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Yellow head virus (YHV) is a pathogen which causes high mortality in penaeid shrimp. Previous studies suggested that YHV enters shrimp cells via clathrin-mediated endocytosis. This research investigated the roles of clathrin adaptor protein 2 subunit β (AP-2β) from Penaeus monodon during YHV infection. PmAP2-β was continuously up-regulated more than twofold during 6-36 hpi. Suppression of PmAP2-β significantly reduced YHV copy numbers and delayed shrimp mortality. Quantitative RT-PCR revealed that knockdown of PmAP2-β significantly enhanced the expression level of PmSpätzle, a signaling ligand in the Toll pathway, by 30-fold at 6 and 12 hpi. Moreover, the expression levels of gene components in the Imd and JAK/STAT signaling pathways under the suppression of PmAP2-β during YHV infection were also investigated. Interestingly, anti-lipopolysaccharide factor isoform 3 (ALFPm3) was up-regulated by 40-fold in PmAP2-β knockdown shrimp upon YHV infection. In addition, silencing of PmAP2-β dramatically enhanced crustinPm1 expression in YHV-infected shrimp. Knockdown of ALFPm3 and crustinPm1 significantly reduced shrimp survival rate. Taken together, this work suggested that PmAP2-β-deficiency promoted the Toll pathway signalings, resulting in elevated levels of ALFPm3 and crustinPm1, the crucial antimicrobial peptides in defence against YHV.

Gill-associated virus (GAV) and Mourilyan virus (MoV) can occur at very high prevalence in healthy black tiger shrimp (Penaeus monodon) in eastern Australia, and both have been detected in moribund shrimp collected from mid-crop mortality syndrome (MCMS) outbreaks. Experimental evidence presented here indicates that GAV, but not MoV, is the cause of MCMS. Firstly, in healthy P. monodon used for experimental infections, pre-existing MoV genetic loads were very high (mean >10(9) viral RNA copies μg(-1) total RNA) and did not increase significantly following lethal challenge with an inoculum containing both GAV and MoV. In contrast, GAV genetic loads prior to challenge were low (mean ∼10(5) RNA copies μg(-1) total RNA) and increased >10(4)-fold in moribund shrimp. Secondly, dsRNAs targeted to the GAV RNA-dependent RNA polymerase (RdRp) or helicase gene regions reduced GAV genetic loads, delayed the onset of mortalities and improved survival following challenge. In contrast, dsRNA targeted to the MoV RdRp gene (L RNA) was highly effective in reducing MoV genetic loads, but mortality rates were unaffected. Targeting of the MoV S2 RNA, encoding a small non-structural protein (NSs2), a putative supressor of RNA interference, did not reduce the MoV genetic loads or enhance knockdown of GAV when administered simultaneously with dsRNA targeted to the GAV helicase gene. Overall, the data show that P. monodon can tolerate a high-level MoV infection and that mortalities are associated with GAV infection.

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.

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.