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Flaviviruses, including Zika virus (ZIKV), utilise host mRNA degradation machinery to produce subgenomic flaviviral RNA (sfRNA). In mammalian hosts, this noncoding RNA facilitates replication and pathogenesis of flaviviruses by inhibiting IFN-signalling, whereas the function of sfRNA in mosquitoes remains largely elusive. Herein, we conduct a series of in vitro and in vivo experiments to define the role of ZIKV sfRNA in infected Aedes aegypti employing viruses deficient in production of sfRNA. We show that sfRNA-deficient viruses have reduced ability to disseminate and reach saliva, thus implicating the role for sfRNA in productive infection and transmission. We also demonstrate that production of sfRNA alters the expression of mosquito genes related to cell death pathways, and prevents apoptosis in mosquito tissues. Inhibition of apoptosis restored replication and transmission of sfRNA-deficient mutants. Hence, we propose anti-apoptotic activity of sfRNA as the mechanism defining its role in ZIKV transmission., The function on subgenomic flaviviral RNA (sfRNA) in the mosquito vector is not well understood. Here, Slonchak et al. show that sfRNA affects virus-induced apoptosis and dissemination of ZIKV in Aedes aegypti mosquitoes, suggesting a role of sfRNA in Zika virus replication and transmission.

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. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions., Here the authors describe a simple reverse genetics method that relies on overlapping cDNA fragments for generation of positive-strand viruses including SARS-CoV-2 and characterize them in vitro and in vivo.

Arboviruses cycle between, and replicate in, both invertebrate and vertebrate hosts, which for Zika virus (ZIKV) involves Aedes mosquitoes and primates 1 . 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 2–5 whereby a viral cDNA library was constructed containing all codon substitutions in the C-terminal 204 amino acids of ZIKV envelope protein (E). The cDNA library was transfected into C6/36 (Aedes) and Vero (primate) cells, with subsequent deep sequencing and computational analyses of recovered viruses showing that substitutions K316Q and S461G, or Q350L and T397S, conferred substantial replicative advantages in mosquito and primate cells, respectively. A 316Q/461G virus was constructed and shown to be replication-defective in mammalian cells due to severely compromised virus particle formation and secretion. The 316Q/461G virus was also highly attenuated in human brain organoids, and illustrated utility as a vaccine in mice. This approach can thus imitate evolutionary selection in a matter of days and identify amino acids key to the regulation of virus replication in specific host environments. Refereed/Peer-reviewed

Zika virus (ZIKV) is a re-emerging pathogenic flavivirus, which causes microcephaly in infants and poses a continuing threat to public health. ZIKV, like all other flaviviruses, produces highly abundant noncoding RNA known as subgenomic flaviviral RNA (sfRNA). Herein we utilized wild-type and mutant ZIKV defective in production of sfRNA to elucidate for the first time how production of sfRNA affects all aspects of ZIKV pathogenesis. We found that in mouse pregnancy model of infection sfRNA is required for trans-placental dissemination of ZIKV and subsequent infection of fetal brain. Using human brain organoids, we showed that sfRNA promotes apoptosis of neural progenitor cells leading to profound cytopathicity and disintegration of organoids. We also found by transcriptome profiling and gene network analysis that in infected human placental cells sfRNA inhibits multiple antiviral pathways and promotes apoptosis with STAT1 identified as a key shared factor linking these two interconnected sfRNA activities. We further showed for the first time that sfRNA inhibits phosphorylation and nuclear translocation of STAT1 by a novel mechanism which involves binding to and stabilizing viral protein NS5. This allows accumulation of NS5 at the levels required for efficient inhibition of STAT1 phosphorylation. Thus, we elucidated the molecular mechanism by which ZIKV sfRNA exerts its functions in vertebrate hosts and discovered a co-operation between viral noncoding RNA and a viral protein as a novel strategy employed by viruses to counteract antiviral responses.

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. We have extensively optimized the conditions for SARS-CoV-2 infection and demonstrated the great flexibility of iPA detection using several antibodies and dual-probing with two distinct epitope-specific antibodies. In addition, we showed that iPA could be utilized for ultra-high-throughput viral titration and neutralization assay within 24 h and is amenable to a 384-well format. These advantages will significantly accelerate SARS-CoV-2 research outcomes during this pandemic period.

Similar to other flaviviruses, Zika virus (ZIKV) produces abundant subgenomic flavivirus RNA (sfRNA) derived from the 3’ untranslated region. The molecular mechanisms that determine the functions of sfRNA are currently not completely understood. Here, we created ZIKV mutants deficient in sfRNA production and employed them to investigate the role of this RNA in virus interactions with mammalian and insect hosts. We found that in mosquitoes, sfRNA facilitates virus replication and is required for ZIKV dissemination into saliva and virus transmission. The production of sfRNA was found to have no effect on the RNAi pathway, but instead downregulated the expression of genes involved in the regulation of apoptosis. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) of histological sections from infected mosquitoes confirmed that sfRNA prevents the apoptotic death of infected cells, thus identifying inhibition of apoptosis as a novel mechanism of sfRNA action in mosquitoes. We also found that sfRNA facilitates ZIKV replication in mammalian cells, mice, and human brain organoids. Moreover, ZIKV mutants deficient in sfRNA production were unable to form plaques, cause the death of human brain organoids, or establish infection in the mouse foetal brain. We then found that the proviral activity of sfRNA in mammalian cells relies on its ability to suppress type I interferon signalling. We showed that this is achieved via the inhibition of phosphorylation and the nuclear translocation of STAT1. In addition, we found that the production of sfRNA in the ZIKV infection of human brain organoids is associated with the suppression of multiple genes involved in brain development, indicating that sfRNA can be involved in the disruption of brain development associated with ZIKV infection.

The recent emergence of Zika virus (ZIKV) in Brazil was associated with an increased number of fetal brain infections that resulted in a spectrum of congenital neurological complications known as congenital Zika syndrome (CZS). Herein, we generated de novo from sequence data an early Asian lineage ZIKV isolate (ZIKV-MY; Malaysia, 1966) not associated with microcephaly and compared the in vitro replication kinetics and fetal brain infection in interferon α/β receptor 1 knockout (IFNAR1−/−) dams of this isolate and of a Brazilian isolate (ZIKV-Natal; Natal, 2015) unequivocally associated with microcephaly. The replication efficiencies of ZIKV-MY and ZIKV-Natal in A549 and Vero cells were similar, while ZIKV-MY replicated more efficiently in wild-type (WT) and IFNAR−/− mouse embryonic fibroblasts. Viremias in IFNAR1−/− dams were similar after infection with ZIKV-MY or ZIKV-Natal, and importantly, infection of fetal brains was also not significantly different. Thus, fetal brain infection does not appear to be a unique feature of Brazilian ZIKV isolates.

Rocio virus (ROCV) is an arbovirus belonging to the genus Flavivirus, family Flaviviridae. We present an updated sequence of ROCV strain SPH 34675 (GenBank: AY632542.4), the only available full genome sequence prior to this study. Using next-generation sequencing of the entire genome, we reveal substantial sequence variation from the prototype sequence, with 30 nucleotide differences amounting to 14 amino acid changes, as well as significant changes to predicted 3’UTR RNA structures. Our results present an updated and corrected sequence of a potential emerging human-virulent flavivirus uniquely indigenous to Brazil (GenBank: MF461639).

West Nile virus (WNV) is a neurotropic flavivirus that can cause encephalitis in mammalian and avian hosts. In America, the virulent WNV strain (NY99) is causing yearly outbreaks of encephalitis in humans and horses, while in Australia the less virulent Kunjin strain of WNV strain has not been associated with significant disease outbreaks until a recent 2011 large outbreak in horses (but not in humans) caused by NSW2011 strain. Using chimeric viruses between NY99 and NSW2011 strains we previously identified a role for the non-structural proteins of NY99 strain and especially the NS3 protein, in enhanced virus replication in type I interferon response-competent cells and increased virulence in mice. To further define the role of NY99 NS3 protein in inhibition of type I interferon response, we have generated and characterised additional chimeric viruses containing the protease or the helicase domains of NY99 NS3 on the background of the NSW2011 strain. The results identified the role for the helicase but not the protease domain of NS3 protein in the inhibition of type I interferon signalling and showed that helicase domain of the more virulent NY99 strain performs this function more efficiently than helicase domain of the less virulent NSW2011 strain. Further analysis with individual amino acid mutants identified two amino acid residues in the helicase domain primarily responsible for this difference. Using chimeric replicons, we also showed that the inhibition of type I interferon (IFN) signalling was independent of other known functions of NS3 in RNA replication and assembly of virus particles.

Mosquito-transmitted flavivirus Rocio (ROCV) was responsible for an outbreak of encephalitis in the Ribeira Valley, located in the south coast of Sao Paulo State, Brazil, in 1975–1976. ROCV also causes fatal encephalitis in adult mice. Seroprevalence studies in humans, horses and water buffaloes in different regions of Brazil have suggested that ROCV is still circulating in the country, indicating the risk of re-emergence of this virus. West Nile virus (WNV) is also a mosquito-transmitted encephalitic flavivirus, however, WNV strains circulating in Australia have not been associated with outbreaks of disease in humans and exhibit low virulence in adult mice. To identify viral determinants of ROCV virulence, we have generated reciprocal chimeric viruses between ROCV and the Australian strain of WNV by swapping structural prM and E genes. Chimeric WNV containing ROCV prM-E genes replicated more efficiently than WNV or chimeric ROCV containing WNV prM-E genes in mammalian cells, was as virulent as ROCV in adult mice, and inhibited type I IFN signaling as efficiently as ROCV. The results show that ROCV prM and E proteins are major virulence determinants and identify unexpected function of these proteins in inhibition of type I interferon response.