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. 2008 Oct 25;380(2):213-25.
doi: 10.1016/j.virol.2008.07.005. Epub 2008 Sep 2.

Genetic diversity in the yellow head nidovirus complex

Priyanjalie K M Wijegoonawardane  1 , Jeff A Cowley, Thuy Phan, Richard A J Hodgson, Linda Nielsen, Wansika Kiatpathomchai, Peter J Walker
Affiliations

Genetic diversity in the yellow head nidovirus complex

Priyanjalie K M Wijegoonawardane et al. Virology. .

Abstract

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.

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Figures

Fig. 1
Fig. 1
(A) 1% agarose-TAE gels illustrating RT-nested PCR products amplified from 16 of 19 healthy P. monodon broodstock (lanes 1–19) that were collected from central Thailand in March 2002. A semi-nested one-step RT-PCR and primers 2s12, 2a12 and 2a43 were used to amplify products of 280 and 575 bp. A DNA ladder (lane M) and positive control (YHV) reaction (lane +) are indicated. (B) Unrooted, neighbour-joining phylogenetic tree constructed from a ClustalX multiple alignment of a 231 nt sequence in the ORF1b replicase gene obtained for 25 viruses including the reference strains of YHV (THA-98-Ref) and GAV (AUS-96-Ref). The samples were collected from Thailand, Taiwan, Australia, Vietnam and Malaysia in 2000 and 2001. The sources of viruses and their codes are described in Table 1. Bootstrap values shown at key branch nodes were determined for 1000 replicates. Horizontal branch lengths indicate phylogenetic distance calculated as the number of nucleotide substitutions per total nucleotide residues. The clustering of viruses into three discrete genetic lineages (genotypes 1 to 3) is indicated.
Fig. 2
Fig. 2
Unrooted, neighbour-joining phylogenetic tree constructed from a ClustalX multiple alignment of a 668–671 nt sequence in the ORF1b replicase gene obtained for 57 viruses including the reference strains of YHV (THA-98-Ref) and GAV (AUS-96-Ref). The samples were collected from throughout the Indo-Pacific region from 1997–2004. The sources of viruses and their codes are described in Table 1. Bootstrap values shown at key branch nodes were determined for 1000 replicates. Horizontal branch lengths indicate phylogenetic distance calculated as the number of nucleotide substitutions per total nucleotide residues. The clustering of viruses into the six discrete genetic lineages (genotypes 1 to 6) is indicated.
Fig. 3
Fig. 3
(A) ClustalX multiple alignment of the deduced amino acid sequences of the nucleocapsid proteins encoded in ORF2 of the reference genotype 1 (THA-98-Ref) and genotype 2 (AUS-96-Ref) isolates and representatives of genotypes 3 (VNM-02-H93), 4 (IND-02-H9) and 5 (THA-03-SG21). The more variable sequences in the N- and C-terminal regions are shaded. (B) Neighbour-joining tree constructed from a Clustal X multiple alignment of the nucleotide sequence of the nucleocapsid protein genes. Bootstrap values were calculated for 1000 replicate analyses and horizontal branch lengths are proportional to phylogenetic distances.
Fig. 4
Fig. 4
A ClustalX multiple alignment of amino acid sequences spanning the N-terminal gp116 region of the ORF3 gene of the reference genotype 1 (THA-98-Ref) and genotype 2 (AUS-96-Ref) isolates, and representatives of genotypes 3 (VNM-02-H93), 4 (IND-02-H9) and 5 (THA-03-SG21). The site of proteolytic cleavage of the YHV pp3 polyprotein is indicated. Absolutely conserved (⁎) and similar (: or .) amino acids are indicated according to the similarity groups defined in ClustalX. Conserved cysteine and residues are indicated (+). Potential N-linked glycosylation sites are indicated in bold face and underlined in the alignment and denoted (formula image) on the illustration. Six predicted transmembrane-spanning domains (TM1–TM6) are numbered.
Fig. 5
Fig. 5
(A) Conserved sequences in the intergenic regions upstream of ORF2, ORF3 and ORF4 in the reference genotype 1 (THA-98-Ref) and genotype 2 (AUS-96-Ref) isolates and representatives of genotypes 3 (VNM-02-H93), 4 (IND-02-H9) and 5 (THA-03-SG21). The initiation and termination codons of flanking ORFs are indicated in bold face and underlined. The position in each sequence corresponding to the 5′-terminal adenosine of GAV sgmRNA1 and sgmRNA2 is shaded. The highly conserved 14 nt sequence surrounding the terminal adenosine is underlined, as is the region upstream of ORF4 in which the sequence is partially conserved. Nucleotide substitutions (uridine) in the conserved regions are shaded in black. Identical nucleotides in all aligned sequences are indicated (⁎). (B) WebLogo presentation of nucleotides conserved and less conserved between genotypes 1, 2, 3, 4 and 5 in the TRS elements in IGR1 and in IGR2 as well as between the TRS elements of both IGRs. The cDNA rather than RNA sequence was analysed. (C) Multiple alignment of the putative genotype 2 (GAV) ORF4 gene amino acid sequence with the comparable truncated sequences of genotypes 1, 3, 4 and 5. Amino acids shared in all 5 genotypes are indicated (⁎).
Fig. 6
Fig. 6
Conserved RNA secondary structures predicted using MFOLD to form in the 3′-UTR sequence of genotypes 1, 2, 3 and 4 downstream of the putative ORF4 gene to the genome 3′-polyA tail. Helices 1 to 4 are indicated and the positions of all nucleotide differences that occurred amongst the four genotypes, including compensatory changes that preserved base-pairing in helix 2 and that maintained helix 4 in genotype 1, are indicated (▶).
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