Phylogenetic analysis and characterization of bovine herpesvirus-1 in cattle of China, 2016-2019.

Bovine herpesvirus type 1 (BoHV-1) is one of the most critical pathogens in cattle and is prevalent in China. BoHV-1 is divided into two gene types, BoHV-1.1 and 1.2, which are further differentiated into two subtypes, BoHV-1.2a and 1.2b. However, the phylogenetic analysis of BoHV-1 isolates has not been reported in China. To perform a molecular epidemiological survey based on isolates from cattle in China, 102 lung tissue samples of calves under ten months of age with respiratory disease (BRD) that died from 2016 to 2019 in China were used to isolate BoHV-1 with Madin-Darby bovine kidney (MDBK) cells. Part of the BoHV-1 isolates were applied to the phylogenetic analysis based on the region of the glycoprotein C (gC) gene of BoHV-1. Thirty BoHV-1 isolates were obtained, and the gC gene of 13 isolates was amplified by polymerase chain reaction (PCR) methods and sequenced. The result of the phylogenetic analysis according to the 451-nucleotide portion of the gC gene found that all of 13 isolates belonged to the BoHV-1.2b gene subtype, but these isolates had located two different phylogenetic tree branches. The gC gene sequence homology of isolates in group1 was higher with a reference strain of BoHV-1.2b EVI14 up to 98.0-100%, while in group 2, this was higher with reference strain BoHV-1.2b B589 up to 97.8-99.8%. The deduced amino acid sequence of gC from isolates in group 2 had two amino acid mutations with interference strain BoHV-1.2b K22 or BoHV-1.1 COOPER. The cytopathic effects (CPEs) of BoHV-1 isolates in group 2 were ulcered on the centration like a volcano on MDBK cell, and different from traditional CPEs of BoHV-1. Overall, BoHV-1.2b seems to be the primary strain of BoHV-1 in cattle in China and is also a critical cause of BRD. These BoHV-1.2b isolates had significant genetic variations.

A new molecular method for the rapid subtyping of bovine herpesvirus 1 field isolates.

Bovine herpesvirus 1 (BoHV-1) causes several clinical syndromes in cattle worldwide. There are 3 subtypes of BoHV-1: 1.1, 1.2a, and 1.2b. Several molecular methods are commonly used in the detection and characterization of BoHV-1. Among them, restriction endonuclease analysis (REA) and single-nucleotide polymorphism (SNP) analysis of the complete viral genome allow classification of BoHV-1 into different subtypes. However, developing countries need simpler and cheaper screening assays for routine testing. We designed a standard multiplex PCR followed by a REA assay allowing straightforward subclassification of all BoHV-1 isolates tested into 1.1, 1.2a, and 1.2b subtypes based on the analysis of fragment length polymorphism. Our standard multiplex PCR-REA was used to analyze 33 field strains of BoHV-1 isolated from various tissues. The assay confirmed the subtype identified previously by REA. In addition, non-polymorphic or undigested fragments were sequenced in order to confirm the mutation affecting the RE HindIII site. Our PCR-REA method is an affordable and rapid test that will subtype all BoHV-1 strains.

Molecular and antigenic characterization of bovine herpesvirus type 1 (BoHV-1) strains from cattle with diverse clinical cases in Turkey.

The present study reports the molecular and antigenic characterization of 13 bovine herpesvirus type 1 (BoHV-1) field viruses obtained from cattle with different clinical cases in Turkey between 1992 and 2017. We selected glycoprotein C (gC) of BoHV-1 as a target to detect and/or verify presence of the virus in suspect materials followed by virus isolation (VI) in MDBK cells. In seven out of 13 BoHV-1 positive samples, cytophatic effects (CPEs) were observed in MDBK cell cultures, although only four virus samples reached a sufficient titer to use in phylogenetic assay, restriction endonuclease analysis (REA), and virus neutralization test (VNT). According to the results of sequence analysis of the 13 BoHV-1 positive samples, nine BoHV-1 field viruses were determined as BoHV-1.1 and four as BoHV-1.2. Using REA, we demonstrated that two of our isolated viruses could be categorized as BoHV-1.1 while the other two isolates were BoHV-1.2 subtypes. Differences between the BoHV-1.1 and BoHV-1.2 isolates were also detected in the VNT results by assaying 125 suspected serum samples after testing with isolated (KY748023, KY748022, KY748020, and KY748021) and reference viruses (BoHV-1 Cooper and BoHV-5 Texas 89). These results are indicating the need to correctly identify BoHV-1 field isolates to better understand the epidemiology and pathogenesis of infection. In addition, it would be useful to identify the subtypes circulating in the specific geographical area while determining vaccination preferences.

A molecular survey using a validated real-time PCR assay finds no evidence of bovine alphaherpesvirus 2 in samples from animals with suspected vesicular disease in Brazil between 2014 and 2017.

Bovine alphaherpesvirus 2 (BoHV-2) is the etiologic agent of bovine mammillitis (BM) and pseudo-lumpy skin disease. BM is also important because its clinical presentation can be confused with foot-and-mouth disease (FMD), making it necessary to establish differential diagnoses and perform additional laboratory tests. The objective of this work was to use a validated real-time PCR assay to test for the presence of BoHV-2 in samples from cattle and buffalo with suspected vesicular disease in Brazil. The method could detect the virus at a concentration of 0.5 fg/µL and had 99.4% amplification efficiency, a repeatability error of only 4.1%, and good reproducibility with other reagents. No evidence of BoHV-2 causing vesicular disease in cattle and buffalo was found in this work. This study was able to validate a new methodology for detection of BoHV-2 and evaluate its usefulness for investigating outbreaks of vesicular disease Brazil. The importance of BoHV-2 in cases involving other clinical signs should still be studied using the qPCR developed in this work.

Complete genome sequence of bovine herpesvirus type 1.1 (BoHV-1.1) Los Angeles (LA) strain and its genotypic relationship to BoHV-1.1 Cooper and more recently isolated wild-type field strains.

The Cooper and Los Angeles (LA) strains were the two original respiratory strains of bovine herpesvirus type 1.1 (BoHV-1.1) isolated in the 1950s from cattle with infectious bovine rhinotracheitis. We report the complete genome sequence for the BoHV-1.1 LA strain and compare it to the prototype Cooper strain and six wild-type BoHV-1.1 isolates. A nucleotide sequence divergence of 0.74% was noted across the two complete genomes, caused by 19 single-nucleotide polymorphisms (SNPs) involving 12 genes and insertions/deletions that primarily affected the number of repeats within reiterated repeat regions of the genome. Phylogenetic analysis revealed that Cooper and LA strains are genetically the most ancient strains from which all of the more-recently isolated field strains of BoHV-1.1 evolved.

Whole-genome sequence analysis reveals unique SNP profiles to distinguish vaccine and wild-type strains of bovine herpesvirus-1 (BoHV-1).

Bovine herpesvirus-1 (BoHV-1) is a major pathogen affecting cattle worldwide causing primarily respiratory illness referred to as infectious bovine rhinotracheitis (IBR), along with reproductive disorders including abortion and infertility in cattle. While modified live vaccines (MLVs) effectively induce immune response against BoHV-1, they are implicated in disease outbreaks in cattle. Current diagnostic methods cannot distinguish between MLVs and field strains of BoHV-1. We performed whole genome sequencing of 18 BoHV-1 isolates from Pennsylvania and Minnesota along with five BoHV-1 vaccine strains using the Illumina Miseq platform. Based on nucleotide polymorphisms (SNPs) the sequences were clustered into three groups with two different vaccine groups and one distinct cluster of field isolates. Using this information, we developed a novel SNP-based PCR assay that can allow differentiation of vaccine and clinical strains and help accurately determine the incidence of BoHV-1 and the association of MLVs with clinical disease in cattle.

Herpesvírus bovino tipo 1 (BoHV-1): método de diagnóstico e sua influência na qualidade espermática em touros infectados experimentalmente / Bovine Herpes virus type 1 (BoHV-1): Diagnosis method and its influence on spermatic quality in experimentally infected bulls

O herpesvírus bovino tipo-1 (BoHV-1) é um vírus amplamente distribuído no Brasil e no mundo, havendo um crescente número de estudos envolvendo métodos de diagnóstico e o seu impacto na reprodução animal. O objetivo deste trabalho foi identificar o material genético do BoHV-1 no sêmen de touros infectados experimentalmente por meio da técnica de PCR e avaliar a influência do vírus sobre a qualidade espermática desses animais. A técnica de PCR foi satisfatória, permitindo identificar a presença do material genético do vírus no sêmen de todos os animais a partir de sete dias pós-infecção, com persistência de 21 até 28 dias. Apesar da presença do vírus BoHV-1 por um longo período no sêmen dos animais experimentais, não foram observados efeitos deletérios na qualidade do sêmen fresco e nem após a criopreservação.(AU)

Bovine Herpesvirus type-1 (BoHV-1) is a virus widely distributed in Brazil and worldwide, with a growing number of studies involving diagnostic methods and their impact on animal reproduction. The objective of this work was to identify the genetic material of BoHV-1 in the semen of experimentally infected bulls through the PCR technique, and to evaluate the influence of the virus on the sperm quality of these animals. The PCR technique was satisfactory, allowing for the identification of the presence of the genetic material of the virus in the semen of all the animals from 7 days post infection, with persistence of 21 to 28 days. Despite the presence of the BoHV-1 virus over a long period in the semen of the experimental animals, no deleterious effects were observed on the quality of either fresh semen or semen after the cryopreservation.(AU)

First report of bovine herpesvirus 1 isolation from bull semen samples in China.

Bovine herpesvirus 1 (BoHV-1) infection causes substantial economic losses to the cattle industry worldwide. So far, the isolation of BoHV-1 field virus has not been reported in China. Here, for the first time we report that two isolates of BoHV-1 designated as NJ16-1 and NJ16-2 were obtained from semen samples from breeding bulls in China. Typical cytopathic effect in MDBK cells, detection of viral protein VP16 in western blot analysis, PCR detection of BoHV-1 gB gene proved BoHV-1 infection and subsequent nucleotide sequence analysis showed a 99% identity with BoHV-1 Cooper strain. These results suggest that these isolated viruses are BoHV-1.

Phylogenetic and recombination analysis of the herpesvirus genus varicellovirus.

BACKGROUND: The varicelloviruses comprise a genus within the alphaherpesvirus subfamily, and infect both humans and other mammals. Recently, next-generation sequencing has been used to generate genomic sequences of several members of the Varicellovirus genus. Here, currently available varicellovirus genomic sequences were used for phylogenetic, recombination, and genetic distance analysis. RESULTS: A phylogenetic network including genomic sequences of individual species, was generated and suggested a potential restriction between the ungulate and non-ungulate viruses. Intraspecies genetic distances were higher in the ungulate viruses (pseudorabies virus (SuHV-1) 1.65%, bovine herpes virus type 1 (BHV-1) 0.81%, equine herpes virus type 1 (EHV-1) 0.79%, equine herpes virus type 4 (EHV-4) 0.16%) than non-ungulate viruses (feline herpes virus type 1 (FHV-1) 0.0089%, canine herpes virus type 1 (CHV-1) 0.005%, varicella-zoster virus (VZV) 0.136%). The G + C content of the ungulate viruses was also higher (SuHV-1 73.6%, BHV-1 72.6%, EHV-1 56.6%, EHV-4 50.5%) compared to the non-ungulate viruses (FHV-1 45.8%, CHV-1 31.6%, VZV 45.8%), which suggests a possible link between G + C content and intraspecies genetic diversity. Varicellovirus clade nomenclature is variable across different species, and we propose a standardization based on genomic genetic distance. A recent study reported no recombination between sequenced FHV-1 strains, however in the present study, both splitstree, bootscan, and PHI analysis indicated recombination. We also found that the recently sequenced Brazilian CHV-1 strain BTU-1 may contain a genetic signal in the UL50 gene from an unknown varicellovirus. CONCLUSION: Together, the data contribute to a greater understanding of varicellovirus genomics, and we also suggest a new clade nomenclature scheme based on genetic distances.

Evidence of natural interspecific recombinant viruses between bovine alphaherpesviruses 1 and 5.

Closely related bovine alphaherpesviruses 1 (BoHV-1) and 5 (BoHV-5) co-circulate in certain countries, rendering cattle co-infection possible. This is a prerequisite for BoHV recombination. Here, we report the first identification of homologous recombination between field isolates of BoHV-1 and BoHV-5, two alphaherpesviruses belonging to two distinct species with an average genomic similarity of 82.3%. Three isolates of BoHV-5, previously classified as subtype "BoHV-5b", were phylogenetically studied and analyzed via eight PCR sequencing assays dispersed at regular intervals throughout the genome to discriminate between BoHV-1 and BoHV-5. In the phylogenetic analysis, differences of clustering were found in the UL27 gene which encodes the glycoprotein B (gB). We detected two recombination breakpoints in the open reading frame of the UL27 gene. We compared the amino acid sequences of the gB of BoHV-1.1 and 1.2, BoHV-5a and recombinant formerly named BoHV-5b (chimeric gB) and subsequently performed molecular modeling. All structures were alike and, simultaneously, similar to the chimeric gB. Neutralizing antibodies against BoHV-1, BoHV-5 and recombinant viruses were analyzed via serum virus neutralization test using polyclonal sera and a monoclonal antibody against gB to demonstrate an absence of viral escape for both assays. Our results show that homologous recombination between two related species of ruminant alphaherpesviruses can occur in natural field conditions. We found three recombinant field isolates, previously classified as BoHV-5b subtypes, between BoHV-1 and BoHV-5.

EvaGreen-based Multiplex Real-time PCR Assay for Rapid Differentiation of Wild-Type and Glycoprotein E-Deleted Bovine Herpesvirus-1 Strains.

Bovine herpesvirus-1 (BoHV-1) is an important viral pathogen causing significant economic losses to the cattle industry. Glycoprotein E-deleted marker vaccines form the basis for BoHV-1 control programs widely, wherein detection and differentiation of wild-type and gE-deleted vaccine strains is of crucial importance for proper disease management. In the present study, we report an EvaGreen-based multiplex real-time polymerase chain reaction (EGRT-PCR) assay for rapid differentiation of wild-type and glycoprotein E-deleted strains of BoHV-1. The EGRT-PCR assay could simultaneously detect two viral genes (glycoprotein B and E) and an internal positive control gene (bovine growth hormone- bGH), in a single-tube reaction. The analytical sensitivity of the EGRT-PCR assay was as little as 10 copies of the BoHV-1 DNA per reaction. The modified real-time PCR assay could successfully differentiate wild-type and gE-deleted BoHV-1 strains based on gene specific melting temperatures (Tm) peaks. Our results have shown that the EGRT-PCR developed in this study might prove to be a promising tool in disease management by enabling rapid differentiation of wild-type and gE-deleted strains of BoHV-1.

Complete genomic sequence and comparative analysis of four genital and respiratory isolates of bovine herpesvirus subtype 1.2b (BoHV-1.2b), including the prototype virus strain K22.

Bovine herpesvirus subtype 1.2b (BoHV-1.2b) is associated primarily with bovine infectious pustular vulvovaginitis. We report here the complete genomic sequence of four BoHV-1.2b isolates. The DNA sequence identity of the four genomes is 98.9 %. Differences were primarily in regions containing direct repeats, specifically gene UL36 and the terminal repeat regions immediately flanking gene BICP22. BoHV-1.2b and BoHV-1.1 genomes are similar in size (~135 kb), completely orthologous with respect to regional structure and gene location, and have a 97.5 % DNA sequence homology. The most notable difference is the structure of the DNA replication origin of the two viruses.

Bovine herpesvirus-1: Genetic diversity of field strains from cattle with respiratory disease, genital, fetal disease and systemic neonatal disease and their relationship to vaccine strains.

Bovine herpesvirus-1 (BoHV-1) causes disease in cattle with varied clinical forms. In the U.S. there are two BoHV1 subtypes, BoHV-1.1 and BoHV-1.2b. Control programs in North America incorporate modified live (MLV) or killed (KV) viral vaccines. However, BoHV-1 strains continue to be isolated from diseased animals or fetuses after vaccination. It is possible to differentiate BoHV-1 wild-type from MLV vaccine strains by determining their single nucleotide polymorphism (SNP) patterns through either whole-genome sequencing or PCR sequencing of genomic regions containing vaccine-defining SNPs. To determine the BoHV-1 subtype in clinical isolates and their relationship to MLV strains, 8 isolates from varied clinical disease at three different laboratories in the U.S. were sequenced and phylogenetically analyzed. Five samples were isolated within the past 5 years from New York and 3 were archived samples recovered 35 years prior from Oklahoma and Louisiana. Based on phylogenetic analysis, four of the cases appeared to be due to an MLV vaccine: 3 cases of aborted fetuses and one neonate with systemic BoHV-1 disease. One aborted fetus was from a herd with no reported history of MLV vaccination in two years. The remaining four isolates did not group with any MLV vaccines: two were associated with bovine respiratory disease, one with vulvovaginitis, and a fourth was determined to be a BoHV-1.2b respiratory isolate. Recovery of BoHV-1.1 that is very closely related to an MLV vaccine virus from a herd not receiving vaccines in an extended period prior to its isolation suggests that MLV viruses may remain latent or circulate within herds for long periods.

Prevalence of antibodies against Bubaline herpesvirus (BuHV-1) among Mediterranean water buffalo (Bubalus bubalis) with implications in buffalo trade.

BACKGROUND: Both Bovine herpesvirus (BoHV-1) and Bubaline herpesvirus (BuHV-1) have been reported to cross the species barrier. Antibody seroconversion in glycoprotein E (gE) blocking ELISA during BuHV-1 infection has been documented. Recent diagnostic efforts have focused on the development and application of discriminatory tests to distinguish between infections with BoHV-1 and BuHV-1. OBJECTIVE: To evaluate the impact and distribution of these two infections in water buffalo farms in two regions (Piedmont (n = 3) and Campania (n = 10), Italy) where infectious bovine rhinotracheitis control programs have been implemented. ANIMALS AND METHODS: Sampling was carried out on 13 buffalo farms comprising 1089 animals using specific gE-indirect ELISA's test able to discriminate among BoHV-1 and BuHV-1 infections. RESULTS: 59.0% of animals reacted positive to ELISA (irrespective of whether BoHV-1 or BuHV-1 antigen was used) and 86.4% of these were reactive to BuHV-1 only, whereas 11.8% showed absorbance values for both antigens and were classified as inconclusive. There was a statistically significant age-related difference in BuHV-1 infection rates but not in overall individual (47% vs. 58%) or herd prevalence (100% vs. 90%) of infection between the two regions. CONCLUSION: The low percentage of sera reactive to BoHV-1 (1.8%, 12/643) indicates that BuHV-1 may be the main circulating alphaherpesvirus infection in Mediterranean water buffalo in the two study areas. Since Bubalus bubalis is included in Directive 64/432/EEC on animal health problems affecting intra-community trade in bovine animals, diagnostic testing with nonspecific ELISA for BoHV-1 infection in buffalo may yield false-positive reactions. This scenario could lead to economic losses and hamper buffalo trade and movement, particularly for reproduction purposes.

Alcelaphine Herpesvirus-1 (Malignant Catarrhal Fever Virus) in Wildebeest Placenta: Genetic Variation of ORF50 and A9.5 Alleles.

Alcelaphine herpesvirus-1 (AlHV-1), a causative agent of malignant catarrhal fever in cattle, was detected in wildebeest (Connochaetes taurinus) placenta tissue for the first time. Although viral load was low, the finding of viral DNA in over 50% of 94 samples tested lends support to the possibility that placental tissue could play a role in disease transmission and that wildebeest calves are infected in utero. Two viral loci were sequenced to examine variation among virus samples obtained from wildebeest and cattle: the ORF50 gene, encoding the lytic cycle transactivator protein, and the A9.5 gene, encoding a novel polymorphic viral glycoprotein. ORF50 was well conserved with six newly discovered alleles differing at only one or two base positions. In contrast, while only three new A9.5 alleles were discovered, these differed by up to 13% at the nucleotide level and up to 20% at the amino acid level. Structural homology searching performed with the additional A9.5 sequences determined in this study adds power to recent analysis identifying the four-helix bundle cytokine interleukin-4 (IL4) as the major homologue. The majority of MCF virus samples obtained from Tanzanian cattle and wildebeest encoded A9.5 polypeptides identical to the previously characterized A9.5 allele present in the laboratory maintained AlHV-1 C500 strain. This supports the view that AlHV-1 C500 is suitable for the development of a vaccine for wildebeest-associated MCF.

Differentiation of BHV-1 isolates from vaccine virus by high-resolution melting analysis.

An efficacious bovine herpesvirus type-1 (BHV-1) vaccine has been used for many years. However, in the past few years, abortion and respiratory diseases have occurred after administration of the modified live vaccine. To investigate whether BHV-1 isolates from disease outbreaks are identical to those of the vaccines used, selected regions of the BHV-1 genome were investigated by high-resolution melting (HRM) analysis and PCR-DNA sequencing. When a target region within the thymidine kinase (TK) gene was examined by HRM analysis, 6 out of the 11 isolates from abortion cases and 22 out of the 25 isolates from bovine respiratory disease (BRD) cases had different melting curves compared to the vaccine virus. Surprisingly, when a conserved region within the US6 gene that encodes glycoprotein D (gD) was examined by HRM analysis, 5 out of the 11 abortion isolates and 18 out of the 23 BRD isolates had different melting curves from the vaccine virus. To determine whether SNPs within the coding regions of glycoprotein E (gE) and TK genes can be used to differentiate the isolates from the vaccine virus, PCR-DNA sequencing was used to examine these SNPs in all the isolates. This revealed that only 1 out of 11 of the abortion isolates and 4 out of 24 of the BRD isolates are different in the target region of gE from the vaccine virus, while 5 out of 11 abortion isolates and 4 out of 22 BRD isolates are different in the target region of TK from the vaccine virus. No DNA sequence differences were observed in glycoprotein G (gG) region between disease and vaccine isolates. Our study demonstrated that many disease isolates had genetic differences from the vaccine virus in regions examined by HRM and PCR-DNA sequencing analysis. In addition, many isolates contained more than one type of mutation and were composed of mixed variants. Our study suggests that a mixture of variants were present in isolates collected post-vaccination. HRM is a rapid diagnostic method that can be used for rapid differentiation of clinical isolates from vaccine strains.

Bovine herpesvirus-1: evaluation of genetic diversity of subtypes derived from field strains of varied clinical syndromes and their relationship to vaccine strains.

Bovine herpesvirus-1 (BoHV-1) causes significant disease in cattle. Control programs in North America incorporate vaccination with modified live viral (MLV) or killed (KV) vaccine. BoHV-1 strains are isolated from diseased animals or fetuses after vaccination. There are markers for differentiating MLV from field strains using whole-genome sequencing and analysis identifying single nucleotide polymorphisms (SNPs). Using multiple primer sets and sequencing of products permits association of BoHV-1 isolates with vaccines. To determine association between vaccine virus and strains isolated from clinical cases following vaccination, we analyzed 12 BoHV-1 isolates from animals with various clinical syndromes; 9 corresponded to BoHV-1.1 respiratory group. The remaining three corresponded to BoHV-1.2b, typically found in genital tracts of cattle. Four BoHV-1 isolates were identical to a vaccine strain; three were from post-vaccination abortion episodes with typical herpetic lesions whose dams had received MLV vaccine during pregnancy, and one from a heifer given a related MLV vaccine; Sequences of two respiratory isolates perfectly matched mutations characterizing RLB106 strain, a temperature sensitive mutant used in intranasal and parenteral vaccines. The last three respiratory strains clearly appeared related to a group of MLV vaccines. Previously the MLV vaccines were grouped into four groups based on SNPs patterns. In contrast with above-mentioned isolates that closely matched SNP patterns of their respective MLV vaccine virus, these 3 strains both lacked some and possessed a number of additional mutations compared to a group of MLV vaccine viral genome. Finding BoHV-1.2b in respiratory cases indicates focus should be given BoHV-1.2b as an emerging virus or a virus not recognized nor fully characterized in BRD.

Genetic diversity of 3' region of glycoprotein D gene of bovine herpesvirus 1 and 5.

Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) are closely related alphaherpesviruses of cattle. While BoHV-1 is mainly associated with respiratory/genital disease and rarely associated with neurological disease, BoHV-5 is the primary agent of meningoencephalitis in cattle. The envelope glycoprotein D of alphaherpesviruses (BoHV-1/gD1 and BoHV-5/gD5) is involved in the early steps of virus infection and may influence virus tropism and neuropathogenesis. This study performed a sequence analysis of the 3' region of gD gene (gD3') of BoHV-1 isolates recovered from respiratory/genital disease (n = 6 and reference strain Cooper) or from neurological disease (n = 7); and from seven typical neurological BoHV-5 isolates. After PCR amplification, nucleotide (nt) sequencing, and aminoacid (aa) sequence prediction; gD3' sequences were compared, identity levels were calculated, and selective pressure was analyzed. The phylogenetic reconstruction based on nt and aa sequences allowed for a clear differentiation of BoHV-1 (n = 14) and BoHV-5 (n = 7) clusters. The seven BoHV-1 isolates from neurological disease are grouped within the BoHV-1 branch. A consistent alignment of 346 nt revealed a high similarity within each viral species (gD1 = 98.3 % nt and aa; gD5 = 97.8 % nt and 85.8 % aa) and an expected lower similarity between gD1 and gD5 (73.7 and 64.1 %, nt and aa, respectively). The analysis of molecular evolution revealed an average negative selection at gD3'. Thus, the phylogeny and similarity levels allowed for differentiation of BoHV-1 and BoHV-5 species, but not further division in subspecies. Sequence analysis did not allow for the identification of genetic differences in gD3' potentially associated with the respective clinical/pathological phenotypes, yet revealed a lower level of gD3' conservation than previously reported.

Bovine herpesvirus-1 (BHV-1) - a re-emerging concern in livestock: a revisit to its biology, epidemiology, diagnosis, and prophylaxis.

Bovine herpesvirus-1 (BHV-1) is known to cause several diseases worldwide. It is a double-stranded DNA virus consisting of 33 structural proteins out of which 13 are associated with the envelope. Based on genomic analysis and viral peptide patterns, BHV-1 virus can be divided into several subtypes like BHV-1.1, BHV-1.2, and BHV-1.3. However, all subtypes are antigenically similar. The symptoms of the related diseases are mainly non-life-threatening but have a rather wide host range that limits animal trade. The different modes of transmission as unique feature of this virus and the tendency to cause infection in the early age with latency development in trigeminal and sacral ganglion cause huge economic losses around the world. The virus also affects endangered bovine species like mithun (Bos frontalis) and yak (Poephagus grunniens). The disease can be diagnosed by using conventional procedures (like cell culture, immune-histopathology, and enzyme-linked immunosorbent assay (ELISA)) as well as highly sensitive modern techniques (like nested PCR and southern hybridization) with the virus neutralization test regarded as gold standard. With the currently available diagnostic tests it is not possible to identify animals which have a latent BHV-1 infection. Different types of modern and conventional vaccines are available for immunoprophylaxis. Inactivated vaccines are not as efficacious as modified live virus (MLV) vaccines. Marker vaccines allow the distinction between vaccinated and naturally infected animals. In this review the present status of BHV-1 around the world will be addressed besides the current knowledge with regard to its biology, epidemiology, diagnosis, and prophylaxis.

Bovine herpesvirus-1: comparison and differentiation of vaccine and field strains based on genomic sequence variation.

Bovine herpesvirus-1 (BoHV-1) causes significant disease in cattle including respiratory, fetal diseases, and reproductive tract infections. Control programs usually include vaccination with a modified live viral (MLV) vaccine. On occasion BoHV-1 strains are isolated from diseased animals or fetuses postvaccination. Currently there are no markers for differentiating MLV strains from field strains of BoHV-1. In this study several BoHV-1 strains were sequenced using whole-genome sequencing technologies and the data analyzed to identify single nucleotide polymorphisms (SNPs). Strains sequenced included the reference BoHV-1 Cooper strain (GenBank Accession JX898220), eight commercial MLV vaccine strains, and 14 field strains from cases presented for diagnosis. Based on SNP analyses, the viruses could be classified into groups having similar SNP patterns. The eight MLV strains could be differentiated from one another although some were closely related to each other. A number of field strains isolated from animals with a history of prior vaccination had SNP patterns similar to specific MLV viruses, while other field isolates were very distinct from all vaccine strains. The results indicate that some BoHV-1 isolates from clinically ill cattle/fetuses can be associated with a prior MLV vaccination history, but more information is needed on the rate of BoHV-1 genome sequence change before irrefutable associations can be drawn.