Co-infection with Bovine Herpesvirus 4 and Histophilus somni Significantly Extends the Service Period in Dairy Cattle with Purulent Vaginal Discharge.

The aim of the study was to investigate the effect of Bovine Herpesvirus 4 (BoHV-4) and Histophilus (H.) somni on fertility rate of cows in a Hungarian Holstein-Friesian dairy herd with purulent vaginal discharge (PVD). Non-pregnant cows (n = 188) with mature corpus luteum were treated with cloprostenol and 3 days later if they did not show oestrus, were examined by rectal palpation. Animals showing PVD (n = 60/31.9%/) and 14 controls with normal vaginal discharge (Score 0) were randomly selected and further examined by ultrasonography and blood samples were collected for detecting BoHV-4 DNA and transcervical guarded swabs were collected from the uterus for bacteriological examination. Although the majority of the examined animals were infected with BoHV-4 and H. somni including the control animals as well, in group of animals with PVD score 3, fewer animals became pregnant and the duration between the first treatment to pregnancy was significantly extended. Based on these clinical and comparative data, our results confirm that these two microorganisms together may impair important reproductive parameters which may cause large economic losses to dairy farms.

Detection of circovirus in free-ranging brown rats (Rattus norvegicus).

Accidentally found, two poisoned brown rats from Hungary were surveyed for presence of circoviral DNA, using specific nested primers, designed against the rep gene of the virus. Both specimens were positive. The whole genomes were amplified using inverse PCR based on the Rep sequence parts and sequenced by the primer walking method. Genomic analyses revealed that these novel rat viruses, together with tawny owl-associated circovirus reported by Italian researchers in 2022, are sequence variations of the same virus from genus Circovirus. In phylogenetic reconstructions, these circovirus strains detected from brown rats clustered closest to circoviruses derived from faeces samples of various predatory mammals. Molecular data as well as the phylogenetic analyses of the complete derived replication-associated protein and the capsid protein, as well as the prey preference of the host species of the recently described tawny owl-associated virus suggest that brown rat could be the evolutionary adapted host of the viruses described in this paper (brown rat circovirus types 1 and 2) and the previously reported tawny owl-associated virus. Possible pathogenic and zoonotic role of these viruses need further studies.

Pathologic lesions caused by coinfection of Mycoplasma gallisepticum and H3N8 low pathogenic avian influenza virus in chickens.

Chickens were infected under experimental conditions with Mycoplasma gallisepticum and low pathogenic avian influenza (LPAI) strain A/mallard/Hungary/19616/07 (H3N8). Two groups of chickens were aerosol challenged with M. gallisepticum strain 1226. Seven days later, one of these groups and one mycoplasma-free group was challenged with LPAI H3N8 virus; one group without challenge remained as negative control. Eight days later, the birds were euthanized and examined for gross pathologic and histologic lesions. The body weight was measured, and the presence of antimycoplasma and antiviral antibodies was tested before the mycoplasma challenge, before the virus challenge, and at the end of the study to confirm both infections. Chickens in the mycoplasma-infected group developed antibodies against M. gallisepticum but not against the influenza virus. Chickens of the group infected with the influenza virus became serologically positive only against the virus, while the birds in the coinfected group developed antibodies against both agents. The LPAI H3N8 virus strain did not cause decrease in body weight and clinical signs, and macroscopic pathological lesions were not present in the chickens. The M. gallisepticum infection caused respiratory signs, airsacculitis, and peritonitis characteristic of mycoplasma infection. However, the clinical signs and pathologic lesions and the reduction in weight gain were much more significant in the group challenged with both M. gallisepticum and LPAI H3N8 virus than in the group challenged with M. gallisepticum alone.

Analysing the genomes of two tick-borne encephalitis viruses isolated in Hungary in 1952 and 2019.

The genomes of two Tick-borne encephalitis virus (TBEV) strains were fully sequenced and compared to those of known Hungarian strains. One was a laboratory strain (KEM-1) isolated in 1952, which had gone through hundreds of passages both on Vero cell cultures and in laboratory mice, while the other was a recent isolate (2019) from questing female ticks. The laboratory strain formed a monophyletic group with the already published 4 Hungarian strains on the evolutionary tree, located relatively close to Finnish (Kumlinge) and Russian (Absettarov) strains. This KEM-1 strain was phylogenetically distantly related both to the geographically close reference strain Neudörfl and the chronologically close Czech isolates from 1953. The 2019 isolate, KEM-195 was related to TBEV isolates from Southern Slovakia and Styria, and had the longest (328 nucleotides) deletion in its 3'-non-coding region among published sequences of strains of European subtype. Our results show that decades of laboratory passage have not altered the viral genome too much and that at least two distinct branches of TBEV strains circulate in Hungary.

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.

Detection of bovine herpesvirus 4 in CD11b+ leukocytes of experimentally infected rabbits.

The presence and numbers of bovine herpesvirus 4 (BoHV-4) infected CD11b+ leukocytes were investigated during experimental infections of New Zealand White rabbits by Fluorescence Activated Cell Sorter (FACS) analysis. Peripheral blood leukocytes (PBL) were collected every second day, and the cells were stained with phycoerythrin-labelled CD11b-specific mouse monoclonal antibody and fluorescein-conjugated bovine herpesvirus 4-specific mouse monoclonal antibody. The numbers of double-stained cells from PBLs of the control and inoculated groups were measured and compared in FACSTREK analyser. Double-stained cells were detected in the virus-inoculated group on postinoculation days (PID) 2-5 and 9-12. The results indicated that CD11b+ PBLs were permissive for BoHV-4 infection, and are probably the main reservoir of the virus during the latent period. The data did not indicate production of infectious viral particles, but virus-specific proteins were expressed on the surface of CD11b+ cells. The two waves of double-stained cells gave similar results to the PCR assays from serum samples, which showed the presence of viral DNA in the serum on the same days when virus-infected CD11b cells were also present. Productive BoHV-4 infection of mast cells or undifferentiated leukocytes in the bone marrow and the antiviral immune response might be responsible for this periodic appearance of the virus in CD11b+ PBLs and in the serum. The paper provides evidence that CD11b+ PBLs are the main target cell populations in the blood for BoHV-4.

Histological studies of bovine herpesvirus type 4 infection in non-ruminant species.

The pathology of bovine herpesvirus type 4 (BHV-4) infection was studied in cats, rabbits and guinea pigs. Twenty kittens, twenty-two rabbits and ten guinea pigs, some treated with glucocorticoid-were inoculated with a BHV-4 strain of feline origin, via various routes of inoculation (conjunctival, intranasal, peritoneal). Clinical signs were recorded. After euthanizing at different post inoculation days macro- and microscopic changes were observed by necropsy and in hematoxylin-eosin stained histological sections. The presence of the virus in organs was detected by immunohistochemistry and a nested PCR assay. Inclusion bodies and monoclonal antibody-stained cells were found in the conjunctiva, trachea, lungs, spleen and lymph nodes. Most of the lesions were localized to the respiratory and the immune system. The macro- and microscopic lesions and clinical signs were more severe in kittens and guinea pigs. The histological data indicated that cats, especially kittens, were susceptible for BHV-4 and the infection was not confined to the urinary bladder.

Periodic reappearance of bovine herpesvirus type 4 DNA in the sera of naturally and experimentally infected rabbits and calves.

A BHV-4 specific nested PCR was used for the detection of viral DNA in serum samples of rabbits and calves. All animals were followed up for 62 days, blood samples were taken for PCR studies every second day. Maternal infection of calves resulted in the repeated regular reappearance (10-14 days) of the virus (DNA) in serum samples. When PCR positive five-day-old calves were infected with tissue culture adapted virus, the reappearance of the DNA in the serum was shown to be irregular, nevertheless, DNA peaks reappeared during the whole observation period. A PCR negative calf infected at the age of 60 days was found to possess viraemia until p.i.d. 32. In rabbits treated intravenously with BHV-4 the inoculum or a primary viraemia was detected at p.i.d. 2-3 and p.i.d. 14-16. Published data on human herpesviruses suggest, that the target cells might be a pluripotent stem cell population of the bone marrow and differentiated virus-infected cells destroyed by the immune system might be the source of viral DNA detected in the serum. Frequency of DNA reappearance was depended on the age of the infected animals but not on the inoculated amount of BHV-4. The described phenomenon might be part of BHV-4 infection of very young animals.

An experimental study of a concurrent primary infection with bovine respiratory syncytial virus (BRSV) and bovine viral diarrhoea virus (BVDV) in calves.

Experimental infections with bovine respiratory syncytial virus (BRSV) and bovine viral diarrhoea virus (BVDV) were performed to study the effect of concurrent BRSV and BVDV infections. Twelve seronegative calves, in 3 groups, were inoculated on a single occasion with pure BRSV (group A), BRSV and noncytopathogenic BVDV (group B) or mock infected (group C). Mild respiratory symptoms were recorded 4 to 5 days post inoculation (dpi) in group A and group B calves. One calf in group A was severely affected and required medical treatment. In group B, fever (40.7-41.4 degrees C) was prominent 7 to 8 dpi. Only calves in group B were BVDV positive in purified lymphocytes at 2 to 14 dpi and showed increased serum interferon levels, with a peak at 4 dpi, indicating BVDV to be responsible for inducing the rise. BRSV was detected in lung lavage fluids up to 7 dpi for group A calves, compared to 11 dpi for group B and calves in this group also seroconverted later displaying lower BRSV titers. The time lag before an antibody response and the titers recorded in group B, indicated that the duration of BVDV infection in lymphocytes negatively influenced the capacity to mount a BRSV antibody response.

PCR studies on the potential sites for latency of BHV-4 in calves.

The aim of the study was to examine various tissues of experimentally infected calves for the BHV-4 genome so as to detect in which cells the virus persists during the latent phase of the infection. The presence of the bovine herpesvirus type 4 genome was detected by a nested PCR in a variety of tissues collected from two susceptible calves experimentally infected 62 days earlier. Mild clinical signs of bronchitis, an elevated body temperature for 2-3 days, and a slightly increased number of blood leukocytes were observed in both inoculated calves. BHV-4 was demonstrated in seven samples from the 12 different parts of the nervous system tested from each calf (29.1%), from the cornea, from lymph nodes near to the inoculation site, from the gallbladder and from the bone marrow. Thus a member of the predominantly lymphotropic Gammaherpesvirinae subfamily was detected in neural tissue and other organs that have never been associated with persistence.

Genomic and pathogenic studies on a glycoprotein E variant field isolate of bovine herpesvirus 1.

Glycoprotein E-negative (gE-) laboratory strains of bovine herpesvirus 1 (BHV-1) were recently introduced as novel marker vaccines, allowing serological discrimination between vaccinated and naturally infected animals on the basis of lack or presence of antibodies against gE epitopes. The applicability pf this approach is based on the genetic stability of the gE. However, mutant field variants of BHV-1 with a variable response in anti-gE ELISA have been isolated. The molecular characterization of a gE variant field isolate (Salwa strain) is presented here. By comparing the gE nucleotide and amino acid sequences of the Salwa strain with those of the wild strain Jura, ten mutated bases were found in the gE strain of Salwa, six of which alter the amino acid sequence, leading to changes in five amino acids. Both strains caused respiratory disease in experimentally infected calves, but Salwa generated slightly milder signs. Both viruses were excreted in nasal and ocular discharges, and were reactivated by dexamethasone treatment. In conclusion, the rather close similarities observed in the gE gene structure and pathogenicity features of the gE mutant and of the wild strain of BHV-1 confirm the genetic stability of gE. The findings indicate that the Salwa isolate is virulent, but less virulent than wild strains. Our data support the use of gE-negative marker vaccines in eradication programmes.

Bovine herpesvirus type 4: a special herpesvirus (review article).

This paper summarizes the history of and information on bovine herpesvirus type 4 (BoHV-4) from the first isolation to the most recent results. For almost twenty years BoHV-4 has been considered a typical herpes 'orphan' virus, which infects several species but causes no illness. The latest experiments revealed the close relationship of this virus with the immune system and other tissues. The virus was even considered as a possible candidate for a vector vaccine. BoHV-4 as a strange herpesvirus has several features which are not characteristic of other herpesviruses, such as several latency sites, persistence in serum, dividing cells necessary for virus replication, and the wide host range. In addition to describing the main features of the virion, replication, clinical signs, nomenclature problems, this review intends to concentrate on the new and strange results coming out from several laboratories worldwide. It is also suggested that because the virus combines several properties of various herpesvirus subfamilies and because of its close relationship with the immune system, it may deserve further attention as a representative of a potentially new genus within the Gammaherpesvirinae subfamily.

Study of bovine herpesvirus type 1 strains with monoclonal antibodies.

Fourteen strains of bovine herpesvirus type 1 (BHV-1, IBRV) representing all three groups of BHV-1 (BHV-1.1, BHV-1.2, BHV-1.3) were studied by ELISA using 106 monoclonal antibodies (Mabs) produced against BHV-1. On the basis of the ELISA, the Mabs could be divided into three groups. The first group (40 Mabs, 38%) reacted with all strains, the second group (43 Mabs, 41%) with the respiratory and genital strains (BHV-1.1 and BHV-1.2) while the third group (23 Mabs, 22%) only with the respiratory strains. Only 5 out of the antibodies neutralized respiratory and genital strains, and none of them neutralized the encephalitogenic strains (1.3). Three Mabs selected from each of the 3 groups, and the above five neutralizing strains were studied by Western blot. Antibodies of groups 1 and 3, and two neutralizing antibodies bound to a 90k protein (gpIII), whereas members of group 2 and 3 neutralizing antibodies reacted with a 74k and a 130k protein (both gpl). The results indicate that reactivity with monoclonal antibodies is as suitable for the classification of BHV-1 strains as is restriction endonuclease (RE) analysis but it cannot distinguish between subgroups within the groups.

Rickettsiae of the spotted-fever group in ixodid ticks from Hungary: identification of a new genotype ('Candidatus Rickettsia kotlanii').

Three common European 'anthrophilic' ticks, Ixodes ricinus, Haemaphysalis concinna and Dermacentor reticulatus, were collected in Hungary and tested, in assays based on nested PCR, for rickettsiae of the spotted-fever group. Low percentages of I. ricinus (2.7%) and H. concinna (1.0%) and a high percentage of D. reticulatus (26.8%) were found to be infected. The rickettsiae in the ticks were then identified, by sequencing of the genes coding for 16S ribosomal RNA (16S rDNA), citrate synthase (gltA) and the rOmpA outer-membrane protein (ompA), as Rickettsia helvetica, Rickettsia monacensis, Rickettsia sp. RpA4, or what is probably a newly recognized Rickettsia species ('Candidatus Rickettsia kotlanii'). These results raise the possibility that rickettsiae other than Rickettsia slovaca are involved in human disease in Hungary. Current knowledge on the distributions of the rickettsiae of the spotted-fever group that are emerging in Europe is also summarized.

Replication of bovine herpesvirus type 4 in human cells in vitro.

A reference strain (Movár 33/63) of bovine herpesvirus type 4 (BHV-4) was inoculated into 14 different human cell lines and five primary cell cultures representing various human tissues. BHV-4 replicated in two embryonic lung cell lines, MRC-5 and Wistar-38, and in a giant-cell glioblastoma cell culture. Cytopathic effect and intranuclear inclusion bodies were observed in these cells. PCR detected a 10,000-times-higher level of BHV-4 DNA. Titration of the supernatant indicated a 100-fold increase of infectious particles. Since this is the first bovine (human herpesvirus 8 and Epstein-Barr virus related) herpesvirus which replicates on human cells in vitro, the danger of possible human BHV-4 infection should not be ignored.

Role of bovine herpesvirus 4 in bacterial bovine mastitis.

In order to study the role played by bovine herpesvirus 4 (BoHV-4) in bovine mastitis, PCR experiments were performed on a Hungarian dairy herd of 2000 cows. Milk cells were tested with a nested PCR adjusted to detect the virus in the milk. Thirty to forty-one percentage of the udders of 101 cows with bacterial mastitis (Escherichia coli, Streptococcus uberis or Staphylococcus aureus) gave positive results, whereas less than 6% of the milk samples were positive for BoHV-4 from 118 animals with healthy udders. The mastitis status of these 118 healthy cows was followed throughout the milking period; 4.2% had clinical, and 25.42% had subclinical mastitis. By the end of the milking period, more than 90% of the cows shed the virus in their milk, regardless of the bacterial status of the udder. No correlation was found between the virus shed, the somatic cell count, and the bacterial status of the udder. Viral DNA was detected in the wall of the milk duct. These results demonstrate that BoHV-4 neither causes mastitis directly nor plays a role in the initiation of the process, but later, when bacterial infection of the udder occurs, the reactivated virus replicates in the immune cells of the udder and/or in the epithelial cells of the milk ducts and may be responsible for more severe, prolonged mastitis. As mastitis is a crucial problem of milk production, this virus may be considered a possible predisposing factor and also an agent of secondary udder infections in prolonged mastitis cases.

Studies of in vivo distribution of bovine herpesvirus type 4 in the natural host.

The in vivo distribution of bovine herpesvirus type 4 (BHV-4) was examined by testing nasal and conjunctival exudates, peripheral blood leukocytes, and various organs of experimentally infected calves. For virus detection, a nested PCR assay, virus isolation, and immunohistochemistry were applied. The nervous system and the muscles were free of viral DNA. Liver and intestinal lymph nodes contained low amounts of virus (less than two copies per 1 microgram of cellular DNA). Intestinal, tonsil, thymus, and kidney tissues contained more viral DNA copies (5 to 50 copies per 1 microgram of cellular DNA). The highest amounts of BHV-4 DNA (50 to 500 copies per 1 microgram of cellular DNA) were found in the spleen, lungs, trachea, and nasal epithelium. Amplification of DNA from blood lymphocytes through postinoculation (p.i.) day 48 proved that the virus started to replicate in these cells immediately after inoculation of the calves and that intensive virus growth took place during the 7 to 8 weeks of the infection. The number of virus-infected lymphocytes reached the maximum on p.i. days 22 to 26 and slowly declined thereafter. Virus-infected cells were found only in the spleen on p.i. day 48 by immunohistochemistry. Western blotting (immunoblotting) detected signs of an immune response against 9 of the 29 BHV-4 proteins.