High-throughput quantitation of SARS-CoV-2 antibodies in a single-dilution homogeneous assay.

SARS-CoV-2 emerged in late 2019 and has since spread around the world, causing a pandemic of the respiratory disease COVID-19. Detecting antibodies against the virus is an essential tool for tracking infections and developing vaccines. Such tests, primarily utilizing the enzyme-linked immunosorbent assay (ELISA) principle, can be either qualitative (reporting positive/negative results) or quantitative (reporting a value representing the quantity of specific antibodies). Quantitation is vital for determining stability or decline of antibody titers in convalescence, efficacy of different vaccination regimens, and detection of asymptomatic infections. Quantitation typically requires two-step ELISA testing, in which samples are first screened in a qualitative assay and positive samples are subsequently analyzed as a dilution series. To overcome the throughput limitations of this approach, we developed a simpler and faster system that is highly automatable and achieves quantitation in a single-dilution screening format with sensitivity and specificity comparable to those of ELISA.

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.