Detection Of Bovine Viral Diarrhea Virus Research Articles

Simultaneous detection of bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) using recombinase polymerase amplification

Bovine viral diarrhea virus (BVDV) is considered to be the most common agent of severe diarrhea in cattle worldwide, causing fever, diarrhea, ulcers, and abortion. Bovine herpesvirus 1 (BoHV-1) is also a major bovine respiratory disease agent that spreads worldwide and causes extensive damage to the livestock industry. Recombinase polymerase amplification (RPA) is a novel nucleic acid amplification method with the advantages of high efficiency, rapidity and sensitivity, which has been widely used in the diagnosis of infectious diseases. A dual RPA assay was developed for the simultaneous detection of BVDV and BoHV-1. The assay was completed at a constant temperature of 37 °C for 30 min. It was highly sensitive and had no cross-reactivity with other common bovine viruses. The detection rate of BVDV RPA in clinical samples (36.67%) was higher than that of PCR (33.33%), the detection rate of BoHV-1 RPA and PCR were equal. Therefore, the established dual RPA assay for BVDV and BoHV-1 could be a potential candidate for use as an immediate diagnostic.

Establishment and application of a rapid diagnostic method for BVDV and IBRV using recombinase polymerase amplification-lateral flow device.

Bovine Viral Diarrhea Virus (BVDV) and Infectious Bovine Rhinotracheitis Virus (IBRV) are the two most prevalent infectious diseases in cattle. They both can cause persistent infection and immunosuppression, resulting in significant economic losses in the livestock industry. Therefore, rapid detection of early BVDV and IBRV infections is crucial. In this study, a method for the rapid detection of BVDV and IBRV was established by using recombinase polymerase amplification (RPA) combined with lateral flow device (LFD). By optimizing the temperature and time conditions of the RPA reaction, the sensitivity, specificity, and clinical performance were evaluated. The results indicated that the RPA reaction could be completed at 40°C within 25 min. The LOD for BVDV and IBRV by RPA-LFD were 5.1 × 101 copies/μL and 6.65 × 101 copies/μL, respectively, with no cross-reactivity observed with other viruses such as CSFV, BRSV, BPIV3, BRV, and BCoV. Testing of 32 clinical samples showed consistent results between RPA-LFD and qPCR. The RPA-LFD method established in this study can be used for the rapid clinical detection of BVDV and IBRV, which providing a rapid and convenient molecular biology approach for on-site rapid detection and epidemiological investigations. Simultaneously, it offers technical support for the prevention and control of these viruses.

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.

Comparative molecular detection of BVDV in cattle herds related to districts and seasons of year in Thi-qar province

Bovine viral diarrhea virus (BVDV) is a complicated ruminant pathogen that naturally infects cattle, goats, sheep, buffalo, pigs, camelids, and wild ruminants. BVDV infection causes serious financial losses, either directly due to high rates of mortality and morbidity brought on by fatal mucosal disease and immunosuppression. From September 2022 to May 2023, blood samples (225) were randomly collected from unvaccinated cattle from various regions within Thi-Qar province, Iraq. 5?-untranslated region (UTR) amplification by nested RT-PCR results showed that the distribution of bovine viral diarrhea virus in Thi-Qar province was 19.11% (43 out of 225). These results show no statistically significant differences in the bovine viral diarrhea virus distribution between geographical regions and study months (P >0.05).

A longitudinal study of bovine viral diarrhea virus in a semi-closed management dairy cattle herd, 2020-2022.

Bovine viral diarrhea virus (BVDV) brings great economic loss to the cattle industry worldwide. Developing a control/prevention strategy requires the prior assessment of certain epidemiological parameters. To determine the BVD incidence rate and associated risk factors, a dairy cattle herd in the eastern region of Saudi Arabia was monitored between 2020 and 2022. Nasal swabs (n = 190), rectal swabs (n = 190), and sera (n = 190) were collected from 79 cows in this herd. Collected sera and swabs were tested using the commercially available ELISAs for the BVDV antibodies and antigens, respectively. Collected sera were also tested for the presence of BVDV nucleic acids using commercial real-time RT-PCR kits. Our data show BVDV seroprevalence (18.8%, 15%, and 8.2%) in the tested animals in 2020-2022, respectively. None of the collected nasal swabs, rectal swabs, or sera tested positive for the BVDV antigen, whereas 10.1%, 10%, and 18.1% of the tested sera were positive for BVDV nucleic acid in 2020-2022, respectively. The incidence rate was estimated at 0.02446 new cases/year despite the detection of BVDV in seronegative animals on single or two occasions at ≥6-month intervals. Young calves and bulls remained apparently unexposed to BVDV despite their presence with BVDV-infected females, with no significant physical separation. Both seropositivity and nucleic acid detectability showed significant positive and negative correlations, respectively, with reproductive performance. Collectively, the present study provides useful clues about the transmissibility of BVDV in the presence of possibly persistently infected animals. To the best of our knowledge, this is the first longitudinal study of BVDV in the Eastern Region of Saudi Arabia. Further detailed characterization of the circulating BVDVs is encouraged.

Serological Detection of SRMV, Bovine Viral Diarrhea Virus, BVDV and BEFV in Camels (Camelus dromedarius) in Southwest Iran

Background: Small ruminant morbillivirus (SRMV), bovine viral diarrhea virus (BVDV), bovine herpes virus-1 (BHV-1), and bovine ephemeral fever virus (BEFV) are among the most important viruses of farm animals. Objectives: This study aimed at the serological detection of SRMV, BVDV, BHV-1, and BEFV in the camel population of Khuzestan Province, located in southwest Iran. Methods: A total of 150 camel blood samples were randomly collected from free-ranging, seemingly healthy camels of both sexes and various ages in 8 different regions of Khuzestan Province, Iran. The sera were tested for SRMV, BVDV, BHV-1, and BEFV with serum neutralization test. Results: The seropositive samples for SRMV, BVDV, and BHV-1 were 1(0.67%), 7(4.67%), and 11(7.33%), respectively. There was no seroconversion against BEFV in the serum samples. Conclusion: The camel population of Khuzestan Province is subclinically infected with SRMV, BVDV, and BHV-1 and could play a significant role in the epizootiology of these viral diseases in this region, which is very important for the control and eradication programs.

Sensitive Detection of BVDV Using Gold Nanoparticle-Modified Few-Layer Black Phosphorus with Affinity Peptide-Based Electrochemical Sensor

The lethality of the bovine viral diarrhea virus (BVDV) in cattle involves inapparent infection and various, typically subclinical, syndromes. Cattle of all ages are vulnerable to infection with the virus. It also causes considerable economic losses, primarily due to reduced reproductive performance. In the absence of treatment that can completely cure infected animals, detection of BVDV relies on highly sensitive and selective diagnosis methods. In this study, an electrochemical detection system was developed as a useful and sensitive system for the detection of BVDV to suggest the direction of diagnostic technology through the development of conductive nanoparticle synthesis. As a countermeasure, a more sensitive and rapid BVDV detection system was developed using the synthesis of electroconductive nanomaterials black phosphorus (BP) and gold nanoparticle (AuNP). To increase the conductivity effect, AuNP was synthesized on the BP surface, and the stability of BP was improved by using dopamine self-polymerization. Moreover, its characterizations, electrical conductivity, selectivity, and sensitivity toward BVDV also have been investigated. The BP@AuNP-peptide-based BVDV electrochemical sensor exhibited a low detection limit of 0.59 copies mL-1 with high selectivity and long-term stability (retaining 95% of its initial performance over 30 days).

The effect of fibroblast growth factor 21 on a mouse model of bovine viral diarrhea.

Previously, we researched that bovine viral diarrhea virus (BVDV) induced a very significant increase in fibroblast growth factor 21 (FGF21) expression in mouse liver and that FGF21 was increased in the peripheral blood of BVD cattle and BVD mice. To determine the role of FGF21 in relieving clinical symptoms and inhibiting the intestinal damage caused by BVDV in BVD development in mice, BALB/c mice were intraperitoneally injected with cytopathic biotype (cp) BVDV-LS01 (isolated and identified by our group) to establish a BVD mouse model. The role of FGF21 in the BVD mouse model was investigated by injecting the mice with FGF21. The animals were divided into control, BVDV challenge, BVDV + FGF21, BVDV + FGF21Ab (anti-FGF21 antibody), and BVDV + IgG (immunoglobulin G) groups. The stool consistency, the degree of bloody diarrhea, histopathological changes, inflammatory cell infiltration, weight loss percentage, and detection of BVDV in the feces of the mice were examined, and the pathological changes and inflammatory cytokine expression were analyzed. The results showed that after BVDV challenge, the average BVD mouse model score of the BVDV mice was 11.6 points. In addition to mild diarrhea and tissue damage, BVDV was detected in the stools of 13 BVDV mice. Only two mice in the control group had scores (both, 1 point each). The comprehensive scoring results demonstrated the successful establishment of the BVD mouse model. FGF21 alleviated the clinical symptoms in the BVD mice and significantly improved weight loss. Furthermore, FGF21 inhibited the BVDV-induced leukocyte, platelet, and lymphocyte reduction while inhibiting the expression of BVDV-induced inflammatory factors. In the BVD mice, FGF21 promoted duodenal epithelial cell proliferation, thereby significantly improving the damage to the cells. In conclusion, FGF21 exerted a good therapeutic effect on the BVD mouse model.

Detection of bovine viral diarrhea virus and bovine herpes virus type 1 in cattle with and without endometritis.

This study aimed to investigate the potential presence of bovine herpes virus type 1 (BHV-1) and bovine viral diarrhea virus (BVDV) in cattle uteri that did not display any clinical and macroscopic signs of infection. Virus detection involved polymerase chain reaction (PCR) test, double immunohistochemistry (IHC), and double immunofluorescence (IF). One hundred cornu uterus samples were collected from cattle aged 1 year and older. The BVDV was detected by PCR or by double IHC/IF in the collected samples from slaughterhouses in Kayseri city (Central Anatolia, Türkiye) from 2021 - 2022. By contrast, BHV-1 was detected by PCR and double IHC/IF at a rate of 16.00% and 21.00%, respectively. In the IHC and IF detection, BHV-1 was detected in endometrial epithelial cells and in some mononuclear cells in the lamina propria, periglandular areas and myometrium. Although no macroscopic lesion was found in the BHV-1-positive samples (n = 21), histopathological detection showed that two had acute endometritis, eight had subacute endometritis, eight had chronic endometritis and the three others showed no signs of endometritis. This prevalence study demonstrated for the first time that even while BVDV could not be detected in the samples, BHV-1 posed a critical potential reproductive risk in pregnant animals, as it can specifically cause abortions when it resides in cattle uteri that do not show clinical or macroscopic and even microscopic signs of infection. Additionally, this study was the first to combine PCR and double IHC/IF for BHV-1 and BVDV detection in cattle uteri.

Caracterización del virus de la diarrea viral bovino subtipo 1b aislado de un caso de la enfermedad de las mucosas

This report describes a fatal case of mucosal disease in a two-year-old bull. For causal agent detection, scab, whole blood, and feces samples were tested by RT-PCR, PCR, ELISA, and viral isolation. RT-PCR positive amplification was obtained in blood samples for bovine viral diarrhea virus (BVDV). Viral isolation from the scab samples confirmed BVDV as the causative agent of the clinical manifestations. Subsequently, genetic characterization based on phylogenetic analysis of three partial sequences revealed the presence of BVDV subgenotype 1b in analyzed samples. Due to the development of clinical manifestation named mucosal disease, these findings suggest the detection of BVDV persistently infected (PI) bull; therefore, these results demonstrate the importance of establishing BVDV control programs that rely on testing the presence of PI in cattle from Mexico.

Molecular Investigation of Bovine Viral Diarrhea Virus, Bovine Herpes Virus-1 and Bovine Herpes Virus-4 Infections in Abortion Cases of Cattle in Van District, Turkey

Abortions, fetal mummification, calf anomalies, and infertility problems constitute most of the reproductive problems in cattle. Viruses play a significant role in the cause of these cases. In cattle, these agents are known as primary abortion agents and the most common of these agents are Bovine Viral Diarrhea Virus (BVDV), Bovine Herpes Virus Type 1 (BoHV-1), and Bovine Herpes Virus Type 4 (BoHV-4). The objective of this research is to determine the potential role of BVDV, BoHV-1, and BoHV-4 as viral abortion agents in cattle housed in the Van district. For this, a total of 115 animal specimens (blood, serum, vaginal swab, vaginal fluid discharge, nasal swab, and abortion material) from 100 abortion, early embryonic deaths, and infertility cases in cattle over the age of 2-5 years old were collected. All samples for detection of BVDV, BoHV-1 and BoHV-4 genomes were tested by the Polymerase Chain Reaction (PCR) technique using specific primers encoding Panpesti 5'-UTR, Glycoprotein C (gC) and Glycoprotein B (gB) genes, respectively. Result out of the samples tested, 41.73% were positive for BVDV and all samples were negative for BoHV-1 and BoHV-4. In conclusion, the presence of BVDV in cattle in the Van region and its role in the occurrence of abortion cases was emphasized for the first time. It is necessary to the consideration of viral abortions and determine the etiology of abortion cases and genital system problems. According to this, we need to focus on the detection of persistently infected (PI) animals for prevention and control of infection and the most effective way of vaccinating susceptible populations.

Development of an in-house Indirect ELISA for detection of bovine viral diarrhoea virus antibodies in bovine sera

Bovine viral diarrhoea virus (BVDV) infection is a worldwide distributed animal disease. BVDV is the causal agent of congenital defects, diarrhea, reproductive failure, and contaminating biological products. Virus Neutralization test (VNT) as a gold standard method is used for detection of BVDV. Although this assay is very sensitive and specific, it has disadvantages including requires to an experienced person and cell culture facilities. VNT is time-consuming. It is important to design a method that does not have the mentioned disadvantages. So, in-house indirect enzyme linked immunosorbent assay (i-ELISA) was developed for laboratories where it is not possible to perform VNT. The system was made using NADL strain of BVDV and MDBK cell line. This ELISA system was compared with a commercial ELISA kit using 99 bovine sera. Coefficient of variation (CV) was calculated 3.9% and 4.8% for the positive and negative control, respectively for the designed i-ELISA system. The sensitivity, specificity, and accuracy of i-ELISA system was 88%, 53.6%, and 70.7% respectively. Based on our result correlation between in- house and commercial ELISA kit for detection of antibody against BVDV in bovine sera was significant (Kappa coefficient =0.41, p < 0.05). Results of the present study suggested that an in-house ELISA as an affordable and confident system for primary screening of the sera used for biological product.

First study on genetic variability of bovine viral diarrhea virus isolated from Sapera dairy goats with reproductive disorders in Yogyakarta, Indonesia.

Background and Aim:Bovine viral diarrhea (BVD) virus (BVDV) is an important viral pathogen of cattle that can infect diverse artiodactyl species. The clinical manifestations caused by BVDV in heterologous hosts, as they do in cattle, vary, although respiratory and reproductive failures are commonly reported. BVDV infections commonly result in reproductive failure in goats, with abortion being the primary clinical sign. In central Java, Indonesia, BVDV infection has been reported in two clinically healthy local goat species, and the testing indicated infection by BVDV Type 1. However, the genetic diversity of viruses has not been described in healthy or ill goats. The objectives of the present study were as follows: (1) To investigate the genetic variation of BVDV isolated from Sapera dairy goats with naturally occurring reproductive disorders in Yogyakarta, Indonesia, using the 5’ untranslated region (5’ UTR) and (2) to study the possible correlation between reproductive disorders and the presence of BVDV in the flock.Materials and Methods:Blood samples were collected in October 2021 from 39 goats that had been individually reported to have reproductive disorders. The serum samples were subjected to molecular detection and genetic characterization of BVDV based on the 5’ UTR of the viral genome, followed by sequencing and phylogenetic analyses. Viral isolation was performed on BVDV-positive samples to analyze the viral biotypes.Results:BVDV infection was detected in five out of 39 female goats. The clinical status of the BVDV-infected goats was abortion (n=2), metritis (n=1), and repeated breeding (n=2). All antigen-positive samples were confirmed as BVDV type 1a (BVDV-1a) and noncytopathic (NCP)-BVDV biotype.Conclusion:The BVDV-1a and NCP biotypes are the main subtypes and biotypes present in Sapera dairy goats exhibiting reproductive failure. This result is consistent with previous results in dairy cattle in Yogyakarta. The reported results can facilitate the design of methods for the prevention and control of BVD circulating in Indonesia.

Diagnóstico e análise filogenética do vírus da diarreia viral bovina em bovinos (Bos taurus) e búfalos (Bubalus bubalis) da região amazônica e Sudeste do Brasil

ABSTRACT: Bovine viral diarrhea virus (BVDV) is a highly infectious pathogen that affects bovines worldwide leading to great economic impact. Although Brazil has the largest commercial cattle population throughout the world and an increasing buffalo breeding industry, the country has no control or eradication program for BVDV. In this perspective, the aim of this study was to evaluate the occurrence of BVDV in cattle and buffaloes from two Brazilian states. Four different ELISA tests were performed and confirmed by virus neutralization testing (VNT). The presence of BVDV antibodies in the serum or plasma from 77 cattle from six herds (ELISA-1 and ELISA-4) and from 89 buffaloes from three herds (ELISA-1 through ELISA-4) was detected. Extraction of viral RNA was performed from the serum or plasma samples for the detection of BVDV by RT-PCR analysis. Amplified nucleotide sequences were used to construct a phylogenetic tree. In cattle, ELISA-1 detected 49.4% of seropositive animals, while ELISA-4 detected 37.7%. In buffaloes, ELISA-1 failed to detect any seropositive animals, while ELISA-2 and ELISA-3 detected 20.2% of seropositive animals, and ELISA-4 detected 21.3%. Eight of the nine herds tested had seropositive animals. The rate of PCR positive animals was 6.5% in cattle and 9% in buffaloes. Subtype 1d was found in cattle, and subtypes 1d and 1f were found in buffaloes. This is the first-time subtype 1f has been reported in Brazil. The absence of a control and eradication program seems to be favoring the spread of BVDV in the Brazilian herds. In addition, the improvement of diagnostic strategies for BVDV in buffaloes are required.

A lateral flow dipstick combined with reverse transcription recombinase polymerase amplification for rapid and visual detection of the BVDV and BPIV3

Bovine respiratory disease complex (BRDC) is a serious disease affecting feedlot cattle in China and likely other places worldwide. Bovine viral diarrhea virus (BVDV) and bovine parainfluenza virus type 3 (BPIV3) are principally responsible for causing BRDC, and are a major strain to the industrial economy. Eradication of these viruses/disease requires swift viral identification and treatment. Hence, this study established a fast and easy procedure of BVDV and BPIV3 identification that employs reverse transcription recombinase polymerase amplification (RT-RPA) and lateral flow dipstick (LFD), and uses primers and lateral flow (LF) probe targeting the 5′-UTR gene of BVDV and phosphoprotein P gene of BPIV3, respectively. Our assay was able to successfully amplify BVDV and BPIV3 RNA within 25 min at 35 °C using RT-RPA, with products visible on the LFD within 5 min at room temperature (RT). The lowest detection limits were 50 RNA molecules for BVDV and 34 RNA molecules for BPIV3 per reaction. We also demonstrated that the established dual RT-RPA LFD assay was precise and targeted, harboring excellent potential to become an onsite molecular diagnostic tool in the detection of BVDV and BPIV3. This method can detect BVDV (Pestivirus A, B) and BPIV3, and exhibit no cross-reaction with other viruses like the classical swine fever virus (CSFV) and infectious bovine rhinotracheitis virus (IBRV). The assay performance was further assessed with clinical samples, and demonstrated good performance in comparison to real-time RT-PCR (RT-qPCR). Moreover, the RT-RPA LFD assay was comparitively rapid and required minimal training.

Establishment and Preliminary Application of Multiplex Fluorescent Quantitative PCR for Simultaneous Detection of BVDV, BRV and BCV

Background: In this study, we aimed to establish a multiplex fluorescence quantitative polymerase chain reaction (PCR) method for the identification and detection of bovine viral diarrhea virus (BVDV), bovine rotavirus (BRV) and bovine coronavirus (BCV). Methods: Based on the highly conserved sequences of BVDV E2 gene, BRV VP6 gene and BCV N gene in GenBank, specific primers were designed to amplify the target gene fragments of each virus and the reaction conditions and system were optimized. Multiple fluorescence quantitative methods were established by fluorescence quantitative PCR. Result: The minimum detection limits of plasmid standards for BVDV, BRV and BCV by multiplex fluorescence quantitative PCR were 1.19×102 copies/μL, 3.89×101 copies/μL and 3.74×101 copies/μL, respectively. The lowest sensitivity of the established method was 100 times higher than that of conventional PCR and had high sensitivity. Furthermore, BVDV, BRV and BCV were amplified specifically, with no cross-reactivity with Escherichia coli (E. coli), Salmonella and infectious bovine rhinotracheitis virus (IBRV). The intra-and inter-group coefficients of variation were less than 1%, showing good assay repeatability. Using the established method and ordinary multiplex PCR to simultaneously detect 150 clinical diarrheal disease material samples, the coincidence rate of samples with mixed infection of the three viruses was 83.3%. The results showed that the multiplex fluorescent quantitative PCR detection method established in this study provides a rapid, sensitive and specific technique for clinical diagnosis and epidemiological monitoring of BVDV, BRV and BCV.

The Principles of the Voluntary Programme for the Control and Elimination of Bovine Viral Diarrhoea Virus (BVDV) From Infected Herds in Slovenia.

In Slovenia, the control of bovine viral diarrhoea virus (BVDV) infections started in 1994. Since 2014, a voluntary programme has been running according to the national rules that prescribe the conditions for recognising, acquiring, and maintaining a BVDV-free status for an individual herd. The principle is based on periodical laboratory testing and preventive measures that need to be strictly implemented in a herd. Between 2014 and 2020, a total of 348 herds were included in BVDV antibody testing, and 25.0% of tested herds were detected to be BVDV antibody positive. To recognise the BVDV-free status of the herd, the breeder should provide two consecutive tests with intervals of at least 6 months in all animals in the age from 7 to 13 months, with negative results for BVDV antibodies in ELISA. The BVDV-free status of the herd can be maintained by implementing preventive measures and can be renewed each year with one laboratory test in the age group of animals from 7 to 13 months for antibodies in ELISA. During the 7 years of the voluntary programme, 236 herds were included in the detection of BVDV in individual herds by real-time RT-PCR method and the elimination of positive animals from herds. In 71 (31.3%) herds, at least one BVDV-positive animal was detected, with the identification of a total of 267 persistently infected (PI) animals, representing an average of 2.9% of tested animals. The cost of testing for an average herd, recognised as BVDV-negative, and maintaining its BVDV-free status within the implemented voluntary programme, was €97.64/year, while for the average positive herd, the laboratory costs for elimination of BVDV were €189.59/year. Only limited progress towards eradication at the national level has been achieved in Slovenia since 2014.

Characterization of a Cytopathogenic Reporter CSFV.

Cytopathogenic (cp) pestiviruses frequently emerge in cattle that are persistently infected with the bovine viral diarrhea virus (BVDV) as a consequence of RNA recombination and mutation. They induce apoptosis in infected tissue cultures, are highly attenuated in the immunocompetent host, and unable to establish persistent infections after diaplacental infections. Cp strains of BVDV have been used as naturally attenuated live vaccines and for species-specific plaque reduction tests for the indirect serological detection of BVDV. Here, we present a genetically engineered cp strain of the classical swine fever virus (CSFV). Cytopathogenicity of the strain was induced by the insertion of ubiquitin embedded in a large NS3 to NS4B duplication. The CSFV RNA genome was stabilized by the inactivation of the NS2 autoprotease, hindering the deletion of the insertion and the reversion to a wild-type genome. Additional insertion of a mCherry gene at the 5′-end of the E2 gene allowed fluorescence-verified plaque reduction assays for CSFV, thus providing a novel, cost-efficient diagnostic tool. This genetically stabilized cp CSFV strain could be further used as a basis for potential new modified live vaccines. Taken together, we applied reverse genetics to rationally fixate a typical cp NS3 duplication in a CSFV genome.

Comparison of milk production of dairy cows vaccinated with a live double deleted BVDV vaccine and non-vaccinated dairy cows cohabitating in commercial herds endemically infected with BVD virus.

Daily milk production and reproductive performance of cows vaccinated with a live double-deleted Bovine Viral Diarrhoea Virus (BVDV) vaccine were compared to those of non-vaccinated cows, cohabitating in endemic BVDV herds. All animals in the treatment group were vaccinated on study day 0 irrespective of lactation or gestation status, while control animals did not receive any treatment. 1463 animals were enrolled in the study from four different farms in three different countries (UK, Italy, France). Endemic presence of BVDV in study herds was demonstrated by the detection of BVDV in the bulk tank milk, and seroconversion was evaluated at the beginning of the study. For individual animals, the day of calving was taken to be the start of lactation for the calculation of days in milk (DIM). The standard lactation period of 305 days was divided into three periods: early lactation (EL, from DIM 8 to DIM 102), mid lactation (ML, from DIM 103 to DIM 204 and late lactation (LL, from DIM 205 to DIM 305). For each farm and each lactation period, a mixed model statistical analysis was performed with daily milk production as response, and group, day as well as the interaction between those two factors as fixed factors. Chi-square test was used to compare abortion rate and prolonged inter-oestrous interval rate between treatment and control groups. A significant increase in milk production in the vaccinated group was observed in farms 1 (1.023 L/day) and 3 (0.611 L/day) during EL (p<0.001) and in farm 2 (1.799 L/day) during ML (P<0.001). In addition, at farm 2, vaccinated cows produced more milk than non-vaccinated cows starting from 80 DIM. No differences were found between groups in abortion rates or prolonged inter-oestrous interval rates. Data demonstrate that cows in herds endemically infected with BVDV and vaccinated with live double-deleted BVDV vaccine produce more milk; the difference in milk production occurs during early lactation.

Detection methods and characterization of bovine viral diarrhea virus in aborted fetuses and neonatal calves over a 22-year period.

Detection of bovine viral diarrhea virus (BVDV) in aborted fetus samples is often difficult due to tissue autolysis and inappropriate sampling. Studies assessing different methods for BVDV identification in fetal specimens are scarce. The present study evaluated the agreement between different diagnostic techniques to detect BVDV infections in specimens from a large number of bovine aborted fetuses and neonatal deaths over a period of 22years. Additionally, genetic, serological, and pathological analyses were conducted in order to characterize BVDV strains of fetal origin. Samples from 95 selected cases from 1997 to 2018 were analyzed by antigen-capture ELISA (AgELISA), nested RT-PCR (RT-nPCR), and real-time RT-PCR (RT-qPCR). In addition, amplification and sequencing of the 5'UTR region were performed for phylogenetic purposes. Virus neutralization tests against the BVDV-1a, BVDV-1b, and BVDV-2b subtypes were conducted on 60 fetal fluids of the selected cases. Furthermore, the frequency and severity of histopathological lesions were evaluated in BVDV-positive cases. This study demonstrated that RT-nPCR and RT-qPCR were more suitable than AgELISA for BVDV detection in fetal specimens. However, the agreement between the two RT-PCR methods was moderate. The BVDV-1b subtype was more frequently detected than the BVDV-1a and BVDV-2b subtypes. Neutralizing antibodies to any of the three subtypes evaluated were present in 94% of the fetal fluids. Microscopically, half of the BVDV-positive cases showed a mild non-suppurative inflammatory response. These results emphasize the need to consider different methods for a diagnostic approach of BVDV associated to reproductive losses.