Each of the eight simian hemorrhagic fever virus minor structural proteins is functionally important.
ABSTRACT: The simian hemorrhagic fever virus (SHFV) genome differs from those of other members of the family Arterivirus in encoding two adjacent sets of four minor structural protein open reading frames (ORFs). A stable, full-length, infectious SHFV-LVR cDNA clone was constructed. Virus produced from this clone had replication characteristics similar to those of the parental virus. A subgenomic mRNA was identified for the SHFV ORF previously identified as 2b. As an initial means of analyzing the functional relevance of each of the SHFV minor structural proteins, a set of mutant infectious clones was generated, each with the start codon of one minor structural protein ORF mutated. Different phenotypes were observed for each ortholog of the pairs of minor glycoproteins and all of the eight minor structural proteins were required for the production of infectious extracellular virus indicating that the duplicated sets of SHFV minor structural proteins are not functionally redundant.
Project description:The simian hemorrhagic fever virus (SHFV) genome differs from those of other members of the family Arteriviridae in encoding three papain-like one proteases (PLP1?, PLP1? and PLP1?) at the 5' end and two adjacent sets of four minor structural proteins at the 3' end. The catalytic Cys and His residues and cleavage sites for each of the SHFV PLP1s were predicted and their functionality was tested in in vitro transcription/translation reactions done with wildtype or mutant polyprotein constructs. Mass spectrometry analyses of selected autoproteolytic products confirmed cleavage site locations. The catalytic Cys of PLP1? is unusual in being adjacent to an Ala instead of a Typ. PLP1? cleaves at both downstream and upstream sites. Intermediate precursor and alternative cleavage products were detected in the in vitro transcription/translation reactions but only the three mature nsp1 proteins were detected in SHFV-infected MA104 cell lysates with SHFV nsp1 protein-specific antibodies. The duplicated sets of SHFV minor structural proteins were predicted to be functionally redundant. A stable, full-length, infectious SHFV-LVR cDNA clone was constructed and a set of mutant infectious clones was generated each with the start codon of one of the minor structural proteins mutated. All eight of the minor structural proteins were found to be required for production of infectious extracellular virus. SHFV causes a fatal hemorrhagic fever in macaques but asymptomatic, persistent infections in natural hosts such as baboons. SHFV infections were compared in macrophages and myeloid dendritic cells from baboons and macaques. Virus yields were higher from macaque cells than from baboon cells. Macrophage cultures from the two types of animals differed dramatically in the percentage of cells infected. In contrast, similar percentages of myeloid dendritic cells were infected but virus replication was efficient in the macaque cells but inefficient in the baboon cells. SHFV infection induced the production of pro-inflammatory cytokines, including IL-1?, IL-6, IL-12/23(p40), TNF-? and MIP-1?, in macaque cells but not baboon cells.
Project description:Simian hemorrhagic fever virus (SHFV) has caused lethal outbreaks of hemorrhagic disease in captive primates, but its distribution in wild primates has remained obscure. Here, we describe the discovery and genetic characterization by direct pyrosequencing of two novel, divergent SHFV variants co-infecting a single male red colobus monkey from Kibale National Park, Uganda.The viruses were detected directly from blood plasma using pyrosequencing, without prior virus isolation and with minimal PCR amplification. The two new SHFV variants, SHFV-krc1 and SHFV-krc2 are highly divergent from each other (51.9% nucleotide sequence identity) and from the SHFV type strain LVR 42-0/M6941 (52.0% and 51.8% nucleotide sequence identity, respectively) and demonstrate greater phylogenetic diversity within SHFV than has been documented within any other arterivirus. Both new variants nevertheless have the same 3' genomic architecture as the type strain, containing three open reading frames not present in the other arteriviruses.These results represent the first documentation of SHFV in a wild primate and confirm the unusual 3' genetic architecture of SHFV relative to the other arteriviruses. They also demonstrate a degree of evolutionary divergence within SHFV that is roughly equivalent to the degree of divergence between other arterivirus species. The presence of two such highly divergent SHFV variants co-infecting a single individual represents a degree of within-host viral diversity that exceeds what has previously been reported for any arterivirus. These results expand our knowledge of the natural history and diversity of the arteriviruses and underscore the importance of wild primates as reservoirs for novel pathogens.
Project description:Members of the order Nidovirales express their structural protein ORFs from a nested set of 3' subgenomic mRNAs (sg mRNAs), and for most of these ORFs, a single genomic transcription regulatory sequence (TRS) was identified. Nine TRSs were previously reported for the arterivirus Simian hemorrhagic fever virus (SHFV). In the present study, which was facilitated by next-generation sequencing, 96 SHFV body TRSs were identified that were functional in both infected MA104 cells and macaque macrophages. The abundance of sg mRNAs produced from individual TRSs was consistent over time in the two different cell types. Most of the TRSs are located in the genomic 3' region, but some are in the 5' ORF1a/1b region and provide alternative sources of nonstructural proteins. Multiple functional TRSs were identified for the majority of the SHFV 3' ORFs, and four previously identified TRSs were found not to be the predominant ones used. A third of the TRSs generated sg mRNAs with variant leader-body junction sequences. Sg mRNAs encoding E', GP2, or ORF5a as their 5' ORF as well as sg mRNAs encoding six previously unreported alternative frame ORFs or 14 previously unreported C-terminal ORFs of known proteins were also identified. Mutation of the start codon of two C-terminal ORFs in an infectious clone reduced virus yield. Mass spectrometry detected one previously unreported protein and suggested translation of some of the C-terminal ORFs. The results reveal the complexity of the transcriptional regulatory mechanism and expanded coding capacity for SHFV, which may also be characteristic of other nidoviruses.
Project description:Simian hemorrhagic fever virus is an arterivirus that naturally infects species of African nonhuman primates causing acute or persistent asymptomatic infections. Although it was previously estimated that 1% of baboons are SHFV-positive, more than 10% of wild-caught and captive-bred baboons tested were SHFV positive and the infections persisted for more than 10 years with detectable virus in the blood (100-1000 genomes/ml). The sequences of two baboon SHFV isolates that were amplified by a single passage in primary macaque macrophages had a high degree of identity to each other as well as to the genome of SHFV-LVR, a laboratory strain isolated in the 1960s. Infection of Japanese macaques with 100PFU of a baboon isolate consistently produced high level viremia, pro-inflammatory cytokines, elevated tissue factor levels and clinical signs indicating coagulation defects. The baboon virus isolate provides a reliable BSL2 model of viral hemorrhagic fever disease in macaques.
Project description:SHFV is a member of a new virus family which includes the genus arterivirus. We have cloned and sequenced 6,314 nt from the 3' end of the SHFV genome. This sequence encompasses nine complete ORFs which is three additional ORFs as compared to the other arteriviruses. We have numbered these ORFs 2a, 2b, 3, 4, 5, 6, 7, 8 and 9. At the 5' end of this sequence is a partial ORF (ORF 1b) of 1590 nt and at the 3' end is a poly(A) tract preceded by a 76 nt noncoding region. The coding capacity for each of the SHFV ORFs as well as the potential mass, pI and number of N-linked glycosylation sites for each of the encoded peptides was determined.
Project description:Key biological properties such as high genetic diversity and high evolutionary rate enhance the potential of certain RNA viruses to adapt and emerge. Identifying viruses with these properties in their natural hosts could dramatically improve disease forecasting and surveillance. Recently, we discovered two novel members of the viral family Arteriviridae: simian hemorrhagic fever virus (SHFV)-krc1 and SHFV-krc2, infecting a single wild red colobus (Procolobus rufomitratus tephrosceles) in Kibale National Park, Uganda. Nearly nothing is known about the biological properties of SHFVs in nature, although the SHFV type strain, SHFV-LVR, has caused devastating outbreaks of viral hemorrhagic fever in captive macaques. Here we detected SHFV-krc1 and SHFV-krc2 in 40% and 47% of 60 wild red colobus tested, respectively. We found viral loads in excess of 10(6)-10(7) RNA copies per milliliter of blood plasma for each of these viruses. SHFV-krc1 and SHFV-krc2 also showed high genetic diversity at both the inter- and intra-host levels. Analyses of synonymous and non-synonymous nucleotide diversity across viral genomes revealed patterns suggestive of positive selection in SHFV open reading frames (ORF) 5 (SHFV-krc2 only) and 7 (SHFV-krc1 and SHFV-krc2). Thus, these viruses share several important properties with some of the most rapidly evolving, emergent RNA viruses.
Project description:Simian hemorrhagic fever virus (SHFV) is an arterivirus that causes severe disease in captive macaques. We describe two new SHFV variants subclinically infecting wild African red-tailed guenons (Cercopithecus ascanius). Both variants are highly divergent from the prototype virus and variants infecting sympatric red colobus (Procolobus rufomitratus). All known SHFV variants are monophyletic and share three open reading frames not present in other arteriviruses. Our data suggest a need to modify the current arterivirus classification.
Project description:Arteriviruses are enveloped positive-strand RNA viruses for which the attachment proteins and cellular receptors have remained largely controversial. Arterivirus particles contain at least eight envelope proteins, an unusually large number among RNA viruses. These appear to segregate into three groups: major structural components (major glycoprotein GP5 and membrane protein [M]), minor glycoproteins (GP2a, GP3, and GP4), and small hydrophobic proteins (E and the recently discovered ORF5a protein). Biochemical studies previously suggested that the GP5-M heterodimer of porcine reproductive and respiratory syndrome virus (PRRSV) interacts with porcine sialoadhesin (pSn) in porcine alveolar macrophages (PAM). However, another study proposed that minor protein GP4, along with GP2a, interacts with CD163, another reported cellular receptor for PRRSV. In this study, we provide genetic evidence that the minor envelope proteins are the major determinant of arterivirus entry into cultured cells. A PRRSV infectious cDNA clone was equipped with open reading frames (ORFs) encoding minor envelope and E proteins of equine arteritis virus (EAV), the only known arterivirus displaying a broad tropism in cultured cells. Although PRRSV and EAV are only distantly related and utilize diversified transcription-regulating sequences (TRSs), a viable chimeric progeny virus was rescued. Strikingly, this chimeric virus (vAPRRS-EAV2ab34) acquired the broad in vitro cell tropism of EAV, demonstrating that the minor envelope proteins play a critical role as viral attachment proteins. We believe that chimeric arteriviruses of this kind will be a powerful tool for further dissection of the arterivirus replicative cycle, including virus entry, subgenomic RNA synthesis, and virion assembly.
Project description:The family Arteriviridae presently includes a single genus Arterivirus. This genus includes four species as the taxonomic homes for equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), porcine respiratory and reproductive syndrome virus (PRRSV), and simian hemorrhagic fever virus (SHFV), respectively. A revision of this classification is urgently needed to accommodate the recent description of eleven highly divergent simian arteriviruses in diverse African nonhuman primates, one novel arterivirus in an African forest giant pouched rat, and a novel arterivirus in common brushtails in New Zealand. In addition, the current arterivirus nomenclature is not in accordance with the most recent version of the International Code of Virus Classification and Nomenclature. Here we outline an updated, amended, and improved arterivirus taxonomy based on current data. Taxon-specific sequence cut-offs are established relying on a newly established open reading frame 1b phylogeny and pairwise sequence comparison (PASC) of coding-complete arterivirus genomes. As a result, the current genus Arterivirus is replaced by five genera: Equartevirus (for EAV), Rodartevirus (LDV + PRRSV), Simartevirus (SHFV + simian arteriviruses), Nesartevirus (for the arterivirus from forest giant pouched rats), and Dipartevirus (common brushtail arterivirus). The current species Porcine reproductive and respiratory syndrome virus is divided into two species to accommodate the clear divergence of the European and American "types" of PRRSV, both of which now receive virus status. The current species Simian hemorrhagic fever virus is divided into nine species to accommodate the twelve known simian arteriviruses. Non-Latinized binomial species names are introduced to replace all current species names to clearly differentiate them from virus names, which remain largely unchanged.
Project description:The N-terminal region of simian hemorrhagic fever virus (SHFV) nonstructural polyprotein 1a is predicted to encode three papain-like proteases (PLP1?, PLP1?, and PLP1?). Catalytic residues and cleavage sites for each of the SHFV PLP1s were predicted by alignment of the SHFV PLP1 region sequences with each other as well as with those of other arteriviruses, and the predicted catalytic residues were shown to be proximal by homology modeling of the SHFV nsp1s on porcine respiratory and reproductive syndrome virus (PRRSV) nsp1 crystal structures. The functionality of the predicted catalytic Cys residues and cleavage sites was tested by analysis of the autoproteolytic products generated in in vitro transcription/translation reactions done with wild-type or mutant SHFV nsp1 constructs. Cleavage sites were also analyzed by mass spectroscopy analysis of selected immunoprecipitated cleavage products. The data showed that each of the three SHFV PLP1s is an active protease. Cys63 was identified as the catalytic Cys of SHFV PLP1? and is adjacent to an Ala instead of the canonical Tyr observed in other arterivirus PLP1s. SHFV PLP1? is able to cleave at both downstream and upstream nsp1 junction sites. Although intermediate precursor polyproteins as well as alternative products generated by each of the SHFV PLP1s cleaving at sites within the N-terminal region of nsp1? were produced in the in vitro reactions, Western blotting of SHFV-infected, MA104 cell lysates with SHFV nsp1 protein-specific antibodies detected only the three mature nsp1 proteins.SHFV is unique among arteriviruses in having three N-terminal papain-like protease 1 (PLP1) domains. Other arteriviruses encode one or two active PLP1s. This is the first functional study of the SHFV PLP1s. Analysis of the products of in vitro autoprocessing of an N-terminal SHFV nonstructural 1a polypeptide fragment showed that each of the three SHFV PLP1s is active, and the predicted catalytic Cys residues and cleavage sites for each PLP1 were confirmed by testing mutant constructs. Several unique features of the SHFV PLP1s were discovered. The SHFV PLP1? catalytic Cys63 is unique among arterivirus PLP1s in being adjacent to an Ala instead of a Trp. Other arterivirus PLP1s cleave only in cis at a single downstream site, but SHFV PLP1? can cleave at both the downstream nsp1?-nsp2 and upstream nsp1?-nsp1? junctions. The three mature nsp1 proteins were produced both in the in vitro reactions and in infected cells.