Bovine Viral Diarrhea Virus Isolates Research Articles

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

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

Testing and identification of bovine viral diarrhea virus isolates recovered in Russia between 2019 and 2022

Studying the agents of bovine viral diarrhea (BVD) is an important task given the high probability of new isolate introduction into the Russian Federation, as well as the need to take into account the genotype and subgenotype of the virus circulating in a herd when developing vaccines and diagnostic kits for the infection. During the work performed, 6 BVD virus isolates were recovered and identified. The recovery of these isolates in the lamb testicle cell subculture revealed that Bashkiria/2019, Kirov/2020 and Samara/2020 isolates belong to non-cytopathic bovine viral diarrhea virus biotypes, Chelyabinsk/2021 isolate demonstrated the characteristic cytopathic effect in the monolayer and was classified as a cytopathic variant of thevirus, the adaptation of Belgorod/2021 and Udmurtiya/2020 isolates to this cell system was not possible. The study also identified the species of the recovered isolates. Based on the analysis of the nucleotide sequence of genome 5’-untranslated region (5’-UTR) fragment, these isolates were classified as belonging to three genotypes of the virus. The phylogenetic analysis showed that Chelyabinsk/2021 and Udmurtiya/2020 isolates belong togenotype2 and demonstrate, respectively, 98% and99% homology with reference 890 strain of BVD virus. The recovered Bashkiria/2019, Samara/2020, Kirov/2020 isolates were classified as belonging to subtypes 1i, 1f and1b ofgenotype1, and Belgorod/2021 solate represents genotype 3 of the virus. The findings from the study confirm the presence of all three genotypes of bovine viral diarrhea virus in the Russian Federation and reiterate the need for the development of specific prevention and diagnosis tools for the disease.

Multivariate analysis reveals that BVDV field isolates do not show a close VN-based antigenic relationship to US vaccine strains

ObjectiveEvaluate bovine viral diarrhea virus (BVDV) antigenicity by using virus neutralization titers (VNT) analyzed using the principal component analysis (PCA) from antisera generated against US-based vaccine strains against both US-origin field isolates and non-US-origin field isolates.ResultsData from both independent analyses demonstrated that several US-origin and non-US-origin BVDV field isolates appear to be antigenically divergent from the US-based vaccine strains. Results from the combined analysis provided greater insight into the antigenic diversity observed among BVDV isolates. Data from this study further support genetic assignment into BVDV subgenotypes, as well as strains within subgenotypes is not representative of antigenic relatedness. PCA highlights isolates that are antigenically divergent from members of the same species and subgenotype and conversely isolates that belong to different subgenotypes have similar antigenic characteristics when using antisera from US-based vaccine isolates.

The role of molecular-genetic techniques in BVDV eradication in Lower Austria.

A voluntary bovine viral diarrhoea virus (BVDV) control programme, which later became a compulsory eradication programme, based on the Swedish model was introduced in Lower Austria in 1997. The persistently infected animals were detected by Ag-ELISA and all samples were re-tested by the improved single-tube RT-PCR, employing panpestivirus primers targeting the 5'-UTR of the virus genome. In 2010, the BVDV eradication programme, which became compulsory from 2004, reached the final stage with only five remaining infected herds in which BVDV was difficult to eradicate. To resolve the problem in those herds, a molecular epidemiology approach was used. No differences in the spectrum of BVDV-1 subgenotypes at the beginning and at the final stage of eradication programme were found. The genetic study revealed the importance of human risk factor when finishing an eradication programme. Molecular epidemiology was also used to analyse BVDV isolates associated with re-introductions to BVDV-free herds.

Isolation of BVDV-1a, 1m, and 1v strains from diarrheal calf in china and identification of its genome sequence and cattle virulence.

Bovine viral diarrhea virus (BVDV) is an important livestock viral pathogen responsible for causing significant economic losses. The emerging and novel BVDV isolates are clinically and biologically important, as there are highly antigenic diverse and pathogenic differences among BVDV genotypes. However, no study has yet compared the virulence of predominant genotype isolates (BVDV-1a, 1b, and 1m) in China and the emerging genotype isolate BVDV-1v. The serological relationship among these genotypes has not yet been described. In this study, we isolated three BVDV isolates from calves with severe diarrhea, characterized as BVDV-1a, 1m, and novel 1v, based on multiple genomic regions [including 5-untranslated region (5'-UTR), Npro, and E2] and the phylogenetic analysis of nearly complete genomes. For the novel genotype, genetic variation analysis of the E2 protein of the BVDV-1v HB-03 strain indicates multiple amino acid mutation sites, including potential host cell-binding sites and neutralizing epitopes. Recombination analysis of the BVDV-1v HB-03 strain hinted at the possible occurrence of cross-genotypes (among 1m, 1o, and 1q) and cross-geographical region transmission events. To compare the pathogenic characters and virulence among these BVDV-1 genotypes, newborn calves uninfected with common pathogens were infected intranasally with BVDV isolates. The calves infected with the three genotype isolates show different symptom severities (diarrhea, fever, slowing weight gain, virus shedding, leukopenia, viremia, and immune-related tissue damage). In addition, these infected calves also showed bovine respiratory disease complexes (BRDCs), such as nasal discharge, coughing, abnormal breathing, and lung damage. Based on assessing different parameters, BVDV-1m HB-01 is identified as a highly virulent strain, and BVDV-1a HN-03 and BVDV-1v HB-03 are both identified as moderately virulent strains. Furthermore, the cross-neutralization test demonstrated the antigenic diversity among these Chinese genotypes (1a, 1m, and 1v). Our findings illustrated the genetic evolution characteristics of the emerging genotype and the pathogenic mechanism and antigenic diversity of different genotype strains, These findings also provided an excellent vaccine candidate strain and a suitable BVDV challenge strain for the comprehensive prevention and control of BVDV.

Genetic features of bovine viral diarrhea virus subgenotype 1c in newborn calves at nucleotide and synonymous codon usages.

Bovine viral diarrhea virus (BVDV), serving as an important pathogen for newborn calves, poses threat to reproductive and economic losses in the cattle industry. To survey the infection rate and genetic diversity of BVDV in newborn calves in northern China, a total of 676 sera samples of newborn calves were collected from four provinces between 2021 and 2022. All sera samples were individually detected for BVDV infection by RT-PCR and ELISA. Our results showed that the overall serological rate was 9.76% (66/676) and the average positive rate of BVDV RNA was 8.14% (55/676) in the newborn calves. Eight BVDV strains were successfully isolated from RT-PCR positive sera samples, and four isolates displayed the cytopathic effect (CPE). Based on phylogenetic tree at the genome level, the eight strains were classified into subgenotype 1c. Moreover, the BVDV isolates had a close genetic relationship with the GSTZ strain at either nucleotide or codon usage level. Interestingly, in comparison of synonymous codon usage patterns between the BVDV isolates with CPE and ones without CPE, there were four synonymous codons (UCG, CCC, GCA, and AAC) which displayed the significant differences (p < 0.05) at codon usage pattern, suggesting that synonymous codon usage bias might play a role in BVDV-1c biotypes. In addition, the usage of synonymous codons containing CpG dinucleotides was suppressed by the BVDV-1c isolates, reflecting one of strategies of immune evasion of BVDV to its host. Taken together, our study provided data for monitoring and vaccination strategies of BVDV for newborn calves in northern China.

Molecular Diversity and Histopathological Findings of Novel Bovine Viral Diarrhea Virus Strains Isolated from Bull Semen

Bovine viral diarrhea virus (BVDV) is one of the most common viral pathogens affecting the cattle industry worldwide. The present study aimed to molecularly characterize BVDV isolates that are currently circulating in breeding bulls farmed with cattle suffering from reproductive disorders, and also to assess the consequences of BVDV infection on bulls’ semen quality and conception, and its pathological effects on the structure of testicular tissue and spermatozoa. For this purpose, semen, serum, and testicular samples were collected from four breeding bulls in four private dairy farms in the governorates of Kafr-El Sheik, Beni-Suef, Giza, and Assuit, in Egypt from April 2019 to May 2020. An evaluation of sperm abnormalities was carried out by assessing the integrity of the plasma and acrosomal membranes where severe damage and abnormalities were found. Ultrastructure analysis of the spermatozoa by transmission electron microscopy revealed the presence of a swollen plasma membrane with segmented outer acrosomal membrane of spermatozoa and vacuolar degenerated mitochondria. Histopathological examination of testicular and epididymal tissues indicated moderate to severe degenerative effects of virus infection on seminiferous tubules with hypospermatogenesis. By detection of virus antigen in the serum samples using ELISA, bulls were identified as persistently infected with BVDV. Virus isolation revealed four noncytopathic (NCP-BVDV) strains that were confirmed by fluorescent antibody technique (FAT) and amplification of the 5′ untranslated genomic region (5’UTR) and molecularly typed by amplification of the Erns glycoprotein region. Isolates’ Phylogenetic analysis revealed two subgenotypes: BVDV-1b (Genbank accession numbers; LC634512, LC634513, LC634515) and BVDV-1d (LC634516). According to the knowledge of the authors of the present study, the circulation of the BVDV-1d subgenotype is not reported in Egypt. Therefore, it would be of great importance to track circulating strains in specific countries for successful vaccination programs or accurate diagnostic tests, and this necessitates regular updates.

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.

Endemic infections of bovine viral diarrhea virus genotypes 1b and 2a isolated from cattle in Japan between 2014 and 2020.

Bovine viral diarrhea virus (BVDV) is a causative agent of bovine viral diarrhea. In Japan, a previous study reported that subgenotype 1b viruses were predominant until 2014. Because there is little information regarding the recent epidemiological status of BVDV circulating in Japan, we performed genetic characterization of 909 BVDV isolates obtained between 2014 and 2020. We found that 657 and 252 isolates were classified as BVDV-1 and BVDV-2, respectively, and that they were further subdivided into 1a (35 isolates, 3.9%), 1b (588, 64.7%), 1c (34, 3.7%), and 2a (252, 27.7%). Phylogenetic analysis using entire E2 coding sequence revealed that a major domestic cluster in Japan among BVDV-1b and 2a viruses were unchanged from a previous study conducted from 2006 to 2014. These results provide updated information concerning the epidemic strain of BVDV in Japan, which would be helpful for appropriate vaccine selection.

Use of multivariate analysis to evaluate antigenic relationships between US BVDV vaccine strains and non-US genetically divergent isolates

Bovine viral diarrhea virus (BVDV) comprises two species, BVDV-1 and BVDV-2. But given the genetic diversity among pestiviruses, at least 22 subgenotypes are described for BVDV-1 and 3-4 for BVDV-2. Genetic characterization is generally accomplished through complete or partial sequencing and phylogeny, but it is not a reliable method to define antigenic relationships. The traditional method for evaluating antigenic relationships between pestivirus isolates is the virus neutralization (VN) assay, but interpretation of the data to define antigenic relatedness can be difficult to discern for BVDV isolates within the same BVDV species. Data from this study utilized a multivariate analysis for visualization of VN results to analyze the antigenic relationships between US vaccine strains and field isolates from Switzerland, Italy, Brazil, and the UK. Polyclonal sera were generated against six BVDV strains currently contained in vaccine formulations, and each serum was used in VNs to measure the titers against seven vaccine strains (including the six homologous strains) and 23 BVDV field isolates. Principal component analysis (PCA) was performed using VN titers, and results were interpreted from PCA clustering within the PCA dendrogram and scatter plot. The results demonstrated clustering patterns among various isolates suggesting antigenic relatedness. As expected, the BVDV-1 and BVDV-2 isolates did not cluster together and had the greatest spatial distribution. Notably, a number of clusters representing antigenically related BVDV-1 subgroups contain isolates of different subgenotypes. The multivariate analysis may be a method to better characterize antigenic relationships among BVDV isolates that belong to the same BVDV species and do not have distinct antigenic differences. This might be an invaluable tool to ameliorate the composition of current vaccines, which might well be important for the success of any BVDV control program that includes vaccination in its scheme.

Identification and genotyping of a new subtype of bovine viral diarrhea virus 1 isolated from cattle with diarrhea

In 2019, diarrhea cases occurred on cattle farms in Qionglai and Guang'an, Sichuan Province. Two out of 20 (10%) serum and nasal swab samples were positive when tested using a bovine viral diarrhea virus (BVDV) antigen-capture ELISA kit. Two non-cytopathic strains of BVDV were isolated and named QL1903 and GA190608, respectively. The nucleotide sequences of the genomes of the two isolates were 89.52% identical. Phylogenetic analysis based on the 5'-UTR sequence revealed that the BVDV isolate QL1903 belonged to BVDV subtype 1b, whereas isolate GA190608 clustered with strains HN1814, EN-19, and BJ09_26 in a separate branch, which has tentatively been classified as a new genetic subtype, "1v".

Sequence analysis of the DA domain of glycoprotein E2 of pestiviruses isolated from beef cattle in Southern Brazil.

The envelope glycoprotein E2 of pestiviruses is a major target for neutralizing antibodies. In this study, we analyzed the E2 DA domain of 43 pestiviruses from Southern Brazil. The isolates were identified as Bovine viral diarrhea virus (BVDV) subtypes 1aand 1b orBVDV-2b. Compared to reference strains, the BVDV-1 and -2 isolates had four and two mutationsin the DA domain, respectively. All BVDV-2 isolates had a deletion of residues 724 and 725. All mutated amino acids in the BVDV isolates had the same aa substitution, and all were in previously identified antibody binding sites. It is possible thatan immunity-mediated selection is acting on the pestiviruses circulating in Southern Brazil.

Genetic recombination of bovine viral diarrhea virus subgenotype -1a and -1c in persistently infected dairy cattle

The bovine viral diarrhea virus (BVDV) is a major viral pathogen in cattle worldwide. In Indonesia, diversity in subgenotypes of BVDV-1 has been observed, with the highest proportion of subgenotype -1a, followed by -1c, -1b, and -1d. So far, phylogenetic analysis of BVDV-1 is based on nucleotide sequences of the 5′ UTR and partial NS5B regions. Accuracy in identifying the subgenotype and antigenic type is critical for vaccine development and effective vaccination. The aim of this study was to determine genetic recombination of BVDV through phylogenetic analysis of five different regions (5′ UTR, NPro, E2, NS3, and NS5B) of BVDV in persistently infected dairy cattle. Five isolates were sequenced using next-generation sequencing, and data were analyzed with the CLC Genomic Workbench 9.0 and MEGA-X programs. Phylogenetic analysis based on the 5′ UTR (275 nt), NPro (504 nt), E2 (1,122 nt), NS3 (2,049 nt), and NS5B (2,157 nt) regions indicated that one BVDV isolate from Banyumas, Central Java, could be classified into different subgenotypes based on the E2 region (-1c), but the same subgenotype based on the other four regions (-1a), suggesting the presence of genetic recombination of the BVDV subgenotypes -1a and -1c in persistently infected dairy cattle.

Investigation of viral pathogens in cattle with bovine respiratory disease complex in Inner Mongolia, China

As a multifactor disease, the bovine respiratory disease complex (BRDC) causes high morbidity and mortality that is devastating to the cattle industry. To assess viral infections in beef cattle suffering from respiratory diseases in Inner Mongolia, 302 nasal swabs and serum samples were randomly collected from cattle with mild respiratory symptoms between March 2018 and May 2019. Our results showed that the rate of RT-PCR results positive for nucleic acids of viral pathogens in 6 cities was between 54 and 80%.The rates of bovine viral diarrhea virus (BVDV), bovine herpesvirus 1 (BHV-1), bovine parainfluenza virus type 3(BPIV3), and bovine respiratory syncytial virus(BRSV)infections were 44.70% (135/302), 24.83% (75/302), 5.63% (17/302), and 6.95% (21/302),respectively. There are also 8.94% (27/302) of samples were positive for BVDV and BHV-1, and 3.97% (12/302) of samples were positive for BPIV3 and BRSV. In addition, the RT-PCR products were sequenced, and phylogenetic analysis based on these sequences was performed. The results indicated that: a) all of the BVDV isolates were BVDV-1 and were classified as BVDV-1a (66.67%) and BVDV-1b (33.33%); b) all of the BHV-1 isolates were classified as subtype 1.1; 44.44% of the isolates were closely related to modified live viral vaccine strains, and 55.56% of the isolates were closer to epidemic strains; c) all of the BPIV3 isolates belonged to BPIV3c; d) all of the BRSV isolates were classified into subgroup III. It is suggested that an important cause of respiratory diseases for beef cattle is viral infection, and phylogenetic analysis can help us choose the proper strain to develop a vaccine.

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

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

Changes Introduced in the Open Reading Frame of Bovine Viral Diarrhea Virus During Serial Infection of Pregnant Swine

Bovine viral diarrhea virus (BVDV) is one of the most economically important viruses of cattle, but this pathogen is also able to infect pigs, camelids, and a wide range of domestic and wild ruminants. BVDV isolates circulating in animal populations are genetically and antigenically highly diverse. Acute BVDV infections in cattle cause the introduction of many substitutions in the viral genome. Serial infection of pregnant sheep with a BVDV-1b isolate of bovine origin was also associated with great numbers of substitutions. To our knowledge, genomic changes arising during BVDV infections in swine have not been investigated. The purpose of this study was to investigate the changes occurring in the open reading frame (ORF) of BVDV during serial infection of pregnant swine with a BVDV isolate of bovine origin. The BVDV-1b isolate AU526 was serially passaged in six pregnant gilts, two of which gave birth to live piglets congenitally infected with BVDV. The complete ORF sequences of 14 BVDV isolates obtained from pregnant gilts and their piglets were determined. Their analysis revealed that serial transmission of AU526 in pregnant swine resulted in many genomic changes. All isolates of porcine origin shared 32 nucleotide and 12 amino acid differences with the virus inoculum AU526. These changes were detected after a single passage in pregnant swine and were conserved during the subsequent five passages. Amino acid changes occurred primarily in genomic regions encoding the BVDV structural proteins E2 and Erns. These results suggest that BVDV infections in pregnant swine may contribute significantly to the genetic variability of BVDV and lead to the appearance of adaptive changes.

Phylogenetic analysis and spatial distribution of bovine viral diarrhea virus (BVDV) in dairy cattle from Galicia (NW Spain)

Aim of study: To examine the frequency and diversity of bovine viral diarrhea viruses infecting dairy cattleArea of study: The study was carried out in Galicia (NW Spain), the main dairy cattle area of SpainMaterial and methods: A total of 157 BVDV isolates (from 140 dairy herds) were typed. Typing was based on a 288-bp sequence from the 5′ untranslated region of viral RNA genome. Subsequently, to investigate whether the presence of herds diagnosed with a particular strain was higher in some areas or during some specific time period, data were tested using a Bernouille approachMain results: Of the 157 isolates, 137 (87.3%) were typed as BVDV-1b, 10 (6.4%%) as 1d, 7 (4.4%) as 1e and 2 (1.3%) as 1f. One isolate was assigned to type 1p. Three of the strains found in the study (the three belonging to type 1b) showed significant spatial clustering.Research highlights: This report indicates that BVDV-1b was the predominant species, although there was an important genetic diversity in the study population. Spatial analysis indicated important drawbacks in the application of biosecurity measures, especially as regards purchase of cattle or after the reintroduction of animals from cattle concentration points.

Genetic analysis of NS5B gene from bovine viral diarrhea virus-infected cattle in Central and East Java, Indonesia.

Background and Aim:A previous study divided Indonesian bovine viral diarrhea virus (BVDV)-1 into subgenotypes BVDV-1a to BVDV-1d based on the partial NS5B gene using strain Bega as reference for BVDV-1a. In fact, it is clustered into BVDV-1c with strain Bega-like Australia. BVDV genotyping has been done on isolates from Jakarta, West and Central Java, but East Java isolates have not been genotyped. This study aimed to analyze genetic variability and amino acid residues in the nucleotide-binding pocket of the NS5B gene from infected cattle.Materials and Methods:Samples were obtained from the Sera Bank originating from active and passive surveillance of cattle that had been tested for BVDV antigen from 2013 to 2017. Detection of the p80 antibody and BVDV genotyping was carried out using ELISA and nested-multiplex-polymerase chain reaction (PCR), respectively. We defined 15 nested PCR products for partial sequencing of NS5B. Those field samples were selected from each location and year using proportional calculation as a representative sample. Homological and phylogenetic analyses of the partial NS5B gene were performed using BLAST and MEGA version 6.Results:Based on the phylogenetic tree analysis using 360 nucleotides as the partial NS5B gene, Indonesian BVDV-1 isolates from Central and East Java were subdivided to BVDV-1a (n=9), BVDV-1b (n=1), and BVDV-1c (n=5). In the present study, the homology of BVDV subgenotype -1a, -1b, and -1c was compared to the BVDV GenBank data and found 90-93%, 93%, and 92-95% respectively with the average pairwise distance of 0.207. A point mutation was shown at R283K of all BVDV isolates based on the sequence of three amino acid residues R283, R285, and I287 in the nucleotide-binding pocket as a part of the encoded RNA-dependent RNA polymerase.Conclusion:This study revealed the genetic variability of BVDV infecting cattle in Central Java and East Java, Indonesia, the subtypes BVDV-1a, BVDV-1b, BVDV-1c, and a point mutation at the R283K residue.

Isolation and identification of Bovine Viral Diarrhea Virus (BVDV) from diseased cattle and buffaloes in Sharkia Governorate, Egypt

In the present study, 280 samples including whole blood, nasal swabs, rectal swabs and sera were collected from 70 diseased cattle and buffaloes suspected to be infected with BVDV in Sharkia Governorate, Egypt during the period from July 2013 to June 2014. Animals were suffering from fever, respiratory disorder with nasal lesions, diarrhea and abortion. A total of 210 samples including (70 nasal swabs, 70 rectal swabs and 70 heparinized blood from all animals) were used for isolation of BVDV that was carried out using MDBK tissue culture. Three passages were carried out for each sample. In addition 70 sera samples were used for direct detection of the virus using antigen capture ELISA (Ag-ELISA). The suspected BVDV in tissue cultures (after 3rd passage) was identified by indirect immunofluorescence antibody test (IFAT) and Reverse transcriptase polymerase chain reaction (RT-PCR). Furthermore, nucleotide sequence analysis of amplified 5`UTR gene of the identified BVD nucleic acid was done and compared with published reference sequences on GeneBank. The results of suspected virus isolation revealed that out of 210 examined samples, 12 (5.71%) samples showed clear CPE. The positive results percentage of Ag-ELISA showed that out of 70 examined sera samples, 11 (15.71%) samples were positive. The result of indirect IFA revealed that out of examined 70 samples, 11 (15.71%) samples were positive. Eleven selected samples that showed positive results with IFA were submitted to RT-PCR, and all submitted samples showed positive results of 5`UTR gene amplification. Sequence analysis of 5`UTR gene sequence identified in this study revealed that our isolate was closely related to Denmark BVDV strain (2005). In conclusion, both biotypes cytopathic and non cytopathic BVDV can be isolated from diseased cattle and buffaloes in Sharkia Governorate, Egypt. In addition, Ag-ELISA and IFA can be used for detection of BVDV with the same sensitivity. RT-PCR can be efficiently used for molecular identification BVDV. Further studies are required for better understanding genetic diversity among BVDV cirulating in egyptian farms based on full genome sequencing.

Pathological and virological features of skin lesions caused by BVDV in cattle.

Dermatitis might occur in mucosal disease (MD) caused by bovine viral diarrhea virus (BVDV). This study describes the pathological and virological features of skin lesions associated with BVDV infection in four persistently infected (PI) cattle. Skin samples were reprocessed for histopathology and IHC. BVDV isolates were obtained and were genetically characterized. In addition to upper alimentary system ulcerative lesions, all cattle (one outbreak and three individual cases) presented focal crusty and ulcerative lesions affecting the mucocutaneous and skin-horn junctions, interdigital clefts, pastern, and areas surrounding the dewclaws and diffuse thickened skin within 7-20days of infection. Microscopic analysis revealed parakeratotic hyperkeratosis and single-cell keratinocyte death, accompanied by ballooning degeneration and spongiosis in the epidermis, as well as intraepithelial and subcorneal pustules. IHC showed BVDV antigen in the cytoplasm of keratinocytes undergoing individual cell death. Phylogenetic analysis revealed that the isolates from cattle #1, #2, and #4 belonged to BVDV-1a, whereas that from cattle #3 belonged to BVDV-1d. Cytopathic BVDV was isolated from cattle #2 and #3 (MD), and non-cytopathic BVDV was isolated from cattle #1 and #4. Thus, BVDV infection might cause acute disease, characterized by skin and upper alimentary system ulcerative lesions, in both MD and PI cattle.