Infection of Ruminants, Including Pregnant Cattle, with Bungowannah Virus.

Bungowannah virus is a pestivirus known to cause reproductive losses in pigs. The virus has not been found in other species, nor is it known if it has the capacity to cause disease in other animals. Eight sheep, eight calves and seven pregnant cows were experimentally infected with Bungowannah virus. It was found that sheep and calves could be infected. Furthermore, it was shown that the virus is able to cross the bovine placenta and cause infection of the foetus. These findings demonstrate the potential for species other than pigs to become infected with Bungowannah virus and the need to prevent them from becoming infected.

Serological survey for antibodies against pestiviruses in Wyoming domestic sheep.

Pestiviruses including Bovine viral diarrhea virus type 1 (BVDV-1), BVDV-2 and Border disease virus (BDV) have been reported in both sheep and cattle populations, together with the HoBi-like, an emerging group of pestiviruses. Pestivirus control programs in the United States have focused on the control of BVDV-1 and 2. The incidence of pestivirus infection in sheep in the United States and the risk of transmission between cattle and sheep populations are unknown. The aim of this study was to perform serological surveillance for pestivirus exposure in sheep from an important sheep producing state in the Unites States, Wyoming. For this, sera from 500 sheep, collected across the state of Wyoming (US) in 2015-2016, were examined by comparative virus neutralization assay against four species/proposed species of pestiviruses: BVDV-1, BVDV-2, BDV and HoBi-like virus. Rates of exposure varied between geographic regions within the state. The overall pestivirus prevalence of antibodies was 5.6%. Antibodies were most frequently detected against BVDV-1 (4%), and the highest antibody titers were also against BVDV-1. Data from this study highlights understanding of the dynamics of sheep pestivirus exposure, consideration of reference strains used for VN assays, transmission patterns, and potential vaccination history should be taken into account in implementation of control measures against pestiviruses in sheep and for successful BVDV control programs in cattle.

Natural transmission of bovine viral diarrhoea virus-1c from a persistently infected neonate lamb to naïve sheep and cattle.

This study investigated the transmission of bovine viral diarrhoea virus (BVDV)-1c from a persistently infected (PI) neonate lamb to naïve sheep and cattle using three treatment groups: four naïve ewes and their five lambs, which were copaddocked with the PI lamb; five steers, which were housed in a paddock adjacent to the PI lamb; and five steers, which had direct but limited exposure to the PI lamb. Serum samples were collected and tested for BVDV-specific antibodies. Serum samples from the PI lamb, from day of birth to eight weeks of age, were tested for BVDV-specific antibodies and antigen and submitted for quantitative PCR to determine the viral load present at each week of age. Only one lamb from the copaddocked group developed BVDV-specific antibodies following comingling while all the steers in both the cattle treatment groups remained BVDV antibody negative. Quantitative PCR results from the PI lamb showed lower viral loads from day of birth to six weeks of age, compared with the results at seven and eight weeks of age. This may reflect maternal colostral BVDV antibody concentrations in the neonate lamb or other viral properties.

High Abundance and Genetic Variability of Atypical Porcine Pestivirus in Pigs from Europe and Asia.

Atypical porcine pestivirus (APPV) was recently reported to be associated with neurologic disorders in newborn piglets. Investigations of 1,460 serum samples of apparently healthy pigs from different parts of Europe and Asia demonstrate a geographically wide distribution of genetically highly variable APPV and high APPV genome and antibody detection rates.

Molecular analyses detect natural coinfection of water buffaloes (Bubalus bubalis) with bovine viral diarrhea viruses (BVDV) in serologically negative animals.

Infection of water buffaloes (Bubalus bubalis) with bovine viral diarrhea viruses (BVDV) has been confirmed in several studies by serological and molecular techniques. In order to determine the presence of persistently infected animals and circulating species and subtypes of BVDV we conducted this study on a buffalo herd, whose habitat was shared with bovine cattle (Bossp.). Our serological results showed a high level of positivity for BVDV-1 and BVDV-2 within the buffalo herd. The molecular analyses of blood samples in serologically negative animals revealed the presence of viral nucleic acid, confirming the existence of persistent infection in the buffaloes. Cloning and sequencing of the 5' UTR of some of these samples revealed the presence of naturally mix-infected buffaloes with at least two different subtypes (1a and 1b), and also with both BVDV species (BVDV-1 and BVDV-2).

Pestiviruses infections at the wild and domestic ruminants interface in the French Southern Alps.

In alpine pasture, interspecies transmission has recently been incriminated in the epidemiology of pestivirus infection. The aim of this study was to investigate pestivirus infections in wild and domestic ruminants sharing pastures in the French Southern Alps. Animal sera were screened for pestivirus antibodies against the pestivirus NS3 protein by a commercial blocking enzyme linked immunosorbent assay (ELISA). All 38 domestic herds tested were positive for pestivirus-specific antibodies. Individual sero-prevalence reached 76.5% (95% confidence interval [95% CI]: [74.2-78.8%]) of the 1383 sheep tested. For wild ruminants, 38.7% (95% CI: [33.8-43.9%]) of the 369 chamois tested, 28.7% (95% CI: [17.4-38.1%]) of the 72 roe deer, and 22.2% (95% CI: [6.5-37.9%]) of the 27 mouflons were seropositive. Virus screening was carried out on spleen samples from hunted wild animals (n=160) and from 15 domestic ruminants (clinically suspected to be persistently infected animals), by a conventional reverse transcription-polymerase chain reaction (RT-PCR). Three pestivirus strains were isolated from the sheep samples positive by RT-PCR. The viruses were classified in the BDV-3, BDV-Tunisian and BDV-6 genotypes. For the first time, one strain (RUPI-05 strain) was isolated from an alpine chamois and clustered in the BDV-6 genotype, showing in the 5'-UTR region 92% of identity with the ovine isolate from the same area. Thus, an active circulation of pestiviruses was demonstrated in both wild and domestic ungulates from the French Southern Alps. The results suggest that interspecies transmission between sheep and chamois probably occur.

Pestiviruses.

Pestiviruses cause economically important diseases among domestic ruminants and pigs, but they may also infect a wide spectrum of wild species of even-toed ungulates (Artiodactyla). Bovine viral diarrhea virus (BVDV) and Border disease virus of sheep infect their hosts either transiently or persistently. Cellular and humoral immunotolerance to the infecting strain is a unique feature of persistent infection (PI) by ruminant pestiviruses. Persistence, caused by transplacental infection early in fetal development, depends on virally encoded interferon antagonists that inactivate the host's innate immune response to the virus without globally interfering with its function against other viruses. At epidemiological equilibrium, approximately 1-2% of animals are PI. Successful BVDV control programs show that removal of PI animals results in viral extinction in the host population. The nucleotide sequences of ruminant pestiviruses change little during persistent infection. Nevertheless, they display large heterogeneity, pointing to a long history of virus-host coevolution in which avirulent strains are more successful.

Long-term clinicopathological characteristics of alpacas naturally infected with bovine viral diarrhea virus type Ib.

BACKGROUND: Substantial bovine viral diarrhea virus (BVDV)-related production losses in North American alpaca herds have been associated with BVDV type Ib infection. OBJECTIVES: To classify and differentiate the long-term clinicopathological characteristics of BVDV type Ib infection of alpaca crias, after natural virus exposure. We hypothesized that persistently infected (PI) alpacas specifically demonstrate growth retardation, clinicopathological evidence of opportunistic infections, and early mortality. ANIMALS: Thirty-five crias naturally exposed to BVDV (18 acute, 3 chronic, 14 PIs), and 19 healthy cohort controls of 5 northeastern alpaca farms were prospectively evaluated over 2 years (September 2005-September 2008). METHODS: Observational cohort-control study. RESULTS: Chronically (viremia >3 weeks) and PI crias demonstrated significantly lower birth weights, decreased growth rates, anemia, and monocytosis compared with control animals. Common clinical problems of PI alpacas included chronic wasting, diarrhea, and respiratory disease. Median survival of PI alpacas that died was 177 days (interquartile range, 555) with a case fatality rate of 50% within 6 months of life. Transplacental infection was confirmed in 82% (9/11) of pregnant females on 1 farm, resulting in the birth of 7 PI crias (7/10 deliveries; 1 animal was aborted). Mean gestation at the beginning and end of BVDV exposure was 64 and 114 days, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Natural BVDV type 1b infection during early pregnancy resulted in a high incidence of PI offspring. Although PI alpacas may have distinct clinical characteristics, verification of persistent viremia in the absence of endogenous, neutralizing antibodies is essential to differentiate persistent from chronic infection.

Epidemiology of Pestivirus infection in wild ungulates of the French South Alps.

Inter-species transmission is often incriminated in the epidemiology of Pestivirus diseases. The purpose of this study was to investigate the prevalence of Pestivirus in some mountain wild ungulates and to determine their role in Pestivirus transmission, as mountain pastures are a place where cohabitations between wild and domestic ungulates are particularly high. Between 2003 and 2007, a longitudinal epidemiological study was carried out on hunted ungulates in the French Hautes-Alpes department. Pestivirus-specific antibodies against p80 protein (also named NS3) common to all Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus (BDV) were found in 45.9% (95% confidence interval [CI95%]: 40.5-51.3%) of the 343 tested chamois (Rupicapra rupicapra). In addition, mouflons (Ovis gmelinii musimon) were shown for the first time to be strongly infected (61.1%; CI95%: 38.6-83.6) by a Pestivirus. These serological ELISA results were confirmed by comparative virus neutralization tests, performed on seven Pestivirus strains by using 15 seropositive samples. The highest antibody titers were directed against 2 BDV strains (Av and 33s strains), rather than BDV-4, a strain responsible for Pyrenean-chamois epizooties. Virus neutralization tests confirm a BDV circulation in wild ungulates in the French South Alps. However, no Pestivirus RNA was detected by reverse-transcriptase polymerase chain reaction in serum and spleen samples from seronegative animals and no virus was isolated from those samples either. Efforts should be made to improve the protocol in order to be able to isolate and characterize the local strain. Finally, the oldness (age) and femaleness (gender) increase the risk of seroconversion in chamois.

Experimental infections of the porcine foetus with Bungowannah virus, a novel pestivirus.

In 2003 an outbreak of sudden deaths occurred in 2-3-week-old pigs on a piggery in New South Wales, Australia. There was a marked increase in the birth of stillborn pigs and preweaning losses associated with a multifocal non-suppurative myocarditis with myonecrosis. The aim of this study was to amplify any infectious agents present in field material to aid the detection and identification of the causative agent of the porcine myocarditis syndrome (PMC). Foetuses were directly inoculated in utero with tissue extracts from field cases of PMC at 56-60, 70-84 or 85-94 days of gestation and euthanased 7-28 days later. The IgG concentration in foetal sera/body fluids was measured, hearts were examined by light microscopy and selected hearts were examined by electron microscopy. An infectious agent was detected in tissues from cases of PMC and its identification as the novel pestivirus Bungowannah virus has recently been reported (Kirkland et al., 2007). Sow sera, foetal tissues and foetal sera/body fluids were tested for Bungowannah virus RNA by qRT-PCR and antibody by peroxidase-linked assay. Bungowannah virus was detected in numerous organs of the porcine foetus. Following direct foetal exposure it is probable that this virus spreads by direct intra-uterine transmission to adjacent foetuses and by trans-uterine transmission to the dam. Data were obtained for both the replication of the virus in the porcine foetus and the humoral immune response in the foetus and sow.

Experimental infection of white-tailed deer fawns (Odocoileus virginianus) with bovine viral diarrhea virus type-1 isolated from free-ranging white-tailed deer.

The objective of the current study was to elucidate the within-host dynamics of bovine viral diarrhea virus (BVDV) type-1 infection to better understand how this virus could be maintained in white-tailed deer (Odocoileus virginianus, WTD) populations. The BVDV type-1 used in this study was originally isolated from a free-ranging WTD in Indiana. Four fawns were intranasally inoculated with 2 ml BVDV type-1 strain 544 WTD at a 10(6) tissue culture infectious dose (TCID(50))/ml. Two fawns were inoculated with sham inoculum (negative controls). Animals were bled on days -7, 0, 1, 7, and 14 postinoculation (PID) for a complete blood count, chemistry panel, buffy coat (BC), real-time RT-PCR, and virus neutralization (VN). On days 7 and 14 PID, nasal and rectal swabs were obtained for RT-PCR and two of the virus-inoculated fawns and one of the negative controls fawns were euthanized. At necropsy, multiple samples were obtained for histopathology and in situ hybridization (ISH). Quantitative RT-PCR was performed on serum, BC, nasal, and rectal swabs. All animals tested negative for BVDV type 1 neutralizing antibodies on day 0 and animals in the control group remained seronegative throughout the study. No gross lesions were observed at necropsy. BVDV was isolated from lung and pooled lymph nodes from all BVDV-inoculated fawns on days 7 and 14 PID. Infected deer had lymphoid depletion, apoptosis, and lymphoid necrosis in the Peyer's patches and mesenteric lymph nodes. BVDV was detected in lymphoid tissues of infected animals by ISH. No lesions or virus were identified in control fawns. On day 7 PID, samples from two virus-inoculated fawns were positive for BVDV by virus isolation and RT-PCR from BC and nasal swab samples. One fawn was also positive on a rectal swab. Nasal and rectal swabs from all animals were negative on day 14. Results indicate that infection of WTD with BVDV is possible, and leads to histologic lesions in variety of tissues. In addition, virus shedding into the environment through feces and other secretions is likely.

Seroprevalence of antibodies against pestiviruses in small ruminants in The Netherlands.

In this study, a serological survey was performed to determine the prevalence of pestivirus (bovine viral diarrhea virus (BVDV) and border disease virus (BDV)) infected small ruminants herds in the Netherlands. After random selection of sheep farms, a sample size was determined to detect a 5% herd prevalence. 13 out of 29 farms were tested seropositive using an ELISA which detects antibodies directed against the non structural protein 3 (NS3) of pestiviruses. This resulted in a seroprevalence for the Netherlands of 45% [0.36; 0.54]. The within farm prevalence ranged from 4 till 65%. Using a virus neutralization assay, specific anti-BDV antibodies could be detected on two farms, while on one other farm anti-BVDV antibodies were present. On four farms antibodies to both viruses could be detected, on three of these farms antibodies against both viruses were equally present. At five farms that tested positive in the NS3-ELISA we were unable to detect pestivirus neutralizing antibodies in all sera using the VN test. This resulted in an estimated prevalence using the VN for the Netherlands of 28% [0.20; 0.60]. An additional survey in sera from dairy goats revealed that 34 out of 126 farms were serological positive resulting in a seroprevalence of 27% [0.23; 0.31], with a herd prevalence of 32% ranging from 1-100%.

Seroprevalence and characterization of pestivirus infections in small ruminants and new world camelids in Switzerland.

The seroprevalence of pestivirus infections in small ruminants and new world camelids in Switzerland was determined. In 5'059 sera of sheep from 382 herds, 503 sera of goats from 54 herds and 109 sera of alpacas and lamas from 53 herds, population prevalences of 16.1% (sheep), 25.4% (goats) and 4.6% (new world camelids), respectively, were found. In order to determine the source of infection, the serological reactions were further characterized by cross-neutralization against two pestiviruses representing the genotypes BVDV (Bovine Virus Diarrhea Virus)-1 and BDV (Border Disease Virus)-1. Based on the ratio of respective antibody titres, 56.1% of the infections in sheep were induced by a BDV-1, 12.9% by a BVDV-1 and 31.0% by an unresolved pestivirus. In goats, the corresponding proportions were 23.4%, 10.2% and 66.4%, respectively. In Alpacas and Lamas, the source of infection of 1 animal was BDV-1 and that of 4 seropositive animals remained unresolved. In view of the phylogenetic relationship between pestiviruses, the unresolved source of infection is most probably attributable to other pestivirus genotypes circulating in small ruminants and new world camelids. Due to the predominance of pestiviral genotypes other than BVDV-1, the risk of transmission of BVDV from persistently infected small ruminants and new world camelids to cattle appears to be moderate, apart from close direct contact in mixed animal husbandry, communal pasturing and grazing in the Alps.

Regeneration and characterization of a recombinant bovine viral diarrhea virus and determination of its efficacy to cross the bovine placenta.

The capacity of different bovine viral diarrhea virus (BVDV) strains to cause transplacental infection is variable. BVDV strain SD-1 was isolated from a persistently infected heifer. Its genome represents the only reported nucleotide sequence of a noncytopathic viral isolate determined without cell culture passage in the laboratory. Thus, SD-1 might possess biological advantages over other NCP BVDV strains to be used as a model virus for investigation of viral transplacental transmission. To evaluate if a molecularly generated BVDV SD-1 is capable of crossing the bovine placenta efficiently, a full-length cDNA clone of SD-1 was constructed using RT-PCR amplification and standard molecular techniques. In vitro transcripts synthesized from the cDNA template directed the generation of infectious virus in MDBK cells with a transfection efficiency as high as 4.7 x 10(5) FFU/mug RNA. The recovered virus termed ASD1 harbored five silent point mutations engineered as genetic markers and was similar to wild type (wt) SD-1 in viral growth kinetics. As evaluated in the pregnant heifers, ASD1 was capable of crossing the bovine placenta efficiently, suggesting that NCP BVDV SD-1 is a suitable viral backbone for investigation of the role of viral genetic element(s) in viral transplacental transmission by allowing for evaluation of newly created viral mutants.

Febrile response and decrease in circulating lymphocytes following acute infection of white-tailed deer fawns with either a BVDV1 or a BVDV2 strain.

Although commonly associated with infection in cattle, bovine viral diarrhea viruses (BVDV) also replicate in many domestic and wildlife species, including cervids. Bovine viral diarrhea viruses have been isolated from a number of cervids, including mule deer (Odocoileus hemionus), European roe deer (Capreolus capreolus), red deer (Cervus elaphus), white-tailed deer (Odocoileus virginianus), and mouse deer (Tragulus javanicus), but little information is available regarding clinical presentation and progression of infection in these species. In preliminary studies of experimental infection of deer with BVDV, researchers noted seroconversion but no clinical signs. In this study, we infected white-tailed deer fawns that were negative for BVDV and for antibodies against BVDV, with either a type 1 or a type 2 BVDV that had been isolated from white-tailed deer. Fawns were monitored for changes in basal temperature, circulating lymphocytes, and platelets. The clinical progression following inoculation in these fawns was similar to that seen with BVDV infections in cattle and included fever and depletion of circulating lymphocytes. Because free-ranging cervid populations are frequently in contact with domestic cattle in the United States, possible transfer of BVDV between cattle and cervids has significant implications for proposed BVDV control programs.

Transmission of a pestivirus infection in a population of Pyrenean chamois.

Outbreaks of a previously unrecorded disease have recently affected Pyrenean chamois (Rupicapra pyrenaica pyrenaica) populations across the mountain range. A pestivirus was hypothesized to be the cause of this emerging disease and this type of virus can cross the species barrier and be transmitted to or from wildlife. Using an epidemiological survey conducted from 1995 to 2004 at Orlu, France, we characterized the virus and analyzed its transmission. A phylogenetic analysis of viral sequences and virus neutralization tests showed that the virus belonged to the newly described border disease virus-4 group. The increase of seroprevalence with age indicated that infection can occur at any age and resulted in lifelong immunity. Overall, 70.3% of 323 samples were positive for anti-p80 antibodies and 10.2% of 167 samples showed viremia, as demonstrated by either positive ELISA antigen test or RT-PCR. Infection has thus been widespread in this population since 1995, whereas no mass mortality or clinical signs have been observed. Incidence and seroprevalence varied seasonally and according to number of individuals aged less than 2 years old in the population, so viral transmission was dependent on host population age structure. We propose that the virus is now endemic in this population and is likely detrimental for reproduction and juveniles. Further investigation is needed to estimate the impact of pestivirus on host population dynamics and the risk of cross-transmission to farm animals.

Detection of a newly described pestivirus of Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in France.

A pestivirus was detected and characterized in chamois (Rupicapra pyrenaica pyrenaica) originating from the French part of the Pyrenees. Phylogenetic analysis of the pestivirus was done on the basis of a fragment from the 5' noncoding region including 22 published nucleotide sequences of different pestivirus strains. Our strain was grouped within the clade of border disease viruses (BDV). However, it had an intermediate position between clade BDV and classical swine fever viruses representing a basal position to BDV strains of domestic sheep. Our strain was grouped as a sister unit to a novel pestivirus (Chamois-1) recently described from chamois in Spain. Therefore, we postulate that this virus occurs in the entire population of Pyrenean chamois. On the basis of the phylogenetic grouping of this isolate, a postulated cross-species transmission of pestivirus from domestic sheep to chamois via shared pastures seems to be unlikely.

Diva vaccines that reduce virus transmission.

This brief review deals with the effect of diva (Differentiating Infected from VAccinated individuals) vaccines (also termed marker vaccines) on transmission of herpesviruses and pestiviruses in swine and cattle. Pseudorabies and bovine herpesvirus 1 diva vaccines have been demonstrated to reduce transmission of wild-type virus in populations of pigs and cattle in the laboratory as well as in the field. A subunit diva vaccine based on the immunodominant E2 protein of classical swine fever virus that is expressed in the baculovirus system may reduce transmission of wild-type virus among pigs and also transmission from mother to foetuses. A similar diva vaccine against bovine virus diarrhoea infections protected sheep against transplacental transmission of antigenically homologous wild-type virus. Diva vaccines along with their companion diagnostic tests can play a role in control of infections, ultimately leading to eradication of viruses.

Variation in neuropathogenicity in sheep fetuses transplacentally infected with non-cytopathogenic and cytopathogenic biotypes of bovine-virus diarrhoea virus.

Pregnant Merino ewes were inoculated intravenously between days 63 and 65 of gestation with a non-cytopathogenic (ncp) bovine-virus diarrhoea-virus (BVDV) isolate (experiment A). The histomorphological findings and the distribution of viral antigen, as revealed by immunohistochemistry in brains of fetuses from experiment A, were compared with those seen in fetal brains from a previous study (experiment B), in which pregnant ewes had been intravenously infected between days 65 and 68 of gestation with the cytopathogenic (cp) BVDV strain Indiana. The two viruses showed remarkable variations concerning their pathogenicity for the developing fetal brain. The cp BVDV had a much higher neuropathogenic potential than the ncp BVDV and induced severe intracranial malformations in most fetuses. In experiment A, exclusively relatively mild leucoencephalomalacic lesions occurred. Between fetuses of the two experiments, significant differences concerning the distribution of viral antigen and the inflammatory response were found. In the majority of fetal brains from experiment B examined at days 10, 14 and 21 post inoculation (p.i.), antigen-containing differentiated brain cells (neurons, astrocytes, oligodendrocytes) and undifferentiated cells in the periventricular germinal zones were seen throughout the different zones of the developing telencephalon and cerebellum. At 21 days p.i., a marked inflammatory response consisting of brain macrophages and other mononuclear cells occurred in the meninges and in the brain parenchyma of fetuses from experiment B. In brain sections of fetuses infected with ncp BVDV, in contrast to fetuses infected with cp BVDV, viral antigen was not detectable during the early stages (days 10 and 20) p.i., and histopathological lesions were not seen at this stage. At days 41 and 47 p.i., antigen-positive astrocytes and oligodendrocytes were found in the developing white matter of the telencephalon and cerebellum. Furthermore, antigen-containing neurons were seen in the developing cerebral cortex. Cellular infiltrations in fetal brains from experiment A were limited to the leucoencephalomalacic areas in the developing cerebral and cerebellar white matter and consisted exclusively of brain macrophages. Immunohistochemical staining in brain sections of fetuses from both experiments revealed that numerous perivascular cells contained viral antigen, whilst positive endothelial cells were exclusively found in fetuses from experiment A. From the findings of this study it was concluded that the cp BVDV stain used in experiment B has a marked tropism for the fetal brain and both its already differentiated and undifferentiated cell populations, and that the resulting brain lesions primarily are the consequence of a direct cytolysis of these cells.(ABSTRACT TRUNCATED AT 400 WORDS)