Bovine Viral Diarrhea Virus Virus 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.

Quercetin Inhibits Hsp70 Blocking of Bovine Viral Diarrhea Virus Infection and Replication in the Early Stage of Virus Infection.

Bovine viral diarrhea virus (BVDV), a positive-strand RNA virus of the genus Pestivirus in the Flaviviridae family, is the causative agent of viral diarrheal disease in bovine. BVDV has been used as a surrogate model for the hepatitis C virus (HCV) to evaluate the efficacy of antiviral drugs. The plant flavonol quercetin causes multiple health-promoting effects in humans and animals. It can be made into a variety of additives, and it exerts a variety of immunomodulatory effects with the potential to be used as an antiviral agent. However, quercetin's antiviral effect and mechanism of action on BVDV are still unclear. Therefore, this study was designed to evaluate quercetin's effect on BVDV virus replication in vitro and in vivo and elucidate its mechanism of action. A CCK-8 kit was used to analyze the toxicity of the quercetin to the MDBK cells. Western blot, qRT-PCR, TCID50, and histological analysis were used to determine the mechanism of quercetin's anti-BVDV activity. An oxidative stress kit was used to evaluate the effects of quercetin on ROS, antioxidant enzymes, and MDA indexes. The effect of quercetin on IL-2 and IFN-γ in the serum of mice was determined by using an ELISA kit. The results showed that quercetin inhibits Hsp70, blocks BVDV infection in the early stage of virus infection and inhibits BVDV replication by inhibiting oxidative stress or ERK phosphorylation. In addition, quercetin alleviated the decrease in IFN-γ and IL-2 in the serum of BVDV-infected mice. Quercetin ameliorated BVDV-induced histopathological changes. In summary, this study demonstrated for the first time the role of Hsp70 in BVDV infection and the potential application of quercetin in treating BVDV infection.

CRISPR-Cas13a-Based Detection for Bovine Viral Diarrhea Virus.

Bovine Viral Diarrhea Virus (BVDV) is the main pathogen of bovine viral diarrhea disease (BVD), which leads to enormous economic losses in the cattle industry. A sensitive and specific detection for BVDV is advantageous to the control of BVDV. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have been used for detecting virus RNA. In this study, the expression and purification of LwCas13a protein was optimized and the RNase activity of LwCas13a in vitro was verified. CRISPR-LwCas13a system could detect BVDV virus and BVDV RNA with high specificity and simplicity. The detection limit of the LwCas13a system was 103 pM, and there were no cross-reactions with HEK293T and MDBK. In summary, a sensitive, specific, and simple nucleic acid detection method based on CRISPR-Cas13a was developed for BVDV. This method provides a new detection strategy for early diagnosis of BVDV.

Multivariate analysis as a method to evaluate antigenic relationships between BVDV vaccine and field strains

Bovine viral diarrhea virus (BVDV) is comprised of two species, BVDV-1 and BVDV-2, but given the genetic diversity among pestiviruses, at least 21 subgenotypes are described for BVDV-1 and 4 for BVDV-2. Genetic characterization can be achieved through complete or partial sequencing and phylogeny, but antigenic characterization can be difficult to determine due to the antigenic diversity and cross-neutralization that exists among isolates. The traditional method for evaluating antigenic relationships between pestivirus isolates is the virus neutralization (VN) assay, but interpretation of the data to determine antigenic difference can be unclear. Data from this study utilized a multivariate analysis for visualization of VN results to analyze the antigenic relationships between vaccine strains and multiple field isolates. Polyclonal sera were generated against 6 BVDV strains currently contained in vaccine formulations, and each serum was used in VN’s to measure the neutralizing antibody titers against 15 BVDV field isolates characterized as prevalent and divergent subgenotypes in the USA. Principal component analysis (PCA) were performed on the VN assay datasets, and results were interpreted from PCA clustering within the PCA dendrogram and scatter plot. The results demonstrated clustering patterns among isolates suggestive of antigenic differences. While expected, the BVDV-1 and BVDV-2 isolates did not cluster together and had the greatest spatial distribution. In addition, other BVDV isolates had distinct spatial patterns suggesting antigenically divergent isolates. This analysis provides an alternative and more efficient means to analyze large VN datasets to visualize antigenic relationships between pestivirus isolates. This analysis could be beneficial for vaccine development and evaluation of efficacy, since most vaccines cannot fully protect animals from the broad range diversity of BVDV viruses.

Possibility Of Using Urine Samples For Detection Of Bovine Viral Diarrhea Virus In Egyptian Cattle

This study aims to employ urine as an easily accessible sample for the detection of bovine viral diarrhea virus (BVDV). Thus, a periodic scan can be applied easily to reach an efficient control of the disease. A total of 86 different samples collected from 30 animals (dams n=23 and calves n=7) were raised in a farm with 1,200 cattle in the El-Fayoum district in Egypt with a case history of abortions were used for this study. All samples were subjected to virological investigation using indirect fluorescent antibody technique (IFAT) and virus neutralization tests (VNT) to detect BVDV. Isolation of the virus was achieved using MDBK cells. The isolates were confirmed using reverse transcriptase polymerase chain reaction. The BVDV was detected in nasal swabs in 63.33% and 70% using IFAT and VNT. In urine samples, the virus was detected with 46.66% and 43.33% by IFAT and VNT, respectively. BVDV was detected in most of the tissues of aborted fetuses in both techniques. Vaginal swabs revealed positive results in 53.85% and 61.54% by IFAT and VNT, respectively. Isolates were confirmed by RT-PCR by successful amplification of 288bp. Statistical analysis revealed good correlation of urine samples and other samples. In conclusion, the BVDV virus can be isolated from urine samples. To the best of our knowledge, this is the first work that provides an overview of urine usability as an alternative sample for BVD virus diagnostics in Egypt. The recommendation for further studies should elucidate on a large scale population.

Testing and Management Strategies for Effective Beef and Dairy Herd BVDV Biosecurity Programs

Elements of a bovine viral diarrhea virus (BVDV) biosecurity program, associated costs and benefits, and criteria veterinarians and producers can use in considering whether to adopt a program are described in this report. Protection against BVDV infections includes proper immunization, identification and elimination of persistently infected (PI) carrier cattle, and prevention of exposure of cattle at risk to BVDV. The decision to implement a BVDV biosecurity program must be based on a herd-by-herd cost-benefit analysis. Costs include diagnostic testing as well as costs imposed by constraints on production practices, which are necessary to implement the program. Benefits include reduced disease loss, potential added value of replacement breeding animals which can be marketed as test-negative for PI infection, and pregnant females at low risk of carrying a PI fetus. Various herd-level diagnostic strategies have been devised to screen herds for the presence ofBVDV, identify and eliminate PI carrier cattle, or help prevent new exposure of the herd to BVDV. No universally applicable protocol exists for herd level BVDV biosecurity. The specific biosecurity methods used in each herd depend on the type of livestock production system involved. Important variables to consider are the number of animals in the herd, livestock concentration, distribution of animal groups, and desired level of biosecurity assurance. When BVDV virus is known to exist in a herd, control of new infections must take into account the possible presence of PI animals. A single PI animal can serve as the source of virus for a herd, and a stringent herd testing program may be required to identify and eliminate PI animals. Producers must understand the risks of re-exposure occurring in a BVDV-free herd and the importance of continuous management to prevent contact with cattle from other herds. In BVDV-free herds, it is essential to annually test and confirm the BVDV PI-free status of all calves born. Purchased animals should be isolated and tested before being added to the herd to avoid introduction of transiently infected animals. It is strongly recommended that bred heifers from outside sources not be added to the BVDV-free herd. Purchasing tested, non-pregnant replacement heifers is less risky than buying tested pregnant females. If tested, pregnant cattle are purchased, then the offspring must also be tested to confirm their BVDV PI-free status before introduction into the herd.

Impact of passive immunity induced by maternal vaccination on subsequent immunization and disease-sparing in early-weaned beef calves challenged with highly virulent BVDV

Vaccination programs have been developed to limit disease associated with bovine viral diarrhea virus (BVDV) infection. The ultimate goal of BVDV vaccination is to induce immunity that prevents viral replication after infection; however, passively acquired BVDV-specific neutralizing antibodies can impact immunity and vaccine responses in the young calf. The purpose of this study was to examine the impact of passively-derived antibody on vaccine response and mitigation of disease in early weaned beef calves whose passive immune status was documented, and when vaccination and challenge were separated by only 5 days.

Efficacy of multivalent, modified- live virus (MLV) vaccines administered to early weaned beef calves subsequently challenged with virulent Bovine viral diarrhea virus type 2.

BackgroundVaccination of young calves against Bovine viral diarrhea virus (BVDV) is desirable in dairy and beef operations to reduce clinical disease and prevent spread of the virus among cattle. Although protection from clinical disease by multivalent, modified-live virus (MLV) vaccines has been demonstrated, the ability of MLV vaccines to prevent viremia and viral shedding in young calves possessing passive immunity is not known. The purpose of this study was to compare the ability of three different MLV vaccines to prevent clinical disease, viremia, and virus shedding in early weaned beef calves possessing maternal immunity that were vaccinated once at 45 days prior to challenge with virulent BVDV 2.ResultsAt 45 days following vaccination, calves that received vaccines B and C had significantly higher BVDV 1 and BVDV 2 serum antibody titers compared with control calves. Serum antibody titers for BVDV 1 and BVDV 2 were not significantly different between control calves and calves that received vaccine D. Following BVDV 2 challenge, a higher proportion of control calves and calves that received vaccine D presented viremia and shed virus compared with calves that received vaccines B and C. Rectal temperatures and clinical scores were not significantly different between groups at any time period. Calves that received vaccines B and C had significantly higher mean body weights at BVDV 2 challenge and at the end of the study compared with control calves.ConclusionsModerate to low maternally-derived BVDV antibody levels protected all calves against severe clinical disease after challenge with virulent BVDV 2. Vaccines B and C induced a greater antibody response to BVDV 1 and BVDV 2, and resulted in reduced viremia and virus shedding in vaccinated calves after challenge indicating a greater efficacy in preventing virus transmission and reducing negative effects of viremia.

Immunogenicity of an inactivated Chinese bovine viral diarrhea virus 1a (BVDV 1a) vaccine cross protects from BVDV 1b infection in young calves

Bovine viral diarrhea virus (BVDV) 1a and 1b strains are the predominant subgenotypes in China. Because of the genetic and antigenic variability among different BVDV strains, a vaccine effective in one region may fail to protect against infections caused by different virus strains in another region. No BVDV vaccine developed with the predominant strains in China are available. In this study, the immunogenicity of an inactivated Chinese BVDV 1a NM01 vaccine strain was evaluated by challenging with a Chinese BVDV 1b JL strain. Ten 2–4-month-old calves were intramuscularly vaccinated with a single dose of the vaccine strain and boosted with same dose three weeks after the first vaccination, with five mock immunized calves serving as a control group. The average titer of neutralization antibody to BVDV 1a and BVDV 1b of immunized calves reached 1:410 and 1:96, respectively, at 21 days post the second vaccination. Twenty-one days post the second vaccination, all calves were challenged with strain JL. The clinical signs, such as the temperature and leukopenia of the immunized calves and viral shedding, were significantly less than the mock immunized calves after challenging with the virulent BVDV 1b strain, indicating that the BVDV 1a vaccine strain elicited efficacious protection against the endemic BVDV 1b strain in China. To the best of our knowledge, this is the first report of an inactivated BVDV vaccine which demonstrated effective cross-protection against BVDV type 1b infection in China.

Mixed Triple: Allied Viruses in Unique Recent Isolates of Highly Virulent Type 2 Bovine Viral Diarrhea Virus Detected by Deep Sequencing

In February 2013, very severe acute clinical symptoms were observed in calves, heifers, and dairy cattle in several farms in North Rhine Westphalia and Lower Saxony, Germany. Deep sequencing revealed the coexistence of three distinct genome variants within recent highly virulent bovine viral diarrhea virus type 2 (BVDV-2) isolates. While the major portion (ca. 95%) of the population harbored a duplication of a 222-nucleotide (nt) segment within the p7-NS2-encoding region, the minority reflected the standard structure of a BVDV-2 genome. Additionally, unusual mutations were found in both variants, within the highly conserved p7 protein and close to the p7-NS2 cleavage site. Using a reverse genetic system with a BVDV-2a strain harboring a similar duplication, it could be demonstrated that during replication, genomes without duplication are generated de novo from genomes with duplication. The major variant with duplication is compulsorily escorted by the minor variant without duplication. RNA secondary structure prediction allowed the analysis of the unique but stable mixture of three BVDV variants and also provided the explanation for their generation. Finally, our results suggest that the variant with duplication plays the major role in the highly virulent phenotype. This study emphasizes the importance of full-genome deep sequencing in combination with manual in-depth data analysis for the investigation of viruses in basic research and diagnostics. Here we investigated recent highly virulent bovine viral diarrhea virus isolates from a 2013 series of outbreaks. We discovered a unique special feature of the viral genome, an unstable duplication of 222 nucleotides which is eventually deleted by viral polymerase activity, leading to an unexpectedly mixed population of viral genomes for all investigated isolates. Our study is of high importance to the field because we demonstrate that these insertion/deletion events allow another level of genome plasticity of plus-strand RNA viruses, in addition to the well-known polymerase-induced single nucleotide variations which are generally considered the main basis for viral adaptation and evolution.

Assessment of vaccine efficacy in early-weaned beef calves challenged with bovine viral diarrhea virus (BVDV)

Early weaning of beef calves is a management strategy that beef cattle producers can implement to reduce their forage needs and maintain the cow herd during severe drought. To ensure the economic success of early weaning, adequate vaccination protocols must be implemented. Vaccination of young calves against BVDV is a key management strategy to prevent economic losses associated with disease caused by BVDV in beef herds. The purpose of this study was to evaluate the efficacy of different BVDV vaccines for protecting calves against virulent BVDV challenge 45 days after vaccination, and the effect of BVDV vaccination on weight gain of early-weaned beef calves that become acutely infected with BVDV.

Efficacy of a non-adjuvanted, modified-live virus vaccine in calves with maternal antibodies against a virulent bovine viral diarrhea virus type 2a challenge seven months following vaccination

In the presence of maternal antibodies, 26 calves three days of age were either vaccinated (n=15) with a single dose of a non-adjuvanted, pentavalent, modified-live vaccine containing bovine herpes virus-1, bovine respiratory syncytial virus, bovine viral diarrhea virus 1a (BVDV1a), bovine viral diarrhea virus 2a (BVDV2a), and bovine parainfluenza-3, or sham vaccinated with sterile saline (n=11). At approximately seven months post-vaccination, when vaccinates and non-vaccinates were seronegative to BVDV1a and BVDV2a antibodies, all calves were intranasally challenged with a virulent BVDV2a (strain 1373). Clinical signs of BVDV infection were monitored beginning three days prior to challenge and concluded on day 21 after challenge. Calves vaccinated in the face of maternal antibodies (IFOMA) were less impacted by the challenge. Based on clinical parameters, vaccinates had reduced clinical scores, rectal temperature, weight loss, and mortality compared to sham-vaccinated animals. Vaccinated animals had higher levels of circulating white blood cells and fewer animals were viremic than sham-vaccinated animals. This study demonstrated that a calf as young as three days of age when vaccinated IFOMA can be protected against a virulent BVDV2a challenge with a non-adjuvanted, pentavalent vaccine.

Field Study

Bovine viral diarrhea virus (BVDV) is a major pathogen involved in the bovine respiratory disease complex (BRD). Immunosuppression induced by BVDV is thought to contribute to BRD, exacerbating secondary bacterial and viral pneumonias. Calves persistently infected (PI) with BVDV maintain the virus in the cattle population and serve as the primary source of transmission within and between herds. The impact that PI calves have on overall feedlot calf health and performance has been shown to be significant. In this field study, calves from one farm, but from different BVDV exposure backgrounds, were monitored after entry into a feedlot in which constant exposure to BVDV virus occurred via presence of PI calves. The objective was to determine if different pre-weaning BVDV exposures mitigated adverse consequences of constant BVDV exposure in a feedlot setting.

Evaluation of a commercial bovine viral diarrhea virus vaccine in nonpregnant female alpacas ( Vicugna pacos)

Bovine viral diarrhea virus (BVDV) is an emerging pathogen in alpacas and many questions still persist regarding disease mechanisms and control strategies. The purpose of this study was to evaluate a commercial BVDV vaccine for safety and efficacy in alpacas. Five nonpregnant alpacas were vaccinated with a modified-live BVDV vaccine and challenged 25 days post-immunization by nasal and ocular inoculation with a BVDV Type 1b strain isolated from a confirmed BVDV persistently infected alpaca. Two nonpregnant alpacas served as non-vaccinated controls and were similarly challenged. Results indicated that BVDV virus could not be detected from the vaccinated alpacas but was detected in the unvaccinated alpacas. Results suggest that administration of modified-live BVDV vaccine protected the alpacas in this study from experimental challenge and no adverse effects from the vaccine were observed.

Comparing Cytopathic and Noncytopathic Bovine Viral Diarrhea Virus (BVDV) Vaccines

Bovine viral diarrhea virus (BVDV) is a major pathogen of cattle causing severe respiratory and reproductive disease. Since BVDV infects through the oral-nasal route, increasing mucosal immunity should decrease BVDV infection and disease. This makes parenteral vaccines that can traffic antigens to mucosal lymphoid tissue more advantageous for generating mucosal immunity. This study was aimed at the distribution of bovine viral diarrhea virus (BVDV) in tissues following vaccination with one of three commercial vaccines: two vaccines containing cytopathic (CP) BVDV viruses and one vaccine containing noncytopathic (NCP) BVDV virus.

Alteration in ultrastructural morphology of bovine embryos following subzonal microinjection of bovine viral diarrhea virus (BVDV)

The aim of the present study was to evaluate the development and ultrastructure of preimplantation bovine embryos that were exposed to bovine viral diarrhea virus (BVDV) in vitro. The embryos were recovered from superovulated and fertilized Holstein-Friesian donor cows on day 6 of the estrous cycle. Compact morulae were microinjected with 20 pl of BVDV suspension (10(5.16) TCID(50)/ml viral stock diluted 1:4) under the zona pellucida (ZP), then washed in SOF medium and cultured for 24-48 h. Embryos were evaluated for developmental stages and then processed immunocytochemically for the presence of viral particles, using fluorescent anti-BVDV-FITC conjugate. Ultrastructure of cellular organelles was analysed by transmission electron microscopy (TEM).After microinjection of BVDV under the ZP, significantly more (p<0.001) embryos (83.33%) were arrested at the morula stage compared with the intact control (30.33%). Immunocytochemical analysis localized the BVDV-FITC signal inside the microinjected embryos. TEM revealed: (i) the presence of virus-like particles in the dilated endoplasmic reticulum and in cytoplasmic vacuoles of the trophoblast and embryoblast cells; (ii) the loss of microarchitecture: and (iii) abnormal disintegrated nuclei, which lacked reticular structure and the heterochromatin area. In all, the embryo nuclear structure was altered and the microarchitecture of the nucleolus had disappeared when compared with the nuclei from control embryos. Dilatation of the intercellular space and the loss of the intercellular gap junctions were often observed in bovine BVDV-exposed embryos. These findings provide evidence for the adverse effect of BVDV virus on the development of bovine embryos, which is related to irreversible changes in the ultrastructure of cell organelles.

Fenbendazole stimulates interferon secretion in calves during viral infection

A total of six trials (five with live animals and one in vitro study) were conducted to evaluate the effects of fenbendazole (FBZ) on the immune response in stressed feedlot cattle. FBZ was given to all treatment calves at the recommended label dosage of 2.3 mg/lb (5 mg/kg) body weight. In Trial 1, treatment with FBZ did not affect serum neutralizing (SN) virus titers or viral shedding following inoculation with live, avirulent bovine herpes virus (BHV-1), but did increase titers for interferon (IFN) in nasal secretions at one, four and seven days post-inoculation. In Trial 2, FBZ-treatment reduced viral shedding and increased IFN titer in nasal secretions on days 4, 7 and 11 following inoculation with live, virulent bovine viral diarrhea virus (BVDV). In Trial 3, treatment with FBZ increased average daily gain ( P =0.11) and increased IFN titer in nasal secretions at two ( P =0.12) and seven ( P =0.07) days following intranasal vaccination with avirulent BHV-1. In naturally infected calves, control and FBZ-treated calves had greater nasal secretions of IFN compared to calves treated with morantel tartrate, levamisole, or thiabendazole (Trial 4). Treatment with FBZ did not increase weight gain or interferon secretions in response to vaccination with IBR and BVD viruses (Trial 5). In cell cultures treated with bluetongue virus, FBZ increased IFN titers. FBZ appears to stimulate IFN secretion in calves during experimental infection with either BHV-1 or BVDV (Trial 6).

157 VIRAL RISK ASSESSMENT OF BOVINE OOCYTES HARVESTED FROM ABATTOIR ORIGIN

Bovine oocytes derived from abattoir origin are mainly used for artificial reproductive techniques in both agricultural and biomedical applications. Regulatory agencies have expressed concern for potential transmission of adventitious viruses by sourcing bovine oocytes from abattoir origin. To evaluate this concern, a viral risk assessment was conducted on batch samples collected from follicular fluid, cumulus cells, oocytes, and Day 8 embryos. These batch samples were collected from ovaries on seven randomly selected days in a 2-week period and they were frozen and stored at –80°C until tested. All samples were tested by 9 Code of Federal Regulations (9CFR) part 113.53c (animal viral testing) at a GLP compliant laboratory (American BioResearch Laboratories, Sevier, TN, USA). The 9CFR viral testing includes bovine viral diarrhea virus (BVDV), bovine parvovirus, bovine adenovirus type 3 and 5, bovine rabies virus, bovine bluetongue virus, bovine respiratory syncytial virus, bovine reovirus, viral cytopathic effect, and hemadsorption on permissive cell cultures. Batch samples were also tested for BVDV and bovine leukemia virus (BLV) by polymerase chain reaction (PCR) and follicular fluids for BVDV antibody neutralization activity at an accredited diagnostic laboratory (Animal Disease Research and Diagnostic Laboratory, SDSU, Brookings, SD, USA). The 9CFR viral testing results on all the batch samples were negative. The BVDV PCR test had a low positive (37.89 cycles) with one follicular fluid batch sample (1/7) and a low positive (37.9 cycles) on all cleaved embryos (7/7). However, BVDV virus isolation was negative for both batch samples by 9CFR testing. The BLV PCR had a positive follicular batch sample (1/7), with all other samples testing being negative. BVD serum neutralization antibody assay demonstrated that all follicular fluid had significant titers of 1:128–1:1024. Although some of the viral particles may have been detected in follicular fluid and cleaved embryos by PCR, none of the batch samples collected were positive for viral growth in this study. The BVDV PCR indicated low levels of BVDV RNA. It is speculated that the positive BVDV PCR results on cleaved embryos could possibly be attributed to the use of irradiated fetal calf serum (contaminated BVDV virus) used in culture media. Follicular fluids also have high titers to BVDV which may have neutralized the virus. These results indicate that virus-free bovine oocytes can be derived from the abattoir. Thus, with appropriately applied quality assurance testing, abattoir-origin oocytes might be used effectively in agricultural and biomedical applications. Table 1. Summary of test results on batch samples from abattoir-derived oocytes

Packaged replicons of bovine viral diarrhea virus are capable of inducing a protective immune response

Bovine viral diarrhea virus (BVDV) replicons with deletions within the capsid, E RNS or E1 encoding region were constructed and efficiently packaged with a helper cell line. High titres of packaged replicons were observed as early as 24 h after transfection, whereas no virus progeny could be detected after transfection of non-complementing cells. Infection of bovine cell cultures with rescued viruses resulted in one cycle of replication without release of infectious virus particles, and no genetic reversion of the generated viruses was detected. Packaged replicons with a deletion within the capsid-coding region were characterized in vivo in immunization and challenge trials. Following immunization of calves with the replication-deficient virus, neither virus shedding nor viremia was detected. After challenge infection with virulent BVDV, all vaccinates were completely protected from disease as measured by the absence of viremia and shedding of challenge virus, which indicated that a ‘sterilizing immunity’ could be induced with the generated replication-deficient packaged replicons.

Truncated E2 of bovine viral diarrhea virus (BVDV) expressed in Drosophila melanogaster cells: A candidate antigen for a BVDV ELISA

A simple and reliable indirect enzyme-linked immunosorbent assay for detection of antibodies directed against a major bovine viral diarrhea virus (BVDV) immunogen, the E2 glycoprotein (tE2-ELISA), has been developed using the recombinant C-terminal truncated E2 glycoprotein (tE2) expressed in a Drosophila melanogaster system. This strategy demonstrated that tE2 is secreted efficiently in the supernatant, no purification steps are necessary, it is easy to produce and carries out the post translational modifications necessary to preserve its native conformation. Preliminary analysis of 183 cattle serum samples using tE2-ELISA showed a 98% specificity and a 100% sensitivity compared with the standard homologous BVDV virus neutralization test. The results also showed that the tE2 is immunoreactive because the conformation and antigenicity of the original E2 are maintained to a large extent. To our knowledge this is the first study report of the recombinant tE2 of BVDV expressed in D. melanogaster system as an antigen for ELISA.