Bovine Viral Diarrhea Research Articles

Novel Pestiviruses Detected in Cattle Interfere with Bovine Viral Diarrhea Virus Diagnostics.

Since the start of the mandatory nationwide bovine viral diarrhea (BVD) eradication program in Germany in 2011, the number of persistently infected (PI) animals has decreased considerably, resulting in a continuous decrease in seroprevalence. The increasingly BVD-naive cattle population could facilitate spillover infections with non-BVDV ruminant pestiviruses. Here, we report two cases in which novel pestiviruses were isolated from cattle; in both cases, the whole genome sequence showed the highest level of identity to strain "Pestivirus reindeer-1". Both novel viruses gave positive results in BVDV diagnostic test systems, confirming that cross-reactivity is an important issue in pestivirus diagnostics. In the first case, the pestivirus was probably transmitted from sheep kept with the affected cattle, suggesting that the co-housing of small ruminants and cattle is a risk factor. The source of infection could not be determined in the second case. The occurrence of these two cases in independent cattle holdings within a relatively short time frame suggests that it would be useful to determine the presence of pestiviruses in small ruminants or even wild ruminants to better assess risk factors, especially for BVDV-free populations.

Bergamottin Inhibits Bovine Viral Diarrhea Virus Replication by Suppressing ROS-Mediated Endoplasmic Reticulum Stress and Apoptosis.

Bovine viral diarrhea virus (BVDV) is one of the most important etiological agents that causes serious economic losses to the global livestock industry. Vaccines usually provide limited efficacy against BVDV due to the emergence of mutant strains. Therefore, developing novel strategies to combat BVDV infection is urgently needed. Bergamottin (Berg), a natural furanocoumarin compound, possesses various pharmaceutical bioactivities, but its effect on BVDV infection remains unknown. The present study aimed to investigate the antiviral effect and underlying mechanism of Berg against BVDV infection. The results showed that Berg exhibited an inhibitory effect on BVDV replication in MDBK cells by disrupting the viral replication and release, rather than directly inactivating virus particles. Mechanistically, Berg inhibits BVDV replication by suppressing endoplasmic reticulum (ER) stress-mediated apoptosis via reducing reactive oxygen species (ROS) generation. Studies in vivo demonstrated that oral gavage of Berg at doses of 50 mg/kg and 75 mg/kg significantly reduced the viral load within the intestines and spleen in BVDV-challenged mice. Furthermore, histopathological damage and oxidative stress caused by BVDV were also mitigated with Berg treatment. Our data indicated that Berg suppressed BVDV propagation both in vitro and in vivo, suggesting it as a promising antiviral option against 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.

Recombinant Subunit Vaccine Candidate against the Bovine Viral Diarrhea Virus.

Multivalent live-attenuated or inactivated vaccines are often used to control the bovine viral diarrhea disease (BVD). Still, they retain inherent disadvantages and do not provide the expected protection. This study developed a new vaccine prototype, including the external segment of the E2 viral protein from five different subgenotypes selected after a massive screening. The E2 proteins of every subgenotype (1aE2, 1bE2, 1cE2, 1dE2, and 1eE2) were produced in mammalian cells and purified by IMAC. An equimolar mixture of E2 proteins formulated in an oil-in-water adjuvant made up the vaccine candidate, inducing a high humoral response at 50, 100, and 150 µg doses in sheep. A similar immune response was observed in bovines at 50 µg. The cellular response showed a significant increase in the transcript levels of relevant Th1 cytokines, while those corresponding to the Th2 cytokine IL-4 and the negative control were similar. High levels of neutralizing antibodies against the subgenotype BVDV1a demonstrated the effectiveness of our vaccine candidate, similar to that observed in the sera of animals vaccinated with the commercial vaccine. These results suggest that our vaccine prototype could become an effective recombinant vaccine against the BVD.

Prospective approaches to target bovine viral diarrhea virus and hepatitis C virus using miRNA-based inhibitors

Prospective approaches to target bovine viral diarrhea virus and hepatitis C virus using miRNA-based inhibitors

Transplacental Infections Associated with Macavirus in Aborted Bovine Fetuses.

The Macavirus genus, Gammaherpesvirinae subfamily, Herpesviridae family, contains ovine gammaherpesvirus 2 (OvGHV2), the cause of sheep-associated malignant catarrhal fever (SA-MCF). Members of the Macavirus genus associated with the development of malignant catarrhal fever (MCF) in their respective hosts share the 15A antigenic epitope, are conserved within the DNA polymerase gene and are collectively referred to as the malignant catarrhal fever virus (MCFV) complex. The ability of MCFV and/or OvGHV2 to produce abortions in ruminants is currently unknown, with little documentation of infections by these agents in bovine fetuses. This report presents the findings observed due to the detection of OvGHV2 DNA and MCFV tissue antigens in aborted bovine fetuses from southern Brazil. Four aborted bovine fetuses from three farms, located in a geographical region of Paraná State with elevated immunohistochemical (IHC) prevalence of MCFV tissue antigens, with gestational ages varying between 78 to 208 days were investigated. Significant gross and histopathological alterations were not observed in any of these fetuses. An IHC assay using the 15A-monoclonal antibody (15A-MAb), which is based on the 15A antigenic epitope of Macavirus, identified MCFV tissue antigens in multiple organs from two fetuses (#1 and #4); however, positive immunoreactivity to the 15A-MAb IHC assay was not detected in Fetus #2 and #3. Molecular testing amplified OvGHV2 DNA only from the myocardium and lungs of Fetus #1 that had positive intracytoplasmic immunoreactivity to the 15A-MAb IHC assay in these tissues. Furthermore, infections by Leptospira spp. were confirmed by molecular assays in fetuses #1, #3, and #4, while PCR detected Neospora caninum in the myocardium of Fetus #2. Additionally, molecular assays to identify well-known fetopathy agents of cattle, including bovine viral diarrhea virus, bovine alphaherpesvirus 1, Histophilus somni, and Listeria monocytogenes, did not amplify the nucleic acids of these pathogens. PCR assays to identify bovine gammaherpesvirus 6 (BoGHV6), another Macavirus known to infect cattle in Brazil, were unsuccessful. These findings confirmed that the 15A-MAb IHC assay can be efficiently used to detect MCFV antigens in organs of aborted bovine fetuses. The identification of MCFV antigens with the simultaneous detection of OvGHV2 DNA confirmed that Fetus #1 was infected by OvGHV2 and added to the few descriptions of this infection in aborted fetuses of ruminants worldwide. Moreover, the IHC detection of MCFV in multiple organs of Fetus #4, without the molecular detection of OvGHV2 or BoGHV6, may suggest that this fetus was infected by a Macavirus that was not previously diagnosed in cattle herds from Brazil. These findings strongly suggest that OvGHV2 and MCFV can produce transplacental infections in cattle.

Prevalence of bovine respiratory viruses in cattle calves in Kangra district of Himachal Pradesh

Respiratory diseases causing pneumonia are the 2nd leading cause of mortality in bovine calves and are primarily caused by viral pathogens. The present study was undertaken in cattle calves under twelve months of age for detecting four respiratory viruses, viz. bovine herpes virus-1 (BoHV-1), bovine viral diarrhoea virus (BVDV), bovine respiratory syncytial virus (BRSV), and bovine parainfluenza virus type 3 (BPI3), using a commercially available multiscreen antigen ELISA kit. A total of 30 cattle calves necropsied at the Department of Veterinary Pathology, DGCN COVAS, CSKHPKV, Palampur, from June 2021 to June 2022 were screened. The samples of lung tissues and nasal turbinates were collected and stored at –20°C until further processing. The collected tissue samples were homogenised and processed as per the standard protocol. Among the 30 necropsied samples, BoHV-1 and BRSV were detected in 17/30 (56.67%) samples, BPI3 was detected in 14/30 (46.67%) samples, and BVDV was detected in 8/30 (26.67%) samples. 5/30 (16.67%) samples were found positive for all four viruses. Moreover, three viruses were concurrently detected in 4/30 samples (13.33%), and two viruses were present in 10/30 samples (33.33%). Additionally, a single virus was detected in 4/30 samples (13.33%). In conclusion, the present investigation reveals the substantial presence of respiratory viruses in the respiratory tract of cattle calves. The result indicates a complex pattern of co-infections of different viruses, emphasising the need for effective surveillance and management strategies to address the diverse viral dynamics affecting bovine respiratory health.

Detection of IgM antibodies against bovine viral diarrhea virus using IgM capture ELISA on farms with persistently infected cattle

Bovine viral diarrhea (BVD) is a serious disease in cattle and causes economic losses in the livestock industry. Bovine viral diarrhea virus (BVDV) is the causative agent of BVD and spreads among herds via persistently infected (PI) animals that shed large amounts of the virus throughout their lives. Hence, identifying, and culling PI animals and assessing the immune status against BVDV on farms are important strategies for controlling BVD. Additionally, estimating the time when individuals around PI animals were infected with the virus could also be supportive information to interpret a farm status. We herein constructed a BVDV-specific IgM capture ELISA using recombinant E2 antigen and applied it to detecting BVDV-specific IgM antibodies on farms with identified PI cattle. The IgM ELISA detected anti-BVDV IgM antibodies during approximately 2–3 weeks post infection and identified IgM-positive cattle on two farms with recognized PI cattle. Virus neutralization tests showed that almost all adult cattle had high virus neutralization antibodies against BVDV, and sero-positive and -negative cattle coexisted in young herds. In this situation, most of the IgM-positive cattle were in relatively young animals, implying that BVDV had been recently spreading in these young herds. Thus, our findings demonstrated that detecting IgM antibodies could be useful to know recent BVDV infection on farm on which PI cattle were identified.

Seroprevalence of bovine leukemia virus and association with bovine infectious abortion in Creole breeds from tropical grazing herds in the Colombian Caribbean.

In the Caribbean region of Colombia, the concomitance of endemic infectious agents is a common problem, and coinfections are possible, increasing the complexity of cattle herds' sanitary, reproductive, and productive problems. This study aimed to estimate the seroprevalence of bovine leukemia virus and its association with bovine infectious abortion in grazing Creole breeds from tropical herds in the Colombian Caribbean. For the determination of bovine leukemia virus (BLV), bovine viral diarrhea virus (BVDV), bovine herpes virus-1 (BoHV-1), and Neospora Caninum (NC), the enzyme-linked immunosorbent assay technique was used. Matrix analysis was performed to represent multiple seroprevalence in the same cow. To explore the association between the seroprevalence of BLV and bovine infectious abortion agents, a multivariate logistic regression model was used. The seroprevalence was as follows: BLV 30.78%, BVDV 33.01%, BoHV-1 12.85%, and NC 8.96%. In the multivariate logistic regression model, seroprevalence of BVDV (OR 10.8; 95% CI: 7.5-15.6) and seroprevalence of BoHV-1 (OR 1.8; 95% CI: 1.1-3.0) were associated with the seroprevalence of BLV. Animals infected with BLV are more susceptible to coinfections with BVDV and BoHV-1. Implementing healthy measures against these two immunosuppressive infections could enhance the hygiene of numerous cattle herds. This study was designed as a retrospective cross-sectional study, which limits the ability to confirm that BLV is the primary infection. Further studies to confirm the primary infection of BLV with an active viral coinfection are necessary and the factors associated with these phenomena.

Lively commodities and endemic diseases: Shifting commodity situations and nonhuman disability in cattle and sheep on UK farms

The concept of ‘lively commodities’ captures how aspects of the life of certain entities affect their commodification and exchange within capitalist economic systems. Their status as being, or comprised of, living things matters to their commodification in different ways in particular places and spaces and at particular times. This paper uses the empirical example of diseased farmed animals in the north of England to examine the effects of susceptibility to disease on the process of lively commodification, drawing on conceptualisations of nonhuman disability and relations of care alongside literature on lively commodities, and exploring cases of multi-lifeform co-production of disease. It thus focuses on moments where the liveliness of animals means that commodification ‘goes wrong’, because liveliness means susceptibility to injury and disease, alongside its potential for economic production. The paper focuses on two important endemic conditions affecting UK farming: lameness in cattle and sheep, and bovine viral diarrhoea (BVD) in cattle. These conditions significantly affect animals' welfare and impact on farm productivity. Drawing on qualitative analysis of transcripts from in-depth interviews with 29 farmers and 21 farm advisers (e.g. vets), the paper examines three empirical themes where farming practices are strongly affected by the lively nature of the commodities being produced: first, the anticipatory practice of breeding animals resistant or vulnerable to disease; second, lameness and nonhuman disability; and third, BVD and threats to agricultural biosecurity. The paper concludes by revisiting the concept of lively commodities in situations where farmed animals are diseased, and reflects on the implications of this for their shifting commodity status in particular times and places.

A label-free G-quadruplex aptamer/gold nanoparticle-based colorimetric biosensor for rapid detection of bovine viral diarrhea virus genotype 1.

Bovine viral diarrhea virus (BVDV) is the cause of bovine viral diarrhea disease, one of the most economically important livestock diseases worldwide. The majority of BVD disease control programs rely on the detection and then elimination of persistent infection (PI) cattle, as the continuing source of disease. The main purpose of this study was to design and develop an accurate G-quadruplex-based aptasensor for rapid and simple detection of BVDV-1. In this work, we utilized in silico techniques to design a G-quadruplex aptamer specific for the detection of BVDV-1. Also, the rationally designed aptamer was validated experimentally and was used for developing a colorimetric biosensor based on an aptamer-gold nanoparticle system. Firstly, a pool of G-quadruplex forming ssDNA sequences was constructed. Then, based on the stability score in secondary and tertiary structures and molecular docking score, an aptamer (Apt31) was selected. In the experimental part, gold nanoparticles (AuNPs) with an average particle size of 31.7 nm were synthesized and electrostatically linked with the Apt31. The colorimetric test showed that salt-induced color change of AuNPs from red to purple-blue occurs only in the presence of BVDV-Apt31 complex, after 20 min. These results approved the specificity of Apt31 for BVDV. Furthermore, our biosensor could detect the virus at as low as 0.27 copies/ml, which is an acceptable value in comparison to the qPCR method. The specificity of the aptasensor was confirmed through cross-reactivity testing, while its selectivity was confirmed through plasma testing. The sample analysis showed 90% precision and 94% accuracy. It was concluded that the biosensor was adequately sensitive and specific for the detection of BVDV in plasma samples and could be used as a simple and rapid method on the farm.

Two thiosemicarbazones derived from 1-indanone as potent non-nucleoside inhibitors of bovine viral diarrhea virus of different genotypes and biotypes

Bovine viral diarrhea virus (BVDV) is a widespread pathogen of cattle and other mammals that causes major economic losses in the livestock industry. N4-TSC and 6NO2-TSC are two thiosemicarbazones derived from 1-indanone that exhibit anti-BVDV activity in vitro. These compounds selectively inhibit BVDV and are effective against both cytopathic and non-cytopathic BVDV-1 and BVDV-2 strains. We confirmed that N4-TSC acts at the onset of viral RNA synthesis, as previously reported for 6NO2-TSC. Moreover, resistance selection and characterization showed that N4-TSCR mutants were highly resistant to N4-TSC but remained susceptible to 6NO2-TSC. In contrast, 6NO2-TSCR mutants were resistant to both compounds. Additionally, mutations N264D and A392E were found in the viral RNA-dependent RNA polymerase (RdRp) of N4-TSCR mutants, whereas I261 M was found in 6NO2-TSCR mutants. These mutations lay in a hydrophobic pocket within the fingertips region of BVDV RdRp that has been described as a “hot spot” for BVDV non-nucleoside inhibitors.

Effects of rate of body weight gain during the first trimester of gestation on beef heifer and offspring performance, concentrations of hormones and metabolites, and response to vaccination.

Our study objectives were to evaluate the effects of divergent rates of body weight (BW) gain during early gestation in beef heifers on F0 performance, metabolic and endocrine status, colostrum immunoglobulins, and subsequent F1 calf characteristics, growth performance, concentrations of hormones and metabolites, and response to vaccination. Angus-based heifers (n = 100; BW = 369 ± 2.5kg) were adapted to individual feeding for 14 d and bred using artificial insemination with female-sexed semen. Heifers were ranked by BW assigned to either a basal diet targeting 0.28kg/d gain (LG, n = 50) or the basal diet plus an energy/protein supplement targeting 0.79kg/d gain (MG, n = 50) until d 84 of gestation. Dam BW and blood samples were collected at 6 time points during gestation; body composition was evaluated at d -10 and 84; and fetal measurements were taken on d 42, 63, and 84. At calving (LG, n = 23; MG, n = 23), dam and calf BW were recorded; and colostrum, calf body measurements, and blood samples were collected. Cow-calf pairs were managed on a common diet from calving to weaning, followed by a common postnatal development period for all F1 female offspring. Growth performance, hormone and metabolite profiles, feeding behavior, and reproductive performance were assessed from birth to pre-breeding in F1 heifers. Offspring were vaccinated against respiratory disease and bovine viral diarrhea pathogens on d 62.3 ± 4.13 and 220.3 ± 4.13 post-calving. By design, MG dams were heavier (P < 0.0001) than LG at d 84, and the BW advantage persisted until subsequent weaning of F1 calves. Concentrations of serum IGF-1 and glucose were increased throughout gestation (P < 0.001) for MG dams, whereas concentrations of NEFA were decreased (P < 0.001) in LG dams. Calves from MG dams were 2.14kg heavier (P = 0.03) and had larger chest circumference (P = 0.04) at birth compared with LG cohorts. Heifers from MG dams continued to have greater (P ≤ 0.03) BW gain and feed efficiency during the development period, but no differences were observed (P ≥ 0.13) in body composition, concentrations of hormones and metabolites, feeding behavior, puberty attainment, and response to vaccination in F1 offspring. Hence, early gestation rate of gain impacted BW and concentrations of glucose and IGF-1 throughout gestation in the F0 dam, resulting in altered F1 calf BW and measurements at birth and increased gain and efficiency during the development period.

Preparation of a monoclonal antibody against recombinant LSDV034 protein and its application in detecting lumpy skin disease virus through a competitive enzyme-linked immunosorbent assay (cELISA)

Lumpy skin disease (LSD) is a highly contagious disease caused by lumpy skin disease virus (LSDV) in bovines. Rapid and accurate diagnosis is very important to controll it. However, current commercial detection kits need to be improved in terms of sensitivity or specificity. This study aimed to develop a novel diagnostic competitive enzyme-linked immunosorbent assay (cELISA) based on the newly identified antigen gene LSDV034. The rLSDV034 protein was identified as a potential diagnostic antigen, and it was expressed, purified, and used to immunize BALB/c mice. Using laboratory-prepared indirect ELISA (iELISA), the positive cell lines were screened, and their blocking activity was further verified by competitive ELISA (cELISA). The cell line, 1H7, was chosen to produce mouse ascites, which were purified for a monoclonal antibody (mAb, 5.395 mg/mL). The heavy chain type of the 1H7 mAb was identified as IgG1a, and its light chain subtype was identified as κ. Furthermore, cELISA was developed to detect bovine serum antibodies, with rLSDV034 (4 μg/mL) as the coating antigen and HRP-1H7 mAb (1:300) as the competitive antibody. The cutoff value of cELISA was 55%, based on 32 negative bovine serum samples. The analytical sensitivity was 1:8, and no cross-reaction was detected with bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), Pasteurella multocida (P. multocida), or Mycoplasma bovis (M. bovis) from the serum samples. The diagnostic sensitivity and specificity of cELISA were 98.46% (95% confidence interval, CI: 91.7–100) and 100% (95% CI: 89.1–100), respectively, based on the analysis of 30 LSDV-infected bovine serum samples, 35 GTPV-vaccinated samples, and 32 negative samples. The overall coincidence of the cELISA with the virus neutralization test (VNT) reached 98.97% (95% CI: 94.4–100). Furthermore, we used cELISA to analyze 230 clinical bovine serum samples (including 59 infected and 171 vaccinated samples) and found that the serum positivity rates of the immunized samples (on d 60 postimmunization) and infected samples were 77.78% (95% CI: 70.8–83.8%) and 71.19% (95% CI: 57.9–82.2), respectively. These results indicate that the developed cELISA is promising for detecting serum antibodies in naturally infected or vaccinated cattle.

Seroprevalence of Bovine Viral Diarrhea Virus in Wild Pigs (Sus scrofa) in 17 States in the USA.

Wild pigs (Sus scrofa) are among the most detrimental invasive species in the USA. They are damaging to crops and agriculture, pose a public health risk as reservoirs of zoonotic pathogens, and may also spread disease to livestock. One pathogen identified in wild pigs is bovine viral diarrhea virus (BVDV), a virus that causes an economically important disease of cattle (Bos taurus and Bos indicus). We sought to determine the BVDV seroprevalence in wild pigs in 17 states across the US and to determine whether age category, sex, or location were associated with a positive antibody titer. Serum samples from 945 wild pigs were collected from 17 US states. Virus neutralization assays were performed to determine antibody titers against BVDV-1b and BVDV-2a. Total BVDV seroprevalence for the study area was 5.8% (95% confidence interval [CI], 4.11-8.89). Seroprevalence across all evaluated states was determined to be 4.4% (95% CI, 2.48-6.82) for BVDV-1b and 3.6% (95% CI, 1.54-5.60) for BVDV-2a. The seroprevalence for individual states varied from 0% to 16.7%. There was no statistical difference in median antibody titer for BVDV-1b or BVDV-2a by sex or age category. State seroprevalences for both BVDV-1b and BVDV-2a were associated with wild pig population estimates for those states.

Influence of bovine pestivirus heterogeneity on serological responses to 10 different commercial vaccine formulation

Bovine Pestivirus typically involves one or more organ systems, with clinical manifestations ranging from mild to severe fatal systemic illness that lead to significant reproductive, productive, and economic losses. Vaccines face the challenge of addressing the significant variability of pestiviruses, which affects the interaction between viral antigens and the immune system’s ability to provide protection. This study aimed to evaluate the serological responses against bovine viral diarrhea virus 1 (Pestivirus A) and Pestivirus B induced by 10 commercial vaccines, including one recombinant (vaccine E), two modified live (MLV multivalent, vaccine I, and MLV monovalent, vaccine J), and seven killed vaccines (KLV, vaccines A to H). Additionally, we evaluated the cross-reactivity between Pestivirus A and B from vaccines and HoBi-like pestivirus (Pestivirus H). In Phase 1, guinea pigs were used to screen for non-MLVs. They were divided into nine groups (n=6 each) and received two doses (⅕ of bovine dose) of eight different non-MLV on Days 0 and 21. Serum samples were collected on Days 0 and 30 for serological analyse. In Phase 2, Holstein × Gir heifers (n= 45) were divided into five groups, comprising 6–9 animals. They were vaccinated either once with MLVs or twice with the top non-MLVs screened in Phase 1. Serum samples were harvested on d0 (vaccination day) and d60 (60 days after the first dose) for MLV and non-MLV. Specific antibody titers were assessed virus neutralization (VN) and transformed in log2 for statistical analysis using PROC-MIXED. Significant effects were observed for vaccine groups, time points, and their interactions concerning neutralizing antibodies against Pestivirus A and B in both Guinea pigs and heifers. The Phase 1 study revealed serological responses against Pestivirus A exclusively in non-MLV D (85.33±13.49) and E (72.00±19.26). In the bovine study, the KLD vaccine D (72.00±15.10), recombinant vaccine E (90.66±25.85), and MLV I (170.66±28.22) resulted in an average of neutralizing antibodies against Pestivirus A that exceeded the protective threshold (≥ 60). However,individual analysis of heifers showed a higher frequency of animals presenting titers of Pestivirus A Ab surpassing 32 following vaccination with MLV I and J. None of the vaccine formulations in either study elicited a protective immune response against Pestivirus B or demonstrated cross-reactivity against Pestivirus H.

Genetic analyses of the structural protein E2 bovine viral diarrhea virus isolated from dairy cattle in Yogyakarta, Indonesia.

Bovine viral diarrhea (BVD), a highly pathogenic ribonucleic acid (RNA) virus, causes devastating financial losses and reproductive deaths among dairy cattle in Yogyakarta and globally. This study aimed to identify point mutations within the E2 structural protein of the acquired BVD virus (BVDV) isolates using genetic analysis. The study period shows that we performed the research in 2023. We collected 118 serum samples from 2019 to 2023, among which only 10 BVDV positive were used and 108 were negative lacking the BVDV antigen. An anti-Erns monoclonal antibody-coated protein was used in indirect antigen capture enzyme-linked immunosorbent assay (I-ACE) to detect the BVD antigen present in positive BVDV serum specimens. In the initial step of the two-step reverse transcription polymerase chain reaction, the enzyme (superscript III reverse transcriptase) and the primer (random hexamer) were used to convert the RNA of the BVDV into complementary deoxyribonucleic acid (cDNA) during the process of reverse transcription. The final step involved the amplification of the E2 gene of the resultant BVDV cDNA through gene-specific primers (E2_fwd: 5'-TGGTGGCCTTATGAGAC-3' and P7_rev: 5'-CCCATCATCACTATTTCACC-3') and enzyme (platinum taq DNA polymerase high fidelity). For conducting Sanger sequencing, those 3 BVDV-1-positive isolates (about 2.6% of all isolates) were selected as a typical specimen for each site and year between 2019 and 2023 using a proportional computation. Therefore, only two BVDV isolates with complete genomes were chosen to perform their homological and genetic analysis based on the E2 gene by means of Blast and MEGA Version 11 in addition to the Bioedit 7.2.5 program. By applying phylogenetic analysis relying on the E2 gene, a sum of 1011 nucleotides of the BVDV-1 isolates derived from each of the two BVDV-1 Indonesian isolates (n = 2) and its 23 reference BVDV strains were acquired from the National Center for Biotechnology Information (NCBI) database. The findings of the genetic analysis inside the phylogenetic tree revealed that the two BVDV Indonesian isolates were clustered into BVDV-1a subgenotype, while the reference BVDV strains were clustered into the five BVDV subgenotype, BVDV-1a (n = 6), BVDV-1b (n = 3), BVDV-1c (n = 11), BVDV-1m (n = 1), and BVDV-1n (n = 2). The branch exists in phylogenetic tree located before the division of our two BVDV isolates was divided into two branches with the same maximum bootstrap values of 99%, indicating a high degree of confidence, was seen. Next, we observed the branch near our study samples, which displayed the bootstrap value of 100, indicating that our 02 isolates were identical. In both isolates, V11 BVDV1/Indonesia/Yogyakarta/2023 and V16 BVDV1/Indonesia/Yogyakarta/2023 with GenBank accession numbers PP836388 and PP836389, respectively, conserved D7E residues were mutated as well as cysteine changed/altered into serine (S) was identified at amino acid position 201. We identified two isolates of BVDV belonging to the BVDV-1a subgenotype. Our findings indicate that the conserved D7E residues of isolates V11 BVDV1/Indonesia/Yogyakarta/2023 and V16 BVDV1/Indonesia/Yogyakarta/2023 were altered. The Indonesian BVDV isolates exhibited a cysteine to serine mutation at amino acid position 201, leads to vaccination failure, range of animal's host will increase, and diagnostic kit will not be effective.

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.

Infection status of bovine viral diarrhea virus in Korean native cattle in Jeonbuk State, Korea

Infection status of bovine viral diarrhea virus in Korean native cattle in Jeonbuk State, Korea

Coding-complete genome sequence of a bovine viral diarrhea virus isolate NX2023 from a calf in China.

The coding-complete genome sequence of bovine viral diarrhea virus (BVDV) isolate NX2023 that originated from a calf in China was determined. Phylogenetic analysis showed that the NX2023 strain belongs to the BVDV-1d subgenotype.