Comparison of 11 herpesvirus isolates from tortoises using partial sequences from three conserved genes

Abstract

Herpesviruses are an important cause of epidemic disease in tortoises. There are at least two serologically distinct herpesviruses capable of infecting tortoises. Methods for the diagnosis of herpesvirus infections in tortoises include virus isolation and a number of different PCRs. We have compared 11 virus isolates collected from various species in different countries over several years using sequences from three different viral genes. During this study we used four different PCR protocols described for the diagnosis of herpesvirus infections in tortoises. The protocols used included two based on portions of the DNA polymerase gene, one targeting the UL5 homologue, and one targeting the UL39 homologue. Comparison of the methods showed that the tortoise herpesvirus-specific protocols were all serotype specific. Sequences of the obtained amplicons were compared with one another and with sequences of herpesviruses available in GenBank. The sequence alignments showed that the tortoise herpesviruses were most closely related to members of the subfamily Alphaherpesvirinae. They also showed that the tortoise isolates could be clearly divided into two genogroups.

Introduction

Herpesviruses cause an important epidemic disease in tortoises that seems to occur worldwide. It has been reported in association with diphtheroid-necrotizing stomatitis in Europe, the USA and South Africa and is often associated with high mortality rates. Infections appear to be most common in the European tortoises Testudo hermanni, T. horsfieldii, T. graeca and T. marginata, but have also been found in other species such as Gopherus agassizii, Goechelone pardalis and Geochelone chilensis (McArthur et al., 2002).

There are a number of tests available for the diagnosis of herpesvirus infections in tortoises. These include serological tests, histological and direct virus detection methods. Direct virus detection has been carried out using virus isolation and polymerase chain reaction (PCR). Four different PCR protocols have been used for the amplification of portions of the genome of tortoise herpesviruses. The first PCR to be used to detect herpesviruses in tortoises targeted the DNA polymerase gene (Van Devanter et al., 1996, Une et al., 2000). This PCR was developed for the detection of a wide range of herpesviruses from many different animal species and uses degenerate primers and a nested format. Shortly after the successful use of this PCR for tortoise herpesviruses, Murakami et al. (2001) developed a tortoise herpesvirus-specific PCR targeting the same gene. Two other PCRs that have been published for the detection of herpesviruses in tortoises are based on the UL5 (DNA helicase/primase associated protein gene) homologue (Teifke et al., 2000) and the homologue of the ribonucleotide reductase gene (UL39) (Origgi et al., 2004).

Little is known about the classification or characterization of tortoise herpesviruses. Some work has been done, comparing 16 tortoise herpesviruses from different species and isolated in different years, on the basis of serology and restriction enzyme digestion patterns of viral DNA. In that study, plasma from herpesvirus-infected tortoises was used in neutralization tests with different herpesvirus isolates. One of the isolates studied was not neutralized by any of the sera used. This isolate also showed distinct differences to the other isolates in the restriction enzyme profiles (Marschang et al., 2001). Recent partial characterization of a herpesvirus from a California desert tortoise (G. agassizii) based on partial sequences of the DNA polymerase gene and the UL39 homologue showed that this virus differed from other tortoise herpesviruses (Johnson et al., 2005). Information available so far supports the classification of tortoise herpesviruses in the subfamily Alphahepesvirinae of the family Herpesviridae (Teifke et al., 2000, Une et al., 2000, Origgi et al., 2004). But so far, only DNA sequences from a limited number of tortoise herpesvirus isolates have been made available. These include the partial sequences of the DNA polymerase gene and the UL39 homologue from a California desert tortoise (GenBank accession nos. AY916792 and DQ027825), partial sequences from the DNA polymerase gene from a tortoise from Japan (GenBank accession no. AB047545) and from the UL5 homologue from a tortoise from the United Kingdom (GenBank accession no. AY188757) as well as partial sequences from the UL36 and UL40 homologues and complete sequences from the UL37, UL38 and UL39 homologues from a tortoise from the USA (GenBank accession no. AY338245).

The objective of this work was to compare tortoise herpesviruses isolated from different species in different years and different countries. Further, the four PCR methods described for the detection of herpesviruses in tortoises were compared.