Differential expression of cyclins mRNA in neural tissues of BoHV-1- and BoHV-5- infected cattle.

Bovine alphaherpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are closely related alphaherpesviruses. BoHV-5 causes non-suppurative meningoencephalitis in calves. BoHV-1 is associated with several syndromes and, occasionally, can cause encephalitis. Although both viruses are neurotropic and they share similar biological properties, it is unknown why these alphaherpesviruses differ in their ability to cause neurological disease. Neural tissue samples were collected from BoHV-1- and BoHV-5-intranasally inoculated calves during acute infection, latency and reactivation and the levels of cyclins mRNA expression were analyzed by qRT-PCR. Striking differences in the levels of cyclins mRNA were particularly detected in trigeminal ganglion (TG). The expression levels of cyclins in TG during BoHV-5 latency suggest that these viruses utilize different strategies to persist in the host. It is apparent that a relationship between virus loads and cyclin mRNA levels can be established only during acute infection and other factors might be involved in the regulation of cell cycle components during BoHV latency and reactivation. Bovine alphaherpesviruses neuropathogenicity might be influenced by the differential control of cell cycle components by these herpesviruses. This is the first report on BoHV-5 modulation of cyclins expression in neural tissues from its natural host.

Functional analysis of the latency-related gene of bovine herpesvirus types 1 and 5.

Bovine herpesvirus type 1 and type 5 (BoHV-1 and BoHV-5) are two alphaherpesviruses that affect cattle with two different syndromes. While BoHV-1 mainly produces respiratory symptoms, BoHV-5 is highly neuropathogenic and responsible for meningoencephalitis in young cattle. The latency-related (LR) gene, which is not conserved between these two herpesviruses, is the only viral gene abundantly expressed in latently infected neurons. The antiapoptotic action of this gene has been demonstrated during acute infection and reactivation from latency and seems to be mainly mediated by a LR protein (ORF-2) which is truncated in amino acid 51 in the case of BoHV-5. In this work, we show that the BoHV-5 LR gene is less efficient at cell survival and apoptosis inhibition in transient as well as in established neuronal cell lines compared to its BoHV-1 homolog. We hypothesize that the BoHV-5 LR gene may have novel functions that are lacking in the BoHV-1 LR gene and that these differences may contribute to its enhanced neuropathogenesis.

Design, biological activity and signaling pathway of bovine consensus omega interferon expressed in Pichia pastoris.

The bovine IFN-ω (BoIFN-ω) multigene family is located on chromosome 8, which has 14 potential functional genes and 10 pseudogenes. After aligning 14 BoIFN-ω subtypes and assigning the most frequently occurring amino acids in each position, one artificial consensus BoIFN-ω (CoBoIFN-ω) gene was designed, optimized and synthesized. Then, CoBoIFN-ω was expressed in Pichia pastoris, which was demonstrated to have 3.94-fold and 14.3-fold higher antiviral activity against VSV on MDBK cells than that of BoIFN-ω24 and BoIFN-ω3, respectively. Besides this, CoBoIFN-ω was confirmed to have antiviral activity against VSV on BL, BT, PK-15 cells, and against BEV, BHV-1, BPIV3 on MDBK cells. Additionally, CoBoIFN-ω could bind with bovine type I IFN receptors, and then activate the promoters of NF-κB, ISRE and BoIFN-ß, and induce the transcription of ISGs and expression of Mx1 and NF-κB p65, which suggested CoBoIFN-ω exerts antiviral activity via activation of the JAK-STAT signaling pathway. Overall, this research on CoBoIFN-ω not only extends and improves consensus IFN research, but also reveals that CoBoIFN-ω has the potential to be used in the therapy of bovine viral diseases.

The bovine herpesvirus 1 regulatory proteins, bICP4 and bICP22, are expressed during the escape from latency.

Following acute infection of mucosal surfaces by bovine herpesvirus 1 (BoHV-1), sensory neurons are a primary site for lifelong latency. Stress, as mimicked by the synthetic corticosteroid dexamethasone, consistently induces reactivation from latency. Two viral regulatory proteins (VP16 and bICP0) are expressed within 1 h after calves latently infected with BoHV-1 are treated with dexamethasone. Since the immediate early transcription unit 1 (IEtu1) promoter regulates both BoHV-1 infected cell protein 0 (bICP0) and bICP4 expressions, we hypothesized that the bICP4 protein is also expressed during early stages of reactivation from latency. In this study, we tested whether bICP4 and bICP22, the only other BoHV-1 protein known to be encoded by an immediate early gene, were expressed during reactivation from latency by generating peptide-specific antiserum to each protein. bICP4 and bICP22 protein expression were detected in trigeminal ganglionic (TG) neurons during early phases of dexamethasone-induced reactivation from latency, operationally defined as the escape from latency. Conversely, bICP4 and bICP22 were not readily detected in TG neurons of latently infected calves. In summary, it seems clear that all proteins encoded by known BoHV-1 IE genes (bICP4, bICP22, and bICP0) were expressed during early stages of dexamethasone-induced reactivation from latency.

Bovine herpesviruses induce different cell death forms in neuronal and glial-derived tumor cell cultures.

Oncolytic viruses have the ability to infect tumor cells and leave healthy cells intact. In this study, bovine herpesvirus 1 (BHV1; Los Angeles, Cooper, and SV56/90 strains) and bovine herpesvirus 5 (BHV5; SV507/99 and GU9457818 strains) were used to infect two neuronal tumor cell lineages: neuro2a (mouse neuroblastoma cells) and C6 (rat glial cells). BHV1 and BHV5 strains infected both cell lines and positively correlated with viral antigen detection (p < 0.005). When neuro2a cells were infected by Los Angeles, SV507/99, and GU9457818 strains, 40 % of infected cells were under early apoptosis and necroptosis pathways. Infected C6 cells were >40 % in necroptosis phase when infected by BHV5 (GU9457818 strain). Blocking caspase activation did not interfere with cell death. However, when necroptosis was blocked, 60-80 % of both infected cells with either virus switched to early apoptosis pathway with no interference with virus replication. Moreover, reactive oxygen species production and mitochondrial membrane dysfunction were detected at high levels in both infected cell lines. In spite of apoptosis and necroptosis blockage, tumor necrosis factor alpha (TNFA) and virus transcription were positively correlated for all viral strains studied. Thus, these results contribute to the characterization of BHV1 and BHV5 as potential oncolytic viruses for non-human cells. Nonetheless, the mechanisms underlying their oncolytic activity in human cells are still to be determined.

A bovine herpesvirus 1 pUL51 deletion mutant shows impaired viral growth in vitro and reduced virulence in rabbits.

Bovine herpesvirus 1 (BoHV-1) UL51 protein (pUL51) is a tegument protein of BoHV-1 whose function is currently unknown. Here, we aimed to illustrate the specific role of pUL51 in virion morphogenesis and its importance in BoHV-1 virulence. To do so, we constructed a BoHV-1 bacterial artificial chromosome (BAC). We used recombinant BAC and transgenic techniques to delete a major part of the UL51 open reading frame. Deletion of pUL51 resulted in severe viral growth defects, as evidenced by lower single and multi-step growth kinetics, reduced plaque size, and the accumulation of non-enveloped capsids in the cytoplasm of infected cells. Using tagged BoHV-1 recombinant viruses, it was determined that the pUL51 protein completely co-localized with the cis-Golgi marker protein GM-130. Taken altogether, pUL51 was demonstrated to play a critical role in BoHV-1 growth and it is involved in viral maturation and egress. Moreover, an in vivo analysis showed that the pUL51 mutant exhibited reduced virulence in rabbits, with no clinical signs, no nasal shedding of the virus, and no detectable serum neutralizing antibodies. Therefore, we conclude that the BoHV-1 pUL51 is indispensable for efficient viral growth in vitro and is essential for virulence in vivo.

Isolation and characterization of BoHV-1 from seropositive cows after inducing artificial stress in West Bengal, India.

Infectious Bovine Rhinotracheitis (BoHV-1) is the most important emerging disease of cattle in India. With an aim to reactivate BoHV-1 from latently infected sero-positive cattle for molecular characteristics of the isolates prevalent in tropical and sub-tropical countries like India and further epidemiological investigations on IBR infections this study had been conducted. Artificial stress with dexamethasone at the dose rate of 0.1 mg kg(-1) body weight for 5 consecutive days was induced in BoHV-1 sero-positive cows. Then isolation from nasal swabs was attempted in Madin Darby Bovine Kidney (MDBK) cell line to find out the prevalent strain in India. The virus was isolated from all the three cows. All the three isolates were typed as BoHV-1.2 (Strain India 4, India 5 and India 6). The reactivation obtained in this study with dexamethasone suggests the usefulness of BoHV-1 cow latency model for epidemiological investigations on BoHV-1 infections in tropical and sub-tropical countries like India, Pakistan etc.

In vitro replication of bovine herpesvirus types 1 and 5.

The aim of this work was to study the in vitro replication of bovine herpesvirus types 1 and 5 (BoHV-1 and 5) at the beginning and end of the logarithmic growth phase of Madin-Darby Bovine Kidney (MDBK) cells. The replication kinetics and size of lysis and infection plaques of the field isolates 09/210 (BoHV-1) and 97/613 (BoHV-5) and the reference strains BoHV-1.1 Los Angeles 38 (LA38), BoHV-1.1 Cooper, BoHV-5a N569 and BoHV-5b A663 were evaluated. The highest mean virus titre was recorded for N569, followed by LA38 and 97/613. For most of the viruses, the virus titre values increased from 24 h post-infection (hpi) up to 48 hpi and then, they remained unchanged up to 72 hpi. However, the virus titre for 09/210 was significantly lower and a slight, steady increase was observed from 24 to 72 hpi. Furthermore, the largest lysis and infection plaques were recorded for 97/613 and LA38, respectively. According to this work, it is evident that there is a relationship between the replication of BoHV and the multiplication stage of MDBK cells. The results of this study contribute to the understanding of the replication behaviour in cell cultures of several strains of BoHV, which is critical for the rational design of in vitro experiments and vaccine production.

The nucleolus and viral infection.

The nucleolus is a subnuclear structure of eukaryocytes. It was thought that nucleolus only participates in the biogenesis and processing of rRNA. However, more and more evidence shows that it has many other functions, such as tRNA precursor processing, stress sensing and it is also involved in gene silencing, senescence and cell cycle regulation. Here, we summarize the recent understandings about the nucleolar functions, the regulation of nucleolar localization of proteins and the role that the nucleolus plays in virus infection, in which some related studies of Herpes simplex virus type 1 (HSV-1) US11, UL24 and bovine herpesvirus-1 infected cell protein 27 (BICP27) carried out in our lab will also be included.

Bovine herpesvirus 5 BICP0 complements the bovine herpesvirus 1 homolog.

Bovine herpesvirus 1 (BoHV-1) and BoHV-5 are closely related (82% amino acid identity) but differ strongly in neuropathogenesis. The immediate-early gene for BICP0 is less conserved (70% amino acid identity) and may contribute to a dissimilar phenotype. A peculiar difference is a guanosine hexamer in the BICP0-1 gene which aligns with only five guanosines in the BICP0-5 gene and therefore results in a frameshift in the latter open reading frame. Thus, the C-terminal amino acid sequence (residues 643-676 of BICP0-1 vs. 655-720 of BICP0-5) is completely different. We introduced the BICP0-5 frameshift into the BoHV-1 genome cloned as a bacterial artificial chromosome (BoHV-1 BAC) using the Red recombination system with galK selection and counterselection. Transfection of MDBK cells with the resulting BAC produced recombinant virus that replicated like wild type BoHV-1 in vitro. Attempts to exchange the entire BICP0-1 gene by the BoHV-5 homolog using the same approach failed repeatedly. Therefore, we cotransfected purified BICP0(-)/galK(+)-BoHV-1 BAC DNA with a recombination plasmid coding for BICP0-5 with or without a HA tag into MDBK cells. BoHV-1 recombinants expressing the respective proteins were characterized. In vitro, all recombinants grew to similar titers as the parental viruses, which demonstrates that BICP0-5 compensates for the growth defect of BICP0(-)/galK(+)-BoHV-1 and functionally complements BICP0-1 of BoHV-1. We conclude that BICP0 may be suitable to positively select BoHV-1 recombinants with deletions or insertions of additional genes of interest.

Protein display by bovine herpesvirus type 1 glycoprotein B.

Glycoprotein B (gB) of bovine herpesvirus 1 (BHV-1), a major component of the viral envelope, is essential for membrane fusion during entry and cell-to-cell spread. It is cleaved in the trans-Golgi network by the proprotein convertase furin. Integration of the open reading frame (ORF) encoding a mutated gB with a second furin cleavage site and mature boIFN-alpha as intervening peptide between the amino-terminal (NH(2)) and carboxy-terminal (COOH) gB subunits yielded recombinant BHV-1/gB2FuIFN-alpha which, unexpectedly, express gB with an enlarged NH(2)-subunit of 90kDa. Here we show that boIFN-alpha-specific antibodies bind to the 90kDa gB subunit and efficiently neutralize BHV-1/gB2FuIN-alpha infectivity. We also show that inactivated BHV-1/gB2FuIN-alpha virions induce an antiviral state in cells incubated with UV-inactivated particles. These results demonstrate that the 90kDa protein is a NH(2)-subunit/boIFN-alpha fusion protein whose boIFN-alpha domain is biologically active. To verify that BHV-1 gB is suitable for the display of (glyco)proteins on the surface of virions we constructed BHV-1 recombinants expressing within gB the first 273 amino acids of the NH(2)-subunit (HA1) of avian influenza haemagglutinin, either flanked by two furin cleavage sites or with only one cleavage site between a gB/NH(2)_HA1 fusion protein and the COOH subunit. The resulting recombinant BHV-1/gB2FuHA1 expressed gB from which 55kDa HA1 was excised and secreted. In contrast, gB from BHV-1/gB_NH(2)HA1 infected cells retained HA1 as fusion protein with the NH(2)-subunit. Immunoblotting and neutralization analyses revealed that HA1 is incorporated into the envelope BHV-1/gB/NH(2)_HA1 particles and exposed to the exterior of virions. Thus, this novel approach enables display of polypeptides and (glyco)proteins of at least 273 amino acids on viral particles which is of particular interest for development of novel diagnostics and vaccines as well as for, e.g. gene therapy applications especially when biologically active ligands need to be presented.

A UL47 gene deletion mutant of bovine herpesvirus type 1 exhibits impaired growth in cell culture and lack of virulence in cattle.

Tegument protein VP8 encoded by the U(L)47 gene of bovine herpesvirus type 1 (BHV-1) is the most abundant constituent of mature virions. In the present report, we describe the characterization of U(L)47 gene-deleted BHV-1 in cultured cells and its natural host. The U(L)47 deletion mutant exhibited reduced plaque size and more than 100-fold decrease in intracellular and extracellular viral titers in cultured cells. Ultrastructural observations of infected cells showed normal maturation of BHV-1 virions in the absence of VP8. There was no evidence for a change in immediate-early gene activator function of VP16 in the U(L)47 deletion mutant virus-infected cells, since bovine ICP4 mRNA and protein levels were similar to those in the wild-type and revertant virus-infected cells throughout the course of infection. Whereas VP16, glycoprotein C (gC), gB, and VP5 were expressed to wild-type levels in the U(L)47 deletion mutant-infected cells, the gD and VP22 protein levels were significantly reduced. The reduction in gD protein was associated with increased turnover of the protein. Furthermore, some of the analyzed early and late proteins were expressed with earlier kinetics in the absence of VP8. Extracellular virions of the U(L)47 deletion mutant contained reduced amounts of gD, gB, gC, and VP22 but similar amounts of VP16 compared to those of wild-type or revertant virus particles. In addition, the U(L)47 gene product was indispensable for BHV-1 replication in vivo, since no clinical manifestations or viral shedding were detected in the U(L)47 deletion mutant-infected calves, and the virus failed to induce significant levels of humoral and cellular immunity.

Major tegument protein VP8 of bovine herpesvirus 1 is phosphorylated by viral US3 and cellular CK2 protein kinases.

The UL47 gene product, VP8, is one of the major tegument proteins of bovine herpesvirus 1 (BoHV-1) and is subject to phosphorylation. Analysis of protein bands co-immunoprecipitated with VP8 from BoHV-1-infected cells by mass spectroscopy suggested that VP8 interacts with two protein kinases: cellular CK2 and viral US3. CK2 is a highly conserved cellular protein, expressed ubiquitously and known to phosphorylate numerous proteins. The US3 gene product is one of the viral kinases produced by BoHV-1 during infection. Interactions of CK2 and US3 with VP8 were confirmed outside the context of infection when FLAG-VP8 was expressed alone or co-expressed with US3-haemagglutinin tag in Cos-7 cells. Furthermore, VP8 and US3 were found to co-localize in the nucleus during viral infection. To explore the significance of these interactions, an in vitro kinase assay was performed, which demonstrated that VP8 is heavily phosphorylated by CK2. In the presence of the highly specific CK2 kinase inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), phosphorylation of VP8 was significantly reduced. Phosphorylation of VP8 was also inhibited by the presence of kenpaullone, a less specific CK2 inhibitor, but not by protein kinase CK1 or protein kinase C inhibitors. When VP8 and US3 were both included in the kinase assay in the presence of DMAT, phosphorylation of VP8 was again observed. Autophosphorylation of US3 was also detected and was not inhibited by DMAT. Based on these results, it is proposed that VP8 interacts with cellular CK2 and viral US3 in BoHV-1-infected cells, and is in turn subject to kinase activities associated with both of these proteins.

Characterization of the nuclear and nucleolar localization signals of bovine herpesvirus-1 infected cell protein 27.

Bovine herpesvirus-1 infected cell protein 27 (BICP27) was detected predominantly in the nucleolus. The open reading frame of BICP27 was fused with the enhanced yellow fluorescent protein (EYFP) gene to investigate its subcellular localization in live cells and BICP27 was able to direct monomeric, dimeric or trimeric EYFP exclusively to the nucleolus. By constructing a series of deletion mutants, the putative nuclear localization signal (NLS) and nucleolar localization signal (NoLS) were mapped to (81)RRAR(84) and (86)RPRRPRRRPRRR(97) respectively. Specific deletion of the putative NLS, NoLS or both abrogated nuclear localization, nucleolar localization or both respectively. Furthermore, NLS was able to direct trimeric EYFP predominantly to the nucleus but excluded from the nucleolus, whereas NoLS targeted trimeric EYFP primarily to the nucleus, and enriched in the nucleolus with faint staining in the cytoplasm. NLS+NoLS directed trimeric EYFP predominantly to the nucleolus with faint staining in the nucleus. Moreover, deletion of NLS+NoLS abolished the transactivating activity of BICP27 on gC promoter, whereas deletion of either NLS or NoLS did not. The study demonstrated that BICP27 is a nucleolar protein, adding BICP27 to the growing list of transactivators which localize to the nucleolus.

Characterization of interspecific recombinants generated from closely related bovine herpesviruses 1 and 5 through multiple PCR sequencing assays.

Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) are closely related alphaherpesviruses infecting cattle. In countries where both viruses circulate, co-infection of cattle is likely. It was shown that recombination occurs at a high frequency in cattle infected dually with two BoHV-1 mutants. In addition, interspecific recombinants are generated in cell culture co-infected with BoHV-1 and BoHV-5. Even if the process of interspecific recombination appears inefficient relative to intraspecific recombination, BoHV-1 and BoHV-5 may give rise to interspecific recombinants in co-infected cattle. Since molecular tools for differentiating BoHV-1 from BoHV-5 are limited and do not allow to localize recombination events between these closely related virus species, 13 PCR sequencing assays were developed to discriminate between BoHV-1 and BoHV-5 at regular intervals throughout the entire respective viral DNA genomes. These assays were used to determine the genetic background of two interspecific BoHV-1/-5 recombinants generated previously. The two crossover points where recombination events occurred between the parental strains were determined. This study provides a detailed analysis of two interspecific recombinant viruses generated in vitro from closely related alphaherpesviruses infecting the same natural host. It demonstrates that recombination can occur within very short fragments of sequence homology. This finding raises questions about the mechanisms involved in the strands exchange and resolution step of the homologous recombination used by herpesviruses. This method will allow monitoring generation of recombinants between closely related herpesvirus species both in vitro and in vivo.

Critical assessment of an in vitro bovine respiratory organ culture system: a model of bovine herpesvirus-1 infection.

A bovine in vitro organ culture (BIVOC) system was evaluated as a model to study host and pathogen events during the course of bovine herpesvirus-1 infection. Upper respiratory tract epithelium, from slaughtered animals, was cultured in an air-liquid interface system and integrity, viability, and TNF-alpha gene expression of tissue explants were monitored over 72h in the presence or absence of infection by bovine herpesvirus type 1 (BHV-1). Uninfected explants maintained viability and integrity over the 72h time course although histological signs of degeneration were first visible from 24h of culture. Explants were productively infected with BHV-1 and typical, dose dependent, cytopathic changes were observed in response to infection. Regulation of TNF-alpha gene expression in uninfected explants varied over time and was region-specific but there was significant down-regulation of TNF-alpha gene expression at 2h post-infection when compared to uninfected controls at the same time point. Taking caveats into consideration the BIVOC system shows promise as a tool for analysis of immediate or early events in host-pathogen interaction.

Coinfection with two closely related alphaherpesviruses results in a highly diversified recombination mosaic displaying negative genetic interference.

Phylogenetic studies of the emergence and spread of natural recombinants in herpesviruses infecting humans and animals have been reported recently. However, despite an ever-increasing amount of evidence of recombination in herpesvirus history, the recombination process and the consequences on the genetic diversity of the progeny remain poorly characterized. We addressed this issue by using multiple single-nucleotide polymorphisms (SNPs) differentiating the two subtypes of an alphaherpesvirus, bovine herpesvirus 1 (BoHV-1). Analysis of a large sample of progeny virions obtained in a single growth cycle of coinfected BoHV-1 strains provided a prospective investigation of the recombination dynamics by using SNPs as recombination markers. We found that the simultaneous infection with two closely related herpesviruses results in a highly diversified recombination mosaic. From the analysis of multiple recombinants arising in the progeny, we provide the first evidence of genetic interference influencing the recombination process in herpesviruses. In addition, we report striking differences in the levels of recombination frequency observed along the BoHV-1 genome. With particular emphasis on the genetic structure of a progeny virus population rising in vitro, our data show to which extent recombination participates to the genetic diversification of herpesviruses.

A simple method of infecting rabbits with Bovine herpesvirus 1 and 5.

This report describes an alternative technique to inoculate rabbits and to reproduce infection by Bovine herpesvirus 1 and 5. First, the nostrils are anaesthetized by aspersion with local anaesthetic. A few seconds later, and after proving the insensitivity of the zone, the rabbits are put on their back legs with their nostrils upwards and the inoculum is introduced slowly into each nostril by using disposable droppers. Clinical signs, viral isolation from nasal swabs, histological lesions found, positive polymerase chain reaction and antibodies production confirm the infection. This very simple and bloodless technique, where the animals are exposed to minor distress, may be useful for evaluating the virulence of BoHV-1 and BoHV-5 strains, to study the establishment of latent virus infection and to test the potential of experimental vaccines or properties of antiviral drugs. It may be also suitable for experimental infection with other respiratory viruses in this animal model.

Envelope protein Us9 is required for the anterograde transport of bovine herpesvirus type 1 from trigeminal ganglia to nose and eye upon reactivation.

In this study, the authors examined the role of bovine herpesvirus type 1 (BHV-1) Us9 in the anterograde transport of the virus from trigeminal ganglia (TG) to nose and eye upon reactivation from latency. During primary infection, both BHV-1 Us9-deleted and BHV-1 Us9-rescued viruses replicated efficiently in the nasal and ocular epithelium. However, upon reactivation from latency, only the BHV-1 Us9-rescued virus could be isolated in the nasal and ocular shedding. By real-time polymerase chain reaction, comparable DNA copy numbers were detected in the TGs during latency and reactivation for both the viruses. Therefore, Us9 is essential for reactivation of the virus in the TG and anterograde axonal transport from TG to nose and eye.

Interferon-alpha genes from Bos and Bubalus bubalus.

Interferon-a genes were cloned from six breeds of three species of two genera (three Chinese native cattle breeds of yellow cattle, wild yak and HuanHu domestic yak, one European breed of Holstein cow, and two water buffalo breeds of FuAn water buffalo and FuZhong water buffalo) by direct PCR. The PCR products were directly inserted into the expression vector to be sequenced and expressed. Sequence analysis showed that IFN-a genes of six clones were composed of 498 nucleotides, encoding a mature polypeptide with 166 amino acids. Compared with the published BoIFN-a subtypes, the IFN-a gene of Holstein cow had only one point mutation with the BoIFN-aA subtype. The IFN-a gene of yellow cattle was similar to the BoIFN-aD subtype with amino acid identity of 97.0% and may be considered as a new subtype, namely, BoIFN-aD1. The other four IFN-a genes, cloned from wild yak and HuanHu domestic yak, FuAn water buffalo, and FuZhong water buffalo, represented four new subtypes, namely, BoIFN-aI, BoIFN-aJ, BuIFN-a1, and BuIFN-a2, respectively. Each of the six clones was expressed in E. coli with molecular weight of approximately 20 kDa by SDS-PAGE and Western blot analyses. Antiviral activity assays showed that the six recombinant IFN-a (rIFN-a) all exhibited 1,000 times higher antiviral activity in the MDBK/VSV cell line than in the CEF/VSV one. Moreover, the rIFN-as could inhibit infectious bovine rhinotracheitis virus replication in the MDBK cell line using CPE inhibition method. The results suggested that rIFN-as a potential agent for clinical application against virus diseases in cattle industry.