Detection of Laem-Singh virus in cultured Penaeus monodon shrimp from several sites in the Indo-Pacific region.

Laem-Singh virus (LSNV) is a positive-sense single-stranded RNA (ssRNA) virus that was recently identified in Penaeus monodon shrimp in Thailand displaying signs of slow growth syndrome. A total of 326 shrimp collected between 1998 and 2007 from countries in the Indo-Pacific region were tested by RT-PCR for evidence of LSNV infection. The samples comprised batches of whole postlarvae, and lymphoid organ, gill, muscle or pleopod tissue of juvenile, subadult and adult shrimp. LSNV was not detected in 96 P. monodon, P. japonicus or P. merguiensis from Australia or 16 P. monodon from Fiji, Philippines, Sri Lanka and Mozambique. There was no evidence of LSNV infection in 73 healthy juvenile P. vannamei collected during 2006 from ponds at 9 locations in Thailand. However, LNSV was detected in each of 6 healthy P. monodon tested from Malaysia and Indonesia, 2 of 6 healthy P. monodon tested from Vietnam and 39 of 40 P. monodon collected from slow-growth ponds in Thailand. A survey of 81 P. monodon collected in 2007 from Andhra Pradesh, India, indicated 56.8% prevalence of LSNV infection but no clear association with disease or slow growth. Phylogenetic analysis of PCR amplicons obtained from samples from India, Vietnam, Malaysia and Thailand indicated that nucleotide sequence variation was very low (>98% identity) and there was no clustering of viruses according to site of isolation or the health status of the shrimp. The data suggests that LSNV exists as a single genetic lineage and occurs commonly in healthy P. monodon in parts of Asia.

Homologous genetic recombination in the yellow head complex of nidoviruses infecting Penaeus monodon shrimp.

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.

A virulent isolate of yellow head nidovirus contains a deformed envelope glycoprotein gp116.

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.

RNA transcription analysis and completion of the genome sequence of yellow head nidovirus.

Yellow head virus (YHV) is a pathogen of the black tiger shrimp (Penaeus monodon) and, with gill-associated virus (GAV), is one of two known invertebrate nidoviruses. We describe sequences of the large replicase gene (ORF1a) and 5'- and 3'-terminal UTRs, completing the 26,662 nt sequence of the YHV genome. ORF1a (12,219 nt) encodes a approximately 462,662 Da polypeptide containing a putative 3C-like protease and a putative papain-like protease with the canonical C/H catalytic dyad and alpha+beta fold. The read-through pp1ab polyprotein contains putative uridylate-specific endoribonuclease and ribose-2'-O-methyl transferase domains, and an exonuclease domain incorporating unusual dual Zn2+-binding fingers. Upstream of ORF1a, the 71 nt 5'-UTR shares 82.4% identity with the 68 nt 5'-UTR of GAV. The 677 nt 3'-terminal region contains a single 60 nt ORF, commencing 298 nt downstream of ORF3, that is identical to N-terminal coding region of the 249 nt GAV ORF4. Northern blots using RNA from YHV-infected shrimp and probes directed at ORF1a, ORF1b, ORF2 and ORF3 identified a nested set of 3'-coterminal RNAs comprising the full-length genomic RNA and two sub-genomic (sg) mRNAs. Intergenic sequences upstream of ORF2 and ORF3 share high identity with GAV, particularly in the conserved domains predicted to mediate sgmRNA transcription.

Structural and antigenic analysis of the yellow head virus nucleocapsid protein p20.

Yellow head virus (YHV) is an invertebrate nidovirus that is highly pathogenic for marine shrimp. Nucleotide sequence analysis indicated that the YHV ORF2 gene encodes a basic protein (pI = 9.9) of 146 amino acids with a predicted molecular weight of 16,325.5 Da. The deduced amino acid sequence indicated a predominance of basic (15.1%), acidic (9.6%) and hydrophilic polar (34.3%) residues and a high proportion proline and glycine residues (16.4%). The ORF2 gene was cloned and expressed in Escherichia coli as a M(r) = 21 kDa His(6)-protein that reacted with YHV nucleoprotein (p20) monoclonal antibody. Segments representing the four linear quadrants of the nucleoprotein were also expressed in E. coli as GST-fusion proteins. Immunoblot analysis using YHV polyclonal rabbit antiserum indicated the presence of linear epitopes in all except the V(37)-Q(74) quadrant. Immunoblot analysis of the GST-fusion proteins and C-terminally truncated segments of the nucleoprotein allowed mapping of YHV monoclonal antibodies Y19, Y20 and YII4 to linear epitopes in the acidic domain between amino acids I(116) and E(137). The full-length nucleoprotein was expressed at high level in E. coli and was easily purified in quantity from the soluble cell fraction by Ni(+)-NTA affinity chromatography.

The gene encoding the nucleocapsid protein of Gill-associated nidovirus of Penaeus monodon prawns is located upstream of the glycoprotein gene.

The ORF2 gene of Gill-associated virus (GAV) of Penaeus monodon prawns resides 93 nucleotides downstream of the ORF1a-ORF1b gene and encodes a 144-amino-acid hydrophilic polypeptide (15,998 Da; pI, 9.75) containing 20 basic (14%) and 13 acidic (9%) residues and 19 prolines (13%). Antiserum to a synthetic ORF2 peptide or an Escherichia coli-expressed glutathione S-transferase-ORF2 fusion protein detected a 20-kDa protein in infected lymphoid organ and gill tissues in Western blots. The GAV ORF2 fusion protein antiserum also cross-reacted with the p20 nucleoprotein in virions of the closely related Yellow head virus. By immuno-gold electron microscopy, it was observed that the ORF2 peptide antibody localized to tubular GAV nucleocapsids, often at the ends or at lateral cross sections. As GAV appears to contain only two structural protein genes (ORF2 and ORF3), these data indicate that GAV differs from vertebrate nidoviruses in that the gene encoding the nucleocapsid protein is located upstream of the gene encoding the virion glycoproteins.

Identification and analysis of gp116 and gp64 structural glycoproteins of yellow head nidovirus of Penaeus monodon shrimp.

Yellow head virus (YHV) is a major agent of disease in farmed penaeid shrimp. YHV virions purified from infected shrimp contain three major structural proteins of molecular mass 116 kDa (gp116), 64 kDa (gp64) and 20 kDa (p20). Two different staining methods indicated that the gp116 and gp64 proteins are glycosylated. Here we report the complete nucleotide sequence of ORF3, which encodes a polypeptide of 1666 amino acids with a calculated molecular mass of 185 713 Da (pI=6.68). Hydropathy analysis of the deduced ORF3 protein sequence identified six potential transmembrane helices and three ectodomains containing multiple sites for potential N-linked and O-linked glycosylation. N-terminal sequence analysis of mature gp116 and gp64 proteins indicated that each was derived from ORF3 by proteolytic cleavage of the polyprotein between residues Ala(228) and Thr(229), and Ala(1127) and Leu(1128), located at the C-terminal side of transmembrane helices 3 and 5, respectively. Comparison with the deduced ORF3 protein sequence of Australian gill-associated virus (GAV) indicated 83 % amino acid identity in gp64 and 71 % identity in gp116, which featured two significant sequence deletions near the N terminus. Database searches revealed no significant homology with other proteins. Recombinant gp64 expressed in E. coli with and without the C-terminal transmembrane region was shown to react with antibody raised against native gp64 purified from virions.

Complete ORF1b-gene sequence indicates yellow head virus is an invertebrate nidovirus.

We report the sequence of an 8503 nucleotide (nt) region of the genome of yellow head virus (YHV) encompassing the open reading frame (ORF) 1b gene. Comparison with the sequence of Australian gill-associated virus (GAV) indicated that the region, comprising approximately 30% of the YHV genome, commences 268 nt upstream of the putative ORF1a termination codon and continues through ORF1b to a site 30 nt downstream of the ORF2 initiation codon. YHV ORF1a and ORF1b overlap by 37 nt. MFOLD analysis of the overlap and downstream region predicted a 131 nt folding structure (deltaG = -47.3 kcal mol(-1)) with potential to form an RNA pseudoknot. The structure resides 3 nt downstream of a ribosomal frame-shift 'slippery' sequence (AAAUUUU) and a -1 frame-shift at this site would extend the ORF1 polyprotein by 2616 amino acids (299322 Da). In ORF1b, YHV shares 88.9% amino acid sequence identity with GAV and includes conserved polymerase, metal ion binding, helicase and other domains (Motifs 1 and 3) characteristic of nidoviruses. Compared to GAV, the YHV non-coding region linking the ORF 1b and ORF2 genes contains a 263 nt insertion. However, the region contains a conserved core sequence of 46 nucleotides (84.8% identity) that includes a stretch of 20 identical nucleotides surrounding a sub-genomic RNA transcription termination site. The data confirms the taxonomic placement of YHV in the Nidovirales and supports biological and topographical evidence that YHV and GAV may be classified as distinct species.