Penaeus monodon shrimp collected from across the Indo-Pacific region during 1997-2004 were screened for the presence of yellow head-related viruses. Phylogenetic analyses of amplified ORF1b gene segments identified at least six distinct genetic lineages (genotypes). Genotype 1 (YHV) was detected only in shrimp with yellow head disease. Genotype 2 (GAV) was detected in diseased shrimp with the less severe condition described as mid-crop mortality syndrome and in healthy shrimp from Australia, Thailand and Vietnam. Other genotypes occurred commonly in healthy shrimp. Sequence comparisons of structural protein genes (ORF2 and ORF3), intergenic regions (IGRs) and the long 3'-UTR supported the delineation of genotypes and identified both conserved and variant structural features. In putative transcription regulating sequences (TRSs) encompassing the sub-genomic mRNA 5'-termini, a core motif (5'-GUCAAUUACAAC-3') is absolutely conserved. A small (83 nt) open reading frame (ORF4) in the 3'-UTR of GAV is variously truncated in all other genotypes and a TRS-like element preceding ORF4 is invariably corrupted by a A>G/U substitution in the central core motif (5'-UU(G/U)CAAC-3'). The data support previous evidence that ORF4 is a non-functional gene under construction or deconstruction. The 3'-UTRs also contain predicted 3'-terminal hairpin-loop structures that are preserved in all genotypes by compensatory nucleotide substitutions, suggesting a role in polymerase recognition for minus-strand RNA synthesis.

Yellow head virus (YHV) is a highly virulent pathogen of penaeid shrimp. An isolate obtained from Penaeus vannamei during a yellow head disease outbreak in February 2006 in Ratchaburi Province, Thailand was purified following passage in experimentally infected shrimp. SDS-PAGE of purified virions indicated that envelope glycoprotein gp116 in the Ratchaburi/2006 isolate was smaller and relatively less abundant than in the Chachoengsao/1998 YHV reference strain. The variant gp116 reacted poorly in immunoblots with a gp116 mouse monoclonal antibody and a rabbit anti-serum to a baculovirus-expressed, C-terminally truncated, [His](6)-tagged gp116 that reacted strongly with gp116 of the homologous Chachoengsao/1998 strain. The anti-gp116 polyclonal serum also failed to neutralise the infectivity of the Ratchaburi/2006 isolate in in-vivo assays conducted in P. vannamei, but effectively neutralised the infectivity of the reference strain. Sequence analysis of the approximately 6.0 kb structural protein gene region and 3'UTR of the Ratchaburi/2006 isolate indicated >99.9% overall nucleotide identity with the Chachoengsao/1998 strain. However, in Ratchaburi/2006 a deletion in ORF3, corresponding to 54 amino acids near the N-terminal signal peptidase cleavage site of gp116, resulted in the loss of six conserved cysteine residues and two predicted N-glycosylation sites. Analysis of this ORF3 region in 25 viruses representing each of the six genotypes in the yellow head complex identified this modified form of gp116 in two other virulent YHV isolates classified as genotype 1b. The data indicate that, although the deletion causes a significant structural deformation of gp116 which reduces its incorporation into virions and eliminates the major neutralisation sites, the virus remains highly infectious, virulent and fit for survival.

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Yellow head virus (YHV) is a highly virulent pathogen of Penaeus monodon shrimp. It is one of six known genotypes in the yellow head complex of nidoviruses which also includes mildly pathogenic gill-associated virus (GAV, genotype 2) and four other genotypes (genotypes 3-6) that have been detected only in healthy shrimp. In this study, comparative phylogenetic analyses conducted on replicase- (ORF1b) and glycoprotein- (ORF3) gene amplicons identified 10 putative natural recombinants amongst 28 viruses representing all six genotypes from across the Indo-Pacific region. The approximately 4.6 kb genomic region spanning the two amplicons was sequenced for three putative recombinant viruses from Vietnam (genotype 3/5), the Philippines (genotype 5/2) and Indonesia (genotype 3/2). SimPlot analysis using these and representative parental virus sequences confirmed that each was a recombinant genotype and identified a recombination hotspot in a region just upstream of the ORF1b C-terminus. Maximum-likelihood breakpoint analysis predicted identical crossover positions in the Vietnamese and Indonesian recombinants, and a crossover position 12 nt upstream in the Philippine recombinant. Homologous genetic recombination in the same genome region was also demonstrated in recombinants generated experimentally in shrimp co-infected with YHV and GAV. The high frequency with which natural recombinants were identified indicates that genetic exchange amongst genotypes is occurring commonly in Asia and playing a significant role in expanding the genetic diversity in the yellow head complex. This is the first evidence of genetic recombination in viruses infecting crustaceans and has significant implications for the pathogenesis of infection and diagnosis of these newly emerging invertebrate pathogens.

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