African monkey species are natural hosts of simian hemorrhagic fever virus (SHFV) and develop persistent, asymptomatic infections. SHFV was previously shown to also cause a rapid onset fatal hemorrhagic fever disease in macaques. Infection of macaques with a new isolate of SHFV from persistently infected baboon sera, that showed high nucleotide identity with the lab strain LVR, resulted in viremia, pro-inflammatory cytokine and tissue factor production, and symptoms of coagulation defects. Primary macrophages and myeloid dendritic cell cultures from disease-susceptible macaques efficiently replicated SHFV and produced pro-inflammatory cytokines, including IL-6 and TNF-α, as well as tissue factor. Cells from disease resistant baboons produced low virus yields and the immunomodulatory cytokine IL-10. IL-10 treatment of macaque cells decreased IL-6 levels but had no effect on TNF-α levels, tissue factor or virus production suggesting that IL-10 plays a role in modulating immunopathology in disease-resistant baboons but not in regulating the efficiency of virus replication. SHFV is a member of the family Arteriviridae. The SHFV genome encodes 8 minor structural proteins. Other arteriviruses encode 4 minor structural proteins. Amino acid sequence comparisons suggest that the four additional SHFV minor structural proteins resulted from gene duplication. A full-length infectious clone of SHFV was constructed and produced virus with replication kinetics comparable to the parental virus. Mutant infectious clones, each with the start codon of one of the minor structural proteins substituted, were analyzed. All eight SHFV proteins were required for infectious virus production. The SHFV nonstructural polyprotein is processed into the mature replicase proteins by several viral proteases including papain-like cysteine proteases (PLPs). Only one or two PLP domains are present in other arteriviruses but SHFV has three PLP domains. Analysis of in vitro proteolytic processing of C- and N-terminally tagged polyproteins indicated that the PLP in each of the three SHFV nsp1 proteins is active. However, the nsp1α protease is more similar to a cysteine protease than a PLP. Analysis of the subcellular localization of the three SHFV nsp1 proteins indicated they have divergent functions.

African monkey species are natural hosts of simian hemorrhagic fever virus (SHFV) and develop persistent, asymptomatic infections. SHFV was previously shown to also cause a rapid onset fatal hemorrhagic fever disease in macaques. Infection of macaques with a new isolate of SHFV from persistently infected baboon sera, that showed high nucleotide identity with the lab strain LVR, resulted in viremia, pro-inflammatory cytokine and tissue factor production, and symptoms of coagulation defects. Primary macrophages and myeloid dendritic cell cultures from disease-susceptible macaques efficiently replicated SHFV and produced pro-inflammatory cytokines, including IL-6 and TNF-α, as well as tissue factor. Cells from disease resistant baboons produced low virus yields and the immunomodulatory cytokine IL-10. IL-10 treatment of macaque cells decreased IL-6 levels but had no effect on TNF-α levels, tissue factor or virus production suggesting that IL-10 plays a role in modulating immunopathology in disease-resistant baboons but not in regulating the efficiency of virus replication. SHFV is a member of the family Arteriviridae. The SHFV genome encodes 8 minor structural proteins. Other arteriviruses encode 4 minor structural proteins. Amino acid sequence comparisons suggest that the four additional SHFV minor structural proteins resulted from gene duplication. A full-length infectious clone of SHFV was constructed and produced virus with replication kinetics comparable to the parental virus. Mutant infectious clones, each with the start codon of one of the minor structural proteins substituted, were analyzed. All eight SHFV proteins were required for infectious virus production. The SHFV nonstructural polyprotein is processed into the mature replicase proteins by several viral proteases including papain-like cysteine proteases (PLPs). Only one or two PLP domains are present in other arteriviruses but SHFV has three PLP domains. Analysis of in vitro proteolytic processing of C- and N-terminally tagged polyproteins indicated that the PLP in each of the three SHFV nsp1 proteins is active. However, the nsp1α protease is more similar to a cysteine protease than a PLP. Analysis of the subcellular localization of the three SHFV nsp1 proteins indicated they have divergent functions.

The flavivirus West Nile virus (WNV) encodes a single polyprotein that is processed into three structural and seven nonstructural proteins. Various WNV bicistronic replicons that direct cap-dependent translation of an N-terminal viral capsid or capsid/Renilla luciferase fusion protein as well as IRES-dependent translation of the nonstructural proteins were constructed. An original replicon consisting of the WNV 5' NCR, the 5' 198 nts of the capsid coding sequence, which included the 5' cyclization sequence (Cyc), and an EMCV IRES followed by the WNV nonstructural genes and 3' NCR was generated. Real time qRT-PCR analysis of intracellular levels of this replicon RNA showed a 4 fold increase by 96 hr after transfection of BHK cells. Increasing the distance between the 5' Cyc and IRES by insertion of a 5' IRES flanking sequence alone or together with a Renilla luciferase reporter did not increase RNA replication. Addition of only a reporter decreased RNA replication. The insertion of an extended capsid coding sequence also did not enhance RNA replication, but did enhance both cap- and IRES-dependent translation of replicon RNA, as indicated by immunofluorescence and Western blot analysis. These results suggest the presence of a translation enhancer in the 3' portion of the capsid coding region. Simian hemorrhagic fever virus (SHFV) is a member of the family Arteriviridae, order Nidovirales. SHFV is unique among Nidoviruses in having three instead of two papain-like cysteine protease (PCP) motifs designated alpha, beta, and gamma, within the N-terminal region of its ORF1a. Mutations of putative PCP cleavage sites showed that the most efficient cleavage was by PCP beta at its downstream cleavage site. A large deletion located between the two catalytic residues of PCP alpha was hypothesized to render this protease inactive. However, processing was observed at the cleavage site following PCP alpha. Mutational analyses confirmed that PCP alpha is an inactive protease, and that the cleavage sites downstream of PCP alpha are cleaved by PCP gamma. When the catalytic residues of PCP gamma were mutated, PCP beta was also able to back cleave at these sites. This "back" cleavage is a previously unreported activity for an arterivirus PCP.