Alejandro Rafael Valera

Recombinant MVA Expressing Secreted Glycoprotein D of BoHV-1 Induces Systemic and Mucosal Immunity in Animal Models

Bovine herpesvirus-1 (BoHV-1) infection is distributed worldwide and the development of new tools to fight against this pathogen has become extremely important. In this work a recombinant modified vaccinia virus Ankara (MVA) vector expressing the secreted version of glycoprotein D, MVA-gDs, was obtained and evaluated as a candidate vaccine. First, the correct expression, antigenicity, and N-glycosylation of glycoprotein D were confirmed by molecular techniques. Then MVA-gDs was used as parenteral immunogen in BALB/C mice in which a specific anti-gD humoral immune response was induced and maintained for 7 mo. Two doses of MVA-gDs supplemented with cholera toxin delivered by intranasal immunization induced IgA anti-gD humoral immune responses in nasal and bronchopulmonary washes, as well as IgG anti-gD antibodies in serum samples. In order to evaluate the protection conferred by MVA-gDs immunization, a rabbit BoHV-1 challenge assay was performed. A shorter viral excretion period and a reduction in the number of animals shedding BoHV-1 was observed in the group immunized with recombinant MVA-gDs. In conclusion our data encourage further studies to evaluate MVA-gDs, alone or combined with other immunogens, as a candidate vaccine for BoHV-1.

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.

Identification and molecular characterization of Orf virus in Argentina

Abstract Orf virus (ORFV) is the etiological agent of contagious ecthyma (CE), a pustular dermatitis of sheep and goats. Outbreaks of ORFV have been observed in all geographical regions of the world, including Argentina. The origin and identity of Argentinian ORFVs are unknown, and no comparative or phylogenetic studies of these viruses have been performed. In this study, we described the sequencing and analysis of five ORFV molecular markers: a partial B2L gene (ORF011), VIR (ORF020), an envelope mature protein (ORF109), vIL10 (ORF127), and GIF (ORF117) from two particular Argentinian outbreaks of CE.

A rapid and sensitive diagnosis of bovine leukaemia virus infection using the nested shuttle polymerase chain reaction

Bovine leukaemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL). In Argentina, where a program to eradicate EBL has been introduced, sensitive and reliable diagnosis has attained high priority. Although the importance of the agar gel immunodiffusion test remains unchanged for routine work, an additional diagnostic technique is necessary to confirm cases of sera with equivocal results or of calves carrying maternal antibodies.Utilizing a nested shuttle polymerase chain reaction, the proviral DNA was detected from cows experimentally infected with as little as 5 ml of whole blood from BLV seropositive cows that were nonetheless normal in haematological terms. It proved to be a very sensitive technique, since it rapidly revealed the presence of the provirus, frequently at 2 weeks postinoculation and using a two-round procedure of nested PCR taking only 3 hours. Additionally, the primers used flanked a portion of the viral genome often employed to differentiate BLV type applying BamHI digestion. It is concluded that this method might offer a highly promising diagnostic tool for BLV infection.

Genomic study of Argentinean Equid herpesvirus 1 strains

Equid herpesvirus 1 (EHV-1) infection has a significant economic impact on equine production, causing abortion, respiratory disease, neonatal death and neurological disorders. The identification of specific EHV-1 genes related to virulence and pathogenicity has been the aim of several research groups. The purpose of the present study was to analyze different genomic regions of Argentinean EHV-1 strains and to determine their possible relationship with virulence or clinical signs. Twenty-five EHV-1 Argentinean isolates recovered from different clinical cases between 1979 and 2007 and two reference strains were amplified and sequenced. The sequence alignments were carried out using Clustal X version 1.92 and the putative amino acid sequences were deduced using Bio-Edit version 7.05. Minor changes were observed. No changes that could be involved in the different virulence in the mouse model of three EHV-1 Argentinean strains were found. No genetic variants were observed. The genomic regions analyzed are unsuitable for differentiation between abortigenic strains and those isolated from neonatal deaths.

First isolation and nucleotide comparison of the gag gene of the caprine arthritis encephalitis virus circulating in naturally infected goats from Argentina

Caprine arthritis encephalitis virus (CAEV) has been reported in different countries worldwide, based on serological and molecular detection. In Argentina, the prevalence of CAEV infections is increasing, with goats showing symptoms associated mostly with cachexia and arthritis. Although in Argentina the virus has been detected by serology, it has never been isolated or characterized. Thus, the objectives of this work were to isolate and analyze the nucleotide sequences of the gag gene of Argentine CAEV strains and compare them with those of other SRLVs previously reported. Nucleotide sequence comparison showed homology with CAEV-Co, the CAEV prototype. Phylogenetic analyses showed that the Argentine strains clustered with genotype B, subtype B1. Because the molecular characterization of the gag region is suitable for phylogenetic studies and may be applied to monitor the control of SRLV, molecularly characterizing the Argentine CAEV strains may help develop a proper plan of eradication of CAEV infections.

Systemic infection induced by intranasal inoculation of Bovine herpesvirus 1.1 in pregnant and non-pregnant rabbits

Bovine herpesvirus (BoHV) type 1.1 (BoHV-1.1) causes repeated outbreaks of upper respiratory disease and abortion in cattle. The systemic effects of BoHV-1.1 in rabbits, using intranasal inoculation are reported. Female rabbits were divided into four groups and inoculated with the virus 10 days before mating, and at 15 or 22 days of pregnancy. Studies of the clinical signs, antibody production, virus isolation, and DNA detection as well as histological and immunohistochemical studies were carried out on lungs, kidneys, spleen, placentas, uteri and foetal tissues. All virus-inoculated animals developed respiratory clinical signs and a humoral response. BoHV-1.1 was isolated from nasal swabs and plasma rich in leukocytes, and viral DNA was detected in blood, dead foetuses and placentas. Histopathological lesions were found in the respiratory tract and some placentas and foetuses were immunohistochemically positive. Intranasal inoculation might be useful to study the systemic effects of BoHV-1.1 infection in the rabbit model.