Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b' Is Not Required for In Vitro Virus Replication.

Yingyun Cai,Reed F Johnson,David H O’Connor,Gustavo Palacios,Courtney L Finch,Jens H Kuhn,Steven Mazur,Ying Fang,Sheli R Radoshitzky,Shuiqing Yu,Yanhua Li,Thomas C Friedrich,Peter B Jahrling,Michael Lauck,Laura Bollinger,Elena N Postnikova,Tony L Goldberg

doi:10.3390/v13040632

Abstract

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b’, is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.

Highlights

Simian hemorrhagic fever (SHF) was first identified in 1964 as an acute, almost uniformly lethal disease of Asian macaques (Macaca spp.) in captive colonies in Sukhumi, Georgian Soviet Socialist Republic, USSR, and Bethesda, Maryland, USA [1,2,3]
To construct a DNA-launch infectious clone of simian hemorrhagic fever virus (SHFV), we cloned the cDNA of the entire genome of SHFV variant NIH LVR42-0/M6941 (GenBank #AF180391; RefSeq #NC_003092), flanked by a CMV immediate early enhancer-containing promoter at the 5 end and the hepatitis delta virus 1 (HDV-1) ribozyme sequence at the 3 end to ensure generation of authentic 3 genome sequences, into plasmid pACYC177 (“pCMV-SHFV-NC_003092.2”)
We introduced most of these discrepancies into pCMV-SHFV-NC_003092.2, yielding pCMV-SHFV, and created a derived plasmid thereof, pCMV-SHFV-eGFP, encoding eGFP between open reading frames (ORFs) 1b and ORF 2a’ (Figure 1)

Summary

Introduction

Simian hemorrhagic fever (SHF) was first identified in 1964 as an acute, almost uniformly lethal disease of Asian macaques (Macaca spp.) in captive colonies in Sukhumi, Georgian Soviet Socialist Republic, USSR, and Bethesda, Maryland, USA [1,2,3]. In addition to SHEV, SHFV, PBJV, and KRCV-1, at least seven more simarterivirins have been discovered, often at high titers and sometimes in the presence of other simarterivirins, in serum of apparently healthy African primates of numerous species [9,11,12,13,14]. Many of these simarterivirins may cause SHFV or similar diseases once introduced into a non-natural host such as macaques or, potentially, humans [15]

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Discussion

Conclusion