Bovine Viral Diarrhea Research Articles

Práticas de biosseguridade associadas com infecção do vírus da diarreia viral bovina em rebanhos leiteiros no Brasil

ABSTRACT: This study determined the association between biosecurity practices and the status of bovine viral diarrhea virus (BVDV) in dairy production systems. Approximately 280 herds were screened for BVDV virus detection. Following the screening, 68 herds were selected to identify individual BVDV PI animals using an ear notch biopsy and ELISA-antigen. All offspring of the last generation were tested, and the maternal lineage of positive cases was examined. A questionnaire on BVDV biological risk assessment was completed. A multiple correspondence analysis (MCA) was conducted to determine the association between herds with or without BVDV circulation and biosecurity practices. The MCA revealed that farms with virus circulation lacked knowledge about the disease and wrongly perceived their herds as protected, while farms without virus circulation were aware of the disease but considered their herds unprotected. Vaccination practices differed between positive and negative herds, with positive herds using vaccines only for reproductive diseases and negative herds vaccinating for respiratory and reproductive issues. Biosecurity practices such as frequent visitation, contact between animals of different ages, and annual introduction of new animals were linked to viral circulation, while virus-free herds implemented measures like controlled visitation, no contact between different age groups, and quarantine. Lastly, herds with virus circulation acquired pregnant females without prior testing. This study emphasized the crucial role of biosecurity practices in controlling BVDV in dairy herds. It highlighted the sharper risk perception and better application of biosecurity practices in negative herds compared to BVDV-positive herds.

Antiviral activity of bovine type III interferon against bovine viral diarrhea virus is greatly reduced in bovine turbinate cells due to limited expression of IFN lambda receptor 1 (IL-28Rα).

The antiviral activity of recombinant bovine interferon lambda 3 (bovIFN-λ3) against bovine viral diarrhea virus (BVDV) has been demonstrated in vitro in Madin-Darby bovine kidney cells (MDBK) and in vivo in cattle. However, anti-BVDV activity of bovIFN-λ3 has not been studied in bovine respiratory tract epithelial cells, supposedly a primary target of BVDV infection when entering the host by the oronasal route. Here we investigated the anti-BVDV activity of bovIFN-λ3 in bovine turbinate-derived primary epithelial cells (BTu) using BVDV infection and immunoperoxidase staining, TCID50, RT-qPCR, DNA and transcriptome sequencing, and transfection with plasmids containing the two subunits, IL-28Rα and IL-10Rβ that constitute the bovIFN-λ3 receptor. Our immunoperoxidase staining, RT-qPCR, and TCID50 results show that while BVDV was successfully cleared in MDBK cells treated with bovIFN-λ3 and bovIFN-α, only the latter, bovIFN-α, cleared BVDV in BTu cells. Preincubation of MDBK cells with bovIFN-λ3 before BVDV infection was needed to induce optimal antiviral state. Both cell types displayed intact type I and III IFN signaling pathways and expressed similar levels of IL-10Rβ subunit of the type III IFN receptor. Sequencing of PCR amplicon of the IL-28Rα subunit revealed intact transmembrane domain and lack of single nucleotide polymorphisms (SNPs) in BTu cells. However, RT-qPCR and transcriptomic analyses showed a lower expression of IL-28Rα transcripts in BTu cells as compared to MDBK cells. Interestingly, transfection of BTu cells with a plasmid encoding IL-28Rα subunit, but not IL-10Rβ subunit, established the bovIFN-λ3 sensitivity showing similar anti-BVDV activity to the response in MDBK cells. Our results demonstrate that the sensitivity of cells to bovIFN-λ3 depends not only on the quality but also of the quantity of the IL-28Rα subunit of the heterodimeric receptor. A reduction in IL-28Rα transcript expression was detected in BTu as compared to MDBK cells, despite the absence of spliced variants or SNPs. The establishment of bovIFN-λ3 induced anti-BVDV activity in BTu cells transfected with an IL-28Rα plasmid suggests that the level of expression of this receptor subunit is crucial for the specific antiviral activity of type III IFN in these cells.

Problems of cattle health improvement and protection of population against viral diarrhoea under current conditions of animal husbandry

The article presents the dynamics of the prevalence of bovine viral diarrhoea virus (BVDV) in livestock enterprises of the Ural region in the period 2018-2023. Retrospective and operational analysis of monitoring data showed that the epizootic situation was consistently tense. The share of unfavourable livestock enterprises ranged from 18.2% to 33.3%. The cumulative infection rate in different age groups of cattle ranged from 2.3% to 23.3%. Clinical symptoms of acute course of BVDV-infection in examined cattle were registered in 10.9% of cases; persistent form of BVDV-infection - in 16.5% of cases; subclinical course of BVDV-infection and latent form - in 73.6% of cases. Phylogenetic analysis of BVDV isolates obtained from animal biosamples showed that 81.8% of isolates belong to the Cp-biotype of BVDV, 18.2% of isolates - to the Ncpbiotype of BVDV. It has been established that the intensity of pathogen eradication decreases in livestock enterprises when the regulations of specific prophylaxis are violated, as evidenced by the increase in the number of animals carrying BVDV by 4%; calves with diseases caused by BVDV infection - by 2.5%; cows and heifers with reproductive losses - by 5.7%.

First evaluation of the impact of a targeted subunit vaccine against bovine viral diarrhea virus in feedlot cattle.

Bovine respiratory disease (BRD) is a serious health and economic problem in the beef industry, which is often associated with transportation and caused by different pathogens. In this study, we evaluated the effect of a novel subunit targeted vaccine against bovine viral diarrhea virus (BVDV) in feedlot cattle, a major viral agent of BRD. The core of this novel vaccine is the fusion of the BVDV structural glycoprotein, E2, to a single-chain antibody, APCH, together termed, APCH-E2. The APCH antibody targets the E2 antigen to the major histocompatibility type II molecule (MHC-II) present in antigen-presenting cells. To evaluate the vaccine, 2,992 animals were randomly allocated into two groups, control group (N = 1,491) and treatment group (N = 1,501). Animals of both groups received the routine sanitary plan: two doses of clostridial, respiratory, and rabies vaccines. Animals within the treatment group also received two doses of a targeted subunit vaccine against BVDV. Serum samples were taken on the day of the first inoculation (T0) and 90 d later (T90). Viral circulation was monitored using an anti-P80 ELISA (virus-specific) and immune response was evaluated by anti-E2 ELISA (detects virus and vaccine immune responses). Only animals treated for respiratory disease were considered positive cases of BRD. Results demonstrate that the control group had significantly more animals treated for BRD cases compared to the treatment group (5.9% vs. 3.7%, P = 0.02). The control group had a greater number of animals positive for anti-P80 antibodies and significantly fewer animals positive for anti-E2 antibodies compared to the treatment group (69% vs. 61% and 71% vs. 99%, respectively, P = 0.003), consistent with natural viral circulation within this group. The treatment group, conversely, had fewer animals positive for anti-P80 antibodies and a greater number of animals positive for anti-E2 antibodies, consistent with a robust vaccine-induced antibody response and a reduction of the BVDV circulation within this group. The data indicate the new subunit targeted vaccine induced greater anti-E2 antibodies and reduced the amount of BVD virus circulation within the treatment group leading to a fewer number of animals needing to be treated for BRD.

Combining a lateral flow immunoassay with triplex loop-mediated isothermal amplification for the concurrent identification of three bovine diarrhea syndrome viruses.

Numerous viruses, such as the bovine rotavirus (BRV), the bovine parvovirus (BPV), and the bovine viral diarrhea virus (BVDV), can cause bovine viral diarrhea syndrome. The global livestock industry has been subjected to significant consequences due to this condition. This results in considerable losses and hinders the production of crucial resources such as meat and milk, which are indispensable for sustaining the world's population. It is crucial to develop a quick and precise way of simultaneously detecting BVDV, BRV, and BPV, as they often occur together in mixed infections. A triplex loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) assay that can concurrently detect all three viruses was introduced in this study. The amplification process involved 30 minutes of incubation at 65 °C. The limits of detection (LODs) for BVDV, BRV, and BPV were 2.62 × 101 copies per μL, 2.43 × 101 copies per μL, and 2.50 × 101 copies per μL, respectively. The triplex LAMP-LFD assay was further evaluated in 156 anal swab samples, and the results were in agreement with the results of fluorescence quantitative PCR (qPCR) in more than 99% of the cattle. This assay is expected for the quick identification of triplex viruses in the field because it has high sensitivity and specificity and doesn't depend on laboratory equipment or conditions.

Molecular detection and genotyping of bovine viral diarrhea virus in Selangor, Malaysia.

Bovine viral diarrhea (BVD) disease is a viral infection in cows caused by a single-stranded plus-sense RNA virus of the Pestivirus genus under the Flaviviridae family. The clinical manifestation of BVD mainly includes diarrhea and immunosuppression, thereby exacerbating various respiratory diseases. This study was conducted to detect and molecularly characterize the bovine viral diarrhea disease virus (BVDV) in cattle on selected farms in Selangor, Malaysia. A reverse transcription polymerase chain reaction (RT-PCR) was performed for antigen detection in 253 plasma samples collected from cows using a cross-sectional study design. We selected the 5 untranslated regions (5'-UTR) region and the E2 region to compare the genetic differences between the isolates. One sample was found to be positive (1/253) following RT-PCR targeting the conserved 5'-UTR region of BVDV. Thus, BVDV antigen prevalence was 0.40% (95% confidence interval: 0.0%-2.2%). By targeting the hypervariable E2 region of the isolated virus, UPM/MAL/BVDV/D17, the virus was classified under the subgenotype BVDV-1a. BVDV is present and circulating on selected cattle farms in Selangor, Malaysia. Given the presence of BVDV in several subgenotypes, the screening of all incoming cattle at Malaysia's border is pertinent to prevent the entry of other BVDV subgenotypes into the country.

Resposta anamnéstica contra o vírus da diarreia viral bovina e herpesvírus bovino tipo 1 em novilhas Holandesas jovens vacinadas com quatro diferentes formulações comerciais

ABSTRACT: This study evaluated the vaccine-induced serological response after administering four commercial formulations containing Bovine Viral Diarrhea Virus (BVDV) type-1, BVDV-2, and Bovine Herpesvirus type 1 (BoHV-1) to young heifers with circulating maternal antibody titers. The study also determined the anamnestic response to vaccinations after the complete metabolization of maternal antibodies when the calves reached six months. Eighty-seven Holstein heifers were selected and randomly distributed into four experimental groups based on the vaccine administered. The four vaccine-based experimental groups were as follows: vaccine A (n = 22), vaccine B (n = 22), vaccine C (n = 24), and vaccine D (n = 19), given on the 60th, 90th, and 180th day of life, respectively. Virus-neutralization (VN) tests were performed at baseline (day 60) and 30 days after administering the second (day 120) and third doses (day 210). We analyzed the effect of vaccine group (P ≤ 0.001), day of vaccination (P ≤ 0.001), and group versus vaccine day interaction (P ≤ 0.001) for antibody titers produced against BVDV-1, BVDV-2, and BoHV-1 using the PROC MIXED method (Statistical Analysis System -SAS 9.4). Antibody titers against BVDV-1, BVDV-2, and BoHV-1 were similar at baseline and on day 60 of life. The mean antibody titers were constant and persisted against BVDV-1 in heifers immunized with vaccines A and C. Heifers immunized with vaccine A alone had a similar effect against BVDV-2. Regarding BoHV-1, the antibody titers decreased between days 60 and 210 in groups B, C, and D. The antibody titer for heifers in group A also decreased between days 60 and day 120, and an intense increase in titers was observed on day 210. After being immunized with formulations B, C, and D, the frequency of animals with titers above protective levels for BVDV-1, BVDV-2, and BoHV-1 was very low or null. Indicators of anamnestic response were observed in heifers vaccinated with formulation A only. Therefore, it can be concluded that passive immunity negatively interferes with antibody production after vaccination.

Effect of two different commercial vaccines against bovine respiratory disease on cell-mediated immunity in Holstein cattle.

Bovine respiratory disease (BRD) is a complex illness that impacts the respiratory system of domestic cattle, resulting in significant financial losses for the agriculture industry. Inactivated or modified live (MLV) pathogen vaccines are often used as a management tool to prevent and control BRD effectively. The purpose of this study is to assess the cell-mediated immune response (CMI) induced by two commercially available polyvalent vaccines, namely the MLV (cattle master gold FP) and the inactivated (CATTLEWIN-5K) vaccine. A total of 20 seronegative heifers against 4 BRD viruses, bovine alphaherpisvirus-1 (BoAHV-1), bovine viral diarrhea virus (BVDV BVDV-1: Pesti virus A; BVDV-2: Pesti virus B), bovine respiratory syncytial virus (BRSV) and bovine parainfluenza virus-3 (BPIV3) were chosen for this study. The heifers were divided into three groups. The first group (n = 6) received no vaccination and was kept as a control. The second and third groups (seven heifers each) were vaccinated twice with either an MLV or inactivated vaccine. The gene expression level of interleukin-6 (IL-6) and interferon-gamma (INF-γ) was measured using real-time quantitative polymerase chain reaction on the 7th, 14th, 21st, 28th, and 60th days post-vaccination. The results were compared with the control group to study the effectiveness of the vaccines. There was an upregulation in the expression level of IL-6 and INF-γ in both MLV and inactivated vaccinated groups. The level of IL-6 mRNA expression was statistically increased from the 14th and 28th days post-vaccination in MLV and inactivated vaccine groups, respectively. The expression level of INF-γ increased significantly from the 2nd and 4th weeks post-vaccination in the MLV and inactivated vaccine groups, respectively. The mean expression level of IL-6 and INF-γ mRNAs was significantly higher in the MLV vaccine group than in the inactivated vaccine group at each examination time. Both investigated vaccines are efficient in stimulating CMI, particularly with the MLV vaccine showing a higher preponderance in IL-6 and INF-γ.

Investigation of Interleukins and Oxidative Stress Parameters in Cows Naturally Infected with Bovine Viral Diarrhea Virus

Investigation of Interleukins and Oxidative Stress Parameters in Cows Naturally Infected with Bovine Viral Diarrhea Virus

Survey for Detecting Cattle Persistently Infected with Bovine Viral Diarrhea Virus in Dairy Herds in Ibaraki Prefecture

Survey for Detecting Cattle Persistently Infected with Bovine Viral Diarrhea Virus in Dairy Herds in Ibaraki Prefecture

Virus as Teratogenic Agents.

Viral infectious diseases are important causes of reproductive disorders, as abortion, fetal mummification, embryonic mortality, stillbirth, and congenital abnormalities in animals and in humans. In this chapter, we provide an overview of some virus, as important agents in teratology.We begin by describing the Zika virus, whose infection in humans had a very significant impact in recent years and has been associated with major health problems worldwide. This virus is a teratogenic agent in humans and has been classified as a public health emergency of international concern (PHEIC).Then, some viruses associated with reproductive abnormalities on animals, which have a significant economic impact on livestock, are described, as bovine herpesvirus, bovine viral diarrhea virus, Schmallenberg virus, Akabane virus, and Aino virus.For all viruses mentioned in this chapter, the teratogenic effects and the congenital malformations associated with fetus and newborn are described, according to the most recent scientific publications.

Construction of an IFNAR1 knockout MDBK cell line using CRISPR/Cas9 and its effect on bovine virus replication.

The type I interferon (IFN) pathway is important for eukaryotic cells to resist viral infection, as well as an impediment to efficient virus replication. Therefore, this study aims to create an IFNAR1 knockout (KO) Madin-Darby bovine kidney (MDBK) cell line using CRISPR/Cas9 and investigate its application and potential mechanism in increasing viral replication of bovines. The IFNAR1 KO cells showed increased titers of bovine viral diarrhea virus (BVDV) (1.5 log10), with bovine enterovirus and bovine parainfluenza virus type 3 (0.5-0.8 log10). RNA-seq revealed reduced expression of the genes related IFN-I pathways including IFNAR1, STAT3, IRF9, and SOCS3 in IFNAR1 KO cells compared with WT cells. In WT cells, 306 differentially expressed genes (DEGs) were identified between BVDV-infected and -uninfected cells. Of these, 128 up- and 178 down-regulated genes were mainly associated with growth cycle and biosynthesis, respectively. In IFNAR1 KO cells, 286 DEGs were identified, with 82 up-regulated genes were associated with signaling pathways, and 204 down-regulated genes. Further, 92 DEGs were overlapped between WT and IFNAR1 KO cells including ESM1, IL13RA2, and SLC25A34. Unique DEGs in WT cells were related to inflammation and immune regulation, whereas those unique in IFNAR1 KO cells involved in cell cycle regulation through pathways such as MAPK. Knocking down SLC25A34 and IL13RA2 in IFNAR1 KO cells increased BVDV replication by 0.3 log10 and 0.4 log10, respectively. Additionally, we constructed an IFNAR1/IFNAR2 double-knockout MDBK cell line, which further increased BVDV viral titers compared with IFNAR1 KO cells (0.6 log10). Overall, the IFNAR1 KO MDBK cell line can support better replication of bovine viruses and therefore provides a valuable tool for bovine virus research on viral pathogenesis and host innate immune response.

Development of a Synthesized Gene Unique to Lumpy Skin Disease Virus and Its Application in Serological Differentiation of Naturally Infected from Vaccinated Cattle with Attenuated Goat Pox Vaccine

Lumpy skin disease (LSD) is an important infectious disease caused by lumpy skin disease virus (LSDV) in bovine. LSDV, sheep pox virus (SPPV), and goat pox virus (GTPV) from the same genus Capripoxvirus (CaPV) of the Poxviridae family exhibit a nucleotide sequence similarity of up to 97%. Therefore, attenuated vaccines of GTPV and SPPV are often used to vaccinate cattle against LSD. However, available serological testing methods cannot accurately differentiate cattle vaccinated with GTPV from those infected with LSDV, posing a significant risk for disease spread. In this study, we developed a synthesized gene unique to LSDV as a differential antigen to detect serum antibodies specific to LSDV and differentiate naturally infected from vaccinated animals (DIVA). We used it for an in‐house indirect enzyme‐linked immunosorbent assay (iELISA), and no cross‐reaction with positive sera for bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), Mycobacterium bovis (M. Tb), Pasteurella multocida (P. multocida), and Mycoplasma bovis (M. bovis). The cut‐off value (S/P%) was 30% for in‐house iELISA. The corresponding diagnostic specificity was 100% (95% CI: 88.43–100), and the diagnostic sensitivity was 93.3% (95% CI: 77.93–99.18). The intra‐assay coefficient of variation (CV) ranged from 1.08% to 4.11%, and the interassay CV was 0.00%–8.90%. Furthermore, 200 clinical serum samples were examined, in the vaccinated herd, there were no positive samples (0/141) indicating the strong differentiation ability of this method. On the other hand, in the infected herds, the overall positivity was 33.90% (20/59) (95% CI: 22.08–47.39). In summary, a valuable synthesized protein unique to LSDV was developed and showed a promising application in an iELISA with high specificity and sensitivity in differentiating cattle infected with LSDV from those vaccinated with GTPV.

Risk analysis of the spread of cattle viral diarrhea in Kazakhstan

Bovine viral diarrhea (BVD) is the most common infectious disease of cattle and is registered in many countries of the world. The disease causes significant economic damage to livestock, primarily due to a decrease in the reproductive capacity of infected animals. Infection of cattle during pregnancy results in transmission of the infection to the fetus, which can lead to embryonic death or the birth of persistently infected (PI) calves. PI-animals excrete BVDV in their feces and secretions throughout their lives, which is why they are the main pathway of transmission of the virus. Moreover, acute BVDV infection leads to transient viremia and immunosuppression, resulting in an increase in secondary infections. In recent years, outbreaks of BVD cattle have been reported in several regions of Russia and China bordering Kazakhstan, indicating a high risk of introducing the infection into the country. Although Kazakhstan is officially considered free from BVD cattle, there is ample evidence that this infection is present in many regions of the country. However, the lack of a clear understanding of the epizootic situation in the country in terms of BVD cattle does not allow the full use of effective control measures, such as total vaccination of livestock in regions at risk of infection. This article provides data on the epizootic situation in Kazakhstan on BVD cattle, as well as an epidemiological analysis of the risks of the spread of BVD cattle in the country.

An outbreak of Akabane disease in a cattle herd on the Mughan plain, Iran.

In November 2021, an investigation was conducted into an outbreak of abortion, stillbirth, and the birth of calves with congenital abnormalities (arthrogryposis and hydranencephaly) at a dairy farm in Dasht-e-Mughan city, Ardabil province. A total of 70 cows experienced these issues. To determine the cause of the outbreak, post-mortem brain tissue samples were collected from two calves affected by hydranencephaly, which occurred shortly after their birth. Polymerase chain reaction (PCR) testing was conducted for multiple viruses, including bovine viral diarrhea (BVD), border disease, Akabane, Schmallenberg, and bluetongue viruses (BTVs). The samples were positive only for Akabane virus. Serum samples were collected from a group of 60 cattle, consisting of 45 adult cows and 15 younger calves aged between 8 to 10 months. These samples were analyzed to detect the presence of antibodies against the Akabane and Schmallenberg viruses. Both of these viruses are known to be responsible for causing abortion, stillbirth, and congenital abnormalities in calves. Among 45 cows that tested by competitive enzyme-linked immunosorbent assay (cELISA), 26.66% and 33.33% exhibited antibodies against Akabane and Schmallenberg viruses, respectively. Notably, 20.00% of cows co-exhibited antibodies for both viruses. Despite PCR evidence implicating Akabane virus as the principal etiology of clinical signs observed in the affected herd, the high co-seropositivity to Schmallenberg virus, warrants a thorough investigation into potential viral interactions. Further research is required to determine the source of the virus and their transmission routes. This information could facilitate the refinement of disease control strategies and improving the management of reproductive challenges in such affected herds.

Melatonin inhibits bovine viral diarrhea virus replication by ER stress-mediated NF-κB signal pathway and autophagy in MDBK cells.

Bovine viral diarrhea-mucosal disease (BVD-MD) is a contagious disease in cattle, caused by the bovine viral diarrhea virus (BVDV). This virus continues to spread globally, exerting pressure on both public health and the economy. Despite its impact, there are currently no effective drugs for treating BVDV. This study utilized Madin-Darby bovine kidney (MDBK) cells as a model to investigate the antiviral effects of melatonin against Bovine Viral Diarrhea Virus (BVDV) and its connection with endoplasmic reticulum (ER) stress. Our results show that melatonin can suppress BVDV proliferation in MDBK cells by modulating the endoplasmic reticulum (ER) stress-mediated NF-κB pathway and autophagy. Specifically, melatonin alleviated ER stress, inhibited the activation of IκBα and p65, regulated autophagy, and reduced the expression levels of pro-inflammatory cytokines. Further, when we treated BVDV-infected cells with the ER stress inducer thapsigargin, it led to significant activation of the NF-κB pathway and autophagy. Conversely, treating the cells with the ER stress inhibitor 4-phenylbutyric acid reversed these effects. These findings suggest that melatonin exerts its antiviral effects primarily through the PERK-eIF2α-ATF4 of ER stress-mediated NF-κB pathway and autophagy. Overall, our study underscores the potential of melatonin as an effective protective and therapeutic option against BVDV, offering insights into its anti-infective mechanisms.

Analysis of the Properties of Bovine Viral Diarrhea Virus Isolated from the Australian Imported Cattle Herd and the Status of Possession of Neutralizing Antibodies in the Group

Analysis of the Properties of Bovine Viral Diarrhea Virus Isolated from the Australian Imported Cattle Herd and the Status of Possession of Neutralizing Antibodies in the Group

Development and Application of a Rapid Test for Detection of Bovine Viral Diarrhea Virus-specific Antibodies

Development and Application of a Rapid Test for Detection of Bovine Viral Diarrhea Virus-specific Antibodies

Optimization of conditions for cultivation of pathogens of infectious rhinotracheitis and viral diarrhea

This article reflects the results of research on optimizing the conditions of cultivation of strain “ТК-A(VIEV)-В2” of infectious bovine rhinotracheitis (IBR) and strain “ВК-1” of bovine viral diarrhea (BVDV). It was found that the roller cultivation method is most easily applicable and provides the production of viral material with high infectious activity. A comparative analysis of the results of studying the sensitivity of continuous cell cultures to the pathogens of IBR and BVDV of cattle of the latter made it possible to select the MDBK cell line to obtain the maximum titer of viruses. The optimal dose for infecting cell culture with both pathogens was the content of viral particles from 0.2 to 0.5 TCD50/cm3 per cell. The maximum reproduction of viruses was observed using a culture concentration of cells from 110 to 120 thousand cells/cm3 and the duration of their cultivation for 2-3 days. The results obtained can be applied for the further development and creation of preventive and diagnostic tools for IBR and BVDV of cattle.

Bovine viral diarrhea virus Erns protein expressed in Chinese hamster ovary cells and its immunogenicity analysis

The aim of this study was to produce Erns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified Erns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-Erns was constructed based on the gene sequence of BVDV-1 NADL strain. The Erns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-Erns into CHO cells. The expression and purification of the Erns protein was analyzed by SDS-PAGE, the reactivity was determined with anti-His monoclonal antibodies and BVDV positive serum with Western blotting. Immunogenicity analysis of the Erns protein was determined after immunizing New Zealand white rabbits, and the serum antibodies were tested by indirect ELISA (iELISA) and indirect immunofluorescence (IFA). The serum neutralizing titer of the immunized rabbits was determined by virus neutralization test. The concentration of the purified Erns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the Erns protein could be detected with anti-His monoclonal antibodies and anti-BVDV sera. Serum antibodies could be detected by iELISA on the 7th day post-prime immunization, and the antibody level was maintained at a high titer until the 28th day post-immunization. The antibody titer was 1:128 000. Furthermore, the expression of the Erns protein in BVDV-infected MDBK cells could be detected with immunized rabbits sera by IFA. Moreover, antigen-specific neutralizing antibodies of 2.71 log10 was induced in rabbits. In this study, purified BVDV Erns protein was successfully produced using CHO suspension culture system, and the recombinant protein was proved to have a good immunogenicity, which may facilitate the development of BVD diagnosis method and novel subunit vaccine.