Search

'HoBi'-like virus is an atypical group within the Pestivirus genus that is implicated in economic losses for cattle producers due to both acute and persistent infections. Pestivirus strains exist as quasispecies (swarms of individual viruses) in infected animals and the viral populations making up the quasispecies differ widely in size and diversity in each animal. In the present study the viral quasispecies circulating in persistently infected (PI) calves, generated and maintained under experimental conditions using two different 'HoBi'-like strains, was observed over time. An increase in genetic variability and the development of certain mutations was observed over time. Mutations observed included the loss of a putative N-linked glycosylation site in the E2 region and the change of specific residues in E1/E2. It is hypothesized that these changes may be the results on continued adaption of the pestivirus to individual hosts. This is the first study characterizing variation in the viral swarms of animals persistently infected with HoBi-like viruses over time. Studies of the shifts in PI viral swarms will contribute to our understanding of the host and viral mechanisms that function in the maintenance of pestivirus persistent infections.

Bovine viral diarrhea virus (BVDV) is a positive single stranded RNA virus belonging to the Pestivirus genus of the Flaviviridae family. BVDV has a wide host range that includes most ruminants. Noncytopathic (ncp) BVDV may establish lifelong persistent infections in calves following infection of the fetus between 40 and 120 days of gestation. Cytopathic (cp) BVDV strains arise from ncp strains via mutations. The most common cp mutations are insertions of RNA derived from either host or a duplication of viral sequences into the region of the genome coding for the NS2/3 protein. Superinfection of a persistently infected animal with a cp virus can give rise to mucosal disease, a condition that is invariably fatal. A herd of 136 bred 3-year old cows was studied. These cows gave birth to 41 PI animals of which 23 succumbed to mucosal disease. In this study, we characterized the ncp and cp viruses isolated from 13 of these animals. All viruses belonged to the BVDV type 2a genotype and were highly similar. All the cp viruses contained an insertion in the NS2/3 coding region consisting of the sequences derived from the transcript encoding a DnaJ protein named Jiv90. Comparison of the inserted DnaJ regions along with the flanking viral sequences in the insertion 3' end of the 13 cp isolates revealed sequence identities ranging from 96% to 99% with common borders. This suggested that one animal likely developed a cp virus that then progressively spread to the other 12 animals. Interestingly, when the inserted mammalian gene replicated within viral genome, it showed conservation of the same conserved motifs between the different species, which may indicate a role for these motifs in the insertion function within the virus genome. This is the first characterization of multiple cp bovine viral diarrhea virus isolates that spread in a herd under natural conditions.

A novel adenovirus, CeAdV1, was isolated from buffy coat and nasal swab samples collected from two captive white-tailed deer (Odocoileus virginianus) fawns. The isolation was an incidental finding in the course of screening animals for use in a research study on an unrelated pathogen. In the screening process, virus isolation was performed on both nasal swabs and buffy coat samples and cytopathic effect was observed. Electron microscopy revealed viral particles with the shape and morphology of an adenovirus. Next generation sequencing followed by phylogenetic analysis classified this virus to the Mastadenovirus genus. Its sequence was genetically distinct from all other recognized species in this genus, with only 76% sequence identity to its closest genetic match, bovine adenovirus 3 (BAdV3). The virus could be propagated in bovine derived cells but grew to a higher titer in cervid derived cells. Inoculation of white-tailed deer fawns with the isolated virus resulted in pyrexia, depletion of thymus tissue and mild respiratory disease. Comparative serology performed using convalescent sera revealed distinct antigenic differences between the novel cervid adenovirus and BAdV3. A retrospective serological survey of the captive deer herd indicated that this virus had been circulating in the herd for at least 14 years with no report of clinical disease. A survey of serum collected from free ranging mule deer residing in Nevada revealed high serum titers against this novel adenovirus.

Bovine viral diarrhea virus (BVDV) is a widespread bovine pathogen for which there is no specific therapeutic agent. A previous study using 2-(2-benzimidazolyl)-5-[4-(2-imidazolino)phenyl]furan dihydrochloride (DB772) to treat calves persistently infected with BVDV resulted in a decrease in the viral load of infected calves but treatment resulted in the rapid selection of drug-resistant mutant isolates. In this article we describe three mutations found in the mutant isolates associated with in vivo and in vitro resistance to DB772. All three mutations are found in the NS5B which functions as the RNA-dependent-RNA-polymerase during viral replication. Growth curves for the mutant isolates were not largely different from those of wild-type isolates when cultured in the absence of DB772. Thus, DB772 appears to act by binding to the specified domain but binding is disrupted or inhibited by the described mutation.

Infection of susceptible animals with bovine viral diarrhea viruses (BVDV) can result in an array of disease symptoms that are dependent in part on the strain of infecting virus and the physiological status of the host. BVDV are lymphotrophic and exist as two biotypes. Cytopathic BVDV kill cells outright while noncytopathic strains can readily establish persistent infections. The molecular mechanisms behind these different affects are unknown. To gain a better understanding of the mechanisms of disease, serial analysis of gene expression (SAGE), a powerful method for global gene expression analysis, was employed to examine gene expression changes in BVDV-infected BL3 cells, a bovine B-cell lymphosarcoma cell line. SAGE libraries were constructed from mRNA derived from BL3 cells that were noninfected or infected with the cytopathic BVDV2 strain 296c. Annotation of the SAGE data showed the expression of many genes that are characteristic of B cells and integral to their function. Comparison of the SAGE databases also revealed a number of genes that were differentially expressed. Of particular interest was the increased numbers of transcripts encoding proto-oncogenes (c-fos, c-jun, junB, junD) in 296c-infected cells, all of which are constituents of the AP-1 transcriptional activation complex. Real-time RT-PCR confirmed these results and indicated that the actual increases were larger than that predicted by SAGE. In contrast, there was no corresponding increase in protein levels, but instead a significant decrease of c-jun and junB protein levels in the infected BL3 cells was observed. Rather than an increase in transcription of these genes, it appeared that these proto-oncogenes transcripts accumulated in the BVDV2-infected cells.

A unique pestivirus, isolated from a pronghorn antelope (Antilocopra americana), was characterized. Serum neutralization studies suggested that this virus was antigenically related to pestiviruses. Genomic characteristics, unique to pestiviruses, indicated that this virus belongs to the Pestivirus genus. These characteristics included the organization of the 5' untranslated region (5'-UTR), the presence and length of a viral Npro coding region, conservation of cysteine residues in Npro, conservation of predicted amino acid sequences flanking the cleavage sites between viral polypeptides Npro and C and between C and Erns and conservation of predicted hydrophobicity plots of Npro protein. While this data indicated the virus belongs to the Pestivirus genus, phylogenetic analysis in 5'-UTR, Npro and E2 regions suggested that it is the most divergent of the pestiviruses identified to date. This conclusion was also supported by the amino acid identity in coding regions. The corresponding values were much lower for the comparison of pronghorn pestivirus to other pestivirus genotypes than only between previous recognized genotypes. These results suggest the virus isolated from pronghorn antelope represents a new pestivirus genotype. It also represents the only pestivirus genotype first isolated from New World wildlife.

Phylogenetic analysis divides bovine viral diarrhea viruses (BVDV) into two different genotypes (BVDV1 and BVDV2). BVDV1 strains have been further subdivided into two to 11 subgenotypes. Phylogenetic analysis of BVDV2 isolates, however, has not been able to identify discrete subgenotypes. In this study, we identified six South American BVDV2 strains and one North American BVDV2 strain that cluster to a separate genetic group within BVDV2, thus representing a distinct subgenotype. The 5' untranslated region (UTR) sequence homology between these six strains and other BVDV2 from North America, Europe and Asia (81.7%) is lower than the homology used to segregate BVDV1 into BVDV1a and BVDV1b (83.6%). Most nucleotide differences observed between the two subgroups of BVDV2 were concentrated in two regions, which also harbor most of the differences seen between BVDV1a and BVDV1b. To determine if this segregation was real, an additional analysis was performed comparing NS2/3 sequences. Analysis of a conserved sequence located between nucleotides 6670 and 7186 of the NS2/3 coding region also segregated these isolates to a separate group. The sequence homology between the two subgroups (86.3%) was higher than the homology in the 5'UTR (81.7%), with mean sequence homologies of 91 and 87.2% within the proposed subgroups. In contrast to the 5'UTR, alignment of the NS2/3 sequences revealed nucleotide differences distributed across the region. These results demonstrate that BVDV2 isolates cluster to two genetically distinct subgroups within BVDV2. The differences in both the 5'UTR and NS2/3 are consistent and justify this segregation. We suggest that BVDV2 may thereafter be subgenotyped into BVDV2a and BVDV2b. The existence of subgroups within the BVDV2 genotype with genetic heterogeneity similar to that seen among BVDV1 subgroups argues against BVDV2 isolates arising from BVDV1 in a recent evolutionary event. Unless the evolutionary clocks for BVDV1 and BVDV2 isolates tick along at different rates, these results indicate that BVDV2 have existed as long as BVDV1.

Bovine viral diarrhea viruses (BVDV) exist as two biotypes, cytopathic and noncytopathic, based on their ability to produce cytopathology in cultured cells. Cytopathic viruses arise from noncytopathic viruses following genetic alteration (insertions, duplications, rearrangements) within the region encoding the NS2/3 protein. In BVDV genotype 2, the majority of biotype conversions result from the integration of a cellular mRNA into the genomic RNA of a noncytopathic virus within the NS2/3 coding region. The translation of the cellular sequences during viral replication results in the proteolytic cleavage of the nonstructural protein NS2/3 to the NS2 and NS3 proteins with the appearance of the cytopathic phenotype. Here, these cellular sequences were identified as a portion of a cellular mRNA encoding a novel DnaJ protein (bDnaJ1). The 60 amino acid J-domain was identified near the C-terminus of the protein. Potential nuclear localization, farnesylation and hydrophobic transmembrane domains were also identified. Reverse transcription-PCR analysis of the expression of bDnaJ1 in bovine tissues showed that it was expressed in all tissues examined, and additionally, there may be alternate splicing of the transcript, yielding a second form of the bDnaJ1 protein. Northern blot analysis of mock and BVDV2 infected cells indicated that infection by noncytopathic BVDV2 altered the expression level of a bDnaJ1-hybridizing transcript. The increase in expression may represent a stress response to the infection by noncytopathic BVDV.

Bovine viral diarrhea viruses (BVDV) are ubiquitous viral pathogens of cattle with a high degree of sequence diversity amongst strains circulating in livestock herds. The driving force behind change in sequence is not well established but the inaccurate replication of the genomic RNA by a viral RNA polymerase without proof-reading capabilities as well as immune pressure on immunodominant proteins are thought to play major roles. Additionally, it is not clear when the majority of changes are introduced, whether during acute infections with exposure to innate and adaptive immune responses or in establishment of persistent infections (PI) in utero. To examine which generates greater sequence diversity, two groups of viruses were compared. The first was six isolates of a single strain of BVDV-2 that were isolated over greater than a year’s time. These viruses caused a series of severe acute (SA) BVD outbreaks over a large geographic area. Changes in nucleotide sequence were determined by comparison of the sequence of each strain to the six virus consensus sequence. The second group was composed of six BVDV strains isolated from PI calves whose dams were exposed to PI cattle. Changes were identified by comparison of the sequence of the progenitor PI virus to that of the progeny viruses from the single in vivo ‘passage’. The open reading frames (ORF) of the six SA isolates were >99% identical at the nucleotide level with 30% of the changes being nonsynonymous changes. The amount of genetic change increased with time and distance from the original outbreak. Similarly, the PI viruses isolated from single passage PI calves had >99% identity with the progenitor virus. The number of nucleotide changes in these viruses was equal to or greater than that observed in the SA viruses. The majority of the nonsynonymous changes were found in the structural proteins, with 65% of these occurring in the immunodominant E2 protein. Antigenic mapping studies using a monoclonal antibody panel specific for the BVDV E2 protein showed no antigenic differences amongst the six SA viruses, nor between the progenitor and progeny type 1a and type 2 persistent viruses. However, antigenic differences were observed in the two type 1b progeny viruses that possessed the greatest number of amino acid changes. Two antibodies were found to have altered staining patterns. These results suggest that the establishment of a single persistent infection results in more rapid generation of genetic diversity in BVDV strains than a series of acute infections and may contribute to antigenic change in the absence of an immune response. Published by Elsevier B.V.

In 2003 an outbreak of sudden deaths occurred in 3–4-week-old piglets on a farm in New South Wales, Australia. There was a marked increase in the birth of stillborn foetuses. Pathological changes consisted of a multifocal non-suppurative myocarditis. A viral infection was suspected but a wide range of known agents were excluded. A modified sequence independent single primer amplification (SISPA) method was used to identify a novel virus associated with this outbreak. Conserved 5′UTR motifs, the presence of a putative N pro coding region and limited antigenic cross-reactivity with other members of the Pestivirus genus, support the placement of this virus in the Pestivirus genus. Phylogenetic analysis of the 5′UTR, N pro and E2 coding regions showed this virus to be the most divergent pestivirus identified to date.

Bovine viral diarrhea virus (BVDV), a pestivirus of the Flaviviridae family, is an economically important cattle pathogen with a worldwide distribution. Besides the segregation into two distinct species (BVDV1/BVDV2) two different biotypes, a cytopathic (cp) and a noncytopathic (ncp) biotype, are distinguished based on their behavior in epithelial cell cultures. One of the most serious forms of BVDV infection affecting immunocompetent animals of all ages is severe acute BVD (sa BVD) which is caused by highly virulent ncp BVDV2 strains. Previous studies revealed that these highly virulent ncp viruses cause cell death in a lymphoid cell line (BL3) which is not clearly associated with typical apoptotic changes (e.g. PARP cleavage) observed after infection with cp BVDV. To further characterize the underlying molecular mechanisms, we first analyzed the role of the mitochondria and caspases as key mediators of apoptosis. Compared to infection with cp BVDV2, infection with highly virulent ncp BVDV2 resulted in a delayed and less pronounced disruption of the mitochondrial transmembrane potential (DeltaPsi(m)) and a weaker activation of the caspase cascade. In contrast, infection with low virulence ncp BVDV2 showed no significant differences from the uninfected control cells. Since different pro- and anti-apoptotic cellular signaling pathways may become activated upon virus infection, we compared the effect of different BVDV2 strains on cellular signaling pathways in BL3 cells. Stress-mediated p38 MAPK phosphorylation was detected only in cells infected with cp BVDV2. Interestingly, infection with highly virulent ncp BVDV2 was found to influence the phosphoinositide 3-kinase (PI3K)-Akt pathway. This indicates that BL3 cells respond differently to infection with BVDV depending on virulence and biotype.

Bovine viral diarrhea virus (BVDV) infection in cattle can lead to the development of a fatal syndrome called mucosal disease (MD). The induction of MD requires co-existence of two biotypes of BVDV: cytopathic (cp) and non-cytopathic (ncp). Studies have shown that in some cases, cp viruses are generated from ncp viruses by cellular gene insertions, duplications or rearrangements in the viral genome. Cellular ubiquitin gene is the most frequently reported insert in BVDV 1. Here we report the finding of a novel cellular gene insertion (termed cSNCINS) in the NS4B gene accompanied by the duplication of NS3 in a cpBVDV genome. The amino acid sequence of the insert is identical to that of the human SMT3B protein and is 98% similar to that of the human SMT3A protein. Both SMT3B and SMT3A proteins are homologues of the yeast SMT3 protein. The cSNCINS protein is encoded by an open reading frame located on a 1150-bp bovine endometrial cDNA fragment. Our results indicate that the cSNCINS is a bovine homologue of the human SMT3B gene and that insertion of the BSMT3B gene together with duplication of NS3 in the viral genome may account for the cytopathogenicity of this virus.

Bovine viral diarrhoea viruses (BVDV) have recently been segregated into two genotypes, BVDV 1 and BVDV 2. However, the antigenic differences and similarities of BVDV 1 and BVDV 2 remain poorly defined. In this study, the E2 epitopes of two neutralizing monoclonal antibodies (mAbs) produced against an isolate of BVDV 1 were mapped. The mAb 157, previously determined to be broadly cross-reactive to BVDV, was discovered to be BVDV 1-specific, whereas mAb 348 bound to and neutralized BVDV 2. Both mAbs bound to epitopes within the first 192 amino acids of the E2 protein as determined by reactions with a C-terminally truncated E2. To identify critical amino acids affecting these epitopes, mAb escape mutants were selected for sequencing from BVDV 1 and BVDV 2 strains with different (wild-type) mAb binding phenotypes. In addition, the E2 gene of several BVDV were sequenced and the sequences were compared with amino acid changes in mutant viruses. Single nucleotide changes in escape mutants selected with mAb 157 resulted in deduced amino acid changes at E2 positions 9, 32 or 72. Amino acid changes at position 72 also affected the epitope of mAb 348. Alignment of E2 nucleotide sequences revealed that BVDV 2 are missing six nucleotides encoding the equivalent of amino acids 31 and 32 of BVDV 1 and thus, this difference can account for the BVDV 1-specificity of mAb 157. Single nucleotide mutations in mAb 348 escape mutants of BVDV 1 and BVDV 2 resulted in changes in 3 amino acids in the previously described immunodominant 71–74 region (Virology 190, 763–772). A fourth amino acid change observed in a mutant of BVDV 2 extended this region to position 77. Thus, the amino acid changes affecting the conserved epitope of mAb 348 occurred in a short spatial array over only seven amino acids, unlike the described composite epitopes previously mapped to this region.

The genus Pestivirus is composed of hog cholera virus (HCV) [also known as classical swine fever virus (CSFV)], bovine viral diarrhea virus (BVDV), and border disease virus (BDV). Complete sequences have been published for HCV (or CSFV) and the two genotypes of BVDV (BVDV1 and BVDV2). In this study the complete sequence of the border disease virus (BDV), BD31, was determined. BD31 was isolated from a lamb with hairy shaker syndrome and is the BDV type virus offered by ATCC (ATCC VR-996). The genome was 12268 nucleotides long and had a single large open reading frame (ORF) beginning at nucleotide 357 and ending at nucleotide 12045. The sequence identity of the predicted amino acid sequence of BD31 and other published pestivirus sequences varied from 71% to 78%. Phylogenetic analysis of available complete genomic sequences segregated pestiviruses into two branches. One branch contained BD31 and HCV (or CSFV) isolates while the other branch contained BVDV1 and BVDV2 isolates. Pestiviruses from the same branch were similar in the length of the 5′ and 3′ untranslated regions (UTR). When complete genomic sequences were compared among BD31, HCV (or CSFV), BVDV1 and BVDV2, the highest sequence identity was observed in the 5′ UTR. Within the ORF, the highest sequence identity was observed in the genomic region coding for the nonstructural viral polypeptide p80.

The uptake of porcine parvovirus (PPV) into host cells, permissive or nonpermissive for PPV replication, was monitored by autoradiography, immunofluorescent microscopy, and dual parameter flow cytometry. While both permissive and nonpermissive cells selectively took up the light (activated) form of the virus from a mixed population of heavy and light infectious virions, the virus was only replicated in the permissive cell cultures. Transfection of permissive and nonpermissive cells with purified PPV genome was only successful with the permissive host cells. These results suggest that the restriction of PPV to certain host cells is the result of intracellular events rather than selective binding of virus to the host cell surface.