Identification of Bovine Herpesvirus type 5 in nasal swabs of naturally infected calves by Polymerase Chain Reaction

It should be established, whether animals vaccinated intramuscularly (IM) with a live Bovine herpesvirus type 1 (BHV-1) marker vaccine become viremic and/or excrete vaccine virus with nasal discharge. Five cattle, seronegative for BHV-1, were vaccinated with an overdose of the vaccine (Bovilis IBR marker live) via the IM route. Nasal swabs and blood samples were taken at regular intervals and tested for BHV-1 in a virus infectivity assay. In addition, a polymerase chain reaction (PCR) specific for BHV-1 DNA was performed on the blood samples. BHV-1 neutralizing antibody titres were determined in the sera taken prior to the vaccination and four weeks after immunisation. AIl animals were successfully vaccinated as judged by the development of BHV-1 neutralising antibodies. However, all nasal swab samples were tested negative for vaccine virus, and all blood samples were found negative for BHV-1 vaccine virus and BHV-1 specific DNA. From these data it can be concluded that the vaccine virus was not excreted with nasal discharge after IM vaccination and that the vaccinated animals did not have a detectable viremia. Therefore, it is recommended to apply the tested BHV-1 marker live vaccine by the IM route in situations where it is undesirable that the vaccine virus is excreted.

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.

Infectious pneumoenteritis of cattle is etiologically caused by viruses of different families and species. Bovine herpesvirus type 1 — infectious rhinotracheitis virus — is the main and the most dangerous pathogen transmitted by cattle semen. At the same time, recently, according to European scientists’ data, in addition to this pathogen, other herpesviruses have been circulating in cattle groups, in particular, bovine herpesvirused of types 4 and 5. Studies have been conducted using molecular-genetic and bioinformatic methods. Based on the analysis of the genomes of bovine herpesvirus of types 1 (IBR virus), 4 and 5 we developed primers BoHV-1 F/R, which flanks the DNA fragment of the IBR virus with the length of 204 bp, BoHV-4 F/R, which flanks the DNA fragment of bovine herpesvirus type 4 with the length of 615 bp, and BoHV-5 F/R for bovine herpesvirus type 5 DNA amplification with the formation of specific fragments 158 bp in length. The tests demonstrated that primers specific for bovine herpesvirus of types 1, 4 and 5 can be used in multiplex amplification format and hybridized only with specific DNA matrices of bovine herpesviruses. A standard operating procedure ‘Indication of DNA of infectious bovine rhinotracheitis virus and bovine herpesviruses of types 4 and 5 by polymerase chain reaction’ has been developed

Infertility and reproductive infections are the huge problems for the diary management throughout the world. Bovine herpesviruses act an enormous role in these complicated problems. Bovine Herpesvirus Type 1 (BHV-1) is the most outstanding herpesvirus causing genital and uterine tracts infections among other reproductive viral agents, however, Bovine Herpesvirus Type 4 (BHV-4) is also responsible in terms of similar symptoms and diseases. The main aims of the study are both to investigate the underlying potential presence of BHV-4 in subclinical uterine tract infection, and both to perform molecular and recombination analyses. A herd including 25 repeat breeder cows were investigated by BHV-4. Two out of them were determined BHV-4 infected after a series of Polymerase Chain Reactions (PCRs) tests which able to amplify partial Glycoprotein B (gB) and Thymidine Kinase (TK) gene regions. Obtained sequences were analyzed by using phylogenetic and recombinational software, and two Maximum Likelihood (ML) tree have been constructed. To results, novel Turkish BHV-4 sequences fell into Genotype I in both constructed Maximum Likelihood (ML) phylogenetic trees, however, no recombination evidence has been observed in relevant software. This report is the first genotyping study of BHV-4 from Turkey. This study showed that Turkish BHV-4 substantially originated from European strains and might be observed in different clinical presentations. This suggests that BHV-4 should be deeply investigated by further molecular techniques and included in diagnostic panels for reproductive diseases viruses.

Studies on the pathogenicity of bovine herpesvirus type 5 in sheep

AIM: To detect the presence of bovine herpesvirus (BoHV) type 4 in New Zealand dairy cows with clinical metritis.METHODS: Serum samples taken from 92 dairy cows with clinical metritis, each from a different farm, were tested for the presence of antibodies against BoHV-4 using a commercially available, indirect ELISA. Peripheral blood mononuclear cells (PBMC) were collected from 10 BoHV-4 seropositive cows, and PBMC were examined by a pan-herpesvirus nested PCR to detect herpesvirus. PCR products were sequenced directly and a proportion of the PCR products were cloned and sequenced to identify the virus present.RESULTS: Antibodies to BoHV-4 were detected in 23/92 (25%) serum samples. The pan-herpesvirus PCR was positive in 8/10 PBMC samples. Cloning and sequencing identified that all of the eight PCR-positive PBMC contained bovine lymphotropic herpesvirus (BLHV); no BoHV-4 DNA was detected.CONCLUSIONS: This study reports the finding of the presence of apparent antibodies to BoHV-4, and BLHV DNA in New Zealand dairy cows affected by metritis.CLINICAL RELEVANCE: Bovine herpesvirus type 4 and BLHV are reported to have the potential to cause reproduction failure in cows. This is the first report of apparent BoHV-4 antibodies, and BLHV in New Zealand. The importance and epidemiology of these viruses in cattle in New Zealand requires further investigation.

Background: Bovine herpes virus type 1 (BoHV-1) the causative agent of Infectious bovine rhinotracheitis (IBR) is of great concern to dairy farmers and veterinarians due to great economic impact caused by the virus in terms of loss of production and abortion. Therefore, the study was planned to detect the virus circulating in the bovine population of the region under study. This virus is an important pathogen of bovine respiratory diseases. The aim of the present study was to isolate the BoHV-1 virus from the upper respiratory tract of bovines. Methods: A total of 13 nasal swab samples were subjected to virus isolation in Madin Darby Bovine Kidney (MDBK) cells lines. A PCR assay was applied to confirm the BoHV-1 DNA by targeting gI glycoprotein gene in isolates. Result: Total two IBR virus isolates were recovered from 13 nasal swab samples of bovines. Both isolates exhibited cytopathic effects i.e. clumping and rounding of cells. A 468 base pair of amplified product from both isolates confirmed the IBR virus in gI gene specific PCR for BoHV-1. This study concludes that IBR virus exists among cattle population of Punjab and it is present in the upper respiratory tract of infected animal and shed through respiratory route. The PCR detection assay for detection of BoHV-1 from nasal swab samples is considerably more sensitive than virus isolation.

AIM: To genotype bovine herpesvirus type 1 (BHV-1) isolates from cattle in New Zealand. METHODS: Twenty-eight BHV-1 isolates were collected from clinical samples from cattle over 28 years. They were characterised and compared using restriction endonuclease analysis (REA), and polymerase chain reaction (PCR) and DNA sequencing. RESULTS: Twenty-four isolates were classified as bovine herpesvirus subtype 1.2b (BHV-1.2b) by REA. The remaining four isolates were distinct from the others in REA profiles of one of the major enzymes (HindIII) by which the classification was made. However, these four isolates were closely related to others when the REA profiles of other restriction enzymes were studied, and therefore were regarded as divergent strains of BHV-1.2b. All BHV-1 isolates were detectable by PCR, and sequence analysis of selected PCR products did not indicate any significant differences between isolates. CONCLUSION: BHV-1.2b appears to be the predominant strain of BHV-1 in cattle in New Zealand. There was no evidence that more virulent strains of BHV-1, e.g. subtype 1.1 and BHV type 5, are, or have been, present in New Zealand. Genetic variations exist among these BHV-1.2b isolates.

Infectious bovine rhinotracheitis virus (IBRV) is a major pathogen in livestock animals and had led to significant economic losses to the industrial production worldwide. However, IBR symptoms are not life-threatening. The present study was achieved to make seroprevalence and isolation of bovine herpes virus type (BHV-1) from nasal and ocular swabs samples of suspected cows and buffaloes, identification by serological and polymerase chain reaction (PCR). Total of 380 blood samples were collected from suspected 287 cattle and 93 buffaloes in different districts at Beni-suef and El-Fayoum governorates in winter 2017. Nasal and ocular discharge swabs were collected from cattle and buffaloes with clinical respiratory signs (Nasal discharge, cough, lachrymal discharge with or without mild diarrhea and higher body temperature). A total of 106 (27.89%) samples were positive by indirect ELISA and the positivity of 80 (27.87%) samples from cattle and 26 (27.96%) samples from buffaloes located at different centers at Beni-Suif and El-Fayoum governorates. Virus was isolated from nasal and ocular discharges swabs samples and it was adapted in chorioallantoic membrane (CAM) of 11-day-old embryonated chickens eggs and in MDBK cells. Virus-infected CAM showed congestion, edematous vacuole, thickening small foci ranged from 2 to 3 mm in diameter, scattered on CAM membrane and MDBK cell line inoculated blind serial passages at 3rd passage showed characteristic of cytopathic effect (CPE). Identification of virus isolated on CAM and infected cell culture fluid gave precipitation against positive specific anti-BHV-1 immune serum by AGPT and clear blue zone by Dot ELISA, absent of pock lesion in pock reduction test (PRT), and confirmed by PCR with product size of 175 bp. Finally BHV-1 virus was isolated from nasal and ocular discharges of cattle and buffaloes but the further extensive study still need for clear final classification by phylogenic analysis.

OBJECTIVE To evaluate efficacy and duration of immunity of the bovine herpesvirus type 1 (BHV-1) fraction of a trivalent vaccine also containing parainfluenza virus-3 and bovine respiratory syncytial virus fractions administered intranasally (IN) for protection of calves against infectious bovine rhinotracheitis (IBR). DESIGN Controlled challenge study. ANIMALS 120 dairy calves (3 to 8 days old) seronegative for antibody against BHV-1 (experiments 1 and 2) or seropositive for maternally derived antibody against BHV-1 (experiment 3). PROCEDURES In 3 separate experiments, calves were vaccinated IN via 2 nostrils (experiment 1) or 1 nostril (experiments 2 and 3) with a vaccine containing or not containing a BHV-1 fraction. For seronegative calves, the test vaccine contained a minimum immunizing dose of BHV-1; for seropositive calves, it contained a commercial dose of BHV-1. Calves were challenged IN with virulent BHV-1 on day 28 or 193 (seronegative calves) or day 105 (seropositive calves) after vaccination to evaluate vaccine efficacy. Frequency and duration of clinical signs, rectal temperatures, virus shedding, and serologic responses were compared between treatment groups within experiments. RESULTS In all experiments, BHV-1 vaccinated calves had lower frequencies or shorter durations of clinical signs of IBR than did control calves. Following viral challenge, peak rectal temperatures and degrees of virus shedding were lower and serologic responses were higher in vaccinated versus control calves. CONCLUSIONS AND CLINICAL RELEVANCE IN vaccination against BHV-1 protected all calves against clinical IBR disease, regardless of serologic status at the time of vaccination, and suppressed virus shedding. A single dose of this IN vaccine has the potential to protect seronegative calves for at least 193 days and override maternally derived antibody to protect seropositive calves for at least 105 days.

Polymerase chain reaction (PCR) was used to test six different nonbovine ruminant species for five bovine herpesviruses including infectious bovine rhinotracheitis virus (BoHV-1), bovine herpes mammillitis virus (BoHV-2), Movar-type herpesvirus (BoHV-4), bovine herpesvirus type 5 (BoHV-5), and alcelaphine herpesvirus 1 (AlHV-1). Species tested included 56 roe deer (Capreolus capreolus), 66 red deer (Cervus elaphus), 20 fallow deer (Dama dama), 16 mouflon (Ovis musimon), 34 domestic sheep, and 44 domestic goats, which were sampled in Hungary in 2003. Tracheal and popliteal lymph nodes collected from these animals were tested for the presence of the five bovine herpesviruses using three nested (two of which were duplex) PCR assays. Three bovine herpesviruses (BoHV-1, -2, and -4) were detected, whereas no evidence of AlHV-1 or BoHV-5 was observed. Prevalence of BoHV-1 ranged from 12% to 47%, and PCR-positive results were observed in all species tested. BoHV-2 was detected from roe deer, red deer, fallow deer, mouflon, and domestic sheep, and the prevalence in these species ranged from 3% to 50%. BoHV-4 was detected in all species, with prevalence ranging from 12% to 69%. Sequenced PCR products were 99-100% identical to bovine herpesviral sequences deposited in the GenBank.

In a screening of Brazilian plants, extracts from 27 species were assayed in vitro for antiviral activity against bovine and suid herpesviruses type 1. The plants considered promising as source of antiviral substances were those that presented viral inhibition index equal or more than 1.5 meaning a difference of viral titers between treated and untreated infected cells. Out of the 27 plants tested, extracts of Bumelia sertorum, Coffea arabica, Endopleura uchi, Leandra purpurescens, Psidium cattleianum and Uncaria tomentosa showed antiviral activity for both viruses. Extracts of Prunus myrtifolia and Symphyopappus compressus were active only against bovine herpesvirus while those of Bauhinia blakeana, Origanum vulgare, Ricinus communis and Tibouchina mutabilis inhibited only suid herpesvirus. Most of these plants are part of Brazilian folk medicine warranting the ethnopharmacology as an efficient strategy for selecting the plants for antiviral studies. Plants that presented activity against both animal herpesviruses are promising for further studies as antiviral components source. Key words: Brazilian plants, cytotoxicity, suid and bovine herpesviruses type 1, antiviral activity.

Bovine herpesvirus type 5 (BoHV-5) and bovine herpesvirus 1 (BoHV-1) are two closely related viruses. However, BoHV-5 is responsible for fatal meningitis in calves, while BoHV-1 is associated with infectious rhinotracheitis in cattle, and the mechanism by which the two viruses cause different symptoms is not well understood. In this study, we identified 11 microRNA (miRNA) genes, encoded by the BoHV-5 genome, that were processed into 16 detectable mature miRNAs in productive infection as determined by deep sequencing. We found that 6 out of 16 miRNA genes were present as two copies in the internal repeat and terminal repeat regions, resulting in a total of 17 miRNA-encoding loci distributed in both DNA strands. Surprisingly, BoHV-5 shared only one conservative miRNA with BoHV-1, which was located upstream of the origin of replication. Furthermore, in contrast to BoHV-1, no miRNAs were detected in the unique short region and locus within or near the bovine infected-cell protein 0 and latency-related genes. Variations in both the 5' and 3' ends of the reference sequence were observed, resulting in more than one isoform for each miRNA. All of the 16 miRNAs were detectable by stem-loop reverse transcriptase-PCR. The miRNAs with high read numbers were subjected to Northern blot analysis, and all pre-miRNAs and one mature miRNA were detected. Collectively, the data suggest that BoHV-5 encodes a different pattern of miRNAs, which may regulate the life cycle of BoHV-5 and might account for the different pathogenesis of this virus compared with BoHV-1.

Common epitopes of glycoprotein B map within the major DNA-binding proteins of bovine herpesvirus type 2 (BHV-2) and herpes simplex virus type 1 (HSV-1)

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.