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Viruses assigned to the genus Hapavirus form a distinct monophyletic group based on well-supported Maximum Likelihood trees inferred from complete L sequences. Hapaviruses have been isolated primarily from birds and culicine mosquitoes. The hapavirus clade is part of a larger phylogenetic group of arthropod-borne rhabdoviruses with large and complex genomes that also includes ephemeroviruses, tibroviruses and curioviruses. Most hapaviruses contain i) multiple genes between the P gene and M gene encoding proteins that share low levels of sequence homology and appear to have arisen by gene duplication; and ii) a gene between the G and L genes encoding a small class 1a viroporin-like protein.
Virion morphology in ultrathin sections of infected cells has been described for several hapaviruses. For Flanders virus (FLAV), Mossuril virus (MOSV) and Manitoba virus (MANV), long bullet-shaped virions (~200–300 nm x 45–75 nm) have been observed budding at the plasma membrane and/or in association with intracytoplasmic vacuoles (Artsob et al., 1991, Boyd and Whitaker-Dowling 1988, Karabatsos et al., 1973). Wongabel virus (WONV) virions have also been observed budding from the plasma membrane and endoplasmic reticula but are shorter and more cone-shaped (160–180 nm x 80–90 nm). Variations in virion morphology may be due in part to differences in conditions used for fixation and staining of tissues (Gubala et al., 2008).
Hapavirus genomes consist of a single molecule of negative-sense, single-stranded RNA and range from approximately 12.4–15.8 kb (Walker et al., 2015, Gubala et al., 2010, Boyd and Whitaker-Dowling 1988, Gubala et al., 2008, Allison et al., 2014).
The N, P, M, G and L share sequence homology and/or structural characteristics with the cognate proteins of other rhabdoviruses. Few other proteins that are encoded in hapavirus genomes have yet been identified in infected cells. P-M intergenic region proteins (PMIPs) range from 142 to 194 amino acids (16.5–22.3 kDa) and generally share very low but detectable sequence homology with PMIPs in the same virus and in other closely related hapaviruses (Walker et al., 2015). Of the many hapavirus PMIPs, only WONV U3 has been shown to be expressed and characterised functionally, binding to a component of the SWI/SNF chromatin remodelling complex and regulating immune response gene expression (Joubert et al., 2015). Small class 1a viroporin-like proteins encoded in all hapaviruses (except Marco virus (MCOV) range from 102 to 153 amino acids (11.5–17.6 kDa) and feature an N-terminal domain containing large hydrophobic residues, a predicted hydrophobic transmembrane domain and a C-terminal domain that is rich in basic residues (Walker et al., 2015). The 568-amino-acid Ngaingan virus (NGAV) GNS protein is predicted to be a class I transmembrane glycoprotein (unglycosylated mass 65.0 kDa) that shares extensive sequence homology with NGAV G and G of other rhabdoviruses; similar GNS proteins also occur in all ephemeroviruses but phylogenetic inferences suggest that the NGAV GNS protein has evolved either independently or by genetic recombination (Walker et al., 2015, Gubala et al., 2010). The 459 amino acid MCOV U1 protein is also predicted to be a second class I transmembrane protein (unglycosylated mass 52.5 kDa) but it lacks cysteine residues that could form disulphide bonds and appears to be unrelated to either MCOV G or NGAV GNS (Walker et al., 2015). The many other putative hapavirus accessory proteins are predicted to have largely unremarkable structural characteristics.
Hapavirus genomes include five genes (N, P, M, G and L) encoding the structural proteins and multiple additional long ORFs (Figure 1.Hapavirus). The genomes vary considerably in the number and locations of accessory genes but most hapavirus genomes include: i) multiple genes between the P gene and M gene encoding proteins that share low levels of sequence identity and appear to have arisen by gene duplication; and ii) a gene between the G and L genes encoding a small class 1a viroporin-like protein (Walker et al., 2015, Walker et al., 2011, Allison et al., 2014). Several other hapavirus genes appear to have arisen by gene duplication: in NGAV, the G gene and the GNS gene immediately following encode glycoproteins that share extensive sequence homology; and in Joinjakaka virus (JOIV), the U2 and U3 genes, also located between the G gene and L gene, share significant sequence homology. In WONV, Ord River virus (ORV) and Parry Creek virus (PCV), alternative long ORFs in the N genes (Nx or U4) overlap the end of the N ORF and encode homologous proteins; similarly, FLAV and Hart Park virus (HPV) encode homologous proteins in alternative ORFs (also designated Nx) near the end of the N gene.
Many of the additional hapavirus long ORFs occur in novel independent transcriptional units including conserved transcription initiation and transcription termination sequences. Others are located as alternative, overlapping or consecutive ORFs within the structural protein genes or within the novel transcriptional units. Although yet to be tested experimentally, hapaviruses therefore appear to employ various non-canonical strategies to express proteins encoded in these long ORFs. The likely mechanisms include: i) leaky ribosomal scanning; ii) a stop-start mechanism involving a ‘termination upstream ribosome-binding site’ (TURBS); and iii) ribosomal frame shifts featuring a ‘slippery’ sequence followed by a predicted pseudoknot structure (Walker et al., 2015).
Figure 1.Hapavirus. Schematic representation of hapavirus genome organisations. N, P, M, G and L represent ORFs encoding the structural proteins. ORFs are indicated as block arrows. P-M intergenic region protein (PMIP) ORFs are coloured in red; class 1a viroporin-like protein ORFs are coloured in yellow; other colours indicate ORFs encoding homologous proteins. Other alternative ORFs occur in some genes; only ORFs (≥180 nt) that appear likely to be expressed are shown.
Several members of the genus Hapavirus (HPV, FLAV, Mosqueiro virus (MQOV), MOSV, Kamese virus (KAMV)) cross-react strongly in complement-fixation and indirect immunofluorescence tests and have been assigned to the Hart Park serogroup of rhabdoviruses (Calisher et al., 1989, Tesh et al., 1983). Several of the viruses in this serogroup also cross-react weakly in neutralisation tests.
Hapaviruses have been isolated primarily from culicine mosquitoes and passerine birds. HPV and FLAV circulate in mosquito-bird transmission cycles in western and eastern regions of the USA, respectively (Allison et al., 2014). Landjia virus (LJAV) was isolated from a passerine bird (Riparia paludicola) in Africa; other hapaviruses have been isolated from culicine mosquitoes in Africa (MOSV, KAMV), North America (MANV, Gray Lodge virus (GLOV)), Central America (La Joya virus (LJV)), South America (MQOV), Papua New Guinea (JOIV) or Australia (ORV, PCV). WONV and NGAV were each isolated from biting midges (Culicoides spp.) in Australia (Gubala et al., 2010, Gubala et al., 2008). There is evidence of WONV antibodies in sea birds (Humphrey-Smith et al., 1991) and NGAV antibodies in marsupials (wallabies and wallaroos) (Gubala et al., 2010). MCOV was isolated from reptiles (lizards) in South America (Causey et al., 1966).
Viruses assigned to different species within the genus Hapavirus display several of the following characteristics: A) minimum amino acid sequence divergence of 5% in N; B) minimum sequence divergence of 10% in L; C) minimum amino acid sequence divergence of 15% G; D) significant differences in genome organisation as evidenced by numbers and locations of ORFs; E) can be distinguished in virus neutralisation tests; and F) occupy different ecological niches as evidenced by differences in hosts and/or arthropod vectors.
Hapavirus: from Hart Park serogroup which is the well-established antigenic designation of FLAV, HPV and several other members of the genus.
Holmes Jungle virus
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