Clinical Presentation Resembling Mucosal Disease Associated with ‘HoBi’‐like Pestivirus in a Field Outbreak

The genus Pestivirus of the family Flaviviridae consists of four recognized species: Bovine viral diarrhoea virus 1 (BVDV‐1), Bovine viral diarrhoea virus 2 (BVDV‐2), Classical swine fever virus (CSFV) and Border disease virus (BDV). Recently, atypical pestiviruses (‘HoBi’‐like pestiviruses) were identified in batches of contaminated foetal calf serum and in naturally infected cattle with and without clinical symptoms. Here, we describe the first report of a mucosal disease‐like clinical presentation (MD) associated with a ‘HoBi’‐like pestivirus occurring in a cattle herd. The outbreak was investigated using immunohistochemistry, antibody detection, viral isolation and RT‐PCR. The sequence and phylogenetic analysis of 5′NCR, N^pro and E2 regions of the RT‐PCR positive samples showed that four different ‘HoBi’‐like strains were circulating in the herd. The main clinical signs and lesions were observed in the respiratory and digestive systems, but skin lesions and corneal opacity were also observed. MD characteristic lesions and a pestivirus with cytopathic biotype were detected in one calf. The present study is the first report of a MD like presentation associated with natural infection with ‘HoBi’‐like pestivirus. This report describes the clinical signs and provides a pathologic framework of an outbreak associated with at least two different ‘HoBi’‐like strains. Based on these observations, it appears that these atypical pestiviruses are most likely underdiagnosed in Brazilian cattle.     

HoBi‐Like Virus RNA Detected in Foetuses Following Challenge of Pregnant Cows that had Previously Given Birth to Calves Persistently Infected with Bovine Viral Diarrhoea Virus

The ability of ruminant pestivirus including bovine viral diarrhoea virus (BVDV) and the related emerging pestivirus, HoBi‐like virus, to establish persistent infection (PI) following foetal infection is central to keeping these viruses in circulation. Non‐PI dams carrying BVDV PI calves develop high levels of immunity due constantly viral exposure. A study to determine whether the immunity developed following the generation of a BVDV PI is enough to prevent HoBi‐like virus infection of a subsequent foetus was performed. This study consisted of nine pregnant cows, four had birthed BVDV‐1 PI calves in a previous pregnancy, three cows had birthed BVDV‐2 PIs and two had birthed pestivirus negative calves. From this, six pregnant cows were challenged with HoBi‐like virus about day 85 of gestation (four BVDV‐1 and two BVDV‐2 cows) and three non‐challenged cows (negative control). At the day of challenge, the serum neutralizing titres against the homologous BVDV strains of the first inoculation ranged from 1148 to 5793. At day 6 post‐challenge, HoBi‐like RNA was detected in the serum of all four BVDV‐1 cows but not in the two BVDV‐2 cows. The foetuses harvested from five of the exposed dams (three BVDV‐1 and two BVDV‐2 cows) at day 30 post‐challenge were positive for HoBi‐like virus RNA. The sixth cow, BVDV‐1 cow #541, while pregnant at the time of exposure, had no foetus 30 days after exposure. Foetuses from HoBi‐like virus exposed dams were significantly smaller and lighter than control foetuses. HoBi‐like RNA was detected in samples of all challenged foetuses. The identification of viral RNA in the serum of 4 cows at day 6 post‐challenge, as well viral RNA detection in all foetuses 30 days post‐inoculation, indicates that the foetuses of dams with high antibodies titres against BVDV‐1 or BVDV‐2 would not be protected from challenge with a HoBi‐like virus.     

HoBi‐like is the most prevalent ruminant pestivirus in Northeastern Brazil

The ruminant pestiviral species BVDV ‐1, BVDV ‐2 and BDV , along with the putative species HoBi‐like, may cause substantial economic losses in cattle, sheep and goats. Brazil's large size, variable biomes and wide range of ruminant animal production within different geographic regions suggest that the presence and prevalence of ruminant pestivirus may differ by regions within Brazil. This study investigated the genetic diversity of ruminant pestiviruses and determined the frequency of active infections within two states of the Northeast Region of Brazil, Maranhão and Rio Grande do Norte. Serum samples from 16,621 cattle and 2,672 small ruminants from 569 different herds residing in this region were tested by RT ‐PCR followed by DNA sequencing. Seventeen positive cattle were detected (0.1%) from fifteen different herds (2.64%). All isolates were classified as HoBi‐like pestiviruses based on phylogenetic analysis. All small ruminant samples tested negative. The findings presented herein suggest that the Northeast Region of Brazil has a uniquely high prevalence of HoBi‐like viruses. The increasing reports of HoBi‐like viruses detected in cattle in the field suggest that natural infection with these viruses may be more widespread than previously thought. The identification of HoBi‐like viruses as the most prevalent type of ruminant pestivirus circulating in the Northeast Region of Brazil indicates the need for both continued monitoring and determination of the extent of economic losses associated with HoBi‐like virus infections. In addition, it must be taken into account in the choice of diagnostic tests and in vaccine formulations.     

Genetic Diversity of Brazilian Bovine Pestiviruses Detected Between 1995 and 2014

Pestivirus infections in ruminants result in significant economic losses worldwide. The aetiological agents are three species from the genus Pestivirus, family Flaviviridae, including bovine viral diarrhoea virus type 1 (BVDV‐1), BVDV‐2, border disease virus (BDV), and an atypical pestivirus named HoBi‐like pestivirus. In this study, eighty‐nine pestivirus isolates that were collected in Brazil between 1995 and 2014 and that originated from either cattle, fetal bovine serum (FBS) or as cell culture contaminants were genotyped based on a comparison of gene sequences from their 5′ untranslated regions (5′UTR), N‐terminal autoprotease (N^pro) and envelope glycoprotein 2 (E2). Of these isolates, 53.9% of the sequences were genotyped as BVDV‐1, 33.7% as BVDV‐2 and 12.4% as HoBi‐like pestivirus. The prevalence of subgenotypes within the species was as follows: BVDV‐1a (35.9%), BVDV‐2b (31.4%), BVDV‐1b (10.1%), BVDV‐1d (6.7%), BVDV‐2c (2.2%) and BVDV‐1e (1.1%). BVDV‐2c and BVDV‐1e were detected for the first time in Brazil. This study revealed extensive genetic diversity among Brazilian pestivirus isolates, and the combination of pestiviruses that was detected is unique to Brazil. This information may serve as a foundation for designing and evaluating diagnostic tools and in the development of more effective vaccines; therefore, it may potentially contribute to pestivirus control and eradication.     

Evidence for Circulation of Bovine Viral Diarrhoea Virus Type 2c in Ruminants in Southern Italy

Recently, bovine viral diarrhoea virus type 2c (BVDV‐2c) was responsible for a severe outbreak in cattle in northern Europe. Here, we present the results of an epidemiological survey for pestiviruses in ruminants in southern Italy. Pooled serum samples were obtained from 997 bovine, 800 ovine, 431 caprine and eight bubaline farms, and pestiviral RNA was detected by molecular methods in 44 farms consisting of 16 cattle and one buffalo herds and of 21 sheep and six goat flocks. Twenty‐nine and 15 farms were infected by BVDV‐1 and BVDV‐2 strains, respectively. BVDV‐1 strains were recovered mainly from cattle and were heterogeneous, belonging to the subtypes 1b, 1u, 1e, 1g and 1h. In contrast, all BVDV‐2 viruses but two were detected in sheep or goats and were characterized as BVDV‐2c by sequence analysis of 5′UTR. These strains displayed high genetic identity to BVDV‐2c circulating in cattle in northern Europe and were more distantly related to a BVDV‐2c isolate recovered from a cattle herd in southern Italy more than 10 years before. The circulation of a BVDV‐2c in small ruminants suggests the need for a continuous surveillance for the emergence of pestivirus‐induced clinical signs in southern Italian farms.     

Identification of Natural Infections in Sheep/Goats with HoBi‐like Pestiviruses in China

The natural infections of HoBi‐like pestiviruses in cattle have been reported in South America, Europe and Asia. In China, although the detections of HoBi‐like pestivirus have been reported, the epidemiological investigation was limited. From January 2014 to October 2015, several flocks of sheep/goats in Henan province in central China suffered respiratory diseases which were recovered slowly after antibiotics treatment. To test whether it is the HoBi‐like pestivirus caused this symptom, 49 serum samples and 22 nasal swabs were then collected for analysis by serology and RT‐PCR. Serological result revealed that prevalence of pestivirus in small ruminants was 12.2% (6/49) in central China. Sequence analysis of partial 5′‐UTR nucleotides of pestivirus‐positive samples suggested that HoBi‐like pestivirus might have circulated in sheep/goats of China for a period and have evolved into new genotype clusters. It is apparent that the study provides the molecular evidence of natural infections in goat/sheep species with HoBi‐like pestiviruses in China.     

Detection of border disease virus in Mexican cattle

The genus Pestivirus within Flaviviridae is comprised of four recognized species, namely, bovine viral diarrhoea virus 1 (BVDV ‐1), bovine viral diarrhoea virus 2 (BVDV ‐2), border disease virus (BDV ) and classical swine fever virus (CSFV ). BDV , while primarily infecting sheep and goats, has also been reported in cattle and wild animals. Infections of sheep and goats result in economic loss due to abortions and the birth of persistently infected animals that have poor production and reduced life expectancy. In this study, we report the detection of BDV in cattle serum collected as part of pestivirus surveillance programme from six regions of Mexico, where a 67.1% of BVDV seroprevalence was calculated previously. Phylogenetic analyses based on comparison of the 5′UTR region typed the Mexican strains as BDV ‐1. Border disease (BD ) is listed as an exotic disease in Mexico, and the origin of BDV found in these cattle is unclear. This is the first identification of BDV in Mexican cattle.     

HoBi‐Like Pestivirus and Its Impact on Cattle Productivity

The clinical features and economic impact of the infection caused by an emerging group of pestiviruses, namely HoBi‐like pestivirus, in a cattle herd of southern Italy are reported. In 2011, the virus was first associated with respiratory disease, causing an abortion storm after 1 year and apparently disappearing for the following 3 years after persistently infected calves were slaughtered. However, in 2014, reproductive failures and acute gastroenteritis were observed in the same herd, leading to a marked decrease of productivity. A HoBi‐like strain closely related to that responsible for previous outbreaks was detected in several animals. Application of an intensive eradication programme, based on the detection and slaughtering of HoBi‐like pestivirus persistently infected animals, resulted in a marked improvement of the productive performances.     

Serological relationship between a novel ovine pestivirus and classical swine fever virus

The genus Pestivirus comprises globally distributed members of the family Flaviviridae, which cause severe losses in livestock. The most common species of the genus are bovine viral diarrhoea virus type 1 (BVDV‐1) and type 2 (BVDV‐2), classical swine fever virus (CSFV) and border disease virus (BDV). Recently, a novel ovine pestivirus was repeatedly detected in aborted lamb foetuses on a farm located in the Brescia Province (Italy). Complete genome characterization of this isolate showed that it was highly divergent from known pestivirus species and that it was genetically closely related to CSFV. The aim of this study was to determine the serological relatedness between the identified novel pestivirus and BVDV, BDV and CSFV selected strains for which homologous serum was available, by antigenic characterization performed using cross‐neutralization assays. The serological relatedness was expressed as the coefficient of antigenic similarity (R). Both field and specific antisera raised against the ovine pestivirus neutralized the CSFV reference strain Diepholz with titres significantly higher than those specific for the BDV and BVDV strains. Furthermore, the calculated R values clearly indicated that the novel ovine pestivirus is antigenically more related to CSFV than to ruminant pestiviruses, in agreement with the results of the genomic analysis. This would have severe consequences on CSFV serology in the event of a switch to porcine hosts with implications for CSFV surveillance and porcine health management.     

Distinction Between Persistent and Transient Infection in a Bovine Viral Diarrhoea (BVD) Control Programme: Appropriate Interpretation of Real‐Time RT‐PCR and Antigen‐ELISA Test Results

Control of bovine viral diarrhoea (BVD) in Belgium is currently implemented on a voluntary basis at herd level and mainly relies on detection and culling of persistently infected (PI) animals. The present field study was conducted during the winter of 2010/2011 to assess the performances of diagnostic assays used in the testing scheme for BVD as proposed by the two Belgian regional laboratories. Individual blood samples were collected from 4972 animals, and individual samples from the same herd were pooled (maximum of 30 individual samples per pool) and screened for the presence of Bovine Viral Diarrhoea Virus (BVDV)‐specific RNA using a commercial real‐time RT‐PCR test (ADIAGENE). Individual samples from positive pools were then tested in parallel with the same RT‐PCR test and with an antigen‐capture ELISA test (IDEXX) to detect viremic animals. This study demonstrated that individual results differed according to the type of assay used (P < 0.001): 140 animals (2.8%) were positive by RT‐PCR and 72 (1.4%) by antigen‐ELISA. A second blood sample was taken 40 days later from 74 PCR positive animals to detect persistent viremia: 17 (23%) of these were still PCR positive and considered to be PI and the 57 that no longer tested positive were assumed to be transiently infected (TI) animals. All PI animals were positive also by antigen‐ELISA at both time points. Among TI animals, 10 (16%) were positive by antigen‐ELISA at the first but none at the second sampling. A highly significant difference in cycle threshold (C_t) values obtained by RT‐PCR was observed between PI and TI animals. ROC analysis was performed to establish thresholds to confirm with high probability that an animal is PI, based on the result of RT‐PCR test performed on a single individual blood sample.     

Evaluation of bovine viral diarrhoea virus control strategies in dairy herds in Hokkaido,Japan, using stochastic modelling

Bovine viral diarrhoea virus (BVDV ) infection in cattle can result in growth retardation, reduced milk production, reproductive disorders and death. Persistently infected animals are the primary source of infection. In Hokkaido, Japan, all cattle entering shared pastures in summer are vaccinated before movement for disease control. Additionally, these cattle may be tested for BVDV and culled if positive. However, the effectiveness of this control strategy aiming to reduce the number of BVDV ‐infected animals has not been assessed. The aim of this study was to evaluate the effectiveness of various test‐and‐cull and/or vaccination strategies on BVDV control in dairy farms in two districts of Hokkaido, Nemuro and Hiyama. A stochastic model was developed to compare the different control strategies over a 10‐year period. The model was individual‐based and simulated disease dynamics both within and between herds. Parameters included in the model were obtained from the literature, the Hokkaido government and the Japanese Ministry of Agriculture, Forestry and Fisheries. Nine different scenarios were compared as follows: no control, test‐and‐cull strategies based on antigen testing of either calves or only cattle entering common pastures, vaccination of all adult cattle or only cattle entering shared pastures and combinations thereof. The results indicate that current strategies for BVDV control in Hokkaido slightly reduced the number of BVDV ‐infected animals; however, alternative strategies such as testing all calves and culling any positives or vaccinating all susceptible adult animals dramatically reduced those. To our knowledge, this is the first report regarding the comparison of the effectiveness between the current strategies in Hokkaido and the alternative strategies for BVDV control measures.     

Evidence for Tunisian‐Like Pestiviruses Presence in Small Ruminants in Italy Since 2007

The genus Pestivirus, which belongs to the Flaviviridae family, includes ssRNA+ viruses responsible for infectious diseases in pigs, cattle, sheep, goats and other domestic and wild ruminants. Like most of the RNA viruses, pestivirus has high genome variability with practical consequences on disease epidemiology, diagnosis and control. In addition to the officially recognized species in the genus Pestivirus, such as BVDV‐1, BVDV‐2, BDV and CSFV, other pestiviruses have been detected. Furthermore, most of the ruminant pestiviruses show low or absent species specificity observed in serological tests and are able to infect multiple species. Particularly, small ruminants are receptive hosts of the most heterogeneous group of pestiviruses. The aim of this study was to carry out the molecular characterization of pestiviruses isolated from sheep and goats in Sicily, Italy. Phylogenetic analysis of two viral genomic regions (a fragment of 5′‐UTR and the whole N^pro regions) revealed the presence of different pestivirus genotypes in the analysed goat and sheep herds. Two of five viral isolates were clustered with BVDV‐1d viruses, a strain widespread in Italy, but never reported in Sicily. The other three isolates formed a distinct cluster with high similarity to Tunisian isolates, recently proposed as a new pestivirus species. This represents the first evidence for Tunisian‐like pestivirus presence in small ruminants in Italy. Furthermore, one of the isolates was collected from a goat, representing the first isolation of Tunisian‐like pestivirus from this species.     

Evidence of porcine circovirus‐like virus P1 in piglets with an unusual congenital tremor

Outbreaks of trembling and shaking were reported among pigs at two pig farms in Jiangsu Province, China. Serum and tissue samples tested positive for porcine circovirus‐like virus P1 and negative for classical swine fever virus, porcine circovirus type 2, astrovirus and porcine pestivirus using PCR/RT‐PCR and immunohistochemical techniques. High P1 viral genome loads were identified in sera, brain and lymph node tissue samples by qPCR. In addition, one of the most notable pathological changes was dissolution of the nucleus in Purkinje cells. The results of this study provide molecular evidence of an association between congenital tremor in pigs and P1 virus.     

Molecular Characteristics of Bovine Virus Diarrhoea Virus 1 Isolates from Turkey: Approaches for an Eradication Programme

Forty pestivirus isolates sampled from cattle in Turkey between 2002 and 2007 were characterized according to 5′ untranslated region (5′UTR) sequences and autoprotease (N^pro) gene sequences. The sampling of Bovine virus diarrhoea viruses (BVDVs) from 15 farms in five different regions indicated that BVDV 1‐l (18/40, 45%) was the predominant genotype in Turkey; the samples also contained the genotypes 1‐f (10/40, 25%), 1‐b (7/40, 17.5%), 1‐d (3/40, 7.5%), and 1‐a (2/40, 5%), respectively.     

Epidemiological factors and mitigation measures influencing production losses in cattle due to bovine viral diarrhoea virus infection: A meta‐analysis

Infection with bovine viral diarrhoea virus (BVDV) is associated with a loss in productivity in cattle farms. Determining which factors influence monetary losses due to BVDV could facilitate the implementation of mitigation measures to reduce the burden of BVDV. Mixed‐effect meta‐analysis models were performed to estimate the extent to which the costs of mean annual BVDV production losses per animal may be influenced by epidemiological factors such as BVDV introduction risk, initial prevalence, viral circulation intensity and circulation duration (trial 1). Additionally, changes in mean annual BVDV production losses per animal due to specific mitigation measures (i.e., biosecurity, vaccination, testing and culling, cattle introduction or contact with neighbouring cattle herds) were analysed (trial 2). In total, 19 studies were included in the meta‐analysis to assess mean annual BVDV production losses. The mean annual direct losses were determined to be €42.14 per animal (trial 1). The multivariate meta‐regression showed that four of the previously mentioned epidemiological factors significantly influenced the mean annual BVDV production losses per animal. Indeed, the per animal costs increased to €67.19 when these four factors (trial 1) were considered as “high or moderate” compared to “low”. The meta‐regression analysis revealed that implementation of vaccination and biosecurity measures were associated with an 8%–12% and 28%–29% decrease in BVDV production losses on average, respectively, when simulated herds were compared with or without such mitigation measures (trial 2). This reduction of mean annual BVDV production losses per animal due to mitigation measures was partially counteracted when farmers brought new cattle on to farm or allowed contact with neighbouring cattle herds. The influencing mitigation factors presented here could help to guide farmers in their decision to implement mitigation strategies for the control of BVDV at farm level.     

False‐Positive Results in Metagenomic Virus Discovery: A Strong Case for Follow‐Up Diagnosis

A viral metagenomic approach using virion enrichment, random amplification and next‐generation sequencing was used to investigate an undiagnosed cluster of dairy cattle presenting with high persistent fever, unresponsive to anti‐microbial and anti‐inflammatory treatment, diarrhoea and redness of nose and teat. Serum and whole blood samples were taken in the predicted hyperviraemic state of an animal that a few days later presented with these clinical signs. Bioinformatics analysis of the resulting data from the DNA virus identification workflow (a total of 32 757 sequences with average read length 335 bases) initially demonstrated the presence of parvovirus‐like sequences in the tested blood sample. Thorough follow‐up using specific real‐time RT‐PCR assays targeting the detected sequence fragments confirmed the presence of these sequences in the original sample as well as in a sample of an additional animal, but a contamination with an identical genetic signature in negative extraction controls was demonstrated. Further investigation using an alternative extraction method identified a contamination of the originally used Qiagen extraction columns with parvovirus‐like nucleic acids or virus particles. Although we did not find any relevant virus that could be associated with the disease, these observations clearly illustrate the importance of using a proper control strategy and follow‐up diagnostic tests in any viral metagenomic study.     

First Detection of Antibodies Against African Swine Fever Virus in Faeces Samples

African swine fever (ASF) is a viral, highly lethal haemorrhagic disease of swine with no available vaccine or effective treatment. Introduction of ASF into a country triggers immediate restriction measures that cause significant economic losses and threatens spread to neighbouring countries. Wild boar populations have been recently assigned an essential role in the spread of African swine fever virus (ASFV) to European countries. Therefore, effective surveillance and monitoring of wild boar populations is required, but sampling wild boar is logistically challenging and expensive. This study assessed the feasibility of detecting antibodies against ASFV in faeces for later implementation in surveillance and control programmes. Two groups of pigs were experimentally infected with an attenuated ASFV isolate Ken05, and blood, oral fluid and faecal samples were tested for the presence of viral DNA using quantitative real‐time polymerase chain reaction (qPCR) to monitor infection progress. Faecal samples were analysed using two indirect enzyme‐linked immunosorbent assays (ELISAs) based on semipurified viral protein (vp) 72 or purified recombinant vp30 expressed in mammalian cells. Faecal samples from 9 of 10 pigs with non‐haemorrhagic diarrhoea tested positive for antibodies against ASFV using the two ELISA tests that showed a positive correlation. The serum sample results from the two indirect ELISAs were compared against results from the reference ELISA technique and the immunoperoxidase test. Our findings indicate the feasibility of faecal sampling for detecting anti‐ASFV antibodies, which may provide a practical non‐invasive alternative for sampling wild boar populations. In conclusion, the application of these ELISA tests to faecal field samples could be particularly useful to screen for the presence of ASF in field conditions.     

Immunopathologic Changes in the Thymus of Calves Pre‐infected with BVDV and Challenged with BHV‐1

The aim of this work was to investigate the effect of pre‐infection with bovine viral diarrhoea virus (BVDV) on thymus immune cells from calves challenged with bovine herpesvirus 1 (BHV‐1). Twelve Friesian calves, aged 8 to 9 months, were inoculated with non‐cytopathic BVDV‐1. Ten of them were subsequently challenged with BHV‐1 and euthanized in batches of two at 1, 2, 4, 7 or 14 dpi with BHV‐1. The other two calves were euthanized prior to the second inoculation and were used as BVDV‐infected controls. A further 10 calves were inoculated solely with BHV‐1 and euthanized at the same time points. Two calves were not inoculated with any agent and were used as negative controls. Quantitative changes in immune cells were evaluated with immunohistochemical methods to compare coinfected calves and calves challenged only with BHV‐1. The results of this study pointed out BVDV as responsible for the thymic lesions observed in the experiment as well as for the majority of immunopathologic changes, including a downregulation of Foxp3 lymphocytes and TGFβ, which reverted as BVDV was cleared, and an overexpression of medullary CD8+ T cells. However, despite not inducing evident lesions in the thymus, BHV‐1 seemed to prompt some immune alterations. Collectively, these data contribute to the knowledge on the immunopathologic alterations of the thymus during BVDV infections, and its importance in the development of secondary infections.