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Background: The family Mesoniviridae (order Nidovirales) comprises of a group of positive-sense, single-stranded RNA ([+]ssRNA) viruses isolated from mosquitoes., Findings: Thirteen novel insect-specific virus isolates were obtained from mosquitoes collected in Indonesia, Thailand and the USA. By electron microscopy, the virions appeared as spherical particles with a diameter of ~50 nm. Their 20,129 nt to 20,777 nt genomes consist of positive-sense, single-stranded RNA with a poly-A tail. Four isolates from Houston, Texas, and one isolate from Java, Indonesia, were identified as variants of the species Alphamesonivirus-1 which also includes Nam Dinh virus (NDiV) from Vietnam and Cavally virus (CavV) from Côte d'Ivoire. The eight other isolates were identified as variants of three new mesoniviruses, based on genome organization and pairwise evolutionary distances: Karang Sari virus (KSaV) from Java, Bontag Baru virus (BBaV) from Java and Kalimantan, and Kamphaeng Phet virus (KPhV) from Thailand. In comparison with NDiV, the three new mesoniviruses each contained a long insertion (180 - 588 nt) of unknown function in the 5' region of ORF1a, which accounted for much of the difference in genome size. The insertions contained various short imperfect repeats and may have arisen by recombination or sequence duplication., Conclusions: In summary, based on their genome organizations and phylogenetic relationships, thirteen new viruses were identified as members of the family Mesoniviridae, order Nidovirales. Species demarcation criteria employed previously for mesoniviruses would place five of these isolates in the same species as NDiV and CavV (Alphamesonivirus-1) and the other eight isolates would represent three new mesonivirus species (Alphamesonivirus-5, Alphamesonivirus-6 and Alphamesonivirus-7). The observed spatiotemporal distribution over widespread geographic regions and broad species host range in mosquitoes suggests that mesoniviruses may be common in mosquito populations worldwide.

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.

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.

A multiplex RT-nested PCR has been developed to detect and differentiate the closely related prawn viruses, gill-associated virus (GAV) from Australia and yellow head virus (YHV) from Thailand. RT-PCR using primers to conserved sequences in the ORF1b gene amplified a 794 bp region of either GAV or YHV. Nested PCR using a conserved sense primer and either a GAV- or YHV-specific antisense primer to a divergent sequence differentially amplified a 277 bp region of the primary PCR amplicon. Multiplexing the YHV antisense primer with a GAV antisense primer to another divergent sequence allowed the viruses to be distinguished in a single nested PCR. Nested PCR enhanced detection sensitivity between 100- and 1000-fold and GAV or YHV RNA was detectable in approximately 10 fg lymphoid organ total RNA. The multiplex RT-nested PCR was also able to co-detect GAV and YHV RNA mixed over a wide range of concentrations to simulate potential dual-infection states. The robustness of the test was examined using RNA samples from Penaeus monodon prawns infected either chronically or acutely with GAV or YHV and collected at different locations in Eastern Australia and Thailand between 1994 and 1998. GAV- (406 bp) or YHV-specific (277 bp) amplicons were differentially generated in all cases, including five YHV RNA samples in which no primary RT-PCR amplicon was detected. Sequence analysis of GAV and YHV PCR amplicons identified minor variations in the regions targeted by the virus-specific antisense primers. However, none occurred at positions that critically affected the PCR.

Three monoclonal antibodies (MAbs) raised against pathogenic yellow head virus (YHV) from Thailand were tested against tissues of shrimp from Thailand, Australia, Ecuador and India that were purported to be infected with yellow head complex viruses. MAbs V-3-2B and Y-18 were specific to gp116 and gp64 envelope proteins, respectively, while Y-19 was specific to a 20 kDa putative nucleoprotein p20. As a preliminary step, the site of reactivity of the 3 MAbs in YHV was determined by immuno-electron microscopy using ultra-thin sections of YHV-infected shrimp tissue and negatively stained, semi-purified YHV particles. As expected, MAb Y-19 reacted with viral nucleocapsids in ultra-thin sections but not with negatively stained, whole virions; MAb V-3-2B did react with negatively stained, whole virions, but not with virions or nucleocapsids in ultra-thin sections. Unexpectedly, MAb Y-18 did not react with whole or sectioned virions. By immunohistochemistry, MAbs Y-19 and Y-18 reacted with Penaeus monodon tissues infected with either YHV or with gill-associated virus (GAV) from Australia, while MAb V-3-2B reacted with YHV only. In addition, all the YHV and GAV tissue samples gave positive in situ hybridization reactions with a cDNA probe specific to the ORF1b gene of YHV. They also gave expected differential RT-PCR results for YHV and GAV. By contrast, 2 natural Thai shrimp specimens with no gross signs of disease gave similar immunohistochemical reactions and RT-PCR reactions to GAV. However, sequencing of their RT-PCR products showed that they shared 92.7% identity with GAV, but only 79.0% identity with YHV. Although specimens from Ecuador and India displayed histopathology suggestive of YHV infection, they gave negative immunohistochemical reactions with all 3 Mabs, and negative in situ hybridization results. Additional work is required to determine whether a virus from the yellow head complex was responsible for their observed histopathology. These data show that the 3 YHV MAbs could be used in diagnostic situations to differentiate some viruses in the yellow head virus complex.

Chronic and acute gill-associated virus (GAV) infections were examined by in situ hybridization (ISH) using a DNA probe targeting a 779 nucleotide region of the ORF1b-gene. Chronic GAV infections were observed in healthy Penaeus monodon collected from farms and healthy P. esculentus surviving experimental infection. During chronic-phase infections in both species, GAV was detected only in partitioned foci of cells with hypertrophied nuclei (spheroids) within the lymphoid organ. Acute-phase infections were observed in moribund P. monodon and P. esculentus infected experimentally with a high dose of GAV, and in moribund P. monodon collected from farms during outbreaks of disease. During acute experimental infections in P. monodon, ISH detected GAV throughout the lymphoid organ, in gills and in connective tissues throughout the cephalothorax. In moribund P. monodon collected from natural outbreaks of disease, GAV was also detected in the gills and in connective tissues of the cephalothorax, but the distribution of virus within the lymphoid organ varied. In acutely infected P. esculentus, GAV was detected in connective tissues, but was restricted to the inner stromal matrix cells and endothelial cells of intact lymphoid organ tubules. The tissue distribution of GAV identified by ISH suggests that shrimp are able to control and maintain chronic asymptomatic infection by a process involving lymphoid organ spheroids. Acute phase infections and the development of disease appear to be dose-related and involve the systemic distribution of virus in connective tissues throughout the cephalothorax.

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.

We report here a 5596 nt sequence comprising the 3'-end of the (+) ssRNA genome of gill-associated virus (GAV), an invertebrate nidovirus of Penaeus monodon prawns. The sequence extends from a subgenomic RNA start site 35 nt upstream of the 4923 nt ORF3 gene to a 3'-poly(A) tail of the 26235 nt genome of GAV. The putative 1640 amino acid (aa) ORF3 protein (MW = 182049 Da, pI = 6.62) contains 15 potential N-linked glycosylation sites, 15 potential O-linked glycosylation sites and six highly hydrophobic regions predicted to represent transmembrane (TM) domains. Three of the predicted TM domains occur in the amino-terminal 228 aa, two in the central portion, and one near the carboxy-terminus of ORF3. Only one short (83 aa) open reading frame (ORF4) was identified between ORF3 and the 3'-poly(A) tail. Completion of the genome sequence of GAV has revealed a gene organisation unique among nidoviruses in there is no discrete membrane protein gene and that the putative ORF3 spike glycoprotein gene resides downstream of a gene (ORF2) encoding a structural protein associated with nucleocapsids.

Chronic gill-associated virus (GAV) infection is endemic in Penaeus monodon broodstock captured from north-east Queensland in Australia and in farmed shrimp produced from these. We investigated the role of vertical transmission in perpetuating the high prevalence of these chronic GAV infections. Reverse transcription (RT)-nested PCR detected GAV in spermatophores and mature ovarian tissue from broodstock and in fertilized eggs and nauplii spawned from wild-fertilized females. In laboratory-reared P. monodon (> 12 mo old) that had a high mortality rate, RT-nested PCR detected GAV in male spermatophores at levels significantly higher than that detected in the lymphoid organ. By transmission electron microscopy (TEM), GAV virions were detected in spermatophore seminal fluid, but not sperm cells. Histological evidence of hypertrophied cell foci (spheroids) and TEM observation of GAV nucleocapsids and virions in spheroid cells was also found in 100% of lymphoid organs of approximately 1.2 g juvenile P. monodon reared in the laboratory from postlarvae collected from commercial hatcheries. Sequence analysis of PCR amplicons from parental P. monodon and fertilized eggs of artificially inseminated broodstock indicated that GAV associated with eggs can originate from both the male and female parents. Although the female GAV genotype was predominant in eggs, there was some dependence on infection levels in the male and female shrimp as indicated by RT-PCR. RT-nested PCR data on GAV levels in eggs, nauplii, protozoea and PL5 progeny of the artificial matings suggests that vertically transmitted virus is most probably associated with the egg surface.

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.

Sequence analysis of the approximately 20 kb 5'-terminal portion of the ssRNA genome of gill-associated virus (GAV) of Penaeus monodon prawns has previously established that it contains an ORF1a-1b replicase gene equivalent to those of the coronavirus and arterivirus members of the order Nidovirales. Sequence analysis of the remaining approximately 6.2 kb of the GAV genome downstream of ORF1a-1b to a 3'-poly(A) tail has identified two highly conserved intergenic sequences in which 29/32 nucleotides are conserved. Northern hybridization using probes to the four putative GAV ORFs and either total or poly(A)-selected RNA identified two 3'-coterminal subgenomic (sg) mRNAs of approximately 6 kb and approximately 5.5 kb. Primer extension and 5'-RACE analyses showed that the sgmRNAs initiate at the same 5'-AC positions in the central region of the two conserved intergenic sequences. Neither method provided any evidence that the GAV sgmRNAs are fused to genomic 5'-leader RNA sequences as is the case with vertebrate coronaviruses and arteriviruses. Intracellular double-stranded (ds)RNAs equivalent in size to the 26.2 kb genomic RNA and two sgRNAs were also identified by RNase/DNase digestion of total RNA from GAV-infected prawn tissue. The identification of only two sgmRNAs that initiate at the same position in conserved intergenic sequences and the absence of 5'-genomic leader sequences fused to these sgmRNAs confirms that GAV has few genes and suggests that it utilizes a transcription mechanism possibly similar to the vertebrate toroviruses but distinct from coronaviruses and arteriviruses.