Ebola Virus Disease Diagnostics, Sierra Leone: Analysis of Real-time Reverse Transcription-Polymerase Chain Reaction Values for Clinical Blood and Oral Swab Specimens.

During the Ebola virus outbreak of 2013-2016, the Viral Special Pathogens Branch field laboratory in Sierra Leone tested approximately 26 000 specimens between August 2014 and October 2015. Analysis of the B2M endogenous control Ct values showed its utility in monitoring specimen quality, comparing results with different specimen types, and interpretation of results. For live patients, blood is the most sensitive specimen type and oral swabs have little diagnostic utility. However, swabs are highly sensitive for diagnostic testing of corpses.

Andes hantavirus variant in rodents, southern Amazon Basin, Peru.

We investigated hantaviruses in rodents in the southern Amazon Basin of Peru and identified an Andes virus variant from Neacomys spinosus mice. This finding extends the known range of this virus in South America and the range of recognized hantaviruses in Peru. Further studies of the epizoology of hantaviruses in this region are warranted.

The S Genome Segment Is Sufficient to Maintain Pathogenicity in Intra-Clade Lassa Virus Reassortants in a Guinea Pig Model.

Genome reassortment in Lassa virus (LASV) has been reported in nature, but phenotypic consequences of this phenomenon are not well described. Here we characterize, both in vitro and in vivo, reassortment between 2 LASV strains: the prototypic 1976 Josiah strain and a more recently isolated 2015 Liberian strain. In vitro analysis showed that although cis- and trans-acting elements of viral RNA synthesis were compatible between strains, reassortants demonstrated different levels of viral replication. These differences were also apparent in vivo, as reassortants varied in pathogenicity in the guinea pig model of LASV infection. The reassortant variant containing the Josiah S segment retained the virulence of the parental Josiah strain, but the reassortant variant containing the S segment of the Liberian isolate was highly attenuated compared to both parental strains. Contrary to observations in reassortants between LASV and other arenavirus species, which suggest that L segment-encoded factors are responsible for virulence, these studies highlight a role for S segment-encoded virulence factors in disease, and also suggest that inefficient interactions between proteins of heterologous strains may limit the prevalence of reassortant LASV variants in nature.

Virus fitness differences observed between two naturally occurring isolates of Ebola virus Makona variant using a reverse genetics approach.

During the large outbreak of Ebola virus disease that occurred in Western Africa from late 2013 to early 2016, several hundred Ebola virus (EBOV) genomes have been sequenced and the virus genetic drift analyzed. In a previous report, we described an efficient reverse genetics system designed to generate recombinant EBOV based on a Makona variant isolate obtained in 2014. Using this system, we characterized the replication and fitness of 2 isolates of the Makona variant. These virus isolates are nearly identical at the genetic level, but have single amino acid differences in the VP30 and L proteins. The potential effects of these differences were tested using minigenomes and recombinant viruses. The results obtained with this approach are consistent with the role of VP30 and L as components of the EBOV RNA replication machinery. Moreover, the 2 isolates exhibited clear fitness differences in competitive growth assays.

Lassa and Ebola virus inhibitors identified using minigenome and recombinant virus reporter systems.

Lassa virus (LASV) and Ebola virus (EBOV) infections are important global health issues resulting in significant morbidity and mortality. While several promising drug and vaccine trials for EBOV are ongoing, options for LASV infection are currently limited to ribavirin treatment. A major factor impeding the development of antiviral compounds to treat these infections is the need to manipulate the virus under BSL-4 containment, limiting research to a few institutes worldwide. Here we describe the development of a novel LASV minigenome assay based on the ambisense LASV S segment genome, with authentic terminal untranslated regions flanking a ZsGreen (ZsG) fluorescent reporter protein and a Gaussia princeps luciferase (gLuc) reporter gene. This assay, along with a similar previously established EBOV minigenome, was optimized for high-throughput screening (HTS) of potential antiviral compounds under BSL-2 containment. In addition, we rescued a recombinant LASV expressing ZsG, which, in conjunction with a recombinant EBOV reporter virus, was used to confirm any potential antiviral hits in vitro. Combining an initial screen to identify potential antiviral compounds at BSL-2 containment before progressing to HTS with infectious virus will reduce the amount of expensive and technically challenging BSL-4 containment research. Using these assays, we identified 6-azauridine as having anti-LASV activity, and demonstrated its anti-EBOV activity in human cells. We further identified 2'-deoxy-2'-fluorocytidine as having potent anti-LASV activity, with an EC50 value 10 times lower than that of ribavirin.