Yellow head virus (YHV) is highly pathogenic and was identified as the cause of mass mortalities associated with yellow head disease (YHD) that first appeared in Penaeus monodon farmed in Thailand in 1990. By 1992-1993, YHD was widespread throughout the Thai shrimp farming industry, causing losses estimated at ~US$70 million per annum. By the mid 1990s, gross signs consistent with YHD were also being reported in P. monodon farmed in many regions of the Indo-Pacific. Due to its high pathogenicity and economic impact, YHV has been listed as a notifiable pathogen by the OIE and the control of YHD remains a significant concern. At the outset of this study, two genotypic variants of YHV (genotype 1) had been detected in P. monodon in Australia (gill-associated virus, GAV, genotype 2) and Vietnam (genotype 3), suggesting that more variants might exist in other regions. The aim of this study was, therefore, to test the hypothesis that genotypic variants existed in P. monodon from other locations, and if so, to determine their genetic relationships to the three known genotypes. The study also aimed to improve existing PCR diagnostic protocols to accommodate the detection of all genotypes in the YHV complex. Fifty-seven field isolates of YH-complex viruses were detected by RT-PCR in tissues of P. monodon sampled from nine Indo-Pacific countries. Phylogenetic relationships determined for these isolates using a 671 nucleotide (nt) C-terminal region of the ORF1b gene identified 46 isolates that clustered with the three know genotypes and 11 isolates that clustered in at least three distinct new genotypes. All isolates other than genotype 1 (YHV) were detected in tissues of healthy shrimp. Genotype 4 isolates were detected only in shrimp from India and were slightly less distantly related at the nucleotide level to genotype 5 (85.2% identify) than the other genotypes (80.3%-82.3%). Genotype 6 isolates were only detected in shrimp from Mozambique and were least divergent (3.5%) from genotype 2. One each of three genotype 5 isolates was detected in shrimp from Malaysia, Thailand and the Philippines. The genotype 5 isolate from the Philippines was, however, 6.7% and 7% divergent from the other two isolates, respectively. This level of divergence was greater than found between genotypes 2 and 6 and was similar to that found between isolates of genotype 2 and genotype 3 (~6.7%). This suggests that the Philippine genotype 5 isolate might ultimately be considered as the founding member of a seventh genotype. Genotype 5 isolates were slightly more closely related to genotype 4 (~85.2% identity) than the other genotypes (83.4%-84.8% identity). Genotype 1 (YHV) isolates were only detected in Thai shrimp affected by YHD. Genotype 2 isolates were detected in Australian shrimp as well as shrimp from Vietnam and Thailand. Genotype 3 had the broadest geographic range, being detected in four countries in Southeast Asia. The finding of single genotypes in Australia (genotype 2), India (genotype 4) and Mozambique (genotype 6) supports the hypothesis that they have evolved independently in geographically-isolated populations of P. monodon. The detection of multiple genotypes in Vietnam (genotypes 2 and 3), Malaysia (genotypes 2, 3 and 5) and Thailand (genotypes 1, 2, 3 and 5) suggests that these genotypes have been disseminated by movements of infected P. monodon and the trade in live broodstock used for aquaculture. A ~1.3 kb amplicon at the 5’-terminal region of the ORF3 gene was sequenced for 28 field isolates to examine phylogenetic relationships to assess whether there is evidence of recombination between genotypes. The region, corresponding to N-terminus of gp116 envelope glycoprotein, displayed more sequence variation than the ORF1b amplicon. All isolates of the virulent genotype 1 (YHV) possessed a unique sequence (TILAGIPEKE/D) at the N terminus of gp116 adjacent to the site of endo-proteolysis that cleaves gp116 from the ORF3 polyprotein. In some genotype 1 isolates this unique sequence was followed by a 54 aa deletion that was also not present in other genotypes. The potential role of this unique sequence as a virulence determinant for YHV requires further investigation. Phylogenetic relationships deduced using the ORF3 amplicon sequences were similar to those deduced using the ORF1b amplicon sequence except that genotype 4 was more closely related to genotype 2 than was genotype 3. However, only 18 of the 28 isolates included in the analysis of both ORF1b and ORF3 amplicons clustered in consistent lineages and were assigned as the same genotypes. Inconsistent phylogenies were observed for ten isolates of which six clustered as genotype 3 in ORF1b and as genotype 2 in ORF3, two isolates clustered as genotype 3 in ORF1b and as genotype 5 in ORF3, one isolate clustered as genotype 5 in ORF1b and as genotype 2 in ORF3, and one isolate clustered as genotype 5 in ORF1b and as genotype 3 in ORF3. Discrepancies in genotype assignments were only observed to involve permutations of genotypes 2, 3 and 5 and involved isolates from healthy shrimp originating from Southeast Asia. Sequence analysis of the ~3.2 kb region spanned by the ORF1b and ORF3 amplicons of three putative recombinant viruses VNM-02-H258 (genotype 3/5), IDN-04-H10 (genotype 3/2) and PHL-03-H8 (genotype 5/3) indicated that recombination had occurred at a position just upstream of the ORF1b gene 3’-terminus. These data provide the first evidence of genetic recombination for any shrimp virus. The high prevalence of recombinants amongst isolates from Southeast Asia has significant implications for diversification, disease emergence and assignment of genotypes for YH-complex viruses. The region of the genome from the poly[A] tail to the 3’-end of the ORF1b gene (containing all structural protein genes) was sequenced for representative isolates of genotypes 3 and 4. The analysis was conducted to determine whether the evolutionary divergence in the structural protein genes differed significantly from the replicase (ORF1b) gene and to identify conserved motifs likely to be important for protein function and the regulation of RNA transcription and replication. The sequence of the near 3’-terminal genome region of a genotype 5 isolate was also determined to examine whether it possessed an ORF4 gene like genotype 2 or whether it was truncated as in genotypes 1, 3 and 4. Comparisons of the intergenic regions (IGR) upstream of ORF2 and ORF3 identified a conserved sequence 5’-GUCAAUUACACxxAxxUU-3’ surrounding the central adenosine residue corresponding to the 5’-terminus of the sub-genomic (sg)mRNAs that is likely to represent the consensus motif used as a transcription regulatory sequence (TRS). A sequence upstream of ORF4 possessed limited homology to the predicted consensus TRS but A>G/U substitutions (genotypes 2, 3, 4 and 5) or a point deletion (genotype 1) occurred at the central critical adenosine residue. It is possible that these mutations explain why a sgmRNA is not transcribed in abundance to allow translation of an ORF4 protein, and why the apparently redundant ORF4 gene has accumulated nucleotide deletions or insertions interrupting its reading in all genotypes except genotype 2. The 3’-terminal genome sequence of genotypes 1, 2, 3 and 4 downstream of the putative ORF4 gene region was extremely highly conserved and was predicted to form a stable hairpin-loop RNA secondary structure with four bulges. Where nucleotide variations occurred in a genotype, other compensatory changes maintained base-pairing and stability of the structure, suggesting that this region is likely to be important for polymerase recognition of the (+) genomic RNA for transcription of (-) genomic RNA. Conventional and real-time PCR tests for the detection of all genotypes in the YH complex were developed by identifying highly conserved sequences amongst the 57 virus isolates at which primers could be targeted. In the consensus RT-nested PCR, PCR (358 bp) and nested PCR (147 bp) amplicon lengths were kept short to accommodate degraded RNA and pools of two primers were used rather than a single degenerate primer to accommodate all genotypes whist minimizing levels of degeneracy. The consensus real-time PCR used SYBR-Green chemistry and amplified a 147 bp product using single degenerate primers targeted to the same sites as the nested PCR primer pools. Each PCR method detected the RNA of representatives of all six genotypes. The RT-nested PCR was extremely sensitive, detecting down to a single copy of a GAV synthetic RNA. Phylogenetic analysis using the 95 nt sequence bounded by the nested PCR primers generated genotype associations similar to those generated using the 671 nt sequence, allowing the assignment of genotypes from the amplified products. The consensus RT-nested PCR test has been included in the 5th Edition of the OIE Manual of Diagnostic Tests for Aquatic Animals (2006). The consensus real-time PCR was slightly less sensitive than the RT-nested PCR, detecting down to ~125 copies of the GAV synthetic RNA. However, the test generated products with the expected Tm (77.5ºC) with isolates of the six genotypes and showed a linear relationship between input RNA and Ct value up to 109 RNA copies. Thus, due to its ability to accurately quantify and compare viral RNA loads in clinical samples, the test could be used to define the infection status of shrimp in relation to threshold levels associated with disease.

Yellow head virus (YHV) is highly pathogenic and was identified as the cause of mass mortalities associated with yellow head disease (YHD) that first appeared in Penaeus monodon farmed in Thailand in 1990. By 1992-1993, YHD was widespread throughout the Thai shrimp farming industry, causing losses estimated at ~US$70 million per annum. By the mid 1990s, gross signs consistent with YHD were also being reported in P. monodon farmed in many regions of the Indo-Pacific. Due to its high pathogenicity and economic impact, YHV has been listed as a notifiable pathogen by the OIE and the control of YHD remains a significant concern. At the outset of this study, two genotypic variants of YHV (genotype 1) had been detected in P. monodon in Australia (gill-associated virus, GAV, genotype 2) and Vietnam (genotype 3), suggesting that more variants might exist in other regions. The aim of this study was, therefore, to test the hypothesis that genotypic variants existed in P. monodon from other locations, and if so, to determine their genetic relationships to the three known genotypes. The study also aimed to improve existing PCR diagnostic protocols to accommodate the detection of all genotypes in the YHV complex. Fifty-seven field isolates of YH-complex viruses were detected by RT-PCR in tissues of P. monodon sampled from nine Indo-Pacific countries. Phylogenetic relationships determined for these isolates using a 671 nucleotide (nt) C-terminal region of the ORF1b gene identified 46 isolates that clustered with the three know genotypes and 11 isolates that clustered in at least three distinct new genotypes. All isolates other than genotype 1 (YHV) were detected in tissues of healthy shrimp. Genotype 4 isolates were detected only in shrimp from India and were slightly less distantly related at the nucleotide level to genotype 5 (85.2% identify) than the other genotypes (80.3%-82.3%). Genotype 6 isolates were only detected in shrimp from Mozambique and were least divergent (3.5%) from genotype 2. One each of three genotype 5 isolates was detected in shrimp from Malaysia, Thailand and the Philippines. The genotype 5 isolate from the Philippines was, however, 6.7% and 7% divergent from the other two isolates, respectively. This level of divergence was greater than found between genotypes 2 and 6 and was similar to that found between isolates of genotype 2 and genotype 3 (~6.7%). This suggests that the Philippine genotype 5 isolate might ultimately be considered as the founding member of a seventh genotype. Genotype 5 isolates were slightly more closely related to genotype 4 (~85.2% identity) than the other genotypes (83.4%-84.8% identity). Genotype 1 (YHV) isolates were only detected in Thai shrimp affected by YHD. Genotype 2 isolates were detected in Australian shrimp as well as shrimp from Vietnam and Thailand. Genotype 3 had the broadest geographic range, being detected in four countries in Southeast Asia. The finding of single genotypes in Australia (genotype 2), India (genotype 4) and Mozambique (genotype 6) supports the hypothesis that they have evolved independently in geographically-isolated populations of P. monodon. The detection of multiple genotypes in Vietnam (genotypes 2 and 3), Malaysia (genotypes 2, 3 and 5) and Thailand (genotypes 1, 2, 3 and 5) suggests that these genotypes have been disseminated by movements of infected P. monodon and the trade in live broodstock used for aquaculture. A ~1.3 kb amplicon at the 5’-terminal region of the ORF3 gene was sequenced for 28 field isolates to examine phylogenetic relationships to assess whether there is evidence of recombination between genotypes. The region, corresponding to N-terminus of gp116 envelope glycoprotein, displayed more sequence variation than the ORF1b amplicon. All isolates of the virulent genotype 1 (YHV) possessed a unique sequence (TILAGIPEKE/D) at the N terminus of gp116 adjacent to the site of endo-proteolysis that cleaves gp116 from the ORF3 polyprotein. In some genotype 1 isolates this unique sequence was followed by a 54 aa deletion that was also not present in other genotypes. The potential role of this unique sequence as a virulence determinant for YHV requires further investigation. Phylogenetic relationships deduced using the ORF3 amplicon sequences were similar to those deduced using the ORF1b amplicon sequence except that genotype 4 was more closely related to genotype 2 than was genotype 3. However, only 18 of the 28 isolates included in the analysis of both ORF1b and ORF3 amplicons clustered in consistent lineages and were assigned as the same genotypes. Inconsistent phylogenies were observed for ten isolates of which six clustered as genotype 3 in ORF1b and as genotype 2 in ORF3, two isolates clustered as genotype 3 in ORF1b and as genotype 5 in ORF3, one isolate clustered as genotype 5 in ORF1b and as genotype 2 in ORF3, and one isolate clustered as genotype 5 in ORF1b and as genotype 3 in ORF3. Discrepancies in genotype assignments were only observed to involve permutations of genotypes 2, 3 and 5 and involved isolates from healthy shrimp originating from Southeast Asia. Sequence analysis of the ~3.2 kb region spanned by the ORF1b and ORF3 amplicons of three putative recombinant viruses VNM-02-H258 (genotype 3/5), IDN-04-H10 (genotype 3/2) and PHL-03-H8 (genotype 5/3) indicated that recombination had occurred at a position just upstream of the ORF1b gene 3’-terminus. These data provide the first evidence of genetic recombination for any shrimp virus. The high prevalence of recombinants amongst isolates from Southeast Asia has significant implications for diversification, disease emergence and assignment of genotypes for YH-complex viruses. The region of the genome from the poly[A] tail to the 3’-end of the ORF1b gene (containing all structural protein genes) was sequenced for representative isolates of genotypes 3 and 4. The analysis was conducted to determine whether the evolutionary divergence in the structural protein genes differed significantly from the replicase (ORF1b) gene and to identify conserved motifs likely to be important for protein function and the regulation of RNA transcription and replication. The sequence of the near 3’-terminal genome region of a genotype 5 isolate was also determined to examine whether it possessed an ORF4 gene like genotype 2 or whether it was truncated as in genotypes 1, 3 and 4. Comparisons of the intergenic regions (IGR) upstream of ORF2 and ORF3 identified a conserved sequence 5’-GUCAAUUACACxxAxxUU-3’ surrounding the central adenosine residue corresponding to the 5’-terminus of the sub-genomic (sg)mRNAs that is likely to represent the consensus motif used as a transcription regulatory sequence (TRS). A sequence upstream of ORF4 possessed limited homology to the predicted consensus TRS but A>G/U substitutions (genotypes 2, 3, 4 and 5) or a point deletion (genotype 1) occurred at the central critical adenosine residue. It is possible that these mutations explain why a sgmRNA is not transcribed in abundance to allow translation of an ORF4 protein, and why the apparently redundant ORF4 gene has accumulated nucleotide deletions or insertions interrupting its reading in all genotypes except genotype 2. The 3’-terminal genome sequence of genotypes 1, 2, 3 and 4 downstream of the putative ORF4 gene region was extremely highly conserved and was predicted to form a stable hairpin-loop RNA secondary structure with four bulges. Where nucleotide variations occurred in a genotype, other compensatory changes maintained base-pairing and stability of the structure, suggesting that this region is likely to be important for polymerase recognition of the (+) genomic RNA for transcription of (-) genomic RNA. Conventional and real-time PCR tests for the detection of all genotypes in the YH complex were developed by identifying highly conserved sequences amongst the 57 virus isolates at which primers could be targeted. In the consensus RT-nested PCR, PCR (358 bp) and nested PCR (147 bp) amplicon lengths were kept short to accommodate degraded RNA and pools of two primers were used rather than a single degenerate primer to accommodate all genotypes whist minimizing levels of degeneracy. The consensus real-time PCR used SYBR-Green chemistry and amplified a 147 bp product using single degenerate primers targeted to the same sites as the nested PCR primer pools. Each PCR method detected the RNA of representatives of all six genotypes. The RT-nested PCR was extremely sensitive, detecting down to a single copy of a GAV synthetic RNA. Phylogenetic analysis using the 95 nt sequence bounded by the nested PCR primers generated genotype associations similar to those generated using the 671 nt sequence, allowing the assignment of genotypes from the amplified products. The consensus RT-nested PCR test has been included in the 5th Edition of the OIE Manual of Diagnostic Tests for Aquatic Animals (2006). The consensus real-time PCR was slightly less sensitive than the RT-nested PCR, detecting down to ~125 copies of the GAV synthetic RNA. However, the test generated products with the expected Tm (77.5ºC) with isolates of the six genotypes and showed a linear relationship between input RNA and Ct value up to 109 RNA copies. Thus, due to its ability to accurately quantify and compare viral RNA loads in clinical samples, the test could be used to define the infection status of shrimp in relation to threshold levels associated with disease.

Abstract Yellow head virus (YHV) has caused mass mortalities in Penaeus monodon shrimp farmed throughout Southeast Asia since it was first discovered in the early 1990’s. YHV possesses a positive-sense, single-stranded RNA genome and a rod-shaped enveloped virion. Together with the closely related gill-associated virus (GAV) identified in P. monodon shrimp in Australia, it is classified in the genus Okavirus, family Roniviridae within the order Nidovirales. YHV particles contain only three structural proteins, a nucleocapsid (N) protein (p20) protein and two envelope glycoproteins gp116 and gp64. In this study, the glycosylation status of gp116 and gp64 extracted from YHV virions was characterized in detail, including the identification of active N-linked glycosylation sites and the nature of the attached carbohydrates. This was achieved by optimizing and applying a combination of methods that included SDS-PAGE followed by carbohydrate-specific staining of gels or probing of membrane-bound proteins using lectins with different carbohydrate specificities, enzymatic removal of N-linked carbohydrates and a variety of mass spectrometry techniques. In these analyses, it was found that N-linked glycans are the major contributor to the higher estimated mass of gp116 and gp64 by SDS-PAGE compared to those estimated from their deduced amino acid sequences. Neither gp116 nor gp64 were found to posses O-linked glycans. Mannose residues were identified to be the major glycan component of carbohydrates linked to gp116 and gp64 and are possibly the sole component of carbohydrate linked to gp64. Unlike gp64, other glycans such as terminal N-acetyl--D-galactosamine and N-acetyl--D-glucosamine were identified to be attached to gp116. Assuming that glycosylation processes in shrimp mimic those of vertebrates that are known in more detail, the nature of the glycans attached to gp116 suggests that they might be added and modified during the transportation of the protein from the endoplasmic reticulum (ER) to the trans-Golgi network (TGN). Mass spectrometry analyses of tryptic peptides derived from the native glycoproteins and following their enzymatic deglycosylation, generated approximately 81% (gp116) and 66% (gp64) coverage of their predicted amino acid sequences. Detailed mass spectrometry analyses of peptides derived from the deglycosylated proteins identified that most of the potential N-linked glycosylated site in the virion envelope glycoproteins, 6 of 7 present in gp116 and 3 of 4 present in gp64 were identified to be modified by glycans. In gp116, one site was not identified and in gp64 one site was not utilized. As phosphorylation has been shown to affect nucleocapsid protein (N) functioning in vertebrate nidoviruses, SDS-PAGE using two phosphoprotein-specific staining methods, as well as mass spectrometry methods, were employed to examine whether the YHV N protein present in virions is phosphorylated. The protein staining methods provided contradicting results and no phosphate-containing peptides were identified by mass spectrometry. The apparent absence of phosphate in the N protein was also supported by its isoelectric point (pI ~10) determined by isoelectric focusing and two-dimensional electrophoresis (2-DE) analysis, which was very similar to that predicted (pI = 9.98) from its deduced amino acid sequence. Taken together, the data suggest that the YHV N protein encapsulated within virions is not phosphorylated. The RNA-binding capability of the GAV N protein was assessed using an electrophoretic mobility shift assay (EMSA) technique. Full-length and variously truncated forms of the GAV N protein expressed in bacteria were assessed in the assays. It was found that the full-length recombinant N protein bound to RNA in a sequence non-specific manner. Analysis of the five truncated N protein constructs localized the RNA-binding domain to a 50 amino acid sequence in the N-terminal region residing between Met11 and Arg60. A motif rich in proline and arginine residues, which are commonly found in other RNA-binding proteins, occurred in first 18 amino acids of this region. Although RNA-binding was not sequence-specific, the data suggest that this region of the GAV N protein is the most likely site at which it interacts with and nucleates viral genomic RNA during nucleocapsid formation. A synthetic peptide spanning the 18 amino acid of the putative RNA-binding domain was shown to possess RNA-binding properties similar to the recombinant protein fragment. These results indicated that the 18 amino acid, proline and arginine rich motif (MPVRRPLPPQPPRNARLI) in the N-terminal region of the GAV N protein confers its RNA-binding function. Using an immuno-co-precipitation assay, a host protein was found to interact abundantly with the GAV N protein in infected lymphoid organ cells. Mass spectrometry analysis identified the protein as -actin. Immuno-histochemistical double-labeling methods in conjunction with observations made using confocal and electron microscopy revealed that actin and the N protein were co-located in cytoplasm of infected cells. Electron microscopy suggested that interaction of the two proteins occurs before nucleocapsid envelopment within virions, suggesting that -actin might be involved in transporting the N protein or the nucleocapsid from their sites of synthesis to the rough endoplasmic reticulum where the virion acquires its envelopes. In summary, the research described in this thesis has advanced understanding of the YHV/GAV proteome through the identification of the glycosylation sites in the envelope glycoproteins gp116 and gp64, and demonstrating that nucleocapsid protein encapsulated within virion is unlikely to be phosphorylated. Functional studies have also shown that the nucleocapsid protein binds RNA non-specifically through an 18 amino acid domain near its N-terminus and that it binds and co-localizes with -actin in infected cells, suggesting that -actin may play role in trafficking N protein in infected cells.