Involvement of the Wnt pathway in BVDV cytopathogenic strain replication in primary bovine cells.

BACKGROUND: Bovine viral diarrhea virus 1 (BVDV-1) of the pestivirus genus is an economically crippling virus in the cattle industry; this positive RNA virus causes mucosal disease resulting in reproductive losses and other disease syndromes. The pathogenesis mechanism of the disease caused by BVDV infection is not well understood; for a better understanding of in vivo host BVDV-1 interactions, we conducted a transcriptomic study of infected cells at different times post-infection. METHODS: We compared the permissiveness and cellular response of a BVDV-1 cytopathogenic strain on Madin-Darby Bovine Kidney cells (MDBK) and bovine lung primary cells, a model closer to in vivo infection. Then a RNAseq analysis was realized on the infected bovine lung primary cells, at 10 hpi and 30 hpi (hours post-infection), to identify transcriptomic signatures. RESULTS: RNAseq analysis on BVDV-1 infected bovine primary cells showed 2,759 and 5,376 differentially expressed genes at respectively 10 hpi and 30 hpi with an absolute Fold Change ≥ 2. Among the different pathways deregulated, data analysis revealed a deregulation of Wnt signaling pathway, a conserved process that play a critical role in embryogenesis, cellular proliferation, and differentiation as well as in viral responses against viruses such as Influenza or Hepatitis C. We demonstrated here that the deregulation of the Wnt/ßcatenin signaling pathway plays a role in viral replication of BVDV cp strain. Interestingly, we showed that the inhibition of this Wnt pathway using two inhibitors, FZM1 and iCRT14, induced a delay in onset of the establishment of a cytopathic effect of primary cells. CONCLUSIONS: Thereby, this study highlighted a role of the Wnt signaling pathway in the BVDV-1 viral replication in bovine cells, suggesting an interesting option to explore as a new therapeutic target.

Cattle connection: molecular epidemiology of BVDV outbreaks via rapid nanopore whole-genome sequencing of clinical samples.

BACKGROUND: As a global ruminant pathogen, bovine viral diarrhea virus (BVDV) is responsible for the disease Bovine Viral Diarrhea with a variety of clinical presentations and severe economic losses worldwide. Classified within the Pestivirus genus, the species Pestivirus A and B (syn. BVDV-1, BVDV-2) are genetically differentiated into 21 BVDV-1 and four BVDV-2 subtypes. Commonly, the 5' untranslated region and the Npro protein are utilized for subtyping. However, the genetic variability of BVDV leads to limitations in former studies analyzing genome fragments in comparison to a full-genome evaluation. RESULTS: To enable rapid and accessible whole-genome sequencing of both BVDV-1 and BVDV-2 strains, nanopore sequencing of twelve representative BVDV samples was performed on amplicons derived through a tiling PCR procedure. Covering a multitude of subtypes (1b, 1d, 1f, 2a, 2c), sample matrices (plasma, EDTA blood and ear notch), viral loads (Cq-values 19-32) and species (cattle and sheep), ten of the twelve samples produced whole genomes, with two low titre samples presenting 96 % genome coverage. CONCLUSIONS: Further phylogenetic analysis of the novel sequences emphasizes the necessity of whole-genome sequencing to identify novel strains and supplement lacking sequence information in public repositories. The proposed amplicon-based sequencing protocol allows rapid, inexpensive and accessible obtainment of complete BVDV genomes.

SARS-CoV-2 in environmental perspective: Occurrence, persistence, surveillance, inactivation and challenges.

The unprecedented global spread of the severe acute respiratory syndrome (SARS) caused by SARS-CoV-2 is depicting the distressing pandemic consequence on human health, economy as well as ecosystem services. So far novel coronavirus (CoV) outbreaks were associated with SARS-CoV-2 (2019), middle east respiratory syndrome coronavirus (MERS-CoV, 2012), and SARS-CoV-1 (2003) events. CoV relates to the enveloped family of Betacoronavirus (ßCoV) with positive-sense single-stranded RNA (+ssRNA). Knowing well the persistence, transmission, and spread of SARS-CoV-2 through proximity, the faecal-oral route is now emerging as a major environmental concern to community transmission. The replication and persistence of CoV in the gastrointestinal (GI) tract and shedding through stools is indicating a potential transmission route to the environment settings. Despite of the evidence, based on fewer reports on SARS-CoV-2 occurrence and persistence in wastewater/sewage/water, the transmission of the infective virus to the community is yet to be established. In this realm, this communication attempted to review the possible influx route of the enteric enveloped viral transmission in the environmental settings with reference to its occurrence, persistence, detection, and inactivation based on the published literature so far. The possibilities of airborne transmission through enteric virus-laden aerosols, environmental factors that may influence the viral transmission, and disinfection methods (conventional and emerging) as well as the inactivation mechanism with reference to the enveloped virus were reviewed. The need for wastewater epidemiology (WBE) studies for surveillance as well as for early warning signal was elaborated. This communication will provide a basis to understand the SARS-CoV-2 as well as other viruses in the context of the environmental engineering perspective to design effective strategies to counter the enteric virus transmission and also serves as a working paper for researchers, policy makers and regulators.

Detection of bovine viral diarrhea virus genotype 1 in aerosol by a real time RT-PCR assay.

BACKGROUND: As a pestivirus of the Flaviviridae family, bovine viral diarrhea virus (BVDV), has imposed a large burden on animal husbandry worldwide, and such virus can be transmitted mainly through direct contact with other infected animals and probably via aerosols. In the present study, we aimed to develop a real-time RT-PCR method for detection of BVDV-1 in aerosol samples. METHODS: A pair of primers specific for highly conserved regions of the BVDV-1 5'-UTR was designed. The standard curve and sensitivity of the developed assay were assessed based on 10-fold serial dilutions of RNA molecular standard. The specificity of the assay was evaluated with other pestiviruses and infectious bovine viruses. The clinical performance was examined by testing 169 aerosol samples. RESULTS: The results showed that a good linear relationship existed between the standard curve and the concentration of template. The lowest detection limit was 5.2 RNA molecules per reaction. This assay was specific for detection of BVDV-1, and no amplification was found for other pestiviruses such as classical swine fever virus (CSFV), border disease virus (BDV), and common infectious bovine viruses, including BVDV-2, infectious bovine rhinotracheitis virus (IBRV), bovine parainfluenza virus type 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), bovine ephemeral fever virus (BEFV) and bovine coronavirus (BcoV). The assay was highly reproducible with low variation coefficient values (CVs) for intra-assay and inter-assay. A total of 169 aerosol samples collected from six dairy herds were tested using this method. The results showed that the positive detection rate of BVDV-1 was 17.2% (29/169), which was significantly higher compared with the conventional RT-PCR. Additionally, the positive samples (n = 29) detected by real-time RT-PCR were verified by BVDV RPA-LFD, and a concordance rate of 100% was obtained between them. CONCLUSIONS: Taken together, we developed a real-time RT-PCR assay for quantitative analysis of BVDV-1 in aerosol samples, and our finding provided valuable insights into the risk on aerosol transmission of BVDV-1.

Genetic analysis of bovine viral diarrhea virus in pre-weaned native Korean calves.

Bovine viral diarrhea virus (BVDV), a prominent viral pathogen worldwide, causes substantial economic losses in the cattle industry. BVDV comprises two recognized species, BVDV-1 and BVDV-2, and at least 21 subtypes (1a-1u) for BVDV-1 and four subtypes (2a-2d) for BVDV-2 based on its 5'-untranslated region. This study aimed at investigating the prevalence and genetic analysis of BVDV in calf feces in the Republic of Korea (ROK). We collected fecal samples from 635 pre-weaned native Korean calves aged 1-60 days, regardless of diarrhea, and subjected them to RT-PCR and phylogenetic analysis. Thirty-five (5.5%) of the 635 samples were positive for BVDV infection. BVDV was detected in 20, 10, and 5 calves aged 1-20 days, 21-40 days, and 41-60 days, respectively. BVDV was the most frequent in 17 normal feces, followed by 16 diarrheic feces, and 2 hemorrhagic feces. Phylogenetic analysis revealed that 25 samples belonged to BVDV-1b; 1 sample, BVDV-1c; and 9 samples, BVDV-2a. Moreover, the BVDV-1b and BVDV-2a isolates showed genetic variations. BVDV-1b was detected in diarrheic, hemorrhagic, and normal fecal samples. Thus, BVDV-1b is the most prevalent in calves and causes enteric disease with differing severity. BVDV-1c was newly identified in diarrheic calves. Further studies are warranted to elucidate the pathogenesis of BVDV-1c infection and its clinical manifestations. Our results indicate that effective vaccines and control programs against BVDV are required in the ROK.

Identification of a new bovine viral diarrhea virus subtype in the Republic of Korea.

BACKGROUND: Bovine viral diarrhea virus (BVDV) is prevalent in Korean indigenous cattle, leading to substantial economic losses. This study was conducted to investigate the occurrence of BVDV. In 2016, a total of 143 blood samples were collected from asymptomatic Korean indigenous calves younger than 3-months of age from six different farms in the Republic of Korea (ROK). RESULTS: Eighty-seven calves (60.8%, 87/143) were tested positive for BVDV as evaluated by RT-PCR analysis. Phylogenetic analysis based on the 5'-untranslated region was used to classify these cases into three subtypes: BVDV-1b, BVDV-1o, and BVDV-2a. These results showed that BVDV-1b was the predominant subtype, while 2 samples clustered with BVDV-2a. Interestingly, one sample formed a separate group as a potentially new subtype, BVDV-1o. To our knowledge, this is the first report of BVDV-1o infection in Korean native calves. The BVDV-1o subtype identified in this study was closely related to cattle isolates obtained from Japan, indicating that this subtype is a new introduction to the ROK. CONCLUSIONS: This study provides useful information for carrying out epidemiological surveys of BVDV in the ROK and developing a vaccine for future use in the ROK, particularly for the first detection of BVDV-1o in Korean indigenous calves. Further studies are required to investigate the prevalence and pathogenicity of this BVDV-1o subtype.

Pestivirus infection in cattle dairy farms: E2 glycoprotein ELISA reveals the presence of bovine viral diarrhea virus type 2 in northwestern Italy.

BACKGROUND: Bovine viral diarrhea virus (BVDV) types 1 and 2 are members of the Pestivirus genus of the Flaviviridae family. This genus also includes the HoBi-like virus, tentatively classified as BVDV type 3. BVDV-1 is widely distributed in Italy despite the extensive use of BVDV-1-based vaccines, while BVDV-2 and HoBi-like Pestivirus have been detected occasionally. Monitoring the occurrence of sporadic or atypical pestiviruses is a useful approach to evaluate the need for additional vaccine strains that can be used in BVDV control programs. RESULTS: In this study we developed a multiwell antibody ELISA based on the recombinant E2 protein of the three bovine pestiviruses. We evaluated the assay's applicability for surveillance purposes using pooled milk samples, each prepared from a maximum of 35 lactating cows and collected from 176 dairy herds. As expected, the majority of the pooled samples reacted to a greater extent against the BVDV-1 E2 antigen. All three milk pools from a single farm reacted to the BVDV-2 antigen, however. Further analysis using spot tests, antigen detection, and sequence analysis of the 5'-UTR region confirmed the presence of five persistently infected calves carrying a BVDV-2a strain. CONCLUSIONS: This study highlights for the first time that sporadic circulation of BVDV-2 can be predicted by immunoenzymatic methods in the absence of specific vaccination.

Primary surveys on molecular epidemiology of bovine viral diarrhea virus 1 infecting goats in Jiangsu province, China.

BACKGROUND: Bovine viral diarrhea virus (BVDV) is a pathogen of domestic and wildlife animals worldwide and is associated with several diseases. In China, there are many reports about genotyping of BVDV strains originated from cattle and pigs, and some of them focused on the geographical distributions of BVDV. Currently, the goat industry in Jiangsu province of China is under going a rapid expansion. Most of these goat farms are backyard enterprises and in close proximity to pig and cattle farms. However, there was very limited information about BVDV infections in goats. The objective of this study was to assess the frequency of BVDV infections of goats, the relationship of these infections to clinical signs and determine what BVDV genotypes are circulating in Jiangsu province. RESULTS: From 236 goat sera collected from six regions in Jiangsu province between 2011 and 2013, BVDV-1 was identified in 29 samples from the five regions by RT-PCR. The BVDV-1 infections occurred with/without clinical signs. Eight different BVDV-1 strains were identified from these positive samples based on the 5'-untranslated region (5'-UTR) sequences, and further clustered into four BVDV-1 subtypes on the phylogenetic analysis. Three were BVDV-1b, two BVDV-1m, two BVDV-1o, and one BVDV-1p, respectively. CONCLUSIONS: To our knowledge, this is the first report to investigate the occurrence of BVDV and the genotypes of BVDV infecting goats in China. The results indicated that BVDV-1 infections were indeed present and the viruses were with genetic variations in Chinese goat herds. The information would be very useful for prevention and control of BVDV-1 infections in China.

Identification of bovine viral diarrhea virus infection in Saanen goats in the Republic of Korea.

Bovine viral diarrhea virus (BVDV) is one of the most important viral pathogens of livestock and causes substantial economic losses to the livestock industry worldwide. BVDV is not necessarily species specific and is known to infect domesticated and wild ruminants. In the present study, BVDV infection was identified in two Saanen goats from one farm, and two different viral subtypes were found, BVDV-1a and BVDV-2a. Each isolate was closely related to cattle isolates identified in the Republic of Korea. The two sequences obtained in this study were not consistent with border disease virus (BDV). The incidence of BVDV in this farm apparently occurred in the absence of contact with cattle and may be associated with grazing. This study demonstrates that BVDV infection may be possible to transmit among goats without exposure to cattle. Therefore, this result indicates that Saanen goats may act as natural reservoirs for BVDV. This is the first report of BVDV-1a infection in a Saanen goat.

Cross-priming amplification for detection of bovine viral diarrhoea virus species 1 and 2.

AIMS: The aim of the study was the development of cross-priming amplification for ubiquitous detection of bovine viral diarrhoea virus (BVDV) species 1 and 2. METHODS AND RESULTS: Three and five specific primers, respectively, for the detection of BVDV-1 and BVDV-2, were designed on the basis of the sequences of the 5'UTR region. Incubation temperature and reaction time were determined. The optimal incubation conditions using water bath were 63°C for 75 min. Reverse transcription step (RT) was not required. The results were visualized under UV-light as a bright yellow fluorescence in positive samples. Additional method for results interpretation was agarose gel electrophoresis. Positive samples showed the presence of ladder-like banding patterns, formed by harpin-like cross-priming amplification (CPA) products. Sensitivity of CPA was compared with conventional RT-PCR and real-time RT-PCR. The CPA detection limit was 3500 copies for BVDV-1 and 80000 copies for BVDV-2 per reaction. For RT-PCR it was 350 and 80 copies for BVDV-1 and BVDV-2, respectively, and for real-time RT-PCR it was 35 copies for BVDV-1 and 80 copies for BVDV-2. The sensitivity of the developed method is sufficient to detect persistently infected (PI) animals. Positive results were found in 24 of 25 BVDV isolates belonging to species 1 and 2. Additionally, one false-negative result for BVDV-2 was detected. There were no false-positive results in negative samples and in the negative control. Both sets of primers used for the detection of BVDV-1 and BVDV-2 were not able to detect atypical pestiviruses. CPA positive results were confirmed by RT-PCR and real-time RT-PCR. CONCLUSIONS: CPA is a rapid method for the detection of BVDV-1 and BVDV-2 in field samples from PI animals. SIGNIFICANCE AND IMPACT OF STUDY: This is the first report on the application of the CPA method for the detection of BVDV.

Preclinical Efficacy of Cap-Dependent and Independent mRNA Vaccines against Bovine Viral Diarrhea Virus-1.

Bovine viral diarrhea virus (BVDV) is an RNA virus associated with severe economic losses in animal production. Effective vaccination and viral surveillance are urgent for the prevention and control of BVDV infection. However, the application of traditional modified live vaccines and inactivated vaccines is faced with tremendous challenges. In the present study, we describe the preclinical efficacy of two BVDV mRNA vaccines tested in mice and guinea pigs, followed by a field trial in goats, where they were compared to a commercial vaccine (formaldehyde inactivated). The two mRNAs were engineered to express the envelope protein E2 of BVDV-1, the most prevalent subtype across the world, through a 5' cap-dependent or independent fashion. Better titers of neutralizing antibodies against BVDV-1 were achieved using the capped RNA in the sera of mice and guinea pigs, with maximum values reaching 9.4 and 13.7 (by -log2), respectively, on the 35th day post-vaccination. At the same time point, the antibody levels in goats were 9.1 and 10.2 for the capped and capless RNAs, respectively, and there were no significant differences compared to the commercial vaccine. The animals remained healthy throughout the experiment, as reflected by their normal leukogram profiles. Collectively, our findings demonstrate that mRNA vaccines have good safety and immunogenicity, and we laid a strong foundation for the further exploitation of efficient and safe BVDV vaccines.

Pathogenicity assessment of a bovine viral diarrhea virus type 1l (BVDV-1l) strain in experimentally infected calves.

Bovine viral diarrhea is a widespread and economically important viral disease for livestock which can cause clinically diverse manifestations. The number of established BVDV subgenotypes has increased, not only the serological relationships of recently described subgenotypes but virulence and pathogenic characteristics have not yet been mostly elaborated. The dominant BVDV subgenotype in Turkiye was elaborated to be BVDV-1l, that involves more than half of field strains and there is no scientific data to identify the pathogenicity of this strain so far. This study investigated the pathogenicity of a selected field strain (TR-72) from subgenotype BVDV-1l. Experimental infection was implemented by intranasal inoculation of the strain TR-72 (10 ×105.5) to four young calves which were previously not vaccinated and were free both for BVDV antibodies and antigens. Clinical changes as well as blood parameters, body temperature, and viremia were monitored for 14 days. Only mild clinical signs associated with respiratory signs of BVDV infection were observed. Detected clinical signs included nasal discharge, conjunctivitis, cough, fatigue, high rectal temperature reaching 40.7 â„ƒ, and white blood cell counts depression started from the 2nd day and 40.4% decreased between the 12th and 14th days post-infection (poi). The presence of viremia was investigated by virus isolation, RT-PCR, and real-time RT-PCR from blood samples. The efficiency of experimental infection was established not only by observed clinical signs but also by virus isolation from blood leukocytes between the 5th and 8th days poi., virus detection was obtained by real-time PCR between the 3rd - 13th days poi. Besides, the recorded mild clinical signs, high fever, long duration of viremia , and high decrease in blood parameters obtained in this study, it was shown that the noncytopathogenic BVDV-1l strain TR-72 has a moderate virulence in naïve cattle.

Development of a nucleic acid detection method based on the CRISPR-Cas13 for point-of-care testing of bovine viral diarrhea virus-1b.

Bovine viral diarrhea (BVD) is a single-stranded, positive-sense ribonucleic acid (RNA) virus belonging to the genus Pestivirus of the Flaviviridae family. BVD frequently causes economic losses to farmers. Among bovine viral diarrhea virus (BVDV) strains, BVDV-1b is predominant and widespread in Hanwoo calves. Reverse-transcription polymerase chain reaction (RT-PCR) is an essential method for diagnosing BVDV-1b and has become the gold standard for diagnosis in the Republic of Korea. However, this diagnostic method is time-consuming and requires expensive equipment. Therefore, Clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems have been used for point-of-care (POC) testing of viruses. Developing a sensitive and specific method for POC testing of BVDV-1b would be advantageous for controlling the spread of infection. Thus, this study aimed to develop a novel nucleic acid detection method using the CRISPR-Cas13 system for POC testing of BVDV-1b. The sequence of the BVD virus was extracted from National Center for Biotechnology Information (NC_001461.1), and the 5' untranslated region, commonly used for detection, was selected. CRISPR RNA (crRNA) was designed using the Cas13 design program and optimized for the expression and purification of the LwCas13a protein. Madin Darby bovine kidney (MDBK) cells were infected with BVDV-1b, incubated, and the viral RNA was extracted. To enable POC viral detection, the compatibility of the CRISPR-Cas13 system was verified with a paper-based strip through collateral cleavage activity. Finally, a colorimetric assay was used to evaluate the detection of BVDV-1b by combining the previously obtained crRNA and Cas13a protein on a paper strip. In conclusion, the CRISPR-Cas13 system is highly sensitive, specific, and capable of nucleic acid detection, making it an optimal system for the early point-of-care testing of BVDV-1b.

Molecular Characteristics of Bovine Viral Diarrhea Virus Strains Isolated from Persistently Infected Cattle.

In this study, we reported the isolation, identification, and molecular characteristics of nine BVDV strains that were isolated from the serum of persistently infected cattle. The new strains were designated as BVDV TJ2101, TJ2102, TJ2103, TJ2104, TJ2105, TJ2106, TJ2107, TJ2108 and TJ2109. The TJ2102 and TJ2104 strains were found to be cytopathic BVDV, and the other strains were non-cytopathic BVDV. An alignment and phylogenetic analysis showed that the new isolates share 92.2-96.3% homology with the CP7 strain and, thus, were classified as the BVDV-1b subgenotype. A recombination analysis of the genome sequences showed that the new strains could be recombined by the major parent BVDV-1a NADL strain and the minor parent BVDV-1m SD-15 strain. Some genome variations or unique amino acid mutations were found in 5'-UTR, E0 and E2 of these new isolates. In addition, a potential linear B cell epitopes prediction showed that the potential linear B cell epitope at positions 56-61 is highly variable in BVDV-1b. In conclusion, the present study has identified nine strains of BVDV from persistently infected cattle in China. Further studies on the virulence and pathogenesis of these new strains are recommended.

Prevalence of Bovine Viral Diarrhea Virus Infections in Pigs on Jeju Island, South Korea, from 2009-2019 and Experimental Infection of Pigs with BVDV Strains Isolated from Cattle.

On Jeju Island, South Korea, pigs have not been vaccinated against classical swine fever (CSF) since 1999. Analysis of bovine viral diarrhea virus (BVDV) isolated from pigs on Jeju Island between 2009 and 2019 identified five BVDV-1a strains and one BVDV-1b strain. These BVDV types were shown to be the same types as BVDV strains isolated from neighboring cow farms. BVDV antibody-positive pigs (both BVDV-1 and -2) were also detected at 54 of 168 pig farms during this period. In pig infection experiments using BVDV-1a and -2a strains isolated from neighboring cow farms, BVDV-1a was detected in the blood of one of four pigs infected at both 6 and 35 days post-infection (dpi) and in the blood of two of the four pigs at 28 dpi. Pigs showed higher anti-BVDV-1 titers (5.5 ± 1.5 log2) at 35 dpi. BVDV-2a was detected in the blood of one of four pigs infected with this virus at 28 dpi only, and lower antibody titers (2.75 ± 0.75 log2) were seen in these pigs at 35 dpi. While BVDV infection is not particularly pathogenic in pigs, it is still important to monitor porcine BVDV infections due to a differential diagnosis of CSFV.

Identification of differentially expressed gene pathways between cytopathogenic and non-cytopathogenic BVDV-1 strains by analysis of the transcriptome of infected primary bovine cells.

The bovine viral diarrhea virus 1 (BVDV-1), belonging to the Pestivirus genus, is characterized by the presence of two biotypes, cytopathogenic (cp) or non-cytopathogenic (ncp). For a better understanding of the host pathogen interactions, we set out to identify transcriptomic signatures of bovine lung primary cells (BPCs) infected with a cp or a ncp strain. For this, we used both a targeted approach by reverse transcription droplet digital PCR and whole genome approach using RNAseq. Data analysis showed 3571 differentially expressed transcripts over time (Fold Change >2) and revealed that the most deregulated pathways for cp strain are signaling pathways involved in responses to viral infection such as inflammatory response or apoptosis pathways. Interestingly, our data analysis revealed a deregulation of Wnt signaling pathway, a pathway described in embryogenesis, that was specifically seen with the BVDV-1 cp but not the ncp suggesting a role of this pathway in viral replication.

Isolation of BVDV-1a, 1m, and 1v strains from diarrheal calf in china and identification of its genome sequence and cattle virulence.

Bovine viral diarrhea virus (BVDV) is an important livestock viral pathogen responsible for causing significant economic losses. The emerging and novel BVDV isolates are clinically and biologically important, as there are highly antigenic diverse and pathogenic differences among BVDV genotypes. However, no study has yet compared the virulence of predominant genotype isolates (BVDV-1a, 1b, and 1m) in China and the emerging genotype isolate BVDV-1v. The serological relationship among these genotypes has not yet been described. In this study, we isolated three BVDV isolates from calves with severe diarrhea, characterized as BVDV-1a, 1m, and novel 1v, based on multiple genomic regions [including 5-untranslated region (5'-UTR), Npro, and E2] and the phylogenetic analysis of nearly complete genomes. For the novel genotype, genetic variation analysis of the E2 protein of the BVDV-1v HB-03 strain indicates multiple amino acid mutation sites, including potential host cell-binding sites and neutralizing epitopes. Recombination analysis of the BVDV-1v HB-03 strain hinted at the possible occurrence of cross-genotypes (among 1m, 1o, and 1q) and cross-geographical region transmission events. To compare the pathogenic characters and virulence among these BVDV-1 genotypes, newborn calves uninfected with common pathogens were infected intranasally with BVDV isolates. The calves infected with the three genotype isolates show different symptom severities (diarrhea, fever, slowing weight gain, virus shedding, leukopenia, viremia, and immune-related tissue damage). In addition, these infected calves also showed bovine respiratory disease complexes (BRDCs), such as nasal discharge, coughing, abnormal breathing, and lung damage. Based on assessing different parameters, BVDV-1m HB-01 is identified as a highly virulent strain, and BVDV-1a HN-03 and BVDV-1v HB-03 are both identified as moderately virulent strains. Furthermore, the cross-neutralization test demonstrated the antigenic diversity among these Chinese genotypes (1a, 1m, and 1v). Our findings illustrated the genetic evolution characteristics of the emerging genotype and the pathogenic mechanism and antigenic diversity of different genotype strains, These findings also provided an excellent vaccine candidate strain and a suitable BVDV challenge strain for the comprehensive prevention and control of BVDV.

The NADL strain of bovine viral diarrhea virus induces the secretion of IL-1ß through caspase 1 in bovine macrophages.

Bovine viral diarrhea virus (BVDV) infects different cell types including antigen-presenting cells such as macrophages. The infection induces pro-inflammatory cytokines like interleukin 1 beta (IL-1ß), which is necessary to trigger a successful inflammatory response against infections. Several authors have reported differences between IL-1ß gene expression and protein detection in BVDV-infected macrophages. These patterns may be related to inflammasome assembly, which promote the formation of active caspase 1 in order to produce mature IL-1ß molecules. Our goal was to assess BVDV ability to induce the release of IL-ß through a caspase 1 dependent pathway in bovine macrophages. We infected peripheral blood monocyte-derived macrophages using BVDV NADL strain at 0.001, 0.1, 2 and 10 multiplicities of infection (MOI) and we measured IL-1ß at different times 2, 6, 12, 24, 48, 72 h. We found an increase of 1140-2154 pg for a MOI of 10:1 and 2:1 respectively. To inhibit caspase 1, we used either carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Z-VAD) or carbobenzoxy-tyr-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Y-VAD). We found decreased IL-1ß secretion 2154 pg/ml to 854 pg/ml IL-1ß secretion using Y-VAD and we observed decrease from 2154 pg/ml to 22.33 pg/ml with Z-VAD, and this inhibition was followed by diminished viral replication from 2.25 × 107 to 2.1 × 105 CCID50, which suggests that caspase 1-dependent secretion of the IL-1ß active molecule is important for viral replication. This is the first report showing that BVDV infected-bovine macrophages trigger the caspase 1 dependent pathway for IL-1ß activation and that activation increases viral replication.

Failure in dry period vaccination strategy for bovine viral diarrhea virus.

Bovine viral diarrhea is a common disease of cattle and has significant impact on animal welfare worldwide. There are fundamental approaches i.e. elimination of persistently infected animals, vaccination and biosecurity measures for effective control and eradication of BVD virus (BVDV). By this study, the presence of persistent infection with divergent BVDV subgenotype in the calves in a dairy herd having regular vaccination program was investigated. In the herd, vaccinated with a killed whole virion trivalent vaccine (composed of BVDV-1a) during the dry period of the cows, abortion cases were existed in the late autumn 2019. During herd screening by BVDV antigen-ELISA, 2 out of 300 dams were detected positive. Following, by ear notch-based BVDV antigen-ELISA, 30 calves were detected positive. Confirmation of persistent BVDV infection was performed 3 weeks later by testing with antigen-ELISA, where 8 of 9 selected newborn calves were positive for the second time. The entire antigen-ELISA positive samples were subjected to virus isolation on MDBK cell culture and identified as non-cytopathogenic pestiviruses by indirect immunoperoxidase assay. Presence of pestivirus RNA was detected in the 8 isolates by panpestivirus RT-PCR. Analysis of the 5'UTR regions revealed that BVDV-1 r circulate in the herd. Results of this study lead to questioning the efficiency of dry period vaccination strategy against BVDV. But otherwise, vaccination with BVDV-1a can be inefficient for complete protection against BVDV-1 r. Therefore, serological relationship between mentioned subgenotypes or protection by current vaccines against latest field isolates needs to be investigated before development of new BVDV vaccine candidates.

First-time detection of bovine viral diarrhoea virus, BVDV-1, in cattle in Botswana.

Infectious diseases are serious constraints for improving livestock productivity. Bovine viral diarrhoea virus (BVDV) is a virus causing grave economic losses throughout the cattle producing world. Infection is often not apparent, but the virus can also cause respiratory signs, diarrhoea, reproductive problems and immunosuppression. Risk factors for disease transmission include, but are not limited to, herd size, animal trade and grazing on communal pastures. Several prevalence studies have been conducted in southern Africa, but in Botswana the occurrence is largely unknown. In this study, blood samples were obtained from 100 goats from three villages around the capital city, Gaborone. Also, 364 blood samples from cattle around Gaborone, collected as part of another study, were analysed. The detected antibody prevalence was 0% in goats and 53.6% in cattle when using a competitive enzyme-linked immunoassay. Three animals from two different herds were positive for viral nucleic acids on polymerase chain reaction. The two herds with viraemic animals had significantly higher antibody prevalence compared to the other herds. Also, two of the detected viruses were sequenced and found to be most similar to BVDV-1a. To the authors' knowledge, this is the first time that sequencing has been performed on BVDV isolated in Botswana.