Human recombinant interleukin-2 augments in vitro blastogenesis of bovine and porcine lymphocytes

The recent cloning of the human gene encoding interleukin 2 (IL-2) has provided the means for economical production of large quantities of the pure lymphokine for clinical studies. Human recombinant interleukin 2 (HrIL-2) has been reported to have in vitro and in vivo immunomodulating effects in the murine system, suggesting the cloned gene product has cross-species activity. Bovine and porcine peripheral blood lymphocytes were tested for responsiveness to HrIL-2 in a lymphocyte blastogenesis assay. Not only was the HrIL-2 highly stimulatory but it also reconstituted lymphocyte responsiveness to maximal values following incubation with suboptimal concentrations of mitogen plus exogenous lymphokine. These studies suggest that HrIL-2 has the potential of serving as an in vivo modulator of immunoresponsiveness in domestic species. The contribution to food animal medicine will be considerable if administration of the lymphokine results in augmentation of antigen-specific immune responses when applied as an adjuvant, non-specific booster of pre-existing immunity, or for therapy of immunosuppression.     

Encephalitis in aborted bovine fetuses associated with Bovine herpesvirus 1 infection.

Brain tissue from 12 aborted bovine fetuses submitted to the Wisconsin Veterinary Diagnostic Laboratory revealed histologic lesions that consisted of glial nodules and variable degrees of mononuclear inflammation, microhemorrhage, neuronal necrosis, and cerebral cortical cavitation. A diagnosis of Bovine herpesvirus 1 (BHV-1) abortion had been made in all of these cases through multiple testing modalities. Brain tissue from 8 of the 12 fetuses was immunohistochemically stained with a monoclonal antibody specific to BHV-1, and, in 5 fetuses, there was positive intralesional staining of neurons, glial cells, and endothelial cells. This preliminary data suggested that herpesviral infection of brain tissue led to the described neurologic lesions. BHV-1 was then amplified from brain tissue in all 12 of the fetuses and was confirmed by partial sequencing of the thymidine kinase and glycoprotein C genes. To the authors' knowledge, neurologic lesions have not previously been described in BHV-1-infected fetuses, nor has BHV-1 previously been identified in bovine fetal brain tissue. The neurologic histopathology attributed to BHV-1 infection in these cases overlaps with the neurologic lesions produced by Neospora caninum, a common etiologic agent of bovine abortion. Therefore, when bovine fetal neurologic lesions are found, both etiologies should be considered and then distinguished by using additional diagnostic tools.     

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.     

Bovine herpesvirus type 1 infection of bovine bronchial epithelial cells increases neutrophil adhesion and activation

Respiratory infection of cattle with bovine herpesvirus type 1 (BHV-1) predisposes cattle to secondary pneumonia with Mannheimia haemolytica as part of the bovine respiratory disease complex (BRD). One cell type that has received limited investigation for its role in the inflammation that accompanies BRD is the respiratory epithelial cell. In the present study we investigated mechanisms by which BHV-1 infection of respiratory epithelial cells contributes to the recruitment and activation of bovine polymorphonuclear neutrophils (PMNs) in vitro. Primary cultures of bovine bronchial epithelial (BBE) cells were infected with BHV-1 and assessed for cytokine expression by real-time PCR. We found that BHV-1 infection elicits a rapid IL-1, IL-8 and TNF-α mRNA response by BBE cells. Bovine PMNs exhibited greater adherence to BHV-1 infected BBE cells than uninfected cells. The increased adherence was significantly reduced by the addition of an anti-IL-1β antibody or human soluble TNF-α receptor (sTNF-αR). Pre-incubation of bovine PMNs with conditioned media from BHV-1 infected BBE cells increased PMN migration, which was inhibited by addition of an anti-IL-1β antibody, sTNF-αR, or an IL-8 peptide inhibitor. Conditioned media from BHV-1 infected BBE cells activated bovine PMNs in vitro as demonstrated by PMN shape change, production of reactive oxygen species and degranulation. PMNs also exhibited increased LFA-1 expression and susceptibility to M. haemolytica LKT following incubation with BHV-1 infected BBE cell conditioned media. Our results suggest that BHV-1 infection of BBE cells triggers cytokine expression that contributes to the recruitment and activation of neutrophils, and amplifies the detrimental effects of M. haemolytica LKT.     

Proliferative response of mammary gland mononuclear cells to recombinant bovine interleukin-2.

Methods of augmenting bovine mononuclear cell responsiveness during physiological transitions of the udder may enhance resistance of the mammary gland to intramammary infections. Interleukin-2 is required for proliferation of T-lymphocytes and may contribute to B-lymphocyte proliferation. Recombinant bovine interleukin-2 (rBoIL-2) was evaluated as a potential immunoenhancer of bovine mammary gland mononuclear cells. Bovine mononuclear cells were isolated from five primiparous Holstein cows at 14-18 and 28-32 days of involution and at 7-13 days prior to parturition. Bovine blood and mammary gland mononuclear cells were highly responsive to rBoIL-2. Response of mammary gland mononuclear cells to rBoIL-2 was comparable with response of blood mononuclear cells. These data suggest that rBoIL-2 may be an effective immunoenhancer of bovine mononuclear cells during the non-lactating and prepartum periods.     

Detection of Bovine herpesvirus 4 DNA in aborted bovine fetuses

The presence of Bovine herpesvirus 4 (BoHV-4) was investigated by several methods in 24 aborted bovine fetuses. Polymerase chain reaction (PCR) and in situ DNA hybridization proved the presence of BoHV-4 DNA in 7 (29%) of the fetuses. The BoHV-4 genome was detected in the cytoplasm of splenic lymphocytes and monocytes, and sometimes in renal tubular epithelial cells or hepatic Kupffer cells, in all 7 PCR-positive fetuses. However, BoHV-4-specific monoclonal antibody failed to detect viral antigen in the formalin-fixed, paraffin-embedded tissue samples. No bacterial pathogens were found in the tissues of the BoHV-4-positive fetuses. Fungi were detected in 1 sample, and antibody to bovine viral diarrhea virus was detected in another. These results indicate that BoHV-4 could play a role in reproductive disorders of cattle, including abortion.     

Bovine abortion associated with mixed Movar 33/63 type herpesvirus and bovine viral diarrhea virus infection.

A herpesvirus and cytopathogenic bovine viral diarrhea virus were isolated from a bovine fetus aborted in the 6th month of gestation. The herpesvirus was serologically indistinguishable from bovine herpesviruses DN-599 and Movar 33/63.     

Encephalitis induced by bovine herpesvirus 5 and protection by prior vaccination or infection with bovine herpesvirus 1.

Calves were intranasally challenged with bovine herpesvirus 5 (BHV5) and followed for the development of viral infection, clinical encephalitis, histologic lesions in the brain, and viral sequences in the trigeminal ganglia. Calves that were previously vaccinated with bovine herepesvirus 1 (BHV1, n = 4) or previously infected with BHV1 (n = 5) or that had not been exposed to either virus (n = 4) were compared. No calf developed signs of encephalitis, although all calves developed an infection as indicated by nasal secretion of BHV5 and seroconversion to the virus. Histologic lesions of encephalitis consisting of multifocal gliosis and perivascular cuffs of lymphocytes were observed in calves not previously exposed to BHV1. BHV5 sequences were amplified from the trigeminal ganglia of calves previously vaccinated and from calves not previously exposed to BHV1; calves sequentially challenged with BHV1 and later BHV5 had exclusively BHV1 sequences in their trigeminal ganglia. Administration of dexamethasone 28 days after BHV5 challenge did not influence clinical disease or histologic lesions in either previously unexposed calves (n = 2) or previously immunized calves (n = 2), although it did cause recrudescence of BHV5, as detected by nasal virus secretion.     

Analysis of bovine trigeminal ganglia following infection with bovine herpesvirus 1

Following primary infection of the eye, oral cavity, and/or nasal cavity, bovine herpesvirus 1 (BHV-1) establishes latency in trigeminal ganglionic (TG) neurons. Virus reactivation and spread to other susceptible animals occur after natural or corticosteroid-induced stress. Infection of calves with BHV-1 leads to infiltration of lymphocytes in TG and expression of IFN-gamma (interferon-gamma), even in latently infected calves. During latency, virus antigen and nucleic acid positive non-neural cells were occasionally detected in TG suggesting there is a low level of spontaneous reactivation. Since we could not detect virus in ocular or nasal swabs, these rare cells do not support high levels of productive infection and virus release or they do not support virus production at all. Dexamethasone (DEX) was used to initiate reactivation in latently infected calves. Foci of mononuclear or satellite cells undergoing apoptosis were detected 6h after DEX treatment, as judged by the appearance of TUNEL+ cells (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling). BHV-1 antigen expression was initially detected in lymphocytes and other non-neural cells in latently infected calves following DEX treatment. At 24h after DEX treatment, viral antigen expression and nucleic acid were readily detected in neurons. Our data suggest that persistent lymphocyte infiltration and cytokine expression occur during latency because a low number of cells in TG express BHV-1 proteins. Induction of apoptosis and changes in cytokine expression following DEX treatment correlates with reactivation from latency. We hypothesize that inflammatory infiltration of lymphoid cells in TG plays a role in regulating latency.     

Bovine herpesvirus 4 immediate early 2 (Rta) gene is an essential gene and is duplicated in bovine herpesvirus 4 isolate U

The ORF50/Rta gene has been shown to be an essential gene for many gammaherpesviruses. Although the BoHV-4 ORF50/Rta homolog, immediate early gene 2 (IE2), has been shown to activate several BoHV-4 early and late promoters in cotransfection assays, there is no direct proof of its indispensability for progression of the virus to the lytic replication cycle in the context of the viral genome. In the present communication, replication defective BoHV-4-V.test IE2 mutants were efficiently rescued, with respect to production of infectious virus and DNA replication, upon the expression of BoHV-4 ORF50/Rta in trans. Surprisingly, in the course of our studies, we discovered that the IE2 gene is duplicated in the genome of BoHV-4-U.     

Persistent infection of bovine herpesvirus type 4 in bovine endothelial cell cultures

Herpesviruses can establish a persistent infection in the cells and tissues of their natural hosts and thus may produce diseases due to cytolytic infections. We have isolated a herpesvirus from a bovine vascular endothelial cell culture after continuous subculturing. Typical cytopathic changes were observed in bovine endothelial cell cultures 2 days after inoculation of the virus. The virus had an icosahedral nucleocapsid of 100-150 nm in diameter and an envelope. The sequences of some DNA fragments of the virus were highly homologous to those of the bovine herpesvirus type 4 (BHV-4) strains. The DNA restriction maps of the virus and the reference strains of BHV-4, DN 599 and Movar 33/63 were very similar but not identical. Therefore, the newly isolated virus has been designated Taiwan strain. The presence of BHV-4 DNA in apparently normal bovine endothelial cell cultures was shown by Southern blot hybridization with the BamHI fragment of the newly isolated BHV-4 and was further confirmed by digestion of the DNA with BamHI plus AccI. In conclusion, we have demonstrated that BHV-4 persisted in the bovine endothelial cell cultures and continuous subcultures could lead to the production of infectious viral particles.     

Further studies of naturally occurring latent bovine herpesvirus infection

Attempts were made to enhance the isolation rate of latent bovine herpesviruses from trigeminal ganglia by using a fibroblastic fetal bovine kidney cell line, in addition to Madin-Darby bovine kidney cells, and by superinfecting with a temperature-sensitive helper strain of bovine herpesvirus-1 (BHV-1). Four isolates of latent BHV-1 were obtained from 44 pairs of trigeminal ganglia--thus reflecting no increase in isolation rate over that previously observed. Two isolates of a latent bovine herpesvirus-3 strain DN599 were also obtained. Analysis of plaque morphology of BHV-1 isolates obtained in this and a previous study indicated that several biologically heterogeneous strains were capable of establishing latent infections.