Transcriptional analysis of viral mRNAs reveals common transcription patterns in cells infected by five different filoviruses.

Filoviruses are notorious viral pathogens responsible for high-consequence diseases in humans and non-human primates. Transcription of filovirus mRNA shares several common features with transcription in other non-segmented negative-strand viruses, including differential expression of genes located across the viral genome. Transcriptional patterns of Ebola virus (EBOV) and Marburg virus (MARV) have been previously described using traditional, laborious methods, such as northern blots and in vivo labeling of viral mRNAs. More recently, however, the availability of the next generation sequencing (NGS) technology has offered a more straightforward approach to assess transcriptional patterns. In this report, we analyzed the transcription patterns of four ebolaviruses-EBOV, Sudan (SUDV), Bundibugyo (BDBV), and Reston (RESTV) viruses-in two different cell lines using standard NGS library preparation and sequencing protocols. In agreement with previous reports mainly focused on EBOV and MARV, the remaining filoviruses used in this study also showed a consistent transcription pattern, with only minor variations between the different viruses. We have also analyzed the proportions of the three mRNAs transcribed from the GP gene, which are characteristic of the genus Ebolavirus and encode the glycoprotein (GP), the soluble GP (sGP), and the small soluble GP (ssGP). In addition, we used NGS methodology to analyze the transcription pattern of two previously described recombinant MARV. This analysis allowed us to correct our construction design, and to make an improved version of the original MARV expressing reporter genes.

Complete Genome Sequences of Monongahela Hantavirus from Pennsylvania, USA.

Monongahela hantavirus was first identified in deer mice and was later found responsible for hantavirus pulmonary syndrome cases in Pennsylvania and West Virginia in the United States. Here, we report the complete sequences of Monongahela virus S, M, and L genomic segments obtained from a fatal clinical case reported in 1997.

Development of a reverse genetics system for Sosuga virus allows rapid screening of antiviral compounds.

Sosuga virus (SOSV) is a recently discovered zoonotic paramyxovirus isolated from a single human case in 2012; it has been ecologically and epidemiologically associated with transmission by the Egyptian rousette bat (Rousettus aegyptiacus). Bats have long been recognized as sources of novel zoonotic pathogens, including highly lethal paramyxoviruses like Nipah virus (NiV) and Hendra virus (HeV). The ability of SOSV to cause severe human disease supports the need for studies on SOSV pathogenesis to better understand the potential impact of this virus and to identify effective treatments. Here we describe a reverse genetics system for SOSV comprising a minigenome-based assay and a replication-competent infectious recombinant reporter SOSV that expresses the fluorescent protein ZsGreen1 in infected cells. First, we used the minigenome assay to rapidly screen for compounds inhibiting SOSV replication at biosafety level 2 (BSL-2). The antiviral activity of candidate compounds was then tested against authentic viral replication using the reporter SOSV at BSL-3. We identified several compounds with anti-SOSV activity, several of which also inhibit NiV and HeV. Alongside its utility in screening for potential SOSV therapeutics, the reverse genetics system described here is a powerful tool for analyzing mechanisms of SOSV pathogenesis, which will facilitate our understanding of how to combat the potential public health threats posed by emerging bat-borne paramyxoviruses.

Insights into Reston virus spillovers and adaption from virus whole genome sequences.

Reston virus (family Filoviridae) is unique among the viruses of the Ebolavirus genus in that it is considered non-pathogenic in humans, in contrast to the other members which are highly virulent. The virus has however, been associated with several outbreaks of highly lethal hemorrhagic fever in non-human primates (NHPs), specifically cynomolgus monkeys (Macaca fascicularis) originating in the Philippines. In addition, Reston virus has been isolated from domestic pigs in the Philippines. To better understand virus spillover events and potential adaption to new hosts, the whole genome sequences of representative Reston virus isolates were obtained using a next generation sequencing (NGS) approach and comparative genomic analysis and virus fitness analyses were performed. Nine virus genome sequences were completed for novel and previously described isolates obtained from a variety of hosts including a human case, non-human primates and pigs. Results of phylogenetic analysis of the sequence differences are consistent with multiple independent introductions of RESTV from a still unknown natural reservoir into non-human primates and swine farming operations. No consistent virus genetic markers were found specific for viruses associated with primate or pig infections, but similar to what had been seen with some Ebola viruses detected in the large Western Africa outbreak in 2014-2016, a truncated version of VP30 was identified in a subgroup of Reston viruses obtained from an outbreak in pigs 2008-2009. Finally, the genetic comparison of two closely related viruses, one isolated from a human case and one from an NHP, showed amino acid differences in the viral polymerase and detectable differences were found in competitive growth assays on human and NHP cell lines.

Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein.

Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014-2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulness as a tool for screening antiviral compounds and measuring neutralizing antibodies.

Recombinant Marburg viruses containing mutations in the IID region of VP35 prevent inhibition of Host immune responses.

Previous in vitro studies have demonstrated that Ebola and Marburg virus (EBOV and MARV) VP35 antagonize the host cell immune response. Moreover, specific mutations in the IFN inhibitory domain (IID) of EBOV and MARV VP35 that abrogate their interaction with virus-derived dsRNA, lack the ability to inhibit the host immune response. To investigate the role of MARV VP35 in the context of infectious virus, we used our reverse genetics system to generate two recombinant MARVs carrying specific mutations in the IID region of VP35. Our data show that wild-type and mutant viruses grow to similar titers in interferon deficient cells, but exhibit attenuated growth in interferon-competent cells. Furthermore, in contrast to wild-type virus, both MARV mutants were unable to inhibit expression of various antiviral genes. The MARV VP35 mutants exhibit similar phenotypes to those previously described for EBOV, suggesting the existence of a shared immune-modulatory strategy between filoviruses.