Development of genus-specific universal primers for the detection of flaviviruses

[Background] Flaviviruses are representative arboviruses carried by arthropods and/or vertebrates; these viruses can pose a public health concern in many countries. By contrast, it is known that a novel virus group called insect-specific flaviviruses (ISFs) also infects arthropods, although no such virus has yet been isolated from vertebrates. The characteristics of ISFs, which affect replication of human-pathogenic flaviviruses within co-infected mosquito cells or mosquitoes without affecting the mosquitoes themselves, mean that we should pay attention to both ISFs and human-pathogenic flaviviruses, despite the fact that ISFs appear not to be directly hazardous to human health. To assess the risk of diseases caused by flaviviruses, and to better understand their ecology, it is necessary to know the extent to which flaviviruses are harbored by arthropods. [Methods] We developed a novel universal primer for use in a PCR-based system to detect a broad range of flaviviruses. We then evaluated its performance. The utility of the novel primer pair was evaluated in a PCR assay using artificially synthesized oligonucleotides derived from a template viral genome sequence. The utility of the primer pair was also examined by reverse transcription PCR (RT-PCR) using cDNA templates prepared from virus-infected cells or crude supernatants prepared from virus-containing mosquito homogenates. [Results] The novel primer pair amplified the flavivirus NS5 sequence (artificially synthesized) in all samples tested (six species of flavivirus that can cause infectious diseases in humans, and flaviviruses harbored by insects). In addition, the novel primer pair detected viral genomes in cDNA templates prepared from mosquito cells infected with live flavivirus under different infectious conditions. Finally, the viral genome was detected with high sensitivity in crude supernatants prepared from pooled mosquito homogenates. [Conclusion] This PCR system based on a novel primer pair makes it possible to detect arthropod-borne flaviviruses worldwide (the primer pair even detected viruses belonging to different genetic subgroups). As such, an assay based on this primer pair may help to improve public health and safety, as well as increase our understanding of flavivirus ecology

Lung Cytokine Gene Expression is Correlated with Increased Severity of Disease in a Novel H4N8 Influenza Virus Isolated from Shorebirds

The lung cytokine gene expression profiles of mice infected with 2 strains of H4N8 viruses isolated from shorebirds and reference H4 viruses from ducks are compared. Major differences between the two H4N8 strains of shorebirds, one of which causes a severe respiratory disease in mice, are in the PB1 and NS1 genes. In mice with H4N8 virus induced pneumonia, overall expression of TNF-α, IL-6 and IL-12 genes was markedly higher than in mice infected with other H4 viruses tested, although gene expression of type I interferon was not increased until day 4 post viral infection. In contrast, in mice infected with a comparison H4N8 strain, gene expression of type I interferon peaked on day 1 post viral infection. Overall, the cytokine response corresponds with the severity of disease caused by shorebird H4N8 virus. The results obtained in this study provide valuable information to understand the immunopathology induced by a low pathogenic avian influenza virus, which may be useful in preparation for outbreaks of novel influenza A virus.https://www.jstage.jst.go.jp/article/jvms/75/10/75_13-0201/_article/-char/ja

PB2 mutations arising during H9N2 influenza evolution in the Middle East confer enhanced replication and growth in mammals.

Avian influenza virus H9N2 has been endemic in birds in the Middle East, in particular in Egypt with multiple cases of human infections since 1998. Despite concerns about the pandemic threat posed by H9N2, little is known about the biological properties of H9N2 in this epicentre of infection. Here, we investigated the evolutionary dynamics of H9N2 in the Middle East and identified phylogeny-associated PB2 mutations that acted cooperatively to increase H9N2 replication/transcription in human cells. The accumulation of PB2 mutations also correlated with an increase in H9N2 virus growth in the upper and lower airways of mice and in virulence. These mutations clustered on a solvent-exposed region in the PB2-627 domain in proximity to potential interfaces with host factors. These PB2 mutations have been found at high prevalence during evolution of H9N2 in the field, indicating that they have provided a selective advantage for viral adaptation to infect poultry. Therefore, continuous prevalence of H9N2 virus in the Middle East has generated a far more fit or optimized replication phenotype, leading to an expanded viral host range, including to mammals, which may pose public health risks beyond the current outbreaks

Stimulation of interferon-β responses by aberrant SARS-CoV-2 small viral RNAs acting as retinoic acid-inducible gene-I agonists

Summary: Patients with severe COVID-19 exhibit a cytokine storm characterized by greatly elevated levels of cytokines. Despite this, the interferon (IFN) response is delayed, contributing to disease progression. Here, we report that SARS-CoV-2 excessively generates small viral RNAs (svRNAs) encoding exact 5′ ends of positive-sense genes in human cells in vitro and ex vivo, whereas endemic human coronaviruses (OC43 and 229E) produce significantly fewer similar svRNAs. SARS-CoV-2 5′ end svRNAs are RIG-I agonists and induce the IFN-β response in the later stages of infection. The first 60-nt ends bearing duplex structures and 5′-triphosphates are responsible for immune-stimulation. We propose that RIG-I activation by accumulated SARS-CoV-2 5′ end svRNAs may contribute to later drive over-exuberant IFN production. Additionally, the differences in the amounts of svRNAs produced and the corresponding IFN response among CoV strains suggest that lower svRNA production during replication may correlate with the weaker immune response seen in less pathogenic CoVs

Novel Polymerase Gene Mutations for Human Adaptation in Clinical Isolates of Avian H5N1 Influenza Viruses.

A major determinant in the change of the avian influenza virus host range to humans is the E627K substitution in the PB2 polymerase protein. However, the polymerase activity of avian influenza viruses with a single PB2-E627K mutation is still lower than that of seasonal human influenza viruses, implying that avian viruses require polymerase mutations in addition to PB2-627K for human adaptation. Here, we used a database search of H5N1 clade 2.2.1 virus sequences with the PB2-627K mutation to identify other polymerase adaptation mutations that have been selected in infected patients. Several of the mutations identified acted cooperatively with PB2-627K to increase viral growth in human airway epithelial cells and mouse lungs. These mutations were in multiple domains of the polymerase complex other than the PB2-627 domain, highlighting a complicated avian-to-human adaptation pathway of avian influenza viruses. Thus, H5N1 viruses could rapidly acquire multiple polymerase mutations that function cooperatively with PB2-627K in infected patients for optimal human adaptation

Prevalence of PB1 human adaptation mutations in H5N1, H3N2 and H1N1 viruses.

<p>Prevalence of PB1 human adaptation mutations in H5N1, H3N2 and H1N1 viruses.</p

Structural model of the EG/D1 polymerase complex.

<p>Structural model of the EG/D1 heterotrimeric polymerase complex bound to the vRNA promoter. (A) Surface view of the EG/D1 structure color-coded (as described below) according to the domain structure in which the mutations identified in this study were located. Left and right structures differ by 180° in orientation. (B) Surface view of the EG/D1 structure color-coded showing PB2 (light green), PB1 (pink), PA (light blue) and the mutations in this study (red). Left and right structures differ by 180° in orientation. (C) Transparent EG/D1 surface diagram showing the mutations identified in this study (red) located mainly on the PB1 β-ribbon (magenta) and β-hairpin structures (violet) sandwiching the vRNA promoter. Upper and lower structures differ by 90° in orientation.</p

Relationship between polymerase activity and progeny vRNA production in primary human airway cells.

<p>The vRNA-oriented luciferase activity data in Figs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005583#ppat.1005583.g002" target="_blank">2A</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005583#ppat.1005583.g003" target="_blank">3A</a> were plotted against the progeny vRNA production data in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005583#ppat.1005583.s004" target="_blank">S4A and S4B Fig</a> at 37°C (A) and 33°C (B). The cRNA-oriented luciferase activity data in Figs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005583#ppat.1005583.g002" target="_blank">2B</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005583#ppat.1005583.g003" target="_blank">3B</a> were plotted against the progeny vRNA production data in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005583#ppat.1005583.s004" target="_blank">S4A and S4B Fig</a> at 37°C (C) and 33°C (D). The data of the 29 mutations selected to investigate viral production (Figs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005583#ppat.1005583.g003" target="_blank">3</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005583#ppat.1005583.s004" target="_blank">S4</a>) were expressed relative to the results for EG/D1 (wt). Pearson’s correlation coefficient (R) and a linear fit of the data were also calculated.</p