Isolation and molecular characterization of bovine herpesvirus 4 from cattle in mainland China.

Bovine herpesvirus 4 (BoHV-4) is one of the most important of the known viral respiratory and reproductive pathogens of both young and adult cattle. However, BoHV-4 has not been isolated or detected in mainland China prior to this study. In 2019, BoHV-4 strain 512 was isolated from cattle in Heilongjiang Province, China, using MDBK cells, and characterized by PCR, nucleotide sequence analysis, and transmission electron microscopy. Two other unknown herpesvirus strains, BL6010 and J4034, which were isolated from cattle in 2009 in China and stored at -70℃, were also propagated in MDBK cells and identified as BoHV-4 by PCR. Phylogenetic analysis based on partial nucleotide sequences of the thymidine kinase (TK) gene and glycoprotein B (gB) gene for the three isolates indicated that these three Chinese strains belong to BoHV-4 genotype 1. A preliminary virus neutralization test revealed that 64% of the 70 bovine sera (45/70) collected from Inner Mongolia Autonomous Region, China, had anti-BoHV-4 antibodies and that natural BoHV-4 infection occurred in cattle in China. Here, we report for the first time the isolation and molecular characterization of BoHV-4 from cattle in mainland China.

Analysis of the anti-apoptotic v-Bcl2 and v-Flip genes and effect on in vitro programmed cell death of Argentinean isolates of bovine gammaherpesvirus 4 (BoHV-4).

Some viruses encode inhibitory factors of apoptosis during infection to prolong cell viability and then to achieve a higher production of viral progeny or facilitate persistent infections. There is evidence that some gammaherpesviruses, including BoHV-4, carry genes that can both inhibit or induce apoptosis. BoHV-4 possesses two genes (ORF16 and ORF71) that code for proteins with anti-apoptotic functions, such as v-Bcl2 and v-Flip, respectively. Thus, it is relevant to study BoHV-4 in relation to the modulation of apoptosis in infected cells as a strategy for persistence in the host. The objective of this work was to analyze whether variations in v-Flip and v- Bcl2 of six phylogenetically divergent Argentinean isolates of BoHV-4 can influence the capacity of these strains to induce apoptosis in cell cultures. In this study, variations were mainly detected in the v-Flip gene and protein of the BoHV-4 strains belonging to genotype 3. Thus, it is possible to infer that sequence variations could be associated with some BoHV-4 genotype. Induction of apoptosis was not a significant event for any of the genetically distinct local isolates of BoHV-4 and there was not an evident relationship between the variability of both genes with the apoptotic effect of the phylogenetically distinct strains.

Characterization of the first bovine gammaherpesvirus 4 strain isolated from an aborted bovine fetus in Argentina.

Bovine herpesvirus 4 (BoHV-4) is increasingly believed to be responsible for several disorders of the bovine reproductive tract. The first characterization of BoHV-4 in Argentina was from samples from an aborted fetus. Argentinean isolates are highly diverse and are phylogenetically grouped in three genotypes. In this study, we describe the isolation of BoHV-4 from a bovine fetus with a gestational age of 8 months and without macroscopic lesions. Genetic analyses revealed that the isolated strain belongs to genotype 2. This is the first report on the presence of infectious BoHV-4 in tissues from an aborted bovine fetus.

Genetic characterization of bovine herpesvirus 4 (BoHV-4) isolates from Argentine cattle suggests a complex evolutionary scenario.

Bovine herpevsirus 4 (BoHV-4) is a gammaherpesvirus that has been associated with different clinical conditions in cattle. In Argentina, BoHV-4 was detected in diverse bovine samples. The aim of this study was to analyze the genetic relationship of 48 field BoHV-4 strains isolated from cattle in Argentina. According to thymidine kinase (tk) gene sequences, BoHV-4 isolates belong to genotypes 1, 2 and 3. Phylogenetic analyses confirmed the presence of the three previously described viral genotypes. However, some of the studied isolates presented conflicting phylogenetic signals between the studied markers. This suggests a complex evolutionary background, that is a history of recombination, incomplete lineage sorting (deep coalescence) or a combination of these, which requires further study. These potential events make difficult the diagnosis of BoHV-4 from clinical samples of cattle and may pose a significant problem for the control of the virus in the herds.

Bovine herpes virus type-4 infection among postpartum dairy cows in California: risk factors and phylogenetic analysis.

The aims of this study were to determine the prevalence and associated risk factors of bovine herpes virus type-4 (BoHV-4) infection and describe the genetic characteristics and predominant genotypes of the virus in Yolo and Tulare counties, California. A cross-sectional study involving multi-stage sampling technique was used. One hundred and forty-eight post-partum cows were enrolled from 11 dairy farms. Uterine/vaginal samples were collected and tested for BoHV-4 and other co-infecting viruses using real-time PCR. Data were analysed using multilevel logistic mixed-effect model. Phylogenetic analysis of 10 BoHV-4 isolates was conducted by targeting three open reading frames (ORF3, ORF8 and ORF 22) of the viral genome. The prevalence of BoHV-4 infection was 22.3% (33/148), while post-partum metritis was 33.8% (48/142). Strong association was found between BoHV-4 infection and lactation number, lactation stage and post-partum metritis. The odds of being positive for BoHV-4 infection were 6.47 times (95% CI 1.17-35.92; P 240 days). Cows with post-partum metritis were 4.51 times (95% CI 1.27-16.02; P < 0.05) more likely to test positive for BoHV-4 infection compared with those without post-partum metritis. Phylogenetic analysis of BoHV-4 based on sequencing of glycoprotein and thymidine kinase (TK) genes revealed genetic variability of the virus with glycoprotein B genotype 1 and TK genotype 2 as being dominant genotypes. The reported high genetic variability of BoHV-4 indicates the possibility of co-infection with multiple genotypes.

Uterine infection with bovine herpesvirus type 4 in dairy cows.

Diseases of the reproductive tract are a frequent problem in dairy herds. Herpesviruses are uterine pathogens also involved in other clinical diseases; for example, bovine herpesvirus type 4 BoHV-4 induces abortion, enteritis, metritis, pneumonia and vaginitis, but it can also be detected in healthy cows. The role of BoHV-4 in the development of clinical endometritis (CE) or subclinical endometritis (SE) has not clearly been described. Therefore, the objective of this study was to describe the prevalence of uterine BoHV-4 infection and its relationship with clinical, bacteriological and cytological findings in dairy cows 20-30 days after calving. The experiment was performed as a completely randomized block design, with farm (n = 10) as blocking criterion and with cow (n = 397) as the experimental unit. Logistic regression models were used to assess the effect of BoHV-4 infection on CE, SE and reproductive performance. Proportion of cows infected with BoHV-4 was 5.8% (n = 23/397). BoHV-4 was isolated in 11.0% (n = 12/109), 4.8% (n = 4/84) and 3.6% (n = 7/194) of cows diagnosed as CE, SE or healthy, respectively. A logistic model revealed that BoHV-4 infection showed a tendency to increase the risk for CE (AOR = 2.17; p = .10) but significantly reduced both, the odds for artificial insemination within 80 days post-partum (dpp) (AOR = 0.37; p = .035) and for pregnancy within 200 dpp (AOR = 0.13; p = .004). Furthermore, BoHV-4 infection increased the chance for intrauterine infection with Trueperella pyogenes (AOR = 5.55; p < .001) and vice versa (AOR = 5.79, p < .001). In conclusion, BoHV-4 infection is associated with reduced chances for insemination and pregnancy by 200 dpp and showed a trend to be associated with increased risk for CE. Furthermore, BoHV-4 and Trueperella pyogenes infections are strongly related.

Comparative study on the in vitro replication and genomic variability of Argentinean field isolates of bovine herpesvirus type 4 (BoHV-4).

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus, belonging to the Rhadinovirus genus, which is increasingly associated with various problems of the reproductive tract of cattle. In Argentina, analysis of BoHV-4 strains isolated from cervico-vaginal mucus of aborted cows revealed a high genetic divergence among strains, which could be classified in three different groups: Genotype 1 comprises Movar-like strains (European prototype), Genotype 2 includes DN599-like strains (American prototype) and Genotype 3 corresponds to a novel genotype group. Understanding the replication behavior in cell cultures and the molecular characteristics of this pathogen of cattle is critical for the rational design of in vitro experiments. The aim of this work was to quantitatively evaluate the replication properties of different Argentinean BoHV-4 strains and to characterize their phylogenetic relationships. Significant differences were evident among the virus titers of the different BoHV-4 isolates in vitro. The most conserved gene was the major capsid protein (ORF25). The glycoprotein B (gB), glycoprotein H (gH), and thymidine kinsase (TK) genes displayed both synonymous and non-synonymous substitutions, with the highest diversity observed for gB, which displayed amino acid substitutions in 24 out of the 178 positions examined. Strains 09/759, 12/512, and 07/568 presented a deletion encompassing amino acid position 27 to 35, whereas strains 07/435 and 09/227 had a deletion from position 28 to 35. Two strains, 07/435 and 09/227, also displayed the highest divergence compared to the other strains analyzed. This study provides information about the in vitro replication and behavior of nine field isolates of BoHV-4. These findings are relevant since available information on the in vitro growth characteristics of BoHV-4 strains is scarce. The results from this study may also be useful for establishing comparisons with other related viruses.

Prevalence of Bovine herpesvirus type 4 in aborting dairy cows.

Bovine herpesvirus type 4 (BHV-4) is related to many different conditions: infertility, postpartal metritis, vulvovaginitis, mastitis, encephalitis, calf pneumonia, keratoconjunctivitis, cutaneous lesions, digital dermatitis and abortion. In this study a retrospective PCR examination of 100 extracted DNA samples from aborting cows was performed in order to determine: prevalence of BHV-4 in abortive cattle, whether coinfections BHV-4 with other abortifacient pathogens are present in the same sample and to determine the month of gestation when BHV-4 associated abortions were detected. Out of 100 examined samples, the BHV-4 genome was detected in 21 samples (21%). In two samples we detected coinfection of BHV-4 with bovine viral diarrhea virus (BVDV) and in one with Neospora caninum. Most of the BHV-4-associated abortions were detected during the seventh month of gestation. It was concluded that an active BHV-4 infection was present among cows that aborted on the farms examined. The high prevalence of the BHV-4 genome in abortion material suggests that this virus may have cause the abortions. Further studies and examinations are needed to establish causative connection between presence of BHV-4 and abortion.

Potential Pathogenetic Role of Bovine Herpesvirus 4 in Two Dairy Cows with Dermatitis-Pyrexia-Hemorrhagic Syndrome.

Dermatitis, pyrexia, and hemorrhagic syndrome (DPHS) is a rare bovine syndrome of unclear etiology. We describe two DPHS cases, the first to occur in Italy, with clinicopathological findings suggesting a potential pathogenetic role of bovine herpesvirus-4 (BoHV-4).

[Bovine herpesvirus 4 (BoHV-4): general aspects of the biology and status in Argentina]. / Herpesvirus bovino 4 (BoHV-4): aspectos generales de su biología y situación en la República Argentina.

Bovine herpesvirus 4 (BoHV-4) has been isolated from cattle with respiratory infections, vulvovaginitis, mastitis, abortions, endometritis and from apparently healthy animals throughout the world. Although it has not yet been established as causal agent of a specific disease entity, it is primarily associated with reproductive disorders of cattle. This virus can infect a wide range of species, either in vivo or in vitro. Two groups of prototype strains were originated from the first isolates: the DN599-type strains (American group) and the Movar-type strains (European group). In Argentina, BoHV-4 was isolated and characterized in 2007 from vaginal discharge samples taken from cows that had aborted. So far, more than 40 isolates, mainly associated with aborting bovine females have been registered in our country.

Isolation and complete genomic characterization of a Movar 33/63-like Japanese bovine herpesvirus 4 from a calf with respiratory disease.

Bovine herpesvirus 4 (BoHV-4) is an indigenous virus in cattle prevalent mainly in North and South American countries and European countries, but the genomic sequences and genetic characteristics of Japanese strains have not been reported. BoHV-4 is suspected, but not proven, to be associated with various diseases. In the present study, we isolated BoHV-4 from a 10-month-old Japanese Black calf with respiratory symptoms in Japan. To identify the genetic characteristics of the isolate named strain SG20, complete genome sequencing was performed using a combination of next-generation and Sanger sequencing technologies. The complete long unique coding region (LUR) of SG20 was found to comprise 108,819 nucleotides with 41.4% GC content and contain at least 78 open reading frames. It shares 83.4 to 99.3% overall nucleotide identity with six BoHV-4 strains available in the database. The deduced amino acid sequence alignment revealed that SG20 contains genotype 1-specific features of BoHV-4, such as amino acid substitutions and insertions within the glycoprotein B region. Phylogenetic analyzes based on the nucleotide sequences of ORF20 indicated that the virus belonged to genotype 1 (Movar 33/63-like group). The strain was also analyzed using the complete LUR and placed in the same clade as a strain recently isolated from China, but it was distinct from American and European BoHV-4 strains of genotype 1. Although further genomic and epidemiologic information is needed, our results help elucidate the molecular epidemiology of BoHV-4 and provide a foundation for future studies.

Whole Genome Sequence-Based Analysis of Bovine Gammaherpesvirus 4 Isolated from Bovine Abortions.

Bovine gammaherpesvirus 4 (BoGHV4) is a member of the Gammaherspivirinae subfamily, Rhadinovirus genus. Its natural host is the bovine, and it is prevalent among the global cattle population. Although the complete genome of BoGHV4 has been successfully sequenced, the functions of most of its genes remain unknown. Currently, only six strains of BoGHV4, all belonging to Genotype 1, have been sequenced. This is the first report of the nearly complete genome of Argentinean BoGHV4 strains isolated from clinical cases of abortion, representing the first BoGHV4 Genotype 2 and 3 genomes described in the literature. Both Argentinean isolates presented the highest nt p-distance values, indicating a greater level of divergence. Overall, the considerable diversity observed in the complete genomes and open reading frames underscores the distinctiveness of both Argentinean isolates compared to the existing BoGHV4 genomes. These findings support previous studies that categorized the Argentinean BoGHV4 strains 07-435 and 10-154 as Genotypes 3 and 2, respectively. The inclusion of these sequences represents a significant expansion to the currently limited pool of BoGHV4 genomes while providing an important basis to increase the knowledge of local isolates.

Genetic variability of bovine herpesvirus type 4 (BoHV-4) field strains from Turkish cattle herds.

Bovine herpesvirus type 4 (BoHV­4) is a common virus in the world that is detected in clinically ill or in apparently healthy cattle. This study provides a molecular characterization of BoHV­4 strains from 24 cattle some showing respiratory and/or reproductive problems and some without any apparent clinical sign. This study also reported the growth properties of five BoHV­4 field isolates. The 24 sampled cattle came from 13 different herds in 10 provinces collected between 2007 and 2018. Phylogenetic analysis using partially amplified nucleotide sequences of ORF8 genes coding glycoprotein B (n = 24) and ORF3 genes coding thymidine kinase (n = 9), demonstrated genetic variability among the BoHV­4 strains analysed. The partial gB gene sequences clustered in three different genotypes (genotype I, II and III) were located within the genotype I cluster, such as Movar strain. The analysis of the five BoHV­4 strains isolated from vaginal swabs (n = 2), nasal swab (n = 1), and brain samples (n = 2) revealed no significant differences in their growth properties in MDBK cell culture.

Isolation and characterization of bovine herpesvirus 4 (BoHV-4) from a cow affected by post partum metritis and cloning of the genome as a bacterial artificial chromosome.

BACKGROUND: Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus with a Worldwide distribution in cattle and is often isolated from the uterus of animals with postpartum metritis or pelvic inflammatory disease. Virus strain adaptation to an organ, tissue or cell type is an important issue for the pathogenesis of disease. To explore the mechanistic role of viral strain variation for uterine disease, the present study aimed to develop a tool enabling precise genetic discrimination between strains of BoHV-4 and to easily manipulate the viral genome. METHODS: A strain of BoHV-4 was isolated from the uterus of a persistently infected cow and designated BoHV-4-U. The authenticity of the isolate was confirmed by RFLP-PCR and sequencing using the TK and IE2 loci as genetic marker regions for the BoHV-4 genome. The isolated genome was cloned as a Bacterial Artificial Chromosome (BAC) and manipulated through recombineering technology RESULTS: The BoHV-4-U genome was successfully cloned as a BAC, and the stability of the pBAC-BoHV-4-U clone was confirmed over twenty passages, with viral growth similar to the wild type virus. The feasibility of using BoHV-4-U for mutagenesis was demonstrated using the BAC recombineering system. CONCLUSION: The analysis of genome strain variation is a key method for investigating genes associated with disease. A resource for dissection of the interactions between BoHV-4 and host endometrial cells was generated by cloning the genome of BoHV-4 as a BAC.

Comparative analysis of replicative properties of phylogenetically divergent, Argentinean BoHV-4 strains in cell lines from different origins.

Bovine gammaherpesvirus 4 (BoHV4) is a member of the family Herpesviridae. In Argentina, BoHV4 was isolated and characterized in 2007 from samples of aborted cows. Argentinean isolates are highly divergent and are classified as: Genotype 1(Movar-like), Genotype 2 (DN599-like) and Genotype 3 (a novel group). The aim of this study was to comparatively evaluate the biological characteristics of six Argentinean BoHV4 field isolates in cell lines from different origins. All strains induced productive infection in the cell lines used, with different degrees of permissiveness. A direct relationship among the times of appearance of cytopathic effect, the growth kinetics, the size of the lysis plaques and the virulent-like behaviour in vitro could not be established. However, although slight, there are differences in the biological behaviour of the BoHV4 fields isolates analyzed. This variability is independent of their genetic classification but would be conditioned by the nature of the infected cells.

[Detection of bovine herpesvirus 4 DNA in cattle by realtime PCR.]

INTRODUCTION: BoHV-4 is poorly understood. Data on the circulation of the virus among animals and its role in infectious diseases insufficient. Aimes and goals. Development of real-time PCR for detecting the BoHV-4 and studying the frequency of its presence in samples from sick animals. MATERIAL AND METHODS: The nucleotide sequences of the glycoprotein L gene served as a target for amplification. The sequences of reference strains published in GenBank were used to analyze and design the primers. Studies were conducted in 3 regions of Western Siberia on 5 large dairy farms. RESULTS: 27.7% of samples contained the virus. The virus was present as a monoagent in nasal cavity of calves (80.0%), lungs (46.2%) and bronchial lymph nodes (38.5%) in pneumonia. In the cases of diarrhea the virus was detected in 20%, and in cows with gynecological pathology in 10.0%. In respiratory diseases of calves the virus was detected in association with BoHV-1 (21.6%) and BoCV (20.3%), and in gynecological pathology of cows with BVDV1 (6%). DISCUSSION: According to the phylogenetic analysis of 5 identified virus isolates, four belonged to the American branch and one to the European branch. The circulation of American strains occurred in the territory of the Republic of Kazakhstan (1), Tyumen (1) and Novosibirsk (2) regions, and the European - in the Novosibirsk region. CONCLUSION: The search for viruses involved to the infectious pathology, as well as studying the genetic diversity of viruses circulating on a particular farm including imported from other countries, is relevant.

Presence of gammaherpesvirus BoHV-4 in endometrial cytology samples is not associated with subclinical endometritis diagnosed at artificial insemination in dairy cows.

In the past, bovine herpesvirus 4 (BoHV-4) has been suggested to be associated with metritis and endometritis. However, not many field studies investigated the association between BoHV-4 and subclinical endometritis (SCE). In the present study, the association between the intrauterine presence of BoHV-4 and SCE diagnosed during artificial insemination (AI) was examined on two dairy farms in Belgium. An immunoperoxidase monolayer assay (IPMA) and an enzyme-linked immuno sorbent assay (ELISA) were used to screen the serum for anti-BoHV-4 antibodies. A SYBR green based one step real time qPCR was used to detect and quantify BoHV-4 (ORF20) in nasal, uterine and vaginal samples collected at AI. A reverse transcription qPCR (RT-qPCR) was used to detect mRNA (gB) as proof of a productive BoHV-4 infection. BoHV-4 was detected in 39.4% (farm A)/23.8% (farm B) of the nasal samples, 48.5% (farm A)/19.0% (farm B) of the uterine samples and 51.5% (farm A)/42.9% (farm B) of the vaginal samples. Active replication was only detected in farm A in 38.5% of the BoHV-4 positive nasal samples and in 5.9% positive cases of the vaginal samples. The prevalence of SCE diagnosed at AI was 45.5% and 42.9% in farm A and farm B, respectively. The presence of SCE was associated with a reduced pregnancy outcome at artificial insemination (AI) (P＜0.001). The occurrence of SCE at AI was not associated with the presence of latent or productive BoHV4 infections in the uterus nor in the vagina and nose (P＞0.05).

An investigation of the aetiological role of bovine herpesvirus 4 in bovine endometritis.

Uteri from 31 infertile cattle were examined for the presence of bovine herpesvirus 4 (BoHV-4) by nested polymerase chain reaction (PCR). Samples were also tested for bacteria, including chlamydiae and Mycoplasma bovis. BoHV-4 was detected by PCR in 27/31 (87.1%) samples, but the presence and amount of viral DNA was not correlated with histological and bacteriological findings. Arcanobacterium pyogenes, Histophilus somni and Pasteurella multocida were isolated from five cows with endometritis. Chlamydiae were detected in four cases (12.9%), but only two of these had endometritis. The study does not support a role for BoHV-4 as primary agent in bovine endometritis.

Bovine lymphotropic herpesvirus and non-responsive post-partum metritis in dairy herds in the UK.

Bovine lymphotropic herpesvirus (BLHV) was detected for the first time in the UK in December 2005 in a dairy herd suffering from chronic, non-responsive post-partum metritis (NPPM). A small-scale investigation was undertaken in order to determine whether this was an isolated case. Samples of vaginal exudates or vaginal swabs were collected from cows in 13 UK dairy herds with a history of post-partum metritis that had not responded to standard treatment regimes for this condition. Cows in 9/13 herds and 1/13 herds were positive for BLHV and bovine herpesvirus-4, respectively, by pan-herpesvirus polymerase chain reaction. No consistent pattern of infectious agents or nutritional/metabolic factors commonly associated with post-partum metritis was observed at the times of sampling. The detection of BLHV in association with NPPM indicates that further work is warranted to determine the impact this virus has on cattle health.

Sample-based assessment of the microbial etiology of bovine necrotic vulvovaginitis.

A semiquantitative evaluation of potential bacterial pathogens was correlated to the severity of lesions during an outbreak of bovine necrotic vulvovaginitis (BNVV) on an Israeli dairy herd. Bacteriologic examination of 287 vaginal swabs from 104 post-calving heifers showed a highly significant correlation between Porphyromonas levii colony forming unit numbers and the clinical scores of the lesions, when assessed by an ordinal regression statistical model. No such correlation was found for the other bacteria included in the study. Nineteen samples taken for virological examinations resulted negative for bovine herpes viruses 1, 2, 4 and 5. Thus the results of this study substantiate the essential role of P. levii in the etiology of BNVV and indicate that BHV4 is not required as a predisposing factor to the syndrome.