Non-virulence of a recombinant shrimp nidovirus is associated with its non structural gene sequence and not a large structural gene deletion

Abstract

RT-PCR using a commercial kit for yellow head virus (YHV) detection in growth-retarded shrimp yielded an unusual 777 bp amplicon instead of expected amplicons of 277 bp for YHV type-1 (YHV-1) or 406 bp for YHV type-2 (YHV-2). Cloning and sequencing (GenBank EU170438) revealed approximately 80% identity to non-structural (NS) ORF1b sequences of both YHV-1 (GenBank AA083987) and YHV-2 (GenBank AF227196), indicating an atypical YHV type (A-YHV) phylogenetically equidistant from both types. An RT-PCR test specifically designed for A-YHV revealed that it was uncommon and that its occurrence in shrimp culture ponds did not correlate with growth retardation or mortality. By immunohistochemistry with YHV-specific monoclonal antibodies, the A-YHV gave positive reactions for envelope protein gp64 and capsid protein p20, but not for envelope protein gp116, even though gp116 and gp64 originate from a polyprotein of ORF3. Lack of gp116 immunoreactivity correlated with a large ORF3 deletion (GenBank EU123854) in the region of the protein targeted by an MAb against gp116. Transmission electron microscopy of A-YHV-infected shrimp revealed only unenveloped pre-virions. During manuscript revision, information received revealed that typing of YHV isolates based on sequences of ORF1b and ORF3 had yielded several geographical types, including one virulent type (YHV-1b) with an ORF3 deletion sequence that matched the sequence of A-YHV. Using these sequences and an additional A-YHV sequence (EU853170) from the ORF1b typing region, A-YHV potentially represents a recombinant between type 1b and type 5. SDS-PAGE and Western blot analysis revealed that type 1b produced a gp116 deletion protein that did not bind with the MAb or polyclonal Ab to normal gp116. Overall, the information suggested that lack of A-YHV virulence was associated with the NS gene sequence linked to ORF1b rather than the deletion in ORF3.

Glycosylation of gp116 and gp64 envelope proteins of yellow head virus of Penaeus monodon shrimp

“…Most sequence diversity, including block insertions/deletions, occurs near to the gp116 N terminus, generated by cleavage at a signal peptidase type 1 site at TM domain 3 of the ORF3 polyprotein. Two YHV genotype 1 variants from Thailand, one virulent (genotype 1b) (Sittidilokratna et al, 2009) and the other non-virulent (A-YHV) (Gangnonngiw et al, 2009), for which sequence data have been reported recently, both possess a 54 aa deletion in the gp116 Nterminal region that eliminates the N 63 TS and N 44 ST sites, found here to be glycosylated in the YHV genotype 1a strain. Despite sequence differences and the loss of these two glycan-linked sites in gp116 of YHV genotype 1b strains, they retain virulence, albeit somewhat reduced compared with genotype 1a strains (Sittidilokratna et al, 2009).…”

“…Of the three YHV structural proteins, the amino acid sequence of gp116 is most variable amongst the genotypic variants (Gangnonngiw et al, 2009;Sittidilokratna et al, 2009;Wijegoonawardane et al, 2008). Most sequence diversity, including block insertions/deletions, occurs near to the gp116 N terminus, generated by cleavage at a signal peptidase type 1 site at TM domain 3 of the ORF3 polyprotein.…”

“…Despite sequence differences and the loss of these two glycan-linked sites in gp116 of YHV genotype 1b strains, they retain virulence, albeit somewhat reduced compared with genotype 1a strains (Sittidilokratna et al, 2009). It has been postulated that sequence differences elsewhere, possibly in non-structural proteins, are important in conferring virulence of these two variants of YHV genotype 1 (Gangnonngiw et al, 2009). …”

New yellow head virus genotype (YHV7) in giant tiger shrimp Penaeus monodon indigenous to northern Australia

“…The detection of an RT-PCR amplicon and no nested PCR amplicons, as was the case for YHV7, has occurred previously with a YHV1b/YHV5 recombinant virus (Gangnonngiw et al 2009). A similar result has also been observed for an, as yet, unassigned yellow-head-complex virus detected in several provinces of China (Liu et al 2014).…”

Evidence for a novel coding sequence overlapping the 5'-terminal ~90 codons of the Gill-associated and Yellow head okavirus envelope glycoprotein gene

“…As with other members of the order Nidovirales , these viruses have a linear positive-sense ssRNA genome encoding a large replicase polyprotein that is expressed from the genomic RNA (ORF1a and, via ribosomal frameshifting, an ORF1a-ORF1b fusion product), and a number of other proteins - including the structural proteins - which are translated from a nested set of 3'-coterminal sub-genomic RNAs [1-4]. In the case of the okaviruses, the 3' ORFs encode a polyprotein (ORF3; ~1650 codons) that is cleaved to produce three mature proteins (envelope glycoproteins gp116 and gp64, and an ~25 kDa N-terminal fragment), a nucleocapsid (ORF2; ~145 codons), and possibly an additional short ORF (ORF4) encoded by the sequence 3' of ORF3 (Figure 1A) [4-10]. …”