Studies of genetic relationships between bovine, caprine, cervine, and rangiferine alphaherpesviruses and improved molecular methods for virus detection and identification

Abstract

The glycoprotein B (gB) and D (gD) genes from five ruminant alphaherpesviruses, bovine herpesvirus 1 (BHV-1), bovine herpesvirus 5 (BHV-5), caprine herpesvirus 1 (CapHV-1), cervine herpesvirus 1, and rangiferine herpesvirus 1, were partially sequenced. The nucleotide sequence alignments revealed a highly conserved gB gene, with homologies ranging between 87.2 and 99.6%, and a more variable gD gene, with homologies ranging between 71.3 and 98.9%. The phylogenetic analysis of the gB and gD nucleotide and deduced amino acid sequences revealed that BHV-5 is the most closely related virus to the BHV-1 subtype 1 and BHV-1 subtype 2 cluster and that CapHV-1 is the most distantly related virus. The phylogenetic data showed a close relationship of all the studied viruses with suid herpesvirus 1. On the basis of sequence data for the gB gene, a nested PCR combined with restriction enzyme analysis (REA) of the PCR products was developed for the simultaneous detection and identification of the viruses that were studied. Nested primers from highly conserved sequence stretches were selected in order to amplify a region of 294 bp in all five viruses, and a subsequent REA of the PCR products allowed specific identification. A mimic molecule that served as an internal standard of the amplification efficiency was constructed. The practical diagnostic applicability of the assay was evaluated with clinical samples consisting of semen and organ specimens from experimentally infected animals.

FIG. 1

Alignment of the deduced amino acid sequences of a region from the gB and gD proteins in six ruminant alphaherpesviruses. The sequence of PrV is also included in the alignment; the gB sequence is from Robbins et al. (31), and the gD sequence is from Petrovskis et al. (27). A thick line under the sequences indicates highly conserved regions (more than 4 consecutive amino acids).

FIG. 2

Nucleotide and amino acid sequence similarities for the gB and gD regions of ruminant alphaherpesviruses and PrV.

FIG. 3

Phylogenetic trees of ruminant alphaherpesvirus based on the nucleotide sequences of the gB and gD regions. Tree A was generated by the neighbor-joining method, and tree B was generated by the parsimony method. The lengths of the branches in the tree inferred by the neighbor-joining method reflect phylogenetic distances. The numbers on trees A and B indicate the percentage of times that the particular group was predicted after statistical analysis by bootstrapping. PrV was used as the outgroup.

FIG. 4

(A) PCR amplification of ruminant alphaherpesvirus DNA with the mimic molecule and posterior identification by REA. Lane 1, positive PCR product from a ruminant alphaherpesvirus (BHV-1 as a model) before REA; lanes 2 to 6, positive PCR products after REA; lane 2, CapHV-1; lane 3, RanHV-1; lane 4, BHV-1; lane 5, BHV-5; lane 6, CerHV-1. The bands under 100 bp in length are minor residual digestion products. Lanes M, marker, 100-bp ladder. (B) PCR and REA of ruminant alphaherpesvirus DNA amplified directly from trigeminal ganglia. Lanes 2, 6 and 10, BHV-1; lanes 1 and 8, BHV-5; lanes 3 and 4, CapHV-1; lanes 5 and 11, CerHV-1; lanes 7, 9, and 12, RanHV-1. In each lane the upper band corresponds to the digested amplified mimic molecule and the lower band corresponds to the digested amplified viral DNA. Lanes M, marker, 100-bp ladder.

FIG. 5

PCR results for frozen extended semen samples from five bulls experimentally infected with BHV-1. Postinfection days in boldface type indicate the days of dexamethasone treatment. ND, not done. The lower box expresses the results as a diagram.