Proteomic and phosphoproteomic analyses reveal several events involved in the early stages of bovine herpesvirus 1 infection.

Herpesviruses are predicted to express more than 80 proteins during their infection cycle. The proteins synthesized by the immediate early genes and early genes target signaling pathways in host cells that are essential for the successful initiation of a productive infection and for latency. In this study, proteomic and phosphoproteomic tools showed the occurrence of changes in Madin-Darby bovine kidney cells at the early stage of the infection by bovine herpesvirus 1 (BoHV-1). Proteins that had already been described in the early stage of infection for other herpesviruses but not for BoHV-1 were found. For example, stathmin phosphorylation at the initial stage of infection is described for the first time. In addition, two proteins that had not been described yet in the early stages of herpesvirus infections in general were ribonuclease/angiogenin inhibitor and Rab GDP dissociation inhibitor beta. The biological processes involved in these cellular responses were repair and replication of DNA, splicing, microtubule dynamics, and inflammatory responses. These results reveal pathways that might be used as targets for designing antiviral molecules against BoHV-1 infection.

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.

Distribution of bovine alpha-herpesviruses and expression of toll-like receptors in the respiratory system of experimentally infected calves.

This study provides an initial analysis of the toll-like receptors (TLRs) that might be implicated in alpha-herpesvirus infection of the bovine respiratory system. A significant variation in the expression of TLR3 and TLRs 7-9 during bovine herpesvirus type 1 (BoHV-1) and 5 (BoHV-5) acute infections and particularly an up-regulation during viral reactivation in respiratory tissues has been demonstrated. Furthermore, viral distribution in the respiratory tract of BoHV-1- and BoHV-5-infected calves at different stages of the infectious cycle was analysed. The wide distribution of BoHV DNA in the respiratory tract during acute infection was restricted during latent infection and the subsequent reactivation of BoHV-1 and BoHV-5. Overall, the findings presented here contribute to the knowledge on the replication and dissemination of bovine alpha-herpesviruses. Furthermore, some of the immune factors triggered in the host that determine the different outcomes of infection by two closely related pathogens of cattle have been elucidated.

Protection induced by a glycoprotein E-deleted bovine herpesvirus type 1 marker strain used either as an inactivated or live attenuated vaccine in cattle.

BACKGROUND: Bovine herpesvirus type 1 (BoHV-1) is the causative agent of respiratory and genital tract infections; causing a high economic loss in all continents. Use of marker vaccines in IBR eradication programs is widely accepted since it allows for protection of the animals against the disease while adding the possibility of differentiating vaccinated from infected animals.The aim of the present study was the development and evaluation of safety and efficacy of a glycoprotein E-deleted (gE-) BoHV-1 marker vaccine strain (BoHV-1ΔgEßgal) generated by homologous recombination, replacing the viral gE gene with the ß-galactosidase (ßgal) gene. RESULTS: In vitro growth kinetics of the BoHV-1ΔgEßgal virus was similar to BoHV-1 LA. The immune response triggered by the new recombinant strain in cattle was characterized both as live attenuated vaccine (LAV) and as an inactivated vaccine. BoHV-1ΔgEßgal was highly immunogenic in both formulations, inducing specific humoral and cellular immune responses. Antibody titers found in animals vaccinated with the inactivated vaccine based on BoHV-1ΔgEßgal was similar to the titers found for the control vaccine (BoHV-1 LA). In the same way, titers of inactivated vaccine groups were significantly higher than any of the LAV immunized groups, independently of the inoculation route (p < 0.001). Levels of IFN-γ were significantly higher (p < 0.001) in those animals that received the LAV compared to those that received the inactivated vaccine. BoHV-1ΔgEßgal exhibited an evident attenuation when administered as a LAV; no virus was detected in nasal secretions of vaccinated or sentinel animals during the post-vaccination period. BoHV-1ΔgEßgal, when used in either formulation, elicited an efficient immune response that protected animals against challenge with virulent wild-type BoHV-1. Also, the deletion of the gE gene served as an immunological marker to differentiate vaccinated animals from infected animals. All animals vaccinated with the BoHV-1ΔgE ßgal strain were protected against disease after challenge and shed significantly less virus than control calves, regardless of the route and formulation they were inoculated. CONCLUSIONS: Based on its attenuation, immunogenicity and protective effect after challenge, BoHV-1ΔgEßgal virus is an efficient and safe vaccine candidate when used either as inactivated or as live attenuated forms.

Antiherpetic activity of an Agaricus brasiliensis polysaccharide, its sulfated derivative and fractions.

Agaricus brasiliensis is an edible mushroom, traditionally used for the treatment of several diseases. In this paper, a polysaccharide (PLS) from A. brasiliensis, its carboxymethylated (CPLS) and sulfated (SPLS) derivatives, as well as, fractions (F1-F3) obtained from the PLS were investigated for their effect in the replication of herpes simplex virus and bovine herpes virus in HEp-2 cell cultures. The PLS, SPLS and F3 inhibited both virus strains similarly, in a dose-dependent curve. F1, F2 and CPLS did not show significant effect even at higher concentrations. All the compounds showed neither virucidal or viral adsorption inhibition activities nor effect when cells were treated prior to infection. Our study demonstrated that the extracts of A. brasiliensis, can be promising for future antiviral drug design and its biotechnological production is economically feasible.

Immunogenicity of a bovine herpes virus I peptide expressed in tandem copies in attenuated Salmonella.

A live system to release heterologous antigens using an attenuated Salmonella strain was developed. We transformed Salmonella typhimurium LVR03 (S. LVR03) with a recombinant pTECH2 vector encoding 0, 1, 2, and 4 tandem copies of an imunogenic peptide of bovine herpes virus-1 (BoHV-1) glycoprotein D (gD). The system used yielded peptides fused to the non-toxic C fragment of the tetanus toxin (TetC), which has been shown to have adjuvant properties. Inoculation of BALB/c mice with the transformed Salmonella strains gave rise to a mild self-limited infection, with primary replication of bacteria occurring in Peyer's patches, even when the bacteria was administered intranasally. Humoral and cellular immune responses directed against the BoHV-1 antigens were evaluated after oral or intranasal administration of the recombinant bacteria. The results showed that the S. LVR03-dimer vaccine induced specific humoral (IgG in serum and IgG(1) and IgA in saliva), and cellular immune responses (lymphoproliferation and lymphokine secretion), against not only the selected peptide and whole gD, but also against BoHV-1, when administered intranasally. This is the first time Salmonella has been used as an expression vector to induce immunity against BoHV-1. This work demonstrates the feasibility of using this antigen-release system and encourages future experimentation with a bovine experimental model.