Bovine Viral Diarrhea Virus Strains Research Articles

"Fading out" - genomic epidemiology of the last persistently infected BVDV cattle in Germany.

Bovine viral diarrhea virus (BVDV) is one of the most important cattle pathogens worldwide, causing major economic losses and animal welfare issues. Disease eradication programs have been implemented in several countries, including Germany where an obligatory nationwide control program is in force since 2011. As molecular epidemiology has become an essential tool to understand the transmission dynamics and evolution of BVDV, 5' untranslated region (UTR) sequences are generated from viruses present in persistently infected animals since the beginning of the BVDV control program. Here, we report the results of the sequence-based subtyping of BVDV strains found from 2018 through 2022 in calves born in Germany. In 2018, 2019 and 2020, BVDV-1d and-1b were the dominant subtypes and cases were spread throughout the area that was not yet officially declared BVDV-free at that time. In addition, BVDV-1a, -1e, -1f and -1h could rarely be detected. From 2021 onwards, subtype 1d clearly took over the dominance, while the other subtypes could be gradually nearly eliminated from the cattle population. The eradication success not only results in a drastic reduction of cases, but also in a marked reduction of strain diversity. Interestingly, before vaccination has been banned in regions and farms with a disease-free status, two live-vaccine virus strains were repeatedly detected in ear tissue samples of newborn calves (n = 14) whose mothers were immunized during gestation. The field-virus sequences are an important basis for molecular tracing and identification of potential relationships between the last outbreaks in the final phase of the German BVDV eradication program, thereby supporting classic epidemiological investigations. Furthermore, the monitoring of the composition of virus subtypes in the cattle population helps to maintain effective diagnostic methods and control measures and is an early warning system for the introduction of new pestiviruses in the naïve cattle population.

Testing and identification of bovine viral diarrhea virus isolates recovered in Russia between 2019 and 2022

Studying the agents of bovine viral diarrhea (BVD) is an important task given the high probability of new isolate introduction into the Russian Federation, as well as the need to take into account the genotype and subgenotype of the virus circulating in a herd when developing vaccines and diagnostic kits for the infection. During the work performed, 6 BVD virus isolates were recovered and identified. The recovery of these isolates in the lamb testicle cell subculture revealed that Bashkiria/2019, Kirov/2020 and Samara/2020 isolates belong to non-cytopathic bovine viral diarrhea virus biotypes, Chelyabinsk/2021 isolate demonstrated the characteristic cytopathic effect in the monolayer and was classified as a cytopathic variant of thevirus, the adaptation of Belgorod/2021 and Udmurtiya/2020 isolates to this cell system was not possible. The study also identified the species of the recovered isolates. Based on the analysis of the nucleotide sequence of genome 5’-untranslated region (5’-UTR) fragment, these isolates were classified as belonging to three genotypes of the virus. The phylogenetic analysis showed that Chelyabinsk/2021 and Udmurtiya/2020 isolates belong togenotype2 and demonstrate, respectively, 98% and99% homology with reference 890 strain of BVD virus. The recovered Bashkiria/2019, Samara/2020, Kirov/2020 isolates were classified as belonging to subtypes 1i, 1f and1b ofgenotype1, and Belgorod/2021 solate represents genotype 3 of the virus. The findings from the study confirm the presence of all three genotypes of bovine viral diarrhea virus in the Russian Federation and reiterate the need for the development of specific prevention and diagnosis tools for the disease.

Enhanced infectivity of bovine viral diarrhoea virus (BVDV) in arginase-producing bovine monocyte-derived macrophages

ABSTRACT Macrophages are important cells of the innate immunity that play a major role in Bovine Viral Diarrhea Virus (BVDV) pathogenesis. Macrophages are not a homogenous population; they exist in different phenotypes, typically divided into two main categories: classically (pro-inflammatory) and alternatively activated (anti-inflammatory) or M1 and M2, respectively. The role of bovine macrophage phenotypes on BVDV infection is still unclear. This study characterized the interaction between BVDV, and monocyte-derived macrophages (Mo-Mφ) collected from healthy cattle and polarized to an M1 or M2 state by using LPS, INF-γ, IL-4 or azithromycin. Arginase activity quantitation was utilized as a marker of the M2 Mo-Mφ spectrum. There was a significant association between arginase activity and the replication rate of BVDV strains of different genotypes and biotypes. Inhibition of arginase activity also reduced BVDV infectivity. Calves treated with azithromycin induced Mo-Mφ of the M2 state produced high levels of arginase. Interestingly, azithromycin administered in vivo increased the susceptibility of macrophages to BVDV infection ex vivo. Mo-Mφ from pregnant dams and calves produced higher arginase levels than those from non-pregnant adult animals. The increased infection of arginase-producing alternatively activated bovine macrophages with BVDV supports the need to delve into a possible leading role of M2 macrophages in establishing the immune-suppressive state during BVDV convalescence.

First report of bovine viral diarrhea virus subgenotypes 1d and 1e in southern Chile

Bovine viral diarrhea virus (BVDV) affects cattle worldwide causing severe productive and economic loss. In this study, we investigated the subgenotypes of BVDV circulating in cattle samples from the Aysén region, an active cattle breeding area located in southern Chile. Partial amplification of the 5' untranslated region (UTR) was performed by polymerase chain reaction (PCR), and twelve samples were analyzed by Sanger sequencing and phylogenetic analysis. Eight samples were identified as belonging to Pestivirus bovis subgenotype 1e, three to 1-b, and one to 1-d. The phylogenetic analyses performed revealed a marked distance between these now-identified strains and those previously reported in the country. These findings support the need to continually expand the analysis of the variability of the viral phylogeny for the currently circulating BVDV strains and to update the vaccines recommended for this livestock area and surrounding areas.

Immunoinformatic prediction of the pathogenicity of bovine viral diarrhea virus genotypes: implications for viral virulence determinants, designing novel diagnostic assays and vaccines development.

Bovine viral diarrhea virus (BVDV) significantly impacts the bovine industries, both dairy and beef sectors. BVDV can infect various domestic and wild animals, most notably cattle. The dynamic variations among BVDV serotypes due to the continuous genetic diversity, especially in BVDV1 (BVDV1), reduce the effectiveness of the currently available vaccines and reduce the specificity/sensitivity of the diagnostic assays. The development of novel, safe, and effective vaccines against BVDV requires deep knowledge of the antigenicity and virulence of the virus. Previous studies on the antigenicity and the virulence of BVDV serotypes have been mainly focused on one or a few BVDV proteins. While however, little is known about the orchestration of all BVDV in the context of viral virulence and immunogenicity. The main aim of the current study was to do a comparative computational evaluation of the immunogenicity, and virulence for all the encoded proteins of both BVDV1 and BVDV2 and their sub-genotypes. To achieve this goal, 11,737 protein sequences were retrieved from Virus Pathogen Resource. The analysis involved a total of 4,583 sequences after the removal of short sequences and those with unknown collection time. We used the MP3 tool to map the pathogenic proteins across different BVDV strains. The potential protective and the epitope motifs were predicted using the VaxiJen and EMBOSS antigen tools, respectively. The virulence prediction revealed that the NS4B proteins of both BVDV1 and BVDV2 likely have essential roles in BVDV virulence. Similarly, both the capsid (C) and the NS4-A proteins of BVDV1 and the Npro and P7 proteins of BVDV2 are likely important virulent factors. There was a clear trend of increasing predicted virulence with the progression of time in the case of BVDV1 proteins, but that was not the case for the BVDV2 proteins. Most of the proteins of the two BVDV serotypes possess antigens predicted immunogens except Npro, P7, and NS4B. However, the predicted antigenicity of the BVDV1 was significantly higher than that of BVDV2. Meanwhile, the predicted immunogenicity of the immunodominant-E2 protein has been decreasing over time. Based on our predicted antigenicity and pathogenicity studies of the two BVDV serotypes, the sub-genotypes (1a, 1f, 1k, 2a, and 2b) may represent ideal candidates for the development of future vaccines against BVDV infection in cattle. In summary, we identified some common differences between the two BVDV genotypes (BVDV1 and BVDV2) and their sub-genotypes regarding their protein antigenicity and pathogenicity. The data presented here will increase our understanding of the molecular pathogenesis of BVDV infection in cattle. It will also pave the way for developing some novel diagnostic assays and novel vaccines against BVDV in the near future.

NLRP3 Inflammasome Involved with Viral Replication in Cytopathic NADL BVDV Infection and IFI16 Inflammasome Connected with IL-1β Release in Non-Cytopathic NY-1 BVDV Infection in Bovine Macrophages.

Inflammasomes are multiprotein complexes that play a role in the processing of proinflammatory cytokines such as interleukin 1 beta (IL-1β). The secretion of IL-1β in bovine macrophages infected with the bovine viral diarrhea virus (BVDV) cytopathic strain NADL (NADLcp-BVDV) is caspase 1-dependent. In the present study, we found that in macrophages infected with NADL, the NLRP3 inflammasome participated in the maturation of IL-1β as the level decreased from 4629.3 pg/mL to 897.0 pg/mL after treatment with cytokine release inhibitory drug 3 (CRID3). Furthermore, NLRP3 activation has implications regarding viral replication, as there was a decrease in the viral titer until 1 log of a supernatant of macrophages that were inhibited with CRID3 remained. In the case of the non-cytopathic BVDV strain NY-1 (NY-1 ncpBVDV), IL-1β secretion is not affected by NLRP3, but could be related to the IFI16 inflammasome; we found a colocalization of IFI16 with ASC using confocal microscopy in infected macrophages with the NY-1 ncp-BVDV biotype. To relate IFI16 activation to IL-1β release, we used ODN TTAGGG (A151), a competitive inhibitor of IFI16; the results show a decrease in its level from 248 pg/mL to 128.3 pg/mL. Additionally, we evaluated the caspase 1 activation downstream of IFI16 and found a decrease in the IL-1β from 252.9 pg/mL to 63.5 pg/mL when caspase 1 was inhibited with Y-VAD. Our results provide an improved understanding of the mechanisms involved in the viral replication, inflammation and pathogenesis of bovine viral diarrhea.

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.

Immune protection induced by E2 recombinant glycoprotein of bovine viral diarrhea virus in a murine model.

Bovine viral diarrhea virus (BVDV) is considered the most important viral pathogen in ruminants worldwide due to the broad range of clinical manifestations displayed by infected animals. Therefore, infection with BVDV leads to severe economic losses in several countries' beef and dairy industries. Vaccination prevents reproductive failure and gastrointestinal and respiratory disorders caused by BVDV infection. However, considering their limitations, conventional vaccines such as live, attenuated, and killed viruses have been applied. Hence, different studies have described subunit vaccines as an effective and safe alternative for BVDV protection. Therefore, in this study, the ectodomain of E2 (E2e) glycoprotein from NADL BVDV strain was expressed in mammalian cells and used in two vaccine formulations to evaluate immunogenicity and protection against BVDV conferred in a murine model. Formulations consisted of solo E2e glycoprotein and E2e glycoprotein emulsified in adjuvant ISA 61 VG. Five groups of 6 mice of 6-to-8-week-old were immunized thrice on days 1, 15, and 30 by intraperitoneal injection with the mentioned formulations and controls. To evaluate the conferred protection against BVDV, mice were challenged six weeks after the third immunization. In addition, the humoral immune response was evaluated after vaccination and challenge. Mice groups inoculated with solo E2e and the E2e + ISA 61 VG displayed neutralizing titers; however, the E2 antibody titers in the E2e + ISA 61 VG group were significantly higher than the mice group immunized with the solo E2e glycoprotein. In addition, immunization using E2e + ISA 61 VG prevents animals from developing severe lesions in surveyed tissues. Moreover, this group acquired protection against the BVDV challenge, evidenced by a significant reduction of positive staining for BVDV antigen in the lungs, liver, and brain between the experimental groups. Our findings demonstrated that using E2e + ISA 61 VG induces greater BVDV protection by an early humoral response and reduced histopathological lesions and BVDV antigen detection in affected organs, indicating that E2e + ISA 61 VG subunit formulation can be considered as a putative vaccine candidate against BVDV. The efficacy and safety of this vaccine candidate in cattle requires further investigation.

The Involvement of Neutrophil in the Immune Dysfunction Associated with BVDV Infection.

Bovine viral diarrhea virus (BVDV) induces immune dysfunction that often results in a secondary bacterial infection in the infected animals. The underlying mechanism of BVDV-induced immune dysfunction is not well understood. The role of BVDV-infected macrophage-secreted factors was investigated. BVDV-infected monocyte-derived macrophage (MDM) supernatants down-regulated the expression of neutrophil L-selectin and CD18. Regardless of the biotype, phagocytic activity and oxidative burst were downregulated by BVDV-infected MDM supernatants. However, only supernatants from cytopathic (cp) BVDV down-regulated nitric oxide production and neutrophil extracellular traps (NET) induction. Our data suggested that BVDV-induced macrophage-secreted factors caused immune dysfunction in neutrophils. Unlike lymphocyte depletion, the negative impact on neutrophils seems to be specific to cp BVDV biotype. Interestingly the majority of modified live BVDV vaccines are based on cp strain of BVDV.

Characterization of a Highly Virulent Noncytopathic Bovine Viral Diarrhea Virus 2b Isolate Detected in Cattle in Inner Mongolia, China

Bovine viral diarrhea virus (BVDV) infection causes subclinical to severe acute disease in cattle all over the world. Two genotypes of BVDV are recognized, BVDV1 and BVDV2. Currently, the subtypes of BVDV1b and BVDV2a are mainly circulating in China. Here, a newly noncytopathic (ncp) BVDV strain named BVDVNM21 was isolated and identified from cattle. We determined the complete genome sequence of BVDVNM21, phylogenetic analysis of 5′ untranslated region (5′UTR), Npro, and E2 gene, and complete genome showed the virus belongs to BVDV2b subtype and formed an independent clade within BVDV2b. Genome-wide sequence analysis showed that BVDVNM21 had high homology with SD1301 (98.10%) isolated from China. In the experimental infection study of guinea pigs and calves, they all developed similar clinical signs, including a pronounced and prolonged febrile response lasting more than 3 days and pronounced reduction in white blood cells (WBC) and platelets of more than 40%. Moreover, guinea pigs showed more acute infection characteristics after infection, and WBC decreased by more than 50% at 1 day postinfection (dpi), and they recovered in less than 3 days. The results indicated that the BVDVNM21 strain showed high virulence in calves. It was the first confirmed isolation of a highly pathogenic ncpBVDV2b in cattle, as well as the establishment of the BVDV-guinea pig model. This study may provide a basis for further research and control of the prevalence of BVDV2b in China.

Novel bovine viral diarrhea virus (BVDV) virus-like particle vaccine candidates presenting the E2 protein using the SpyTag/SpyCatcher system induce a robust neutralizing antibody response in mice.

Bovine viral diarrhea virus (BVDV) is a pathogen of commercial consequence in cattle. Although many modified live and killed vaccines are commercially available, their drawbacks precipitate the need for new effective vaccines. Virus-like particles (VLPs) are a safe and powerful technology used in several human and veterinary vaccines; however, it is difficult to produce large amounts of BVDV VLPs. In this study, we generated red-spotted grouper nervous necrosis virus (RGNNV) VLPs presenting the BVDV E2 protein (domain I to IIIb) of the Nose (BVDV-1) or KZ-91-CP (BVDV-2) strain by exploiting SpyTag/SpyCatcher technology. Mice immunized twice with 30 μg of RGNNV VLPs conjugated with 10 μg of E2 proteins of the Nose or KZ-91-CP strain with a 14-day interval elicited high (1:512,000 to 1:1,024,000) and moderate (1:25,600 to 1:102,400) IgG titers against E2 proteins of homologous and heterologous strains, respectively. In addition, this prime-boost regimen induced strong (1:800 to 1:3,200) and weak (~1:10) neutralization titers against homologous and heterologous BVDV strains, respectively. Our results indicate that conjugation of the E2 protein to RGNNV VLPs strongly enhances the antigenicity of the E2 protein and that RGNNV VLPs presenting the E2 protein are promising BVDV vaccine candidates.

Bta-miR-2904 inhibits bovine viral diarrhea virus replication by targeting viral-infection-induced autophagy via ATG13.

MicroRNAs (miRNAs) are endogenous small and noncoding RNA molecules (18-25 nt) that can regulate expression of their target genes post-transcriptionally. Previously, using high-throughput sequencing data obtained on a Solexa platform, we found that Bos taurus bta-miR-2904 (miR-2904) was significantly upregulated in Madin-Darby bovine kidney (MDBK) cells infected with bovine viral diarrhea virus (BVDV) strain NADL at 2, 6, and 18 h postinfection (hpi) compared to uninfected MDBK cells. Moreover, miR-2904 overexpression significantly reduced BVDV replication. However, the mechanism by which miR-2904 inhibits viral replication remains unclear. In this study, we used electron microscopy, laser confocal microscopy, dual-luciferase reporter analysis, real-time PCR, and Western blot assays to investigate the effect of the miR-2904 expression on BVDV NADL replication and virus-infection-induced autophagy. The results indicate that miR-2904 inhibits autophagy of MDBK cells by targeting autophagy-related gene 13 (ATG13), and overexpression of miR-2904 inhibited the replication of BVDV NADL.

Replication of standard bovine viral diarrhea strain OregonC24Va induces endoplasmic reticulum stress-mediated apoptosis of bovine trophoblast cells.

Bovine viral diarrhea (BVD) is a worldwide infectious disease caused by bovine viral diarrhea virus (BVDV) infection, which invades the placenta, causes abortion, produces immune tolerance and continuously infects calves, and causes huge economic losses to the cattle industry. The endoplasmic reticulum (ER) is an important organelle in cells, which is prone to ER stress after being stimulated by pathogens, thus activating the ER stress-related apoptosis. Studies have confirmed that BVDV can utilize the ER of its host to complete its own proliferation and stimulate ER stress to a certain extent. However, the role of ER stress in BVDV infecting bovine placental trophoblast cells (BTCs) and inducing apoptosis is still unclear. We are using the cytopathic strain of BVDV (OregonC24Va), which can cause apoptosis of BTCs, as a model system to determine how ER stress induced by BVDV affects placental toxicity. We show that OregonC24Va can infect BTCs and proliferate in it. With the proliferation of BVDV in BTCs, ER stress-related apoptosis is triggered. The ER stress inhibitor 4-PBA was used to inhibit the ER stress of BTCs, which not only inhibited the proliferation of BVDV, but also reduced the apoptosis of BTCs. The ER stress activator Tg can activate ER stress-related apoptosis, but the proliferation of BVDV does not change in BTCs. Therefore, BVDV utilizes the UPR of activated ER stress to promote the proliferation of BVDV in the early stage of infection, and activates the ER stress-related apoptosis of BTCs in the later stage with the virus proliferation to promote the cell apoptosis and further spread of the virus. Our research provides a new theoretical basis for exploring the placental infection and vertical transmission of BVDV.

A Bovine Viral Diarrhea Virus Type 1c Strain in China: Isolation, Identification, and Assessment of Pathogenicity in Rabbits.

Bovine viral diarrhea virus (BVDV) is an important animal pathogen and has a negative economic impact on cattle industries worldwide. In this study, the BVDV strain named BJ175170 was detected, isolated, and identified from cattle in Beijing, China, during herd screening by BVDV antigen-ELISA and indirect immunofluorescence assay (IFA). To investigate its genomic features, the characteristic 5'UTR region of the isolates were sequenced and BLAST analyzed. BVDV BJ175170 belongs to the BVDV-1c subtype, which differs from the Beijing prevalent BVDV strains. The BVDV particles were further observed by transmission electron microscopy (TEM). To evaluate the virulence of the BVDV BJ175170, the BVDV seronegative rabbits were intraperitoneally inoculated with the virus suspension. Blood samples were analyzed for changes in leukocyte number and antibody titer, and tissue samples were taken for histopathology analysis. These data confirmed again that rabbits could act as the reservoir of BVDV, which poses a small but non-zero risk of re-infection for BVDV-free cattle herds. To our knowledge, this is the first report of pathological changes in rabbits after exposure to BVDV-1c subtype, which could act as experimental reference. Meanwhile, the results of this study indicate that rabbits could act as a potential model for studying the mechanism of BVDV in vivo.

Genetic diversity of Bovine Viral Diarrhea Virus in cattle in France between 2018 and 2020.

Bovine Viral Diarrhea Virus (BVDV) is one of the main pathogens that affects ruminants worldwide, generating significant economic losses. Like other RNA viruses, BVDV is characterized by a high genetic variability, generating the emergence of new variants, and increasing the risk of new outbreaks. The last report on BVDV genotypes in France was in 2008, since which there have been no new information. The goal of this study is to determine the genetic diversity of BVDV strains currently circulating in France. To this aim, samples of cattle were taken from different departments that are part of the main areas of livestock production during the years 2018 to 2020. Using the partial sequence of the 5'UTR region of the viral genome, we identified and classified 145 samples corresponding to Pestivirus A and one sample corresponding to Pestivirus D. For the Pestivirus A samples, the 1e, 1b, 1d, and 1l genotypes, previously described in France, were identified. Next, the 1r and 1s genotypes, not previously described in the country, were detected. In addition, a new genotype was identified and was tentatively assigned as 1x genotype. These results indicate an increase in the genetic diversity of BVDV in France.

PSV-A-2 Evaluation of Immune Performance of Calves Born to Cows Vaccinated with a Lifetime Regimen of a Modified-Live or Killed Viral Vaccine When Challenged with Bovine Viral Diarrhea Virus

Abstract Vaccination is the most effective way at preventing bovine viral diarrhea virus (BVDV) infection. Maternal antibodies from colostrum provide immunoglobulins to stimulate the naïve immune system of calves, but these wane within the first 3 months of life. Limited information is available on the quality of colostrum influenced by vaccines, and their subsequent calf protection against BVDV. The objective of this study was to evaluate calf performance against BVDV after consumption of colostrum from cows vaccinated with either a killed viral (KV, n = 7) or modified-live viral vaccination (MLV, n = 9) against BVDV. Calves were weaned at approximately 140 days of age and intranasally challenged with BVDV strain 1373. Clinical scores were evaluated twice daily for 28 days, and blood samples were collected on day -2, 0, 3, 6, 8, 10, 14, and 28 post-infection (dpi) for evaluation of complete blood counts, virus isolation, and metabolites. Colostrum IgG was similar between treatment groups (P = 0.75). Calves that were born to KV cows had increased leukocyte and lymphocyte concentrations compared to MLV calves (P ≤ 0.03). Calves born to MLV cows tended to have higher clinical scores, including higher temperatures, and nasal discharge (P = 0.08). Overall, metabolites were unaffected by treatment (P ≥ 0.8), however, metabolites did differ by dpi. Serum urea nitrogen was lowest at dpi 8 (P = 0.02) and glucose was reduced from dpi -2 through d 14 (P < 0.001). Non-esterified fatty acids were elevated through dpi 14 (P < 0.0001) and haptoglobin peaked on dpi 10 (P < 0.0001). Metabolites were altered by the viral challenge and indicate the nutritional status of the animal was shifted to support the immune response. Overall, vaccination treatment of the cow had little impact on the calf’s ability to mount an immune response.

Genetic features of bovine viral diarrhea virus subgenotype 1c in newborn calves at nucleotide and synonymous codon usages.

Bovine viral diarrhea virus (BVDV), serving as an important pathogen for newborn calves, poses threat to reproductive and economic losses in the cattle industry. To survey the infection rate and genetic diversity of BVDV in newborn calves in northern China, a total of 676 sera samples of newborn calves were collected from four provinces between 2021 and 2022. All sera samples were individually detected for BVDV infection by RT-PCR and ELISA. Our results showed that the overall serological rate was 9.76% (66/676) and the average positive rate of BVDV RNA was 8.14% (55/676) in the newborn calves. Eight BVDV strains were successfully isolated from RT-PCR positive sera samples, and four isolates displayed the cytopathic effect (CPE). Based on phylogenetic tree at the genome level, the eight strains were classified into subgenotype 1c. Moreover, the BVDV isolates had a close genetic relationship with the GSTZ strain at either nucleotide or codon usage level. Interestingly, in comparison of synonymous codon usage patterns between the BVDV isolates with CPE and ones without CPE, there were four synonymous codons (UCG, CCC, GCA, and AAC) which displayed the significant differences (p < 0.05) at codon usage pattern, suggesting that synonymous codon usage bias might play a role in BVDV-1c biotypes. In addition, the usage of synonymous codons containing CpG dinucleotides was suppressed by the BVDV-1c isolates, reflecting one of strategies of immune evasion of BVDV to its host. Taken together, our study provided data for monitoring and vaccination strategies of BVDV for newborn calves in northern China.

Development of an in-house Indirect ELISA for detection of bovine viral diarrhoea virus antibodies in bovine sera

Bovine viral diarrhoea virus (BVDV) infection is a worldwide distributed animal disease. BVDV is the causal agent of congenital defects, diarrhea, reproductive failure, and contaminating biological products. Virus Neutralization test (VNT) as a gold standard method is used for detection of BVDV. Although this assay is very sensitive and specific, it has disadvantages including requires to an experienced person and cell culture facilities. VNT is time-consuming. It is important to design a method that does not have the mentioned disadvantages. So, in-house indirect enzyme linked immunosorbent assay (i-ELISA) was developed for laboratories where it is not possible to perform VNT. The system was made using NADL strain of BVDV and MDBK cell line. This ELISA system was compared with a commercial ELISA kit using 99 bovine sera. Coefficient of variation (CV) was calculated 3.9% and 4.8% for the positive and negative control, respectively for the designed i-ELISA system. The sensitivity, specificity, and accuracy of i-ELISA system was 88%, 53.6%, and 70.7% respectively. Based on our result correlation between in- house and commercial ELISA kit for detection of antibody against BVDV in bovine sera was significant (Kappa coefficient =0.41, p < 0.05). Results of the present study suggested that an in-house ELISA as an affordable and confident system for primary screening of the sera used for biological product.

Molecular Diversity and Histopathological Findings of Novel Bovine Viral Diarrhea Virus Strains Isolated from Bull Semen

Bovine viral diarrhea virus (BVDV) is one of the most common viral pathogens affecting the cattle industry worldwide. The present study aimed to molecularly characterize BVDV isolates that are currently circulating in breeding bulls farmed with cattle suffering from reproductive disorders, and also to assess the consequences of BVDV infection on bulls’ semen quality and conception, and its pathological effects on the structure of testicular tissue and spermatozoa. For this purpose, semen, serum, and testicular samples were collected from four breeding bulls in four private dairy farms in the governorates of Kafr-El Sheik, Beni-Suef, Giza, and Assuit, in Egypt from April 2019 to May 2020. An evaluation of sperm abnormalities was carried out by assessing the integrity of the plasma and acrosomal membranes where severe damage and abnormalities were found. Ultrastructure analysis of the spermatozoa by transmission electron microscopy revealed the presence of a swollen plasma membrane with segmented outer acrosomal membrane of spermatozoa and vacuolar degenerated mitochondria. Histopathological examination of testicular and epididymal tissues indicated moderate to severe degenerative effects of virus infection on seminiferous tubules with hypospermatogenesis. By detection of virus antigen in the serum samples using ELISA, bulls were identified as persistently infected with BVDV. Virus isolation revealed four noncytopathic (NCP-BVDV) strains that were confirmed by fluorescent antibody technique (FAT) and amplification of the 5′ untranslated genomic region (5’UTR) and molecularly typed by amplification of the Erns glycoprotein region. Isolates’ Phylogenetic analysis revealed two subgenotypes: BVDV-1b (Genbank accession numbers; LC634512, LC634513, LC634515) and BVDV-1d (LC634516). According to the knowledge of the authors of the present study, the circulation of the BVDV-1d subgenotype is not reported in Egypt. Therefore, it would be of great importance to track circulating strains in specific countries for successful vaccination programs or accurate diagnostic tests, and this necessitates regular updates.

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.