Evaluation of three alternative methods to the plaque reduction neutralizing assay for measuring neutralizing antibodies to dengue virus serotype 2.

Background: Dengue is a global public health challenge which requires accurate diagnostic methods for surveillance and control. The gold standard for detecting dengue neutralizing antibodies (nAbs) is the plaque reduction neutralization test (PRNT), which is both labor-intensive and time-consuming. This study aims to evaluate three alternative approaches, namely, the MTT-based (or (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) microneutralization assay, the xCELLigence real-time cell analysis (RTCA), and the immuno-plaque assay-focus reduction neutralization test (iPA-FRNT).

Residue K28 of Zika Virus NS5 Protein Is Implicated in Virus Replication and Antagonism of STAT2.

The identification of four potential nonstructural 5 (NS5) residues-K28, K45, V335, and S749-that share the same amino acid preference in STAT2-interacting flaviviruses [Dengue virus (DENV) and Zika virus (ZIKV)], but not in STAT2-non-interacting flaviviruses [West Nile virus (WNV) and/or Yellow fever virus (YFV)] from an alignment of multiple flavivirus NS5 sequences, implied a possible association with the efficiency of ZIKV to antagonize the human signal transducer and activator of transcription factor 2 (STAT2). Through site-directed mutagenesis and reverse genetics, mutational impacts of these residues on ZIKV growth in vitro and STAT2 antagonism were assessed using virus growth kinetics assays and STAT2 immunoblotting. The results showed that mutations at the residue K28 significantly reduced the efficiency of ZIKV to antagonize STAT2.

Establishment of a CPER reverse genetics system for Powassan virus defines attenuating NS1 glycosylation sites and an infectious NS1-GFP11 reporter virus.

Powassan virus (POWV) is an emerging tick-borne Flavivirus that causes lethal encephalitis and long-term neurologic damage. Currently, there are no POWV therapeutics, licensed vaccines, or reverse genetics systems for producing infectious POWVs from recombinant DNA. Using a circular polymerase extension reaction (CPER), we generated recombinant LI9 (recLI9) POWVs with attenuating NS1 protein mutations and a recLI9-split-eGFP reporter virus.

Viable mpox virus in the environment of a patient room.

We conducted a prospective environmental surveillance study to investigate the air, surface, dust, and water contamination of a room occupied by a patient infected with mpox virus (MPXV) at various stages of the illness. The patient tested positive for MPXV from a throat swab and skin lesions. Environmental sampling was conducted in a negative pressure room with 12 unidirectional high efficiency particulate air filter (HEPA) air changes per hour and daily cleaning of the surfaces.

Field trial assessing the antimicrobial decontamination efficacy of gaseous ozone in a public bus setting.

The widespread COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) necessitated measures aimed at preventing the spread of SARS-CoV-2. To mitigate the risk of fomite-mediated transmission, environmental cleaning and disinfection regimes have been widely implemented. However, conventional cleaning approaches such as surface wipe downs can be laborious and more efficient and effective disinfecting technologies are needed.

Rapid Diagnostic Tests for the Detection of the Four Dengue Virus Serotypes in Clinically Relevant Matrices.

The efficient and accurate diagnosis of dengue, a major mosquito-borne disease, is of primary importance for clinical care, surveillance, and outbreak control. The identification of specific dengue virus serotype 1 (DENV-1) to DENV-4 can help in understanding the transmission dynamics and spread of dengue disease. The four rapid low-resource serotype-specific dengue tests use a simple sample preparation reagent followed by reverse transcription-isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology.

Assessing the efficacy of male Wolbachia-infected mosquito deployments to reduce dengue incidence in Singapore: study protocol for a cluster-randomized controlled trial.

Background: Dengue is a severe environmental public health challenge in tropical and subtropical regions. In Singapore, decreasing seroprevalence and herd immunity due to successful vector control has paradoxically led to increased transmission potential of the dengue virus. We have previously demonstrated that incompatible insect technique coupled with sterile insect technique (IIT-SIT), which involves the release of X-ray-irradiated male Wolbachia-infected mosquitoes, reduced the Aedes aegypti population by 98% and dengue incidence by 88%.

Zika virus noncoding RNA cooperates with the viral protein NS5 to inhibit STAT1 phosphorylation and facilitate viral pathogenesis.

All flaviviruses, including Zika virus, produce noncoding subgenomic flaviviral RNA (sfRNA), which plays an important role in viral pathogenesis. However, the exact mechanism of how sfRNA enables viral evasion of antiviral response is not well defined. Here, we show that sfRNA is required for transplacental virus dissemination in pregnant mice and subsequent fetal brain infection.

The distinguishing NS5-M114V mutation in American Zika virus isolates has negligible impacts on virus replication and transmission potential.

During 2015-2016, outbreaks of Zika virus (ZIKV) occurred in Southeast Asia and the Americas. Most ZIKV infections in humans are asymptomatic, while clinical manifestation is usually a self-limiting febrile disease with maculopapular rash. However, ZIKV is capable of inducing a range of severe neurological complications collectively described as congenital Zika syndrome (CZS).

Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance.

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly.

A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses.

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction.

Non-intrusive wastewater surveillance for monitoring of a residential building for COVID-19 cases.

Wastewater-based surveillance for SARS-CoV-2 has been used for the early warning of transmission or objective trending of the population-level disease prevalence. Here, we describe a new use-case of conducting targeted wastewater surveillance to complement clinical testing for case identification in a small community at risk of COVID-19 transmission. On 2 July 2020, a cluster of COVID-19 cases in two unrelated households residing on different floors in the same stack of an apartment building was reported in Singapore.

Assessing the potential of unmanned aerial vehicle spraying of aqueous ozone as an outdoor disinfectant for SARS-CoV-2.

The COVID-19 pandemic has revealed gaps in our understanding of safe, effective and efficient means of disinfecting high use public spaces. Whilst this creates an opportunity for development and application of innovative approaches such as unmanned aerial vehicle (UAV) based disinfection, unregulated outdoor disinfection using chlorine has led to environmental and public health risks. This study has quantified the efficiency, safety and efficacy of UAV-based spraying of aqueous ozone.

An Optimized High-Throughput Immuno-Plaque Assay for SARS-CoV-2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 and is capable of human-to-human transmission and rapid global spread. The rapid emergence and global spread of SARS-CoV-2 has encouraged the establishment of a rapid, sensitive, and reliable viral detection and quantification methodology. Here, we present an alternative assay, termed immuno-plaque assay (iPA), which utilizes a combination of plaque assay and immunofluorescence techniques.

A broadly protective antibody that targets the flavivirus NS1 protein.

There are no approved flaviviral therapies and the development of vaccines against flaviruses has the potential of being undermined by antibody-dependent enhancement (ADE). The flavivirus nonstructural protein 1 (NS1) is a promising vaccine antigen with low ADE risk but has yet to be explored as a broad-spectrum therapeutic antibody target. Here, we provide the structural basis of NS1 antibody cross-reactivity through cocrystallization of the antibody 1G5.

Antigenic Characterization of New Lineage II Insect-Specific Flaviviruses in Australian Mosquitoes and Identification of Host Restriction Factors.

We describe two new insect-specific flaviviruses (ISFs) isolated from mosquitoes in Australia, Binjari virus (BinJV) and Hidden Valley virus (HVV), that grow efficiently in mosquito cells but fail to replicate in a range of vertebrate cell lines. Phylogenetic analysis revealed that BinJV and HVV were closely related (90% amino acid sequence identity) and clustered with lineage II (dual-host affiliated) ISFs, including the Lammi and Nounané viruses. Using a panel of monoclonal antibodies prepared to BinJV viral proteins, we confirmed a close relationship between HVV and BinJV and revealed that they were antigenically quite divergent from other lineage II ISFs.

Protective Efficacy of a Chimeric Insect-Specific Flavivirus Vaccine against West Nile Virus.

Virulent strains of West Nile virus (WNV) are highly neuro-invasive and human infection is potentially lethal. However, no vaccine is currently available for human use. Here, we report the immunogenicity and protective efficacy of a vaccine derived from a chimeric virus, which was constructed using the structural proteins (prM and E) of the Kunjin strain of WNV (WNV) and the genome backbone of the insect-specific flavivirus Binjari virus (BinJV).

A recombinant platform for flavivirus vaccines and diagnostics using chimeras of a new insect-specific virus.

Flaviviruses such as dengue, yellow fever, Zika, West Nile, and Japanese encephalitis virus present substantial global health burdens. New vaccines are being sought to address safety and manufacturing issues associated with current live attenuated vaccines. Here, we describe a new insect-specific flavivirus, Binjari virus, which was found to be remarkably tolerant for exchange of its structural protein genes (prME) with those of the aforementioned pathogenic vertebrate-infecting flaviviruses (VIFs).

Chimeric viruses of the insect-specific flavivirus Palm Creek with structural proteins of vertebrate-infecting flaviviruses identify barriers to replication of insect-specific flaviviruses in vertebrate cells.

Here we report the generation of novel chimeric flaviviruses, which express the prM and E proteins of either dengue or Zika viruses on the genomic backbone of Palm Creek virus (PCV), an insect-specific flavivirus. The chimeric virus particles were antigenically indistinguishable from their parental prM-E donors, but were unable to infect vertebrate cells. An additional chimera (PCV structural genes in the backbone of West Nile virus - WNV/PCV-prME) was also unable to infect vertebrate cells, but transfection with RNA from this virus resulted in detectable RNA replication and translation but no infectious virion production.

West Nile virus infection and interferon alpha treatment alter the spectrum and the levels of coding and noncoding host RNAs secreted in extracellular vesicles.

Background: Extracellular vesicles (EVs) are small membrane vesicles secreted by the cells that mediate intercellular transfer of molecules and contribute to transduction of various signals. Viral infection and action of pro-inflammatory cytokines has been shown to alter molecular composition of EV content. Transfer of antiviral proteins by EVs is thought to contribute to the development of inflammation and antiviral state.

Author Correction: Infectious DNAs derived from insect-specific flavivirus genomes enable identification of pre- and post-entry host restrictions in vertebrate cells.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Determinants of Zika virus host tropism uncovered by deep mutational scanning.

Arboviruses cycle between, and replicate in, both invertebrate and vertebrate hosts, which for Zika virus (ZIKV) involves Aedes mosquitoes and primates. The viral determinants required for replication in such obligate hosts are under strong purifying selection during natural virus evolution, making it challenging to resolve which determinants are optimal for viral fitness in each host. Herein we describe a deep mutational scanning (DMS) strategy whereby a viral cDNA library was constructed containing all codon substitutions in the C-terminal 204 amino acids of ZIKV envelope protein (E).