A new molecular method for the rapid subtyping of bovine herpesvirus 1 field isolates.

Bovine herpesvirus 1 (BoHV-1) causes several clinical syndromes in cattle worldwide. There are 3 subtypes of BoHV-1: 1.1, 1.2a, and 1.2b. Several molecular methods are commonly used in the detection and characterization of BoHV-1. Among them, restriction endonuclease analysis (REA) and single-nucleotide polymorphism (SNP) analysis of the complete viral genome allow classification of BoHV-1 into different subtypes. However, developing countries need simpler and cheaper screening assays for routine testing. We designed a standard multiplex PCR followed by a REA assay allowing straightforward subclassification of all BoHV-1 isolates tested into 1.1, 1.2a, and 1.2b subtypes based on the analysis of fragment length polymorphism. Our standard multiplex PCR-REA was used to analyze 33 field strains of BoHV-1 isolated from various tissues. The assay confirmed the subtype identified previously by REA. In addition, non-polymorphic or undigested fragments were sequenced in order to confirm the mutation affecting the RE HindIII site. Our PCR-REA method is an affordable and rapid test that will subtype all BoHV-1 strains.

First report of isolation and molecular characterization of bubaline herpesvirus 1 (BuHV1) from Argentinean water buffaloes.

Herpesviruses have mainly co-evolved with their hosts for millions of years. However, bovine herpesvirus 1 (BoHV1) and related ruminant alphaherpesviruses have been reported to cross the species barrier. Bubaline herpesvirus 1 (BuHV1) is an alphaherpesvirus closely related to BoHV1 and BoHV5. According to the serological cross-relationships between ruminant alphaherpesviruses, several surveys have studied the occurrence of BoHV1-related virus infection in wild and domestic ruminant species. Recent studies in Argentina showed an increase in serological prevalence against BoHV1 related viruses in water buffaloes (Bubalus bubalis) population. The aim of this study was to investigate the presence of related ruminant alphaherpesvirus in the Argentinean water buffalo population. BuHV1 was successfully isolated from 5 out of 225 buffaloes analyzed. One isolate was obtained from nasal secretions, and the others were from vaginal swabs. The buffaloes belonged to four different farms located in northeastern Argentina. The isolates were characterized as alphaherpesvirus by direct immunofluorescence using FITC-anti-BoHV1 IgG. Restriction analysis performed with BamHI and BstEII on the complete genome showed differences between the isolates and those from BoHV1 and BoHV5 subtypes. Phylogenetic analysis on both UL27 and US6 showed similarity in tree topology. While three of the isolates grouped together with sequences of BoHV5, two other isolates clustered separately. Genetic analysis of eight concatenated sequences from all isolates and references strains showed high nucleotide sequence identity between BuHV1 and BoHV5. While three of the isolates clustered together with the BoHV5 reference strain, the last two isolates were closely related to an Australian BuHV1 strain. To our knowledge, this is the first report on the isolation and molecular characterization of BuHV1 in South America. Phylogenetic analysis suggested that two different BuHV1 lineages circulate in the Argentinean water buffalo population.

Isolation and characterization of bovine parainfluenza virus type 3 from water buffaloes (Bubalus bubalis) in Argentina.

BACKGROUND: Parainfluenza virus type 3 (PIV3) was isolated from dairy buffaloes (Bubalus bubalis) naturally affected with respiratory and reproductive clinical conditions. RESULTS: Examination of nasal and vaginal swabs collected from 12 diseased buffaloes led to the isolation of three paramyxovirus isolates from two animals. Antigenic, morphological and biological characteristics of these three isolates were essentially similar to those of members of the Paramyxoviridae family. Antigenic analysis by direct immunofluorescence and cross neutralization test placed these isolates together with bovine parainfluenza virus type 3 (BPIV3). Nucleotide and amino acid phylogenetic analysis of partial matrix gene sequences of the buffalo isolates and six field BPIV3 isolates from bovines in Argentina were studied. Buffalo isolates were similar to genotype B (BPIV3b) while the six BPIV3 isolates were similar to genotypes A (BPIV3a) and C (BPIV3c). CONCLUSIONS: This is the first characterization of BPIV3 in water buffalo.According to the samples analyzed, in Argentina, the genotype B was found in buffalo and the genotypes A and C were found in cattle.

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.

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.

Characterization of BoHV-5 field strains circulation and report of transient specific subtype of bovine herpesvirus 5 in Argentina.

BACKGROUND: Bovine herpesvirus 5 (BoHV-5) is a member of the subfamily Alphaherpesvirinae responsible for meningo-encephalitis in young cattle. The first case of bovine meningo-encephalitis associated with a herpesvirus infection was reported in Australia. The current geographical distribution of BoHV-5 infection is mainly restricted to South America, especially Brazil and Argentina. Outbreaks of BoHV-5 are regularly observed in Argentina suggesting the circulation of the virus in the bovine population. RESULTS: Seventeen field strains of BoHV-5 isolated from 1984 to now were confirmed by differential PCR and subjected to restriction endonuclease analysis (REA). Viral DNA was cleaved with BstEII which allows the differentiation among subtypes a, b and non a, non b. According to the REA with BstEII, only one field strain showed a pattern similar to the Argentinean A663 strain (prototype of BoHV-5b). All other isolates showed a clear pattern similar to the Australian N569 strain (prototype of BoHV-5a) consistent with the subtypes observed in Brazil, the other South-American country where BoHV-5 is known to be prevalent. The genomic region of subtype b responsible for the distinct pattern was determined and amplified by PCR; specifically a point mutation was identified in glycoprotein B gene, on the BstEII restriction site, which generates the profile specific of BoHV-5b. CONCLUSIONS: This is the first report of circulation of BoHV-5a in Argentina as the prevailing subtype. Therefore the circulation of BoHV-5b was restricted to a few years in Argentina, speculating that this subtype was not able to be maintained in the bovine population. The mutation in the gB gene is associated with the difference in the restriction patterns between subtypes "a" and "b".

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.