Antigen processing and presentation.

Dendritic cells are at the center of immune responses. They are defined by their ability to sense the environment, take up and process antigen, migrate to secondary lymphoid organs, where they present antigens to the adaptive immune system. In particular, they present lipids and proteins from pathogens, which they encountered in peripheral tissues, to T cells in order to induce a specific effector immune response. These complex antigens need to be broken down into peptides of a certain length in association with Major Histocompatibility Complex (MHC) molecules. Presentation of MHC/antigen complexes alongside costimulatory molecules and secretion of proinflammatory cytokines will induce an appropriate immune response. This interaction between dendritic cells and T cells takes place at defined locations within secondary lymphoid organs. In this review, we discuss the current knowledge and recent advances on the cellular and molecular mechanisms that underlie antigen processing and the subsequent presentation to T lymphocytes.

Genomic characterization of canine circovirus associated with fatal disease in dogs in South America.

Canine circovirus (CanineCV) was detected, together with canine parvovirus (CPV), in samples from an outbreak of fatal gastroenteritis in dogs in Argentina. We obtained the full-length genome of this recently discovered virus by overlapping PCR, designated strain UBA-Baires. Sequence analysis revealed a highly conserved genome but also showed several unique mutations in amino acids from the capsid protein that have not been previously reported. Phylogenetic analysis shows that this strain is more closely related to European strains than to viruses detected in North America or Asia. Although the pathogenic role of CanineCV in dogs is still unclear, this study highlights the importance of CanineCV as a coinfecting virus in disease development. To our knowledge, this is the first report of the involvement of CanineCV in severe clinical disease in dogs in South America. Our results expand our information on the geographical extent of this virus and contribute to the understanding of its role in disease.

Comparative study on the in vitro and in vivo properties of two bovine herpesvirus-5 reference strains.

BACKGROUND: Bovine herpesvirus 5 (BoHV-5) is an alphaherpesvirus responsible for meningoencephalitis in young cattle and it is antigenically and genetically related to bovine herpesvirus 1. BoHV-5 outbreaks are sporadic and restricted in their geographical distribution, being mostly detected in the Southern hemisphere. The N569 and A663 strains are prototypes of the "a" and "b" subtypes of BoHV-5, however, scarce information about their in vitro and in vivo properties is currently available. METHODS: For the in vitro comparison between BoHV-5 A663 and N569 strains, viral growth kinetics, lysis and infection plaque size assays were performed. Additionally, an experimental infection of cattle with BoHV-5 A663 and N569 strains was carried out. Viral excretion, development of neurological signs, presence of specific antibodies in serum and nasal swabs and presence of latent BoHV-5 DNA in trigeminal ganglion, were analyzed. Histopathological examination of samples belonging to inoculated animals was also performed. RESULTS: The lytic capacity and the cell-to-cell spread was lower for the A663 strain compared to the N569 strain, however, the production of total infectious viral particles was similar between both strains. Concerning the in vivo properties, the A663 and N569 strains are able to induce similar degrees of pathogenicity in cattle. CONCLUSIONS: Our results show that the A663 strain used in this study is less adapted to in vitro replication in MDBK cells than the N569 strain and, although slight differences were observed, both strains are able to induce a similar degree of virulence in the natural host.

Effect of the US3 protein of bovine herpesvirus 5 on the actin cytoskeleton and apoptosis.

The US3 protein is a unique protein kinase only present in the Alphaherpesvirinae subfamily of the herpesviruses. Studies performed with several alphaherpesviruses demonstrated that the US3 protein is involved in cytoskeleton modifications during viral infection and displays anti-apoptotic activity. However, the US3 protein of BoHV-5 has not been studied up to now. As reported for other alphaherpesviruses, our results showed that BoHV-5 US3 confers resistance against apoptosis and induces cytoskeletal reorganization leading to cell rounding, actin stress fiber breakdown and cell projections that interconnect cells. The expression of a kinase-dead version of BoHV-5 US3 showed that the anti-apoptotic activity and the induction of cell projections are kinase-dependent whereas kinase activity is not absolutely required for actin stress fiber breakdown. Besides, the kinase-dead version of US3, but not the wild type protein, was found excluded from the nucleus. These results constitute the first report on the BoHV-5 US3 functions, and highlight that there are functional differences and similarities among US3 proteins of different alphaherpesviruses.

In vitro-generated interspecific recombinants between bovine herpesviruses 1 and 5 show attenuated replication characteristics and establish latency in the natural host.

BACKGROUND: Interspecific recombinant viruses R1ΔgC and R2ΔgI were isolated after in vitro co-infection with BoHV-1 and BoHV-5, two closely related alphaherpesviruses that infect cattle. The genetic characterization of R1ΔgC and R2ΔgI showed that they are composed of different sections of the parental genomes. The aim of this study was the characterization of the in vivo behavior of these recombinants in the natural host. RESULTS: Four groups of four 3-month-old calves of both genders were intranasally inoculated with either the recombinant or parental viruses. A control group of two animals was also included. Viral excretion and clinical signs were monitored after infection. Histopathological examination of the central nervous system (CNS) was performed and the establishment of latency in trigeminal ganglia was analyzed by PCR. The humoral response was also evaluated using ELISA tests. Three out of four animals from the BoHV-5 infected group excreted virus for 4-10 days. Two calves shed R1ΔgC virus for one day. In R2ΔgI and BoHV-1.2ΔgCΔgI groups, infectious virus was isolated only after two or three blind passages. None of the infected animals developed neurological signs, although those infected with BoHV-5 showed histopathological evidence of viral infection. Latent viral DNA was detected in at least one calf from each infected group. Serum and/or mucosal antibodies were detected in all groups. CONCLUSION: Both BoHV-1/-5 recombinants and the BoHV-1 parental strain are attenuated in calves, although they are able to replicate in animals at low rates and to establish latent infections.

Biology of bovine herpesvirus 5.

Bovine herpesvirus 5 (BoHV-5) is an alphaherpesvirus responsible for meningoencephalitis in young cattle and is closely antigenically and genetically related to bovine herpesvirus 1 (BoHV-1). Both viruses have common aspects in their pathogenesis: (1) they infect epithelial cells at the portal of entry and (2) they establish a latent infection in the sensory nerve ganglia, i.e., the trigeminal ganglia. However, they have different neuroinvasion and neurovirulence capacities. Only in rare cases can BoHV-1 reach the brain of infected cattle. BoHV-5 infection induces different degrees of severity of neurological disease depending on both viral and host factors. Although a case of BoHV-5 associated disease in Europe and some outbreaks in USA and Australia have been reported, the current geographical distribution of BoHV-5 infection is mainly restricted to South America, especially Brazil and Argentina. This review focuses on the genomic characteristics, pathobiology and epidemiology of BoHV-5, in order to provide information on the possible basis of alphaherpesvirus neuropathogenesis.

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.