs B 3 B 4 and B 5 B 6 . A fragment of 221 bp was obtained with the former for BVDV NADL and New York, and with the latter for BVDV UVR420. This isolate was then used as positive control for the type II amplifications. No amplification product was obtained for BDV Aveyron and CSFV. The BVDV strains C24V and 3620/ Han89 were classified in the genotype I (data not shown).

The same results were obtained after Southern blot hybridization performed on the B E B 2 amplification products by using the DIG-labeled genotype-specific probes. The B E B 2 amplification products shown in Fig. 1(A) were hybridized with B 4 DIG probe in Fig. 2 (A) and with B 6 DIG probe in Fig. 2(B) . With the BVDV type I-specific probe, the amplification products from BVDV NADL and New York were detected. The BVDV type II-specific probe reacted only with the BVDV UVR420 isolate. The BDV and CSFV amplification fragments did not react with either BVDV-specific probe.

A total of 107 BVDV positive samples were tested. RNA was extracted from organs or from leukocytes of infected animals. All of the samples contained a pestivirus as demonstrated by a RT-PCR test using the pestivirus-specific primers B 1 B 2 or B E B 2 . The samples were also tested by both genotype-specific PCR and hybridization. The results are presented in Table 2 . Of a total of 64 organs (lungs, intestines, brains), 55 were assessed as genotype I and nine as genotype II. Two out of 39 immunotolerant cattle were infected with a genotype II isolate and two out of four laboratory viruses were also classified as genotype II. As a total, 13 of the 107 isolates tested belonged to the genotype II. Organs positive for infectious bovine rhinotracheitis, bovine parainfluenza type 3, coronavirus and rotavirus were used as controls. No amplification product was obtained (data not shown).

Type II viruses were isolated from 1991 to 1996 in Belgium (Table 3) . Four isolates came from calves with respiratory disease, one from the intestine of a calf. Three viruses were isolated from the brain of animals showing neurological symptoms. Two genotype II viruses were isolated from the blood of immunotolerant animals and only one virus was isolated from a case of haemorrhagic syndrome (Broes et al., 1992) .

The alignment of nucleotide sequence of a 168 bp DNA fragment, flanked by primers B 1 and B 2 is presented in Fig. 3 . The sequences of 10 Belgian type II isolates were compared to other genotype II sequences extracted from a genebank: 890, isolated from a haemorrhagic syndrome outbreak (Pellerin et al., 1994) , CPA virus isolated from cell culture (Harasawa, 1996) , the ovine pestivirus 175 375 isolated in the UK (Vilcek et al., 1997) . Reference BVDV type I strains NADL and New York and three Belgian virus isolated from organs (ZVD278, ZVR711) and from a p.i. calf were also included in the comparison.

The sequence alignment confirmed the classification of all the isolates. The sequence of the region corresponding to the oligonucleotides B 4 and B 6 , represented in Fig. 3 by a box, showed a high degree of conservation inside both genotypes, and were divergent between the genotypes.

A phylogenetic tree was constructed and is presented in Fig. 4 . Genotype II viruses were segregated into two groups. The first englobed eight Belgian isolates which showed very little genetic divergence. In the other group were classified viruses isolated from cell culture (CPA), from the USA, the UK and two Belgian isolates. Interestingly, one of them (BSE921) was nearly identical to the CPA virus.

The ZVD278, ZVR711 and p.i. viruses were classified in genotype I. The ZVD278 isolate showed a strong homology to the New York strain.

RT-PCR tests and sequence analysis of the p20 and E2 regions were used by several authors to classify the pestiviruses. Using these techniques, four genotypes were identified. The CSFV group was restricted to swine, whereas BVDV types I and II included strains isolated from cattle, sheep, goats and wild ruminants. The BDV genotype comprised strains that were isolated from sheep and pigs (Becher et al., 1995 (Becher et al., , 1997 van Rijn et al., 1997; Vilcek et al., 1997) . This classification did not correlate with the hostspecies origin but was supported by serological investigations (Dekker et al., 1995; Paton et al., 1995) . This accurate method cannot however be used for diagnostic work.

Specific primers located in the 5 0 UTR region were used by others for the diagnosis of BVDV infection by RT-PCR. Canal et al. (1996) reported the use of a conserved antisense oligonucleotide and two sense primers, CSFV and ruminant-pestivirus-specific, respectively, to distinguish these viruses. Another set of primers was designed by Sandvik et al. (1997) . PCR fragments from BVDV types I and II originating from different host-species were successfully amplified.

Here, we describe a differential system to discriminate between BVDV and other pestiviruses and to determine the genotype of the BVDV isolates, using conserved regions Fig. 3 . Alignment of nucleotide sequences from the 5 0 UTR of BVDV types I and II isolates. The sequences are flanked by primers B 1 and B 2 . Genotype II sequences are presented above the horizontal line and genotype I below. Sequence of the B4 and B6 oligonucleotides is boxed. located in the 5 0 UTR. The sequence of the primer used for the cDNA synthesis was conserved between the pestiviruses. This cDNA could thus be used in pestivirus-specific but also BVDV-specific PCR. The primers B E and B 2 have already been used by others and were designed to detect all the pestiviruses (Vilcek et al., 1994) . Based on the search for genotype-conserved regions, two primer pairs were selected. This allowed us to determine the genotype of the BVDV isolates.

The RT-PCR tests were validated with reference BVDV, BDV and CSFV strains. With a combination of PCR reactions, the BVDV strains could be differentiated from BDV and CSFV and the genotype of BVD viruses could be determined. No cross-reactivity was detected between the type-specific primers. Then, the PCR assays were used to characterise field isolates that were previously shown BVDV-positive by other techniques. The RNA extraction was performed on organ homogenate extracts or on leukocytes. This excluded a BVDV contamination during cell culture multiplication. Among the 107 samples examined, 94 were classified as genotype I and 13 as genotype II.

From the 13 genotype II viruses that were obtained, five were isolated from organs, two from the blood of p.i. animals and for two other isolates, we were not able to trace the origin of the virus. Only one virus was isolated from a case of hemorrhagic syndrome. Eight BVDV-positive brains were also tested. Three of them harboured genotype II viruses. Our hypothesis is that these were p.i. animals. Indeed, the presence of BVDV in the central nervous system (CNS) has been described and this seems to be an important location for the persistence of BVDV. Primary infection of the CNS most likely occurs across the blood±brain barrier (Fernandez et al., 1989; Hewicker et al., 1990; Hewicker-Trautwein et al., 1992) . Other experiments showed that BVDV was found in the CNS of all p.i. animal tested, without causing obvious cellular destruction (Wo Èhrmann et al., 1992) .

Ten genotype II and three genotype I isolates were further analyzed by sequencing the amplification DNA fragment. Based on pairwise alignment, conservation of the sequences inside the genotype II group was at least 92%. A minimum of 88% homology was found inside the genotype I group. When NADL strain was excluded from the comparison, the homology rose to 95%, suggesting that the Belgian isolates tested were closer to genotype Ib than Ia strains. The homology between genotype I and II was approximately 75%.

The greatest divergence between genotype II isolates was between UVD493 and 890, which were both isolated from a haemorrhagic syndrome.

Our results demonstrate that RT-PCR could be performed on RNA extracted from organ samples. Furthermore, the BVDV genotype could be determined. The same approach has been used by Sullivan and Akkina (1995) . Consensus primers located in the gp48 region were tested on bovine and ovine isolates, whereas combination of specific primers allowed the differentiation between BVDV type I, type II and BDV.

BVDV has been known not only as a veterinary pathogen but also as a common contaminant in cell culture work and in live vaccines for human, bovine and swine use (Harasawa and Tomiyama, 1994; Harasawa, 1995; Harasawa and Mizusawa, 1995) . Genotype II BVDV has been described as a contaminant of FBS (Ridpath et al., 1994) , of cell lines (Harasawa, 1996) , IFN for human use (Harasawa and Sasaki, 1995) and live virus vaccine for human use (Harasawa, 1994) .

The origin of the Belgian type II isolates is not known. The RNA was extracted from organs or leukocytes avoiding a possible contamination of the samples by the cell line or FBS. However, we cannot exclude the introduction of these strains via live vaccine for bovine use.

Epizootical problems due to the circulation of genotype II viruses in Belgium have not yet been encountered. However, these viruses are antigenically different from classical BVDV viruses (Ridpath et al., 1994) . The complete sequence of a genotype II virus has been published showing that the weak homology with type I strains extended through the complete genome. The gp53 protein had less than 70% identity with the type I counterpart (Ridpath and Bolin, 1995) . Recently, genotype II strains have been isolated in Japan. No cross-neutralization was observed between these type II and type I isolates (Nagai et al., 1998) . One should thus take account of these viruses for the development of control programs and vaccines against BVDV.

Further characterization of border disease virus isolates: Evidence for the presence of more than three species within the genus pestivirus

Phylogenic analysis of pestiviruses from domestic and wild ruminants

Syndrome he Âmorragique chez des bovins infecte Âs par le virus de la diarrhe Âe bovine (BVD/MD)

Differentiation of classical swine fever virus from ruminant pestiviruses by reverse transcription and polymerase chain reaction

Border disease in sheep caused by transmission of virus from cattle persistently infected with BVDV

Etudes se Ârologiques et immunologiques re Âalise Âes a Á la suite de l'isolement d'un pestivirus dans un foyer ovina chez des moutons

Molecular cloning and nucleotide sequence of the pestivirus bovine diarrhea virus

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

Six antigenic groups within the genus pestivirus as identified by cross neutralization assays

A comprehensive set of sequence analysis programs for the VAX

The development of an international reference panel of monoclonal antibodies for the differentiation of hog cholera virus from other pestiviruses

Comparative studies of Border disease and closely related virus infections in experimental pigs and sheep

Viral antigen distribution in the central nervous system of cattle persistently infected with bovine diarrhea virus

Comparative analysis of the 5 0 non-coding region of pestivirus RNA detected from live virus vaccines

Evidence of pestivirus RNA in human virus vaccine

Adventitious pestivirus RNA in live virus vaccines against bovine and swine diseases

Demonstration and genotyping of pestivirus RNA from mammalian cell lines

Sequence analysis of the 5 0 UTR of pestivirus RNA demonstrated in interferons for human use

Phylogenetic analysis of pestivirus based on the 5 0 UTR

Immunohistological detection of bovine viral diarrhoea virus antigen in the central nervous system of persistently infected cattle using monoclonal antibodies

Infection of ovine fetal brain cell cultures with cytopathogenic and non-cytopathogenic bovine viral diarrhoea virus

Variation in neuropathogenicity in sheep fetuses transplacentally infected with non-cytopathogenic and cytopathogenic biotypes of BVDV

Rapid characterization of new pestivirus strains by direct sequencing of PCR-amplified cDNA from the 5 0 noncoding region

Comparison of an antigen capture enzyme-linked assay with reverse transcription-polymerase chain reaction and cell culture immunoperoxidase tests for the diagnosis of ruminant pestivirus infections

Presumptive diagnostic differentiation of hog cholera virus from bovine viral diarrhea and border disease viruses by using a cDNA nested-amplification approach

A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences

Rapid detection of bovine viral diarrhea virus by polymerase chain reaction

Nucleotide sequence homology to BVDV 2 in the untranslated region of BVDVs from cattle with mucosal disease or persistent infection in Japan

Infection of pigs and cattle with bovine viral diarrhoea virus on a farm in England

A proposed division of the pestivirus genus using monoclonal antibodies, supported by cross-neutralisation assays and genetic sequencing

Identification of a new group of bovine viral diarrhea virus strains associated with severe outbreaks and high mortalities

Comparison of nucleic acid hybridization and nucleic acid amplification using conserved sequences from the 5 0 noncoding region for detection of bovine viral diarrhea virus

Segregation of bovine viral diarrhea virus into genotypes

The genomic sequence of a virulent bovine viral diarrhea virus (BVDV) from the type 2 genotype: Detection of a large genomic insertion in a noncytopathic BVDV

Characterisation of p20 sequences from a border disease-like pestivirus isolated from pigs. Veterinary Microbiol

Detection and identification of ruminant and porcine pestiviruses by nested amplification of 5 0 untranslated cDNA regions

A nested polymerase chain reaction assay to differentiate pestiviruses

Attempt to discriminate between bovine viral-diarrhea virus strains using polymerase chain reaction

Subdivision of the pestivirus genus based on envelope glycoprotein E2

Genetic identification of pestivirus strain Frijters as a border disease virus from pigs

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

Molecular characterization of ovine pestiviruses

Distribution of bovine diarrhea viral antigens in the central nervous system of cattle with various congenital manifestations

Detection of hog cholera virus and differentiation from other pestiviruses by polymerase chain reaction

The authors thank Hans Vanderhallen for performing the automated sequencing.