BVDV Strains Research Articles

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.

Development and evaluation of a monoclonal antibody-based blocking ELISA to detect antibodies against the E2 protein of bovine viral diarrhea virus-1

With the rapid development of cattle industry, bovine viral diarrhea virus (BVDV) is becoming widespread in China, which causes serious economic losses to the industry. Effective vaccination and viral surveillance are critical for the prevent and control of BVDV infection. In the present study, the immunogenic domain of E2 protein of BVDV-1 was expressed by prokaryotic pET-28a vector. Monoclonal antibodies (mAbs) against E2 protein were prepared and systemically examined by western blot, immunofluorescence assay, blocking ELISA (bELISA) and virus neutralization test (VNT). The mAb 1E2B3, which showed good reactivity and neutralizing activity to BVDV-1 strains, was selected for ELISA establishment. After a series of screening and optimization, a novel bELISA for highly sensitive and specific detection of BVDV-1 antibodies was established, using HRP-labeled 1E2B3 and recombinant E2 protein. ROC analysis of 91 positive and 84 negative reference bovine serum samples yielded the area under the curve (AUC) of 0.9903. A diagnostic specificity of 96.43 % and a sensitivity of 95.6 % were achieved when the cutoff value was set at 24.31 %. There was no cross reaction to the positive sera of classical swine fever virus (CSFV), BVDV-2, border disease virus (BDV), bovine parainfluenza virus type 3 (BPIV3), infectious bovine rhinotracheitis virus (IBRV), foot-and-mouth disease virus (FMDV), Mycoplasma bovis (M.bovis) and Brucella. The total agreement rate of bELISA with VNT was 93.96 % (249/265). In addition, the result of bELISA was positively correlated with neutralizing antibody titer, and the bELISA could well distinguish the serum samples before and after BVDV vaccination. These results indicate that the established bELISA in this study is specific, sensitive, simple and convenient, which provides technical support for the vaccine efficacy evaluation, prevention and control of BVD in the future.

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

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

Prevalence of Bovine Viral Diarrhoea infection in cattle and buffaloes from Andhra Pradesh, India

The present study was directed at assessing the prevalence of Bovine Viral Diarrhoea (BVD) in various districts of Andhra Pradesh. Samples from organized cattle and buffalo farms and also from clinical cases were tested from January 2022 to December 2022. A total of 673 serum samples from five organised cattle and buffalo farms and 86 clinical samples from animals suffering from severe diarrhoea, respiratory problems and reproductive disorders were screened for the presence of antibodies against BVD. Serum samples were tested for antibodies against BVD virus p80-125 protein (NSP2-3) using a commercially available competitive ELISA kit (ID Screen). The results revealed that a total of 442 out of 759 animals (58.23 per cent) were positive for antibodies against Bovine viral diarrhoea virus. Statistically significant association was observed between BVD seropositivity and different risk factors such as district, farm, species and breed. Sample size and Geographical location may have contributed to this result. In two organized farms where seropositivity was high (more than 70%), all the seronegative animals (55) were screened for presence of BVD virus using a commercially available BVD p80 antigen ELISA kit (ID screen) to detect persistently infected animals. As no animal was found positive, all the remaining seropositive animals (414) in these two farms were also tested for BVD antigen on the suspicion of a current transient infection or presence of a seropositive PI due to exposure to a BVDV strain that is antigenically different to the persisting strain. All the animals tested were negative for BVD antigen. Out of all the clinical samples tested, one animal that was antibody positive was found positive for BVD virus also, indicating a transient infection. The high seropositivity in the organized cattle farms without a PI animal might either be due to an earlier transient infection in the herd or transfer of a PI animal away from the herd before testing. The high seropositivity in the state and presence of BVD antigen in the field animal indicates that a circulating BVD virus was present in most parts of the state.

International proficiency trial for bovine viral diarrhea virus (BVDV) antibody detection: limitations of milk serology.

BackgroundControl programs were implemented in several countries against bovine viral diarrhea (BVD), one of the most significant cattle diseases worldwide. Most of the programs rely on serological diagnostics in any phase of the program. For the detection of antibodies against BVD virus (BVDV), neutralization tests as well as a variety of (commercially available) ELISAs are used. Here, test systems applied in various laboratories were evaluated in the context of an international interlaboratory proficiency trial. A panel of standardized samples comprising five sera and five milk samples was sent to veterinary diagnostic laboratories (n=51) and test kit manufacturers (n=3).ResultsThe ring trial sample panel was investigated by nine commercially available antibody ELISAs as well as by neutralization tests against diverse BVDV-1, BVDV-2 and/or border disease virus (BDV) strains. The negative serum and milk sample as well as a serum collected after BVDV-2 infection were mostly correctly tested regardless of the applied test system. A serum sample obtained from an animal immunized with an inactivated BVDV-1 vaccine tested positive by neutralization tests or by total antibody or Erns-based ELISAs, while all applied NS3-based ELISAs gave negative results. A further serum, containing antibodies against the ovine BDV, reacted positive in all applied BVDV ELISAs, a differentiation between anti-BDV and anti-BVDV antibodies was only enabled by parallel application of neutralization tests against BVDV and BDV isolates. For the BVDV antibody-positive milk samples (n=4), which mimicked prevalences of 20% (n=2) or 50% (n=2), considerable differences in the number of positive results were observed, which mainly depended on the ELISA kit and the sample incubation protocols used. These 4 milk samples tested negative in 43.6%, 50.9%, 3.6% and 56.4%, respectively, of all investigations. Overall, negative results occurred more often, when a short sample incubation protocol instead of an over-night protocol was applied.ConclusionsWhile the seronegative samples were correctly evaluated in most cases, there were considerable differences in the number of correct evaluations for the seropositive samples, most notably when pooled milk samples were tested. Hence, thorough validation and careful selection of ELISA tests are necessary, especially when applied during surveillance programs in BVD-free regions.

Bungowannah Pestivirus Chimeras as Novel Double Marker Vaccine Strategy against Bovine Viral Diarrhea Virus.

Marker or DIVA (differentiation of infected from vaccinated animals) vaccines are beneficial tools for the eradication of animal diseases in regions with a high prevalence of the designated disease. Bovine viral diarrhea virus (BVDV)-1 (syn. Pestivirus A) is a flavivirus that infects predominantly cattle resulting in major economic losses. An increasing number of countries have implemented BVDV eradication programs that focus on the detection and removal of persistently infected cattle. No efficient marker or DIVA vaccine is yet commercially available to drive the eradication success, to prevent fetal infection and to allow serological monitoring of the BVDV status in vaccinated farms. Bungowannah virus (BuPV, species Pestivirus F), a related member of the genus Pestivirus with a restricted prevalence to a single pig farm complex in Australia, was chosen as the genetic backbone for a marker vaccine candidate. The glycoproteins E1 and E2 of BuPV were substituted by the heterologous E1 and E2, which are major immunogens, of the BVDV-1 strain CP7. In addition, the candidate vaccine was further attenuated by the introduction of a deletion within the Npro protein coding sequence, a major type I interferon inhibitor. Immunization of cattle with the chimeric vaccine virus BuPV_ΔNpro_E1E2 CP7 (modified live or inactivated) followed by a subsequent experimental challenge infection confirmed the safety of the prototype strain and provided a high level of clinical protection against BVDV-1. The serological discrimination of vaccinated cattle could be enabled by the combined detection of BVDV-1 E2- in the absence of both BVDV NS3- and BVDV Erns-specific antibodies. The study demonstrates for the first time the generation and application of an efficient BVDV-1 modified double marker vaccine candidate that is based on the genetic background of BuPV accompanied by commercially available serological marker ELISA systems.

A novel NS3/4A protease dependent cleavage site within pestiviral NS2.

Pestiviruses like bovine viral diarrhoea virus (BVDV) and classical swine fever virus (CSFV) belong to the family Flaviviridae. A special feature of the Flaviviridae is the importance of nonstructural (NS) proteins for both genome replication and virion morphogenesis. The NS2-3-4A region and its regulated processing by the NS2 autoprotease and the NS3/4A protease plays a central role in the pestiviral life cycle. We report the identification and characterization of a novel internal cleavage in BVDV NS2, which is mediated by the NS3/4A protease. Further mapping using the NS2 of BVDV-1 strain NCP7 showed that cleavage occurs between L188 and G189. This cleavage site represents a novel sequence motif recognized by the NS3/4A protease and is conserved between the pestivirus species A, B and D. Inhibition of this internal NS2 cleavage by mutating the cleavage site did not cause obvious effects on RNA replication or virion morphogenesis in cultured cell lines. Accordingly, this novel internal NS2 cleavage adds an additional layer to the already complex polyprotein processing of Pestiviruses and might further extend the repertoires of the multifunctional NS2. However, unravelling of the functional relevance of this novel processing event in NS2, therefore, awaits future in vivo studies.

Bovine Viral Diarrhea Virus in Cattle From Mexico: Current Status.

Bovine viral diarrhea (BVD) is an infectious disease, globally-distributed, caused by bovine Pestiviruses, endemic of cattle and other ruminant populations. BVD leads to significant economic losses to the cattle industry due to the wide range of clinical manifestations, including respiratory and gastrointestinal diseases and reproductive disorders. Within the Pestivirus genus of the family Flaviviridae three viral species are associated with BVD; Pestivirus A (Bovine viral diarrhea virus 1, BVDV-1), Pestivirus B (Bovine viral diarrhea virus 2, BVDV-2), and Pestivirus H (HoBi-like pestivirus, atypical ruminant pestivirus). These species are subdivided into subgenotypes based on phylogenetic analysis. The extensive genetic diversity of BVDV has been reported for several countries, where the incidence and genetic variation are more developed in Europe than in the Americas. The first report of BVDV in Mexico was in 1975; this study revealed seropositivity of 75% in cows with a clinical history of infertility, abortions, and respiratory disease. Other studies have demonstrated the presence of antibodies against BVDV with a seroprevalence ranging from 7.4 to 100%. Recently, endemic BVDV strains affecting cattle populations started to be analyzed, providing evidence of the BVDV diversity in several states of the country, revealing that at least four subgenotypes (BVDV-1a, 1b, 1c, and 2a) are circulating in animal populations in Mexico. Little information regarding BVD epidemiological current status in Mexico is available. This review summarizes available information regarding the prevalence and genetic diversity viruses associated with BVD in cattle from Mexico.

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

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

About the necessity of including HoBi-like pestiviruses in bovine respiratory and reproductive viral vacines

ABSTRACT: HoBi-like pestiviruses (HoBiPeV) constitute a novel group of bovine pestiviruses, genetically and antigenically related to bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2. Recent data shows that HoBiPeV are endemic among Brazilian cattle, yet bovine reproductive/respiratory vaccines contain only BVDV-1 and BVDV-2 strains. The present study investigated the neutralizing antibody response against these pestiviruses induced by two commercial vaccines (VA = attenuated, VI = inactivated) and by three experimental, replicative, vaccine formulations (VAC1 = monovalent, BVDV-1; VAC2 = bivalent, BVDV-1 + BVDV-2; VAC3 = trivalent, BVDV-1 + BVDV-2 and HoBiPeV). Seronegative beef calves were immunized once (replicative vaccines) or twice (inactivated vaccine) and serum samples were tested by virus-neutralization (VN) 30 days after vaccination (dpv) (replicative vaccines) or 30 days after the second dose (VI). We considered a threshold VN titer of ≥60 indicative of protection against clinical disease. At 30 dpv, VA induced protective titers against BVDV-2 in 7/7 animals (GMT=289.8) and against BVDV-1 and HoBiPeV in 5/7 animals (GMTs=97.5 and 80, respectively). VI induced protective titers against BVDV-1 in 1/7 animal (GMT=16.4), 2/7 animals against BVDV-2 (GMT=53.8) and in none of the calves against HoBiPeV (GMT=12.2). When a pool of sera of each vaccine group was tested against individual Brazilian isolates, VA induced protective titers against 3/7 BVDV-1 isolates, to 9/10 (BVDV-2) and 1/8 (HoBiPeV); VI induced protective titers against 1/7 (BVDV-1), 1/10 (BVDV-2) and none (0/8) HoBiPeV isolates. The experimental vaccine VAC1 induced protective titers against BVDV-1 in 9/9 animals (GMT=320) but in no animal against BVDV-2 or HoBiPeV (GMT<10). VAC2 induced protective titers to BVDV-1 and BVDV-2 in 9/9 animals (GMTs=160 and 640, respectively), and against HoBiPeV in 7/9 animals (GMT=108.5). Finally, VAC3 induced protective titers in all animals against BVDV-1 (GMT=234.3), BVDV-2 (294.9) and HoBiPeV (201.1). Testing the pool of sera against pestivirus isolates, VAC1 induced titers ≥ 60 against 4/7 BVDV-1 but to none BVDV-2/HoBiPeV isolate; VAC2 induced protective titers against 4/7 BVDV-1; 10/10 BVDV-2 and 2/8 HoBiPeV; VAC3 induced protective titers against all BVDV-1, BVDV-2 and HoBiPeV isolates. These results indicate that vaccines composed by BVDV-1+BVDV-2, especially those containing inactivated virus, may not induce serological response against a variety of HoBiPeV isolates. Thus, the need of inclusion of HoBiPeV in vaccine formulations should be considered.

A Synthetic Modified Live Chimeric Marker Vaccine against BVDV-1 and BVDV-2.

Bovine viral diarrhea virus (BVDV), a pestivirus which exists in the two distinct species BVDV-1 (syn. Pestivirus A) and BVDV-2 (syn. Pestivirus B), is the causative agent of one of the most widespread and economically important virus infections in cattle. For economic as well as for animal health reasons, an increasing number of national BVDV control programs were recently implemented. The main focus lies on the detection and removal of persistently infected cattle. The application of efficient marker or DIVA (differentiation of infected from vaccinated animals) vaccines would be beneficial for the eradication success in regions with a high BVDV prevalence to prevent fetal infection and it would allow serological monitoring of the BVDV status also in vaccinated farms. Therefore, a marker vaccine based on the cytopathic (cp) BVDV-1b strain CP7 was constructed as a synthetic backbone (BVDV-1b_synCP7). For serological discrimination of vaccinated from infected animals, the viral protein Erns was substituted by the heterologous Erns of Bungowannah virus (BuPV, species Pestivirus F). In addition, the vaccines were attenuated by a deletion within the type I interferon inhibitor Npro protein encoding sequence. The BVDV-2 vaccine candidate is based on the genetic sequence of the glycoproteins E1 and E2 of BVDV-2 strain CS8644 (CS), which were introduced into the backbone of BVDV-1b_synCP7_ΔNpro_Erns Bungo in substitution of the homologous glycoproteins. Vaccine virus recovery resulted in infectious cytopathic virus chimera that grew to titers of up to 106 TCID50/mL. Both synthetic chimera BVDV-1b_synCP7_ΔNpro_Erns Bungo and BVDV-1b_synCP7_ΔNpro_Erns Bungo_E1E2 BVDV-2 CS were avirulent in cattle, provided a high level of protection in immunization and challenge experiments against both BVDV species and allowed differentiation of infected from vaccinated cattle. Our study presents the first report on an efficient BVDV-1 and -2 modified live marker vaccine candidate and the accompanying commercially available serological marker ELISA system.

Immune response and onset of protection from Bovine viral diarrhea virus 2 infection induced by modified-live virus vaccination concurrent with injectable trace minerals administration in newly received beef calves

Strategies to improve the onset of protective immunity induced by vaccination against respiratory pathogens may have a significant impact on health of newly received beef calves. The objective was to determine if the use of injectable trace minerals (ITM; Se, Zn, Cu, and Mn) concurrent with a modified-live virus (MLV) vaccine enhances the immune response and onset of protection in beef calves challenged with BVDV2 five days after vaccination. Forty-five calves were randomly assigned to one of three groups (15/group): VAC + ITM, received MLV-vaccine and ITM (Multimin®90) subcutaneously (SC); VAC + SAL, received the same vaccine and saline SC; or UNVAC, unvaccinated. Five days after vaccination (d.0), calves were challenged with BVDV2 strain 890. Health status was evaluated and blood samples were collected for leukocyte counts, BVDV1 and 2 serum neutralizing antibodies (SNA), BVDV-PCR, and percentage of CD4+, CD8+, WC1+ and CD25+ T-cells. VAC + ITM had lower health scores than UNVAC (d.8 and 9). VAC + ITM had higher BVDV1 & 2 SNA titers than VAC + SAL and UNVAC on d.21 and 28. Lymphocyte counts decreased in UNVAC but not in VAC + ITM or VAC + SAL (d.3 to 11). CD4+ T-cells significantly decreased in UNVAC and VAC + SAL (d.3). VAC + ITM had higher percentage of CD4+ T-cells than UNVAC (d.3 and 7). VAC + ITM had lower percentage of activated CD4+ and CD8+ T-cells than UNVAC (d.7). In summary, vaccination induced a rapid protection against BVDV2 infection. Administration of ITM was associated with increased SNA response to BVDV1 & 2, enhanced health status, mitigation of CD4+ T-cells decrease, and reduction of T-cell activation in calves challenged with BVDV2 five days after immunization. These results support the strategic use of ITM concurrent with vaccination, especially when a rapid protection is needed in newly received beef calves.

Outbreaks of Severe Acute BVDV in Quebec, Ontario and New York State, Occurring Between 1993 and 1995, Linked to the Same Strain of Type 2 BVDV

In this study we compared the virulence and genetic sequence of four BVDV strains to each other, and to strains isolated in North America from 1989 to 1996.

Serological relationship between a novel ovine pestivirus and classical swine fever virus.

The genus Pestivirus comprises globally distributed members of the family Flaviviridae, which cause severe losses in livestock. The most common species of the genus are bovine viral diarrhoea virus type 1 (BVDV-1) and type 2 (BVDV-2), classical swine fever virus (CSFV) and border disease virus (BDV). Recently, a novel ovine pestivirus was repeatedly detected in aborted lamb foetuses on a farm located in the Brescia Province (Italy). Complete genome characterization of this isolate showed that it was highly divergent from known pestivirus species and that it was genetically closely related to CSFV. The aim of this study was to determine the serological relatedness between the identified novel pestivirus and BVDV, BDV and CSFV selected strains for which homologous serum was available, by antigenic characterization performed using cross-neutralization assays. The serological relatedness was expressed as the coefficient of antigenic similarity (R). Both field and specific antisera raised against the ovine pestivirus neutralized the CSFV reference strain Diepholz with titres significantly higher than those specific for the BDV and BVDV strains. Furthermore, the calculated R values clearly indicated that the novel ovine pestivirus is antigenically more related to CSFV than to ruminant pestiviruses, in agreement with the results of the genomic analysis. This would have severe consequences on CSFV serology in the event of a switch to porcine hosts with implications for CSFV surveillance and porcine health management.

Neutralizing antibody titers against field strains of bovine viral diarrhea virus after vaccination with three commercial vaccines

Modified killed vaccines against bovine viral diarrhea virus (BVDV) are used worldwide. In the present study, the cross-neutralization antibody responses against field strains from seven subgenotypes of BVDV-1 and one subgenotype of BVDV-2 were evaluated using sera obtained by three inactivated commercial vaccines. One vaccine contained both BVDV-1 and BVDV-2 strains, while the others contained only BVDV-1a. Three vaccine groups, each containing five calves, were vaccinated two times with 30-day intervals. The antibody titers were evaluated by virus neutralization assay. The monovalent vaccine induced the highest antibody titers. Significant levels of neutralizing antibody titers were maintained up to the last sampling time. Although one vaccine contained the BVDV-2 strain, the lowest antibody titers were detected against field strains from BVDV-2, BVDV-1b, and BVDV-1r. This study indicated that cross-protective immune responses with the most common international BVDV vaccine strains need to be evaluated with challenge experiments against field strains for efficient protection.

In vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains using the PrimeFlow RNA assay

Bovine viral diarrhea viruses (BVDV), segregated in BVDV-1 and BVDV-2 species, lead to substantial economic losses to the cattle industry worldwide. It has been hypothesized that there could be differences in level of replication, pathogenesis and tissue tropism between BVDV-1 and BVDV-2 strains. Thus, this study developed an in vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains. To this end the competitive dynamics of BVDV-1a, BVDV-1b, and BVDV-2a strains in cell cultures was evaluated by a PrimeFlow RNA assay. Similar results were observed in this study, as was observed in an earlier in vivo transmission study. Competitive exclusion was observed as the BVDV-2a strains dominated and excluded the BVDV-1a and BVDV-1b strains. The in vitro model developed can be used to identify viral variations that result in differences in frequency of subgenotypes detected in the field, vaccine failure, pathogenesis, and strain dependent variation in immune responses.

CRISPR/Cas9-Mediated Knockout of DNAJC14 Verifies This Chaperone as a Pivotal Host Factor for RNA Replication of Pestiviruses.

Pestiviruses like bovine viral diarrhea virus (BVDV) are a threat to livestock. For pestiviruses, cytopathogenic (cp) and noncytopathogenic (noncp) strains are distinguished in cell culture. The noncp biotype of BVDV is capable of establishing persistent infections, which is a major problem in disease control. The noncp biotype rests on temporal control of viral RNA replication, mediated by regulated cleavage of nonstructural protein 2-3 (NS2-3). This cleavage is catalyzed by the autoprotease in NS2, the activity of which depends on its cellular cofactor, DNAJC14. Since this chaperone is available in small amounts and binds tightly to NS2, NS2-3 translated later in infection is no longer cleaved. As NS3 is an essential constituent of the viral replicase, this shift in polyprotein processing correlates with downregulation of RNA replication. In contrast, cp BVDV strains arising mostly by RNA recombination show highly variable genome structures and display unrestricted NS3 release. The functional importance of DNAJC14 for noncp pestiviruses has been established so far only for BVDV-1. It was therefore enigmatic whether replication of other noncp pestiviruses is also DNAJC14 dependent. By generating bovine and porcine DNAJC14 knockout cells, we could show that (i) replication of 6 distinct noncp pestivirus species (A to D, F, and G) depends on DNAJC14, (ii) the pestiviral replicase NS3-5B can assemble into functional complexes in the absence of DNAJC14, and (iii) all cp pestiviruses replicate their RNA and generate infectious progeny independent of host DNAJC14. Together, these findings confirm DNAJC14 as a pivotal cellular cofactor for the replication and maintenance of the noncp biotype of pestiviruses.IMPORTANCE Only noncp pestivirus strains are capable of establishing life-long persistent infections to generate the virus reservoir in the field. The molecular basis for this biotype is only partially understood and only investigated in depth for BVDV-1 strains. Temporal control of viral RNA replication correlates with the noncp biotype and is mediated by limiting amounts of cellular DNAJC14 that activate the viral NS2 protease to catalyze the release of the essential replicase component NS3. Here, we demonstrate that several species of noncp pestiviruses depend on DNAJC14 for their RNA replication. Moreover, all cp pestiviruses, in sharp contrast to their noncp counterparts, replicate independently of DNAJC14. The generation of a cp BVDV in the persistently infected animal is causative for onset of mucosal disease. Therefore, the observed strict biotype-specific difference in DNAJC14 dependency should be further examined for its role in cell type/tissue tropism and the pathogenesis of this lethal disease.

Comparative analysis of adaptive immune responses following experimental infections of cattle with bovine viral diarrhoea virus-1 and an Asiatic atypical ruminant pestivirus

Atypical ruminant pestiviruses are closely related to the two bovine viral diarrhoea virus (BVDV) species, BVDV-1 and BVDV-2. While there is evidence of cross-protective immune responses between BVDV-1 and BVDV-2, despite antigenic differences, there is little information on the antigenic cross-reactivity with atypical ruminant pestiviruses. The aim of this study was therefore to assess the specificity of antibody and T cell responses induced by experimental infection of calves with BVDV-1 strain Ho916, Th/04_KhonKaen (TKK), an Asiatic atypical ruminant pestivirus, or co-infection with both viruses. Homologous virus neutralization was observed in sera from both single virus infected and co-infected groups, while cross-neutralization was only observed in the TKK infected group. T cell IFN-γ responses to both viruses were observed in the TKK infected animals, whereas Ho916 infected calves responded better to homologous virus. Specifically, IFN-γ responses to viral non-structural protein, NS3, were observed in all infected groups while responses to viral glycoprotein, E2, were virus-specific. Broader antigen-specific cytokine responses were observed with similar trends between inoculation groups and virus species. The limited T cell and antibody immune reactivity of Ho916 inoculated animals to TKK suggests that animals vaccinated with current BVDV-1-based vaccines may not be protected against atypical ruminant pestiviruses.

Bovine viral diarrhea virus infection in wild boar

Bovine viral diarrhea (BVD) is one of the most economically important diseases of cattle. With its very high prevalence, cattle kept on pastures become a source of the virus for the wildlife which, due to their susceptibility, then easily can serve as a source for re-infections of cattle. Therefore, we investigated the BVDV infection in Serbian wild boar and assessed the role of wild boar in BVDV epidemiology including possible spreading to domestic species. This study was based on examination of 50 spleen samples which were collected from wild boars located in Eastern Serbia during the hunting season 2016/2017. BVDV genome was detected in 4 of 50 samples (8%). Phylogenetic analysis based on 5′UTR revealed that BVDV strains from wild boars shared 100% identity. Belonging to the BVDV 1f subgenotype, the most common in cattle, we showed that BVDV infections of wild boar occurred as a result of either direct or indirect contact with domestic animals. Therefore, the occurrence of infectious disease in wildlife emphasizes the need to study the pathogens shared by wildlife and domestic animals by investigating the incidence of pathogens and disease patterns of those populations.

Local innate responses and protective immunity after intradermal immunization with bovine viral diarrhea virus E2 protein formulated with a combination adjuvant in cattle

Bovine viral diarrhea virus (BVDV) is one of the most serious pathogens in cattle. Recently, we developed a novel adjuvant platform (TriAdj) that includes a toll-like receptor 3 agonist, poly (I:C); an innate defense regulatory peptide; and water-soluble polymer, poly[di(sodiumcarboxylatoethylphenoxy)]-phosphazene (PCEP). To develop a needle-free intradermal (ID) subunit vaccine, the BVDV type-2 E2 protein was formulated with TriAdj, and immune protection was evaluated in calves against a BVDV-2 strain. Intradermal delivery of E2/TriAdj elicited robust virus neutralizing antibodies and cell-mediated immune responses including CD4+ and CD8+ T-cell responses. The development of CD8+ T-cell responses in vaccinated calves indicates that TriAdj promotes cross-presentation. Upon challenge with virulent BVDV-2, the vaccinated calves showed no weight loss, leukopenia or virus shedding, and almost no temperature increase, in contrast to the control animals, which had severe clinical disease and shed virus for three to six days in nasal fluids and white blood cells. Intradermal vaccination was shown to attract various immune cell populations including dendritic cells, the most important antigen presenting cells. These data demonstrate that ID delivery is suitable as an administration route in cattle and that ID delivered, TriAdj-formulated E2 can protect cattle from BVDV-2.