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 showed a very 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 100 PFU 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.

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.

Simian hemorrhagic fever virus (SHFV) causes a fatal hemorrhagic fever in macaques but an asymptomatic, persistent infection in baboons. To investigate factors contributing to this differential infection outcome, the targets of SHFV infection, macrophages (M⌽s) and myeloid dendritic cells (mDCs), were differentiated from macaque and baboon peripheral blood monocytes and used to compare viral replication and cell responses. SHFV replicated in >90% of macaque M⌽s but in only ϳ10% of baboon M⌽s. Although SHFV infected ϳ50% of macaque and baboon mDCs, virus replication was efficient in macaque but not in baboon mDCs. Both types of macaque cultures produced higher virus yields than baboon cultures. A more efficient type I interferon response and the production of proinflammatory cytokines, including interleukin-1␤ (IL-1␤), IL-6, IL-12/23(p40), tumor necrosis factor alpha (TNF-␣), and macrophage inflammatory protein 1␣ (MIP-1␣), in response to SHFV infection were observed in macaque but not baboon cultures, suggesting less efficient counteraction of these responses by viral proteins in macaque cells. Baboon cultures produced higher levels of IL-10 than macaque cultures both prior to and after SHFV infection. In baboon but not macaque cell cultures, SHFV infection upregulated IL-10R1, a subunit of the IL-10 receptor (IL-10R), and also SOCS3, a negative regulator of proinflammatory cytokine production. Incubation of macaque cultures with human IL-10 before and/or after SHFV infection decreased production of IL-6, IL-1␤, and MIP-1␣ but not TNF-␣, suggesting a role for IL-10 in suppressing SHFV-induced proinflammatory cytokine production in macaques.

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. IMPORTANCESHFV 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.

Simian hemorrhagic fever virus (SHFV) is an understudied arterivirus that typically causes asymptomatic, persistent infections in multiple species of African nonhuman primates (NHPs) which are natural hosts but fatal hemorrhagic fever disease in macaques. SHFV genomes found in different species of African primates have recently been sequenced but no biological data for these viruses has yet been reported. The sequence of one SHFV isolate from a long term persistently infected baboon showed a high degree of identity to the genome of SHFV-LVR, the prototype strain isolated in the 1960s. Similar to what was observed in early studies with the prototype SHFV isolate LVR, infection of Japanese macaques with 100 PFU of the baboon isolate efficiently induced fatal hemorrhagic fever disease in macaques. Consistent with the differential infection outcomes observed in macaques and baboons, SHFV infection of macaque macrophages and dendritic cells is more efficiently than that of baboons and infection induces pro39 inflammatory cytokine production in macaque but not baboon cells. A stable full-length SHFV LVR infectious clone was constructed and basic knowledge about the unique functions of SHFV has been expanded by several recent studies. The SHFV genome encodes extra genes compared to those of other arteriviruses; three instead of two papain-like protease one (PLP1) domains are encoded at the 5’ end and two adjacent sets of four minor structural proteins at the 3’ end. SHFV PLP1α, PLP1β and PLP1γ were each shown to be active proteases in vitro and the three expected nsp1proteins were detected in infected cells. The catalytic Cys of PLP1α is adjacent to an Ala instead of canonical Typ and PLP1γ is unique among arterivirus PLPs in being able to cleave at both downstream and upstream sites. Although the duplicated sets of SHFV minor structural proteins were predicted to be functionally redundant, data obtained with a set of mutant infectious clones, each with the start codon of one of the minor structural proteins mutated, showed that all eight of the minor structural proteins are required for production of infectious extracellular virus.