Specificity and sensitivity of an indirect fluorescent antibody test to detect antibodies against bovine viral diarrhea virus.

Since being reported in 1979 and 2006, indirect fluorescent antibody (IFA) tests have not been reported to detect bovine viral diarrhea virus (BVDV) antibodies to our knowledge. Thus, we re-evaluated the efficacy and usefulness of IFA tests for BVDV serology. We tested 4 combinations of 2 antibody conjugates (fluorescein isothiocyanate [FITC]-conjugated rabbit IgG anti-bovine IgG; rabbit IgG F(ab')2 fragment anti-bovine IgG [F(ab')2 FITC-IgG]) and 2 washing solutions (PBS; carbonate-bicarbonate-buffered saline [CBBS]) to evaluate the specificity of an IFA test for BVDV. We compared the sensitivity of the optimal combination with virus neutralization (VN) tests and an ELISA, and compared IFA with VN titers against different genotype (subgenotype) strains. For the F(ab')2 FITC-IgG/CBBS combination, only 1 of the 156 (0.6%) 4-fold diluted cattle sera resulted in a nonspecific reaction; other combinations led to a much higher incidence (22.9-37.2%). For the F(ab')2 FITC-IgG/CBBS combination, IFA detection rates were identical (36 of 59) for BVDV1 and BVDV2 genotypes, and IFA titers against them were strongly correlated (r = 0.99). The antibody-detection rates of the IFA tests were almost identical to those of VN tests and the ELISA (κ: 0.96 and 0.89, respectively). The IFA titers against 4 strains (BVDV1a, BVDV1j, BVDV2a, and an unidentified strain) were similar, 1,024 to ≥4,096, although the VN titers were different. Thus, our IFA tests were specific and sensitive, and more useful than VN tests given that the IFA tests could evaluate the immune status of cattle using a representative strain, regardless of genotype (subgenotype).

PI3K/AKT mediated De novo fatty acid synthesis regulates RIG-1/MDA-5-dependent type I IFN responses in BVDV-infected CD8+T cells.

Bovine viral diarrhea virus (BVDV) has caused massive economic losses in the cattle business worldwide. Fatty acid synthase (FASN), a key enzyme of the fatty acid synthesis (FAS) pathway, has been shown to support virus replication. To investigate the role of fatty acids (FAs) in BVDV infection, we infected CD8+T lymphocytes obtained from healthy cattle with BVDV in vitro. During early cytopathic (CP) and noncytopathic (NCP) BVDV infection in CD8+ T cells, there is an increase in de novo lipid biosynthesis, resulting in elevated levels of free fatty acids (FFAs) and triglycerides (TG). BVDV infection promotes de novo lipid biosynthesis in a dose-dependent manner. Treatment with the FASN inhibitor C75 significantly reduces the phosphorylation of PI3K and AKT in BVDV-infected CD8+ T cells, while inhibition of PI3K with LY294002 decreases FASN expression. Both CP and NCP BVDV strains promote de novo fatty acid synthesis by activating the PI3K/AKT pathway. Further investigation shows that pharmacological inhibitors targeting FASN and PI3K concurrently reduce FFAs, TG levels, and ATP production, effectively inhibiting BVDV replication. Conversely, the in vitro supplementation of oleic acid (OA) to replace fatty acids successfully restored BVDV replication, underscoring the impact of abnormal de novo fatty acid metabolism on BVDV replication. Intriguingly, during BVDV infection of CD8+T cells, the use of FASN inhibitors prompted the production of IFN-α and IFN-ß, as well as the expression of interferon-stimulated genes (ISGs). Moreover, FASN inhibitors induce TBK-1 phosphorylation through the activation of RIG-1 and MDA-5, subsequently activating IRF-3 and ultimately enhancing the IFN-1 response. In conclusion, our study demonstrates that BVDV infection activates the PI3K/AKT pathway to boost de novo fatty acid synthesis, and inhibition of FASN suppresses BVDV replication by activating the RIG-1/MDA-5-dependent IFN response.

Seroprevalence of reproductive and infectious diseases in cattle: the case of Madre de Dios in the Peruvian southeastern tropics.

OBJECTIVE: The objective of this study was to determine the seroprevalence of reproductive and infectious diseases in tropical cattle in the Tambopata and Tahuamanu Provinces in the department of Madre de Dios, Peru. SAMPLE: 156 bovines from 7 cattle farms were sampled. These farms used exclusive grazing for food and natural mating for reproduction and did not have sanitary or vaccination programs. METHODS: The serum of blood samples was subjected to ELISA with commercial kits for the detection of antibodies against Neospora caninum, Mycobacterium avium subsp paratuberculosis (MAP), Leptospira interrogans, pestivirus bovine viral diarrhea virus-1, retrovirus bovine leukemia virus (BLV), orbivirus bluetongue virus (BTV), and herpesvirus bovine herpes virus-1 (BHV). The data were analyzed by means of association tests with χ2 (P < .05) and Spearman rank correlation (P < .05) in the SPSS v.15.0 software (IBM Corp). RESULTS: A low prevalence of antibodies to L interrogans, N caninum, M avium subsp paratuberculosis, bovine viral diarrhea virus-1 was found, but it was high to BTV, BLV, and BHV (100%, 53.85%, and 72.44%, respectively). The presence of BLV and BHV was higher in the Las Piedras District, bovines less than 5 years old, and cattle with breed characteristics of zebu and crossbred (P < .01). In addition, there was a significant correlation between both infections, showing 83.3% of BLV positivity that were also BHV positive (P < .01). CLINICAL RELEVANCE: The high prevalence of antibodies to BTV, BHV, and BLV could be due to livestock management practices, direct contact with infected animals, and variation of the presence of vectors and natural reservoirs in the context of climate change in the tropics.

Efficacy of H2O2 inactivated bovine virus diarrhoea virus (BVDV) type 1 vaccine in mice.

BACKGROUND: Bovine viral diarrhea (BVD) is an acute febrile infectious disease caused by the bovine viral diarrhea virus (BVDV), which has brought huge economic losses to the world's cattle industry. At present, commercial inactivated BVDV vaccines may cause some adverse reactions during use. This study aims to develop a safer and more efficient inactivated BVDV vaccine. METHODS: Here, we described the generation and preclinical efficacy of a hydrogen peroxide (H2O2) inactivated BVDV type 1 vaccine in mice, and administered it separately with commercial vaccine (formaldehyde inactivated) in mice to study its efficacy. RESULTS: The BVDV type 1 IgG, IFN- γ, IL-4 and neutralizing antibody in the serum of the H2O2 inactivated vaccine group can be maintained in mice for 70 days. The IgG level reached its maximum value of 0.67 on the 42nd day, significantly higher than the commercial formaldehyde inactivated BVDV type 1 vaccine. IFN- γ and IL-4 reached their maximum values on the 28th day after immunization, at 123.16 pg/ml and 143.80 pg/ml, respectively, slightly higher than commercial vaccines, but the effect was not significant. At the same time the BVDV-1 neutralizing antibody titer reached a maximum of 12 Nu on the 42nd day post vaccination. CONCLUSIONS: The H2O2 inactivated BVDV vaccine has good safety and immunogenicity, which provides a potential solution for the further development of an efficient and safe BVDV vaccine.

Comparison of bovine viral diarrhea virus detection methods: Results of an international proficiency trial.

Bovine viral diarrhea virus (BVDV), one of the most important infectious cattle diseases globally, is being combated in multiple countries. The main source for virus transmission within herds and especially to unaffected cattle farms are life-long persistently infected (PI), immunotolerant animals. Therefore, the early identification of PI calves is a major pillar of disease control programs. In addition, rapid and reliable virus identification is necessary to confirm the causative agent in acute clinical cases. Here, we initiated an international interlaboratory proficiency trial in order to evaluate BVDV detection methods. Four ear notch samples and four sera were provided to the participating veterinary diagnostic laboratories (n = 40). Two of the ear notches and two sera contained BVDV and two ear notches and one serum were negative for pestiviruses. The remaining serum was positive for the ovine border disease virus (BDV). The sample panel was analyzed by an ERNS-based ELISA for antigen detection, diverse real-time RT-PCR (RT-qPCR) assays and/or virus isolation. Occasionally, additional typing of the virus strains was performed by sequencing or specific antibody staining of the obtained cell culture isolates. While the antigen ELISA allowed reliable BVDV diagnostics, infectious virus could be isolated only in just under half of the attempts (43.33%). RT-qPCR enabled the sensitive detection of pestiviruses, though an impact of the extraction method on the resulting quantification cycle values was observed. In general, subsequent typing of the detected virus strains is required to differentiate BVDV from BDV infections. In conclusion, for BVDV identification in clinical cases or in the context of disease control, RT-qPCR methods or ERNS antigen ELISAs should be preferentially used.

Blockade of BTLA alone or in combination with PD-1 restores the activation and proliferation of CD8+ T cells during in vitro infection with NCP BVDV.

Bovine viral diarrhea virus (BVDV) infection can result in typical peripheral blood lymphopenia and immune dysfunction. However, the molecular mechanism underlying the onset of lymphopenia remains unclear. B and T lymphocyte attenuator (BTLA) is a novel immune checkpoint molecule that primarily inhibits activation and proliferation of T cells. Blockade of BTLA with antibodies can boost the proliferation and anti-viral immune functions of T cells. Nonetheless, the immunomodulatory effects of BTLA in CD8+ T cells during BVDV infection remain unknown. Therefore, BTLA expression was measured in bovine peripheral blood CD8+ T cells infected with BVDV in vitro. Furthermore, the effects of BTLA or PD-1 blockade on CD8+ T cell activation, proliferation, and anti-viral immunological activities were investigated, as well as expression of signaling molecules downstream of BTLA, both alone and in combination. The results demonstrated that BTLA and PD-1 mRNA and protein levels were considerably increased in CD8+ T cells infected with cytopathic and non-cytopathic (NCP) BVDV. Surprisingly, as compared to blockade of either BTLA or PD-1, blockade of both dramatically increased proliferation and expression of CD25 and p-EKR of CD8+ T cells infected with NCP BVDV. Furthermore, blockade of BTLA, but not PD-1, had no effect on BVDV replication or IFN-γ expression. These findings confirmed the immunomodulatory roles of BTLA during BVDV infection, as well as the synergistic role of BTLA and PD-1 in NCP BVDV infection, thereby providing new insights to promote activation and the anti-viral immunological activities of CD8+ T cells.

Bovine viral diarrhoea viruses from New Zealand belong predominantly to the BVDV-1a genotype.

AIM: To determine which genotypes of bovine viral diarrhoea virus (BVDV) circulate among cattle in New Zealand. METHODS: Samples comprised BVDV-1-positive sera sourced from submissions to veterinary diagnostic laboratories in 2019 (n = 25), 2020 (n = 59) and 2022 (n = 74) from both beef and dairy herds, as well as archival BVDV-1 isolates (n = 5). Fragments of the 5' untranslated region (5' UTR) and glycoprotein E2 coding sequence of the BVDV genome were amplified and sequenced. The sequences were aligned to each other and to international BVDV-1 sequences to determine their similarities and phylogenetic relationships. The 5' UTR sequences were also used to create genetic haplotype networks to determine if they were correlated with selected traits (location, type of farm, and year of collection). RESULTS: The 5' UTR sequences from New Zealand BVDV were closely related to each other, with pairwise identities between 89% and 100%. All clustered together and were designated as BVDV-1a (n = 144) or BVDV-1c (n = 5). There was no evidence of a correlation between the 5' UTR sequence and the geographical origin within the country, year of collection or the type of farm. Partial E2 sequences from New Zealand BVDV (n = 76) showed 74-100% identity to each other and clustered in two main groups. The subtype assignment based on the E2 sequence was the same as based on the 5' UTR analysis. This is the first comprehensive analysis of genomic variability of contemporary New Zealand BVDV based on the analysis of the non-coding (5' UTR) and coding (E2) sequences. CONCLUSIONS AND CLINICAL RELEVANCE: Knowledge of the diversity of the viruses circulating in the country is a prerequisite for the development of effective control strategies, including a selection of suitable vaccines. The data presented suggest that New Zealand BVDV are relatively homogeneous, which should facilitate eradication efforts including selection or development of the most suitable vaccines.

Uncommon bovine viral diarrhea virus subtype 1e associated with abortions in cattle in southern Brazil.

We characterized bovine viral diarrhea virus (BVDV)-related abortions in cattle and identified the species and subgenotypes in the state of Santa Catarina, southern Brazil. Our RT-PCR assay was positive for BVDV in 5 fetuses from different farms; however, 3 of the 5 fetuses were also PCR-positive for Neospora caninum. In the 5 BVDV-positive fetuses, gross lesions included fetal mummification (1), hepatomegaly (1), subcutaneous edema (1), and perirenal edema (1). Predominant histologic lesions included epicarditis and mild-to-moderate lymphoplasmacytic myocarditis (5), mild multifocal lymphoplasmacytic interlobular pneumonia (4), nephrosis associated with moderate multifocal interstitial nephritis (1), moderate multifocal lymphoplasmacytic necrotic hepatitis (1), and mild multifocal lymphoplasmacytic meningitis (1). The amplification products from the Pestivirus 5'UTR region of 4 of the 5 fetuses had 96.3-100% similarity between fetal strains and reference strains. The samples were distributed into 2 branches of the phylogenetic tree; strains UDESC:01, UDESC:02, and UDESC:05 clustered in the BVDV-1e branch, uncommon in the Americas, and strain UDESC:04 clustered in the BVDV-2b branch. The three 1e strains had 96.9-97.4% similarity.

Sporadic bovine encephalopathy caused by Chlamydia pecorum secondary to bovine viral diarrhoea virus infection in calves in South Australia.

BACKGROUND: Despite bovine viral diarrhoea virus and Chlamydia pecorum being important endemic diseases of cattle, there are limited reports of theirco-occurrence. CASE REPORT: Several 12-18-week-old, weaned Hereford calves presented with ill-thriftiness and neurological signs on a mixed cattle and sheep farm in South Australia in July 2021. Immune suppression resulting from transient infection with bovine viral diarrhoea virus (BVDV) is implicated in predisposing to infection with Chlamydia pecorum, the causative agent of sporadic bovine encephalopathy (SBE). Chlamydia spp. are difficult to culture in vitro or definitively identify based on current standard molecular based tests. In this case, diagnosis was confirmed by immunohistochemistry. CONCLUSION: To the authors' knowledge, this case report is the first to document BVDV transient infection occurring in conjunction with SBE. Given the current high prevalence of BVDV on Australian farms, such co-infections may have significant future clinical relevance. This case also highlights the need for appropriate tests, such as immunohistochemistry to demonstrate the causative organism in histological lesions and thus reduce the occurrence of false negative diagnosis.

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.

Pathogenicity assessment of a bovine viral diarrhea virus type 1l (BVDV-1l) strain in experimentally infected calves.

Bovine viral diarrhea is a widespread and economically important viral disease for livestock which can cause clinically diverse manifestations. The number of established BVDV subgenotypes has increased, not only the serological relationships of recently described subgenotypes but virulence and pathogenic characteristics have not yet been mostly elaborated. The dominant BVDV subgenotype in Turkiye was elaborated to be BVDV-1l, that involves more than half of field strains and there is no scientific data to identify the pathogenicity of this strain so far. This study investigated the pathogenicity of a selected field strain (TR-72) from subgenotype BVDV-1l. Experimental infection was implemented by intranasal inoculation of the strain TR-72 (10 ×105.5) to four young calves which were previously not vaccinated and were free both for BVDV antibodies and antigens. Clinical changes as well as blood parameters, body temperature, and viremia were monitored for 14 days. Only mild clinical signs associated with respiratory signs of BVDV infection were observed. Detected clinical signs included nasal discharge, conjunctivitis, cough, fatigue, high rectal temperature reaching 40.7 â„ƒ, and white blood cell counts depression started from the 2nd day and 40.4% decreased between the 12th and 14th days post-infection (poi). The presence of viremia was investigated by virus isolation, RT-PCR, and real-time RT-PCR from blood samples. The efficiency of experimental infection was established not only by observed clinical signs but also by virus isolation from blood leukocytes between the 5th and 8th days poi., virus detection was obtained by real-time PCR between the 3rd - 13th days poi. Besides, the recorded mild clinical signs, high fever, long duration of viremia , and high decrease in blood parameters obtained in this study, it was shown that the noncytopathogenic BVDV-1l strain TR-72 has a moderate virulence in naïve cattle.

Unveiling tetrahydroquinolines as promising BVDV entry inhibitors: Targeting the envelope protein.

Bovine viral diarrhea virus (BVDV) is known to cause financial losses and decreased productivity in the cattle industry worldwide. Currently, there are no available antiviral treatments for effectively controlling BVDV infections in laboratories or farms. The BVDV envelope protein (E2) mediates receptor recognition on the cell surface and is required for fusion of virus and cell membranes after the endocytic uptake of the virus during the entry process. Therefore, E2 is an attractive target for the development of antiviral strategies. To identify BVDV antivirals targeting E2 function, we defined a binding site in silico located in domain IIIc at the interface between monomers in the disulfide linked dimer of E2. Employing a de novo design methodology to identify compounds with the potential to inhibit the E2 function, compound 9 emerged as a promising candidate with remarkable antiviral activity and minimal toxicity. In line with targeting of E2 function, compound 9 was found to block the virus entry into host cells. Furthermore, we demonstrated that compound 9 selectively binds to recombinant E2 in vitro. Molecular dynamics simulations (MD) allowed describing a possible interaction pattern between compound 9 and E2 and indicated that the S enantiomer of compound 9 may be responsible for the antiviral activity. Future research endeavors will focus on synthesizing enantiomerically pure compounds to further support these findings. These results highlight the usefulness of de novo design strategies to identify a novel class of BVDV inhibitors that block E2 function inhibiting virus entry into the host cell.

The activation of liver X receptors in Madin-Darby bovine kidney cells and mice restricts infection by bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV) is prevalent worldwide and is an important pathogen that represents a serious threat to the development of the cattle industry by causing significant economic losses. Liver X receptors (LXRs) are members of the nuclear receptor superfamily and have become attractive therapeutic targets for cardiovascular disease. In the present study, we found that LXRs in both Madin-Darby bovine kidney (MDBK) cells and mice were associated with BVDV infection. GW3965, an agonist for LXRs, significantly inhibited BVDV RNA and protein levels in MDBK cells. In vivo studies in a mouse model also confirmed the inhibitory role of GW3965 in BVDV replication and the ameliorating effect of GW3965 on pathological injury to the duodenum. In vitro investigations of the potential mechanisms involved showed that GW3965 significantly inhibited BVDV-induced increases in cholesterol levels and viral internalization. Furthermore, the antiviral activity of GW3965 was significantly reduced following cholesterol replenishment, thus demonstrating that cholesterol was involved in the resistance of GW3965 to BVDV replication. Further studies indicated the role of ATP-binding cassette transporter A1 (ABCA1) and cholesterol-25-hydroxylase (CH25H) in the antiviral activity of GW3965. We also demonstrated the significant antiviral effect of 25hydroxycholesterol (25HC), a product of the catalysis of cholesterol by CH25H. In addition, the anti-BVDV effects of demethoxycurcumin (DMC), cyanidin-3-O-glucoside (C3G), and saikosaponin-A (SSA), three natural agonizts of LXRs, were also confirmed in both MDBK cells and mice. However, the antiviral activities of these agents were weakened by SR9243, a synthetic inhibitor of LXRs. For the first time, our research demonstrated that the activation of LXRs can exert significant anti-BVDV effects in MDBK cells and mice.

Immune Responses to Influenza D Virus in Calves Previously Infected with Bovine Viral Diarrhea Virus 2.

Bovine viral diarrhea virus (BVDV) induces immunosuppression and thymus depletion in calves. This study explores the impact of prior BVDV-2 exposure on the subsequent immune response to influenza D virus (IDV). Twenty 3-week-old calves were divided into four groups. Calves in G1 and G3 were mock-treated on day 0, while calves in G2 and G4 received BVDV. Calves in G1 (mock) and G2 (BVDV) were necropsied on day 13 post-infection. IDV was inoculated on day 21 in G3 calves (mock + IDV) and G4 (BVDV + IDV) and necropsy was conducted on day 42. Pre-exposed BVDV calves exhibited prolonged and increased IDV shedding in nasal secretions. An approximate 50% reduction in the thymus was observed in acutely infected BVDV calves (G2) compared to controls (G1). On day 42, thymus depletion was observed in two calves in G4, while three had normal weight. BVDV-2-exposed calves had impaired CD8 T cell proliferation after IDV recall stimulation, and the α/ß T cell impairment was particularly evident in those with persistent thymic atrophy. Conversely, no difference in antibody levels against IDV was noted. BVDV-induced thymus depletion varied from transient to persistent. Persistent thymus atrophy was correlated with weaker T cell proliferation, suggesting correlation between persistent thymus atrophy and impaired T cell immune response to subsequent infections.

Multivariate Analysis as a Method to Evaluate Antigenic Relationships between Bovine Viral Diarrhea Virus 1b Isolates and Vaccine Strains.

The antigenicity of bovine viral diarrhea virus (BVDV) has been evaluated using virus-neutralizing titer data analyzed by principal component analysis (PCA) and has demonstrated numerous isolates to be antigenically divergent from US vaccine strains. The lack of BVDV-1b strains in currently licensed vaccines has raised concerns regarding the lack of protection against BVDV-1b field strains. The aim of this study was to evaluate the antigenic diversity of BVDV-1b strains and better understand the breadth of antigenic relatedness using BVDV-1b antisera and antisera from vaccine strains. Results from this analysis demonstrate the antigenic diversity observed among BVDV-1b isolates and genetic assignment into the BVDV-1b subgenotype is not representative of antigenic relatedness. This is demonstrated by BVDV-1b isolates (2280N, SNc, Illc, MSU, and 2337) observed to be as antigenically dissimilar as BVDV-2a isolates when using BVDV-1b antisera. Additionally, when BVDV-1a vaccine antisera was used for comparisons, a greater percentage of BVDV-1b isolates clustered with BVDV-1a vaccine strains as part of PC1, suggesting antigenic relatedness and potentially partial protection. Collectively, data from this study would suggest that while most BVDV-1b isolates are antigenically similar, there are antigenically dissimilar BVDV-1b isolates as determined by the lack of cross-reactivity, which may contribute to the lack of protection.

Assessing the reliability of innovative criteria to certify that cattle are non-Persistently Infected (non-PI) with the Bovine Viral Diarrhoea Virus (BVDV).

Persistently Infected (PI) animals play a central role in the transmission of BVDV infection between cattle herds. Thus, promoting the certification of non-PI animals is a relevant approach for improving control, as it contributes to securing the trade. The objectives of this study were: i) to assess the reliability of diverse certification criteria, and ii) to identify risk factors for erroneous certification. To do so, the proportion of animals wrongly certified as non-PI on the basis of tests performed after the certification date, was calculated for each criterion. The data used were collected in herds located in Brittany, involved in either a clearance process for those that were infected, or in a surveillance process for herds that were BVDV-free. A total of 23 criteria were defined by combining the technical characteristics of the tests (individual vs. pool; single vs. repeated; direct vs. indirect tests), and some pathogenic characteristics of BVDV infection. Overall, the rates of wrongly-certified animals were low (mean: 1.3 10-4). Direct and indirect criteria had equivalent performances. Heifers from birth, and even foetuses in the last third of gestation, are certified, provided that the herd to which they belong has been free of BVDV for more than 2.5 years. The risk for wrong certification increased in the case of PIs present in the herd or its surroundings. The simplicity of the output-based approach described here, and the excellent performance of indirect criteria relying on serological monitoring of BTM, make it particularly interesting, as its use could facilitate trade between countries.

Up-regulated lncRNA CYLD as a ceRNA of miR-2383 facilitates bovine viral diarrhea virus replication by promoting CYLD expression to counteract RIG-I-mediated type-I IFN production.

Bovine viral diarrhea virus (BVDV) is one of the most important pathogens of cattle, causing numerous economic losses to the cattle industry. To date, many potential mechanisms of BVDV evading or subverting innate immunity are still unknown. In this study, an lnc-CYLD/miR-2383/CYLD axis involved in BVDV-host interactions was screened from RNA-seq-based co-expression networks analysis of long noncoding RNAs, microRNAs and mRNAs in BVDV-infected bovine cells, and underlying mechanisms of lnc-CYLD/miR-2383/CYLD axis regulating BVDV replication were explored. Results showed that BVDV-induced up-regulation of the lnc-CYLD competed for binding to the miR-2383, and then promoted CYLD expression, thereby inhibiting RIG-I-mediated type-I interferon (IFN) production, which was subsequently confirmed by treatment with lnc-CYLD overexpression and miR-2383 inhibitor. However, miR-2383 transfection and small interfering RNA-mediated lnc-CYLD knockdown inhibited CYLD expression and enhanced RIG-I-mediated type-I IFN production, inhibiting BVDV replication. In addition, interaction relationship between lnc-CYLD and miR-2383, and colocalization relationship of lnc-CYLD, miR-2383 and CYLD were confirmed by dual-luciferase assay and in situ hybridization assay. Conclusively, up-regulation of the lnc-CYLD as a competing endogenous RNA binds to the miR-2383 to reduce inhibitory effect of the miR-2383 on the CYLD expression, playing an important role in counteracting type-I IFN-dependent antiviral immunity to facilitate BVDV replication.

Pestivirus A Bovine viral diarrhea virus type 1 species genotypes circulating in China and Turkey.

Background: Pestivirus A Bovine viral diarrhea virus type 1 (BVDV-1) is a heterogeneous species within the genus, affecting cattle and other ruminants, with economic impact on livestock production. Aim: The study aimed to update the taxonomy of the Pestivirus A, BVDV-1 species and to verify the clustering of the strains reported as genotype 1v, originating from different countries. Methods: Recently deposited strains from China, Turkey, and Iran have been evaluated by the palindromic nucleotide substitutions (PNS) genotyping method. Results: Based on secondary structure analysis of the 5'-UTR sequences, strains reported as 1v from China were clustered as sub genotype 1.7.3 (1o). Genotype 1.19 (1w) was restricted to China and genotype 1.21 (1v) was present only in Turkey and Iran. Conclusion: The application of the PNS method clarified the taxonomical status of strains, revealing the homonymy of genetically different clusters. Furthermore, these observations indicated geographic segregation in the Pestivirus A species, and confirmed the occurrence of new atypical genetic variants, with potential implications on control and prophylaxis.

Novel genomic targets for proper subtyping of bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2.

Whole-genome phylogenetic analysis, the most suitable strategy for subtyping bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2, is not feasible for many laboratories. Consequently, BVDV isolates/strains have been frequently subtyped based on analysis of single genomic regions, mainly the 5' untranslated region (UTR). This approach, however, may lead to inaccurate and/or poorly statistically supported viral classification. Herein, we describe novel primer sets whose amplicons may be easily sequenced and used for BVDV subtyping. Initially, genomic regions previously described as the most suitable targets for BVDV subtyping were analyzed for design of high-coverage primers. The putative amplicons were analyzed in silico for their suitability to reproduce the phylogenetic classification of 118 BVDV-1 and 88 BVDV-2 complete/near-complete genomes (CNCGs) (GenBank). This analysis was also performed considering the region amplifiable by primers HCV90-368, 324-326 and BP189-389 (5'UTR), which have been used for BVDV diagnosis and/or classification. After confirming the agreement between the analyses of our primers' amplicon versus the CNCGs, we optimized the RT-PCRs and evaluated their performance for amplification of BVDV isolates/strains (n = 35 for BVDV-1; n = 33 for BVDV-2). Among the potential targets for BVDV subtyping, we designed high-coverage primers for NS3-NS4A (BVDV-1) (526 bp amplicon) and NS5B (BVDV-2) (728 bp). The classification based on these regions fully reproduced the subtyping of all CNCGs. On the other hand, subtyping based on the putative amplicons from primers HCV90-368, 324-326 and BP189-389 showed disagreements in relation the CNCG analysis. The NS3-NS4A and NS5B primers also allowed the amplification of all BVDV isolates/strains tested. Finally, we suggest the use of these primers in future phylogenetic and epidemiological studies of BVDVs.

Molecular and serological survey of bovine viral diarrhea virus infection in cattle in Kazakhstan.

The aim of this study was to estimate the occurrence of bovine viral diarrhea virus (BVDV) infection and to assess the population immunity in cattle vaccinated against BVDV in different regions of Kazakhstan. Cattle samples were collected in 12 oblasts (43 districts) of Kazakhstan. A total of 2477 cattle from 114 herds and 21 Bukhara deer (Cervus elaphus bactrianus) were examined by ELISA and conventional RT-PCR. Univariate and multivariate logistic regression analysis was performed to identify risk factors associated with BVDV infection in the country. In total, antibodies against BVDV were found in 79.3% (1965/2477) of all the animals and 92.1% (105/114) of all the herds examined. Seroprevalence in unvaccinated and vaccinated animals was 48.6% (447/920) and 98.7% (1391/1410), respectively. Seroprevalence in deer was 19.1% (4/21). The BVDV RNA was detected in six unvaccinated cattle (0.2%). Sequence analysis of the 5'-untranslated region demonstrated that four of the detected strains belonged to BVDV-1 and two strains to BVDV-2. Regression analysis revealed that age, production type, housing method, farm size, and geographic location were risk factors for BVDV infection in cattle in Kazakhstan. The present data confirm circulation of BVDV-1 and BVDV-2 in Kazakhstan and highlight the need to improve strategies for prevention and control of BVDV infection in the country.