Jeff A. Cowley

The genome of bovine ephemeral fever rhabdovirus contains two related glycoprotein genes.

A 3789 nucleotide region of the bovine ephemeral fever virus (BEFV) genome, located 1.65 kb downstream of the N gene, has been cloned and sequenced. The region contains two long open reading frames (ORFs) which are bounded by putative consensus (AACAGG) and polyadenylation (CATG[A]7) sequences and are separated by an intergenic region of 53 nucleotides. Discrete mRNAs corresponding to each ORF have been identified. The first ORF encodes a polypeptide comprising 623 residues which was identified by peptide sequencing as the virion G protein. The deduced amino acid sequence of the G protein includes putative signal and transmembrane domains and five potential glycosylation sites. The second ORF encodes a polypeptide of 586 amino acids which also has characteristics of a rhabdovirus glycoprotein, including putative signal and transmembrane domains and eight potential glycosylation sites, and appears to correspond to a 90-kDa nonstructural glycoprotein (GNS) identified in BEFV-infected cells (Walker et al. [1991] J. Gen. Virol. 72, 67-74). A database search indicated that both the G and GNS proteins share significant amino acid sequence homology with other rhabdovirus G proteins and with each other. Highest homology scores for each protein were with sigma virus and vesicular stomatitis virus serotypes.

The 3C-Like Proteinase of an Invertebrate Nidovirus Links Coronavirus and Potyvirus Homologs

ABSTRACT Gill-associated virus (GAV), a positive-stranded RNA virus of prawns, is the prototype of newly recognized taxa (genus Okavirus, family Roniviridae) within the order Nidovirales. In this study, a putative GAV cysteine proteinase (3C-like proteinase [3CLpro]), which is predicted to be the key enzyme involved in processing of the GAV replicase polyprotein precursors, pp1a and pp1ab, was characterized. Comparative sequence analysis indicated that, like its coronavirus homologs, 3CLpro has a three-domain organization and is flanked by hydrophobic domains. The putative 3CLpro domain including flanking regions (pp1a residues 2793 to 3143) was fused to the Escherichia coli maltose-binding protein (MBP) and, when expressed in E. coli, was found to possess N-terminal autoprocessing activity that was not dependent on the presence of the 3CLpro C-terminal domain. N-terminal sequence analysis of the processed protein revealed that cleavage occurred at the location 2827LVTHE↓VRTGN2836. The trans-processing activity of the purified recombinant 3CLpro (pp1a residues 2832 to 3126) was used to identify another cleavage site, 6441KVNHE↓LYHVA6450, in the C-terminal pp1ab region. Taken together, the data tentatively identify VxHE↓(L,V) as the substrate consensus sequence for the GAV 3CLpro. The study revealed that the GAV and potyvirus 3CLpros possess similar substrate specificities which correlate with structural similarities in their respective substrate-binding sites, identified in sequence comparisons. Analysis of the proteolytic activities of MBP-3CLpro fusion proteins carrying replacements of putative active-site residues provided evidence that, in contrast to most other 3C/3CLpros but in common with coronavirus 3CLpros, the GAV 3CLpro employs a Cys2968-His2879 catalytic dyad. The properties of the GAV 3CLpro define a novel RNA virus proteinase variant that bridges the gap between the distantly related chymotrypsin-like cysteine proteinases of coronaviruses and potyviruses.

GENOME ORGANIZATION AND TRANSCRIPTION STRATEGY IN THE COMPLEX GNS-L INTERGENIC REGION OF BOVINE EPHEMERAL FEVER RHABDOVIRUS

A 1622 nucleotide region of the bovine ephemeral fever virus (BEFV) genome, located between the second glycoprotein (GNS) gene and the polymerase (L) gene, has been cloned and sequenced in Australian (BB7721) and Chinese (Beijing-1) isolates of the virus. In the Australian isolate, the region contains five long open reading frames (ORFs) organized into three coding regions (alpha, beta and gamma), each of which are bound by a consensus transcription initiation and transcription termination-polyadenylation-like sequences. The alpha coding region contains three long ORFs (alpha 1, alpha 2 and alpha 3). The alpha 1 ORF encodes a 10.6 kDa polypeptide which contains hydrophobic and highly basic regions characteristic of a viroporin. The alpha 2 ORF encodes a 13.7 kDa polypeptide and overlaps the alpha 3 ORF which encodes a 5.7 kDa polypeptide. The beta coding region contains a single long ORF encoding a polypeptide of 12.2 kDa. The gamma coding region, which does not occur in Adelaide River virus (ARV), contains a single long ORF encoding a polypeptide of 13.4 kDa. The Chinese isolate shares 91% nucleotide sequence identity with the Australian isolate. The organization of the alpha, beta and gamma coding regions is preserved and the sequences of the encoded polypeptides are similar to those of BB7721. The major transcription products of the region were identified in BB7721 as polycistronic alpha (alpha 1-alpha 2-alpha 3) and beta-gamma mRNAs. Sequence similarities in the BEFV alpha-beta and beta-gamma gene junctions, and the gamma-L and beta-L gene junctions of BEFV and ARV, suggest that the gamma gene may have evolved from the beta-gene by sequence duplication.

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

Abstract 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 26,662nt sequence of the YHV genome. ORF1a (12,219nt) encodes a ∼462,662Da polypeptide containing a putative 3C-like protease and a putative papain-like protease with the canonical C/H catalytic dyad and α+β 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 71nt 5′-UTR shares 82.4% identity with the 68nt 5′-UTR of GAV. The 677nt 3′-terminal region contains a single 60nt ORF, commencing 298nt downstream of ORF3, that is identical to N-terminal coding region of the 249nt 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.

Multiplex RT-nested PCR differentiation of gill-associated virus (Australia) from yellow head virus (Thailand) of Penaeus monodon.

Abstract 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 794bp 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 277bp 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 ∼10fg 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- (406bp) or YHV-specific (277bp) 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.

Ticks Associated with Macquarie Island Penguins Carry Arboviruses from Four Genera

Macquarie Island, a small subantarctic island, is home to rockhopper, royal and king penguins, which are often infested with the globally distributed seabird tick, Ixodes uriae. A flavivirus, an orbivirus, a phlebovirus, and a nairovirus were isolated from these ticks and partial sequences obtained. The flavivirus was nearly identical to Gadgets Gully virus, isolated some 30 year previously, illustrating the remarkable genetic stability of this virus. The nearest relative to the orbivirus (for which we propose the name Sandy Bay virus) was the Scottish Broadhaven virus, and provided only the second available sequences from the Great Island orbivirus serogroup. The phlebovirus (for which we propose the name Catch-me-cave virus) and the previously isolated Precarious Point virus were distinct but related, with both showing homology with the Finnish Uukuniemi virus. These penguin viruses provided the second and third available sequences for the Uukuniemi group of phleboviruses. The nairovirus (for which we propose the name Finch Creek virus) was shown to be related to the North American Tillamook virus, the Asian Hazara virus and Nairobi sheep disease virus. Macquarie Island penguins thus harbour arboviruses from at least four of the seven arbovirus-containing genera, with related viruses often found in the northern hemisphere.

Penaeus monodon is protected against gill-associated virus by muscle injection but not oral delivery of bacterially expressed dsRNAs.

Gill-associated virus (GAV) is a nidovirus that commonly infects Penaeus monodon (black tiger shrimp) in eastern Australia, causing morbidity and mortalities in the acute stage of disease. Here we explored the possibility of inhibiting GAV replication and disease using double-stranded (ds)RNAs expressed in bacteria and delivered either orally or by muscle injection. To enhance potential RNA interference (RNAi) responses, 5 long dsRNAs were used that targeted open reading frame 1a/1b (ORF1a/b) gene regions and thus only the genomic length RNA. To examine oral delivery, P. monodon were fed pellets incorporating a pool of formalin-fixed bacteria containing the 5 GAV-specific dsRNAs before being injected with a minimal lethal GAV dose. Feeding with the pellets continued post-challenge but did not reduce mortality accumulation and elevation in GAV loads. In contrast, muscle injection of the dsRNAs purified from bacteria was highly effective at slowing GAV replication and protecting shrimp against acute disease and mortalities. In synergy with these data, dsRNA targeted to P. monodon beta-actin mRNA caused 100% mortality following injection, whilst its oral delivery caused no mortality. Findings confirm that injected dsRNA can mount effective RNAi responses in P. monodon to endogenous shrimp mRNA and exogenous viral RNAs, but when delivered orally in bacteria as a feed component, the same dsRNAs are ineffective. The efficacy of the RNAi response against GAV provided by injection of dsRNAs targeted to multiple genome sites suggests that this strategy might have general applicability in enhancing protection against other shrimp single-stranded (ss)RNA viruses, particularly in hatcheries or breeding programs where injection-based delivery systems are practical.

The Gene Encoding the Nucleocapsid Protein of Gill-Associated Nidovirus of Penaeus monodon Prawns Is Located Upstream of the Glycoprotein Gene

ABSTRACT 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.

Characterization of Human Parainfluenza Viruses. I. The Structural Proteins of Parainfluenza Virus 2 and their Synthesis in Infected Cells

Two strains of human parainfluenza virus 2 (HPV2), P2 1972/6 and P2 1980, grow to high titre in MEK3 cells, and their structural proteins and virus-induced protein synthesis have been characterized by gel electrophoresis and immunoprecipitation. Purified viruses contain seven polypeptides, including cellular actin: L (175K mol. wt.), HN (72K to 74K), NP (66K to 67K), F1 (52K to 58K), P (49K), A (44.5K) and M (39K). Virus-induced polypeptide synthesis was first detected at 8 h post-infection with the appearance of NP; other major structural proteins were detected from 10 to 12 h after infection and onwards. The synthesis of both the structural glycoproteins was demonstrated, although proteolytic processing could not be detected. Reproducible differences in the gel migration of the HN, F1 and NP polypeptides were found in whole virus, in infected cells and cells subjected to immunoprecipitation. These differences may reflect genetic diversity within HPV2 and provide a means of probing the molecular epidemiology of these viruses.

Genetic analysis of Black Tiger shrimp (Penaeus monodon) across its natural distribution range reveals more recent colonization of Fiji and other South Pacific islands

The Black Tiger shrimp (Penaeus monodon) has a natural distribution range from East Africa to the South Pacific Islands. Although previous studies of Indo-Pacific P. monodon have found populations from the Indian Ocean and Australasia to differ genetically, their relatedness to South Pacific shrimp remains unknown. To address this, polymorphisms at eight shared microsatellite loci and haplotypes in a 418-bp mtDNA-CR (control region) sequence were examined across 682 P. monodon from locations spread widely across its natural range, including the South Pacific islands of Fiji, Palau, and Papua New Guinea (PNG). Observed microsatellite heterozygosities of 0.82–0.91, allele richness of 6.85–9.69, and significant mtDNA-CR haplotype variation indicated high levels of genetic diversity among the South Pacific shrimp. Analysis of microsatellite genotypes using a Bayesian STRUCTURE method segregated Indo-Pacific P. monodon into eight distinct clades, with Palau and PNG shrimp clustering among others from Southeast Asia and eastern Australia, respectively, and Fiji shrimp clustering as a distinct group. Phylogenetic analyses of mtDNA-CR haplotypes delineated shrimp into three groupings, with shrimp from Fiji again being distinct by sharing no haplotypes with other populations. Depending on regional location, the genetic structures and substructures identified from the genotyping and mtDNA-CR haplotype phylogeny could be explained by Metapopulation and/or Member–Vagrant type evolutionary processes. Neutrality tests of mutation-drift equilibrium and estimation of the time since population expansion supported a hypothesis that South Pacific P. monodon were colonized from Southeast Asia and eastern Australia during the Pleistocene period over 60,000 years ago when land bridges were more expansive and linked these regions more closely.