Abstract
BackgroundSeveral types and subtypes of bovine herpesviruses 1 and 5 (BoHV-1 and BoHV-5) have been associated to different clinical conditions of cattle, making type/subtype differentiation essential to understand the pathogenesis and epidemiology of BoHV infections. BoHV-5 subtyping is currently carried out by BstEII restriction enzyme analysis (REA) of the complete virus genome. This method allowed the description of three subtypes, one of which is the most widespread while the remaining two have so far only been found in South America. The present work describes a multiplex PCR followed by REA for BoHV-5 subtyping.ResultsThe method consists in the simultaneous amplification of glycoprotein B and UL54 gene fragments of 534 and 669 base pairs (bp), respectively, BstEII digestion of amplicons, separation of products in 1% agarose gels, and analysis of fragment length polymorphims. The multiplex PCR detected up to 227 BoHV-5 genome copies and 9.2 × 105 BoHV-5 genome copies when DNA was extracted from purified virus or infected tissue homogenates, respectively. The applicability of multiplex PCR-REA was demonstrated on 3 BoHV-5 reference strains. In addition, subtyping of two new isolates and seventeen previously reported ones (17 BHV-5a and 2 BHV-5b) by this method gave coincident results with those obtained with the classic BstEII REA assay.ConclusionsMultiplex PCR-REA provides a new tool for the fast and simple diagnosis and subtyping of BoHV-5.
Highlights
Several types and subtypes of bovine herpesviruses 1 and 5 (BoHV-1 and Bovine herpesvirus 5 (BoHV-5)) have been associated to different clinical conditions of cattle, making type/subtype differentiation essential to understand the pathogenesis and epidemiology of BoHV infections
BoHV-5 infection and disease appear to be more frequent in Argentina and Brazil, where numerous outbreaks were described in the last decades [7,8,9,10,11]
We report the identification and characterization of two new BoHV-5 isolates from the Argentinean Provinces of Buenos Aires and Chaco
Summary
Several types and subtypes of bovine herpesviruses 1 and 5 (BoHV-1 and BoHV-5) have been associated to different clinical conditions of cattle, making type/subtype differentiation essential to understand the pathogenesis and epidemiology of BoHV infections. BoHV-5 subtyping is currently carried out by BstEII restriction enzyme analysis (REA) of the complete virus genome. This method allowed the description of three subtypes, one of which is the most widespread while the remaining two have so far only been found in South America. BoHV-5 infection induces either a subclinical infection or disease of moderate severity in adult cattle [15] and lethal encephalitis in young animals [6,7,16]. In light of recent reports of bovine herpesviruses isolated from different samples, and responsible for both symptomatic and asymptomatic infections [11,17,18], classical diagnostic methods are not sufficient for a fast and easy identification and subtyping of the infectious virus
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