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Occurrence of bovine herpesvirus type 4 DNA in Argentinean Holstein cattle from Santiago del Estero, Argentina

Bovine herpesvirus type 4 (BoHV-4) is a gamma-herpesvirus that has been isolated from apparently asymptomatic animals and from cattle with a variety of clinical signs. The pathogenic role of BoHV-4 remains unclear and it is unknown whether the virus acts as a primary pathogen or whether it facilitates secondary infections After natural or experimental infections, BoHV-4 can establish latency, mainly in cells of the monocyte/macrophage linage. Latent virus can be reactivated after glucocorticoid treatment or by stress factors. In 2007, BoHV-4 was isolated for the first time in Argentina, from samples of bovine abortions. In the present study, we used viral isolation, nested PCR and restriction endonuclease analysis (REA) to investigate the presence of BoHV-4 in bovine leukocytes from a single herd of dairy cattle with reproductive problems. In this work, we demonstrated that BoHV-4 genome is present in the leukocytes of a high proportion (63.4%) of animals, probably in a latent or persistent state. BoHV-4 was isolated from one out of eleven peripheral blood leukocyte (PBL) samples. By REA we demonstrated the existence of genomic variation among the strains circulating in this particular herd. Furthermore, all PBL samples evaluated in this study differed from the American prototype strain, DN 599. Overall, this work demonstrated that BoHV-4 is present in the leukocyte fraction of dairy cattle and that viral strains present in this herd are genetically divergent. Although BoHV-4 was detected in a herd with a background of reproductive disorders, it is not possible to conclude that the virus is the primary responsible for these conditions.


Introduction
Bovine herpesvirus type 4 (BoHV-4) is a gammaherpesvirus, which was first isolated in Hungary in 1963 from animals with respiratory and ocular disease 1 . Although cattle are the natural host of the virus, several ruminant 2 and non-ruminant species 3 are susceptible to BoHV-4. Sporadic isolates of BoHV-4 from species as diverse as lions, cats and owl monkeys 4,5 have been reported. The virus has also been isolated from apparently asymptomatic animals 6 and from cattle with a variety of clinical signs, including diarrhea, metritis 7,8,9 abortion, vaginitis, mastitis 10 , ulcerative mammillitis and skin lesions 11 . Like other herpesviruses, after natural or experimental infections, BoHV-4 can establish latency 12,13,14,15,16 . Latent virus can be reactivated after glucocorticoid treatment 17,18 or by stress factors.
Immune cells of the monocyte/macrophage linage are the main site of replication and latency of BoHV-4 19,20 . By nested PCR, BoHV-4 DNA was detected in lymphocytes, lymph nodes and nervous tissue of cattle in absence of clinical signs 8 . In experimentally infected cattle, using the same methodology, Egyed et al. 21 identified BoHV-4 DNA in bovine peripheral blood leukocytes (PBLs) at 48 days post-infection.
Although BoHV-4 replicates in several cell lines and produces cytopathic effect (CPE), isolation of the virus may be difficult in certain lymphoid and myeloid cells which are resistant to virus infection or in a number of epithelial cells which are poorly permissive to BoHV-4 infection 22 . In 2007, BoHV-4 was isolated in Argentina from samples of bovine abortions 23 . Later, the virus was isolated from nasal swabs, brain, ovocyte granulose cells and from semen from an artificial insemination center. Furthermore, BoHV-4 was isolated from buffy coat fractions in association with bovine viral diarrhea virus (BVDV) 24 . x Restriction endonuclease analysis (REA) of BoHV-4 isolates allowed the classification of the virus into two main groups, Movar-like (European prototype) or DN 599-like (North American prototype) viruses 2,6,19,25 . Until now, genetic variations of BoHV-4 field isolates have not been documented. Cross-hybridization with the reference strain DN 599 of 8 American BoHV-4 isolates from cattle with a variety of clinical conditions revealed that only one isolate had a restriction pattern clearly different from the reference strain and no correlation could be established between the origin (disease, sample and herd) and the restriction pattern of the isolates 26 . By REA, Verna et al. 27 have shown that there are remarkable differences among BoHV-4 strains circulating in Argentina. The same analysis revealed that most of these isolates are different from the American prototype, DN 599.
D'Onofrio et al. 20 demonstrated that BoHV-4 has tropism for stromal and epithelial cells of the endometrium where it produces rapid CPE. It has been suggested that BoHV-4 recrudescence from latency at the time of parturition is responsible for the development of postpartum endometritis 7,28 , one of the conditions that has been associated with the virus. In addition, Dubuisson et al. 29 showed that BoHV-4 can establish a latent infection in bovine testicles and that the virus can be reactivated by glucocorticoid administration. Therefore, it is apparent that BoHV-4 is a pathogen associated with reproductive problems in cattle.
Nevertheless, the pathogenic role of BoHV-4 remains unclear and it is unknown whether the virus acts as a primary pathogen or whether it facilitates secondary infections. In the present study, we used viral isolation, nested PCR and REA to investigate the presence of BoHV4 in bovine leukocytes from a single herd of dairy cattle with a history of reproductive problems.

Prevalence of BoHV-4 DNA in bovine PBLs
Nested PCR was used to determine the presence of BoHV-4 DNA in PBLs (Figure 1). By this technique, it was possible to demonstrate that 63.4% (33/52) of the animals harbored BoHV-4 DNA in their blood leukocyte fractions. The analysis by animal category revealed that 60% (18/30) of the bulls and 68% (15/22) of the cows were positive for the presence of viral DNA in their PBLs.

Isolation of BoHV-4 from bovine PBLs
Unlike other gamma-herpesviruses, BoHV-4 is able to replicate in a variety of cell lines 22 , including MDBK cells. 30 BoHV-4-positive and -negative PBLs, as determined by nested PCR, were selected for coculture with MDBK cells. Only eleven PBLs samples were adequate to attempt viral isolation. Viral CPE was evident in one PBL sample belonging to a yearling bull after five days of co-culture. CPE is characterized by the presence of areas of cell rounding and lysis, which progressively extends to the complete monolayer ( Figure 2). No CPE was observed in the remaining samples analyzed ( Table 1). The isolation of BoHV-4 in the co-culture was confirmed by direct immunofluorescene using an anti-BoHV-4 monoclonal antibody (Figure 3).

BoHV-4 DNA restriction pattern
Comparison of EcoRI restriction profiles (Figure 4) of BoHV-4 DNA obtained from PBLs revealed the exis-tence of genomic variation among the strains circulating in this particular herd. All the PBL samples evaluated in this study differed from the American prototype strain,

Discussion and conclusions
BoHV-4 has been isolated from asymptomatic animals and from cattle with a wide range of clinical manifestations. In the last years, BoHV-4 has been isolated and/or its genome has been identified in several bovine tissues, especially in cases associated with reproductive disorders such as, endometritis 7,8,9 , abortion 10,23 and vaginitis 10  Therefore, sequencing of these regions from several field isolates will be important to explain the heterogeneity observed. It is likely that these genomic differences are the result of variations in the number of repeated sequences, as it has been described for many herpesviruses, such as, Epstein-Barr 35 , varicella-zoster virus 36 and bovine herpesvirus 37 .
Previous works 7,9 have shown that BoHV-4 can undergo genomic drift and significant geographic variation has also been observed among strains. From our study, it is also possible to infer that there is consid-