Attenuation of SARS-CoV-2 replication and associated inflammation by concomitant targeting of viral and host cap 2'-O-ribose methyltransferases.

The SARS-CoV-2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2'-O-ribose cap needed for viral immune escape. We find that the host cap 2'-O-ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS-CoV-2 replication. Using in silico target-based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti-SARS-CoV-2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co-substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID-19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection-induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID-19.

The anti-influenza virus drug favipiravir has little effect on replication of SARS-CoV-2 in cultured cells.

Favipiravir (T-705, commercial name Avigan), a drug developed to treat influenza virus infection, has been used in some countries as an oral treatment for COVID-19; however, its clinical efficacy in this context is controversial.….

Pathogenesis of Influenza A(H7N9) Virus in Aged Nonhuman Primates.

The avian influenza A(H7N9) virus has caused high mortality rates in humans, especially in the elderly; however, little is known about the mechanistic basis for this. In the current study, we used nonhuman primates to evaluate the effect of aging on the pathogenicity of A(H7N9) virus. We observed that A(H7N9) virus infection of aged animals (defined as age 20-26 years) caused more severe symptoms than infection of young animals (defined as age 2-3 years). In aged animals, lung inflammation was weak and virus infection was sustained. Although cytokine and chemokine expression in the lungs of most aged animals was lower than that in the lungs of young animals, 1 aged animal showed severe symptoms and dysregulated proinflammatory cytokine and chemokine production. These results suggest that attenuated or dysregulated immune responses in aged animals are responsible for the severe symptoms observed among elderly patients infected with A(H7N9) virus.

Identification of a distinct lineage of aviadenovirus from crane feces.

Viruses are believed to be ubiquitous; however, the diversity of viruses is largely unknown because of the bias of previous research toward pathogenic viruses. Deep sequencing is a promising and unbiased approach to detect viruses from animal-derived materials. Although cranes are known to be infected by several viruses such as influenza A viruses, previous studies targeted limited species of viruses, and thus viruses that infect cranes have not been extensively studied. In this study, we collected crane fecal samples in the Izumi plain in Japan, which is an overwintering site for cranes, and performed metagenomic shotgun sequencing analyses. We detected aviadenovirus-like sequences in the fecal samples and tentatively named the discovered virus crane-associated adenovirus 1 (CrAdV-1). We determined that our sequence accounted for approximately three-fourths of the estimated CrAdV-1 genome size (33,245 bp). The GC content of CrAdV-1 genome is 34.1%, which is considerably lower than that of other aviadenoviruses. Phylogenetic analyses revealed that CrAdV-1 clusters with members of the genus Aviadenovirus, but is distantly related to the previously identified aviadenoviruses. The protein sequence divergence between the DNA polymerase of CrAdV-1 and those of other aviadenoviruses is 45.2-46.8%. Based on these results and the species demarcation for the family Adenoviridae, we propose that CrAdV-1 be classified as a new species in the genus Aviadenovirus. Results of this study contribute to a deeper understanding of the diversity and evolution of viruses and provide additional information on viruses that infect cranes, which might lead to protection of the endangered species of cranes.

Characterization of H7N9 influenza A viruses isolated from humans.

Avian influenza A viruses rarely infect humans; however, when human infection and subsequent human-to-human transmission occurs, worldwide outbreaks (pandemics) can result. The recent sporadic infections of humans in China with a previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern owing to the appreciable case fatality rate associated with these infections (more than 25%), potential instances of human-to-human transmission, and the lack of pre-existing immunity among humans to viruses of this subtype. Here we characterize two early human A(H7N9) isolates, A/Anhui/1/2013 (H7N9) and A/Shanghai/1/2013 (H7N9); hereafter referred to as Anhui/1 and Shanghai/1, respectively. In mice, Anhui/1 and Shanghai/1 were more pathogenic than a control avian H7N9 virus (A/duck/Gunma/466/2011 (H7N9); Dk/GM466) and a representative pandemic 2009 H1N1 virus (A/California/4/2009 (H1N1pdm09); CA04). Anhui/1, Shanghai/1 and Dk/GM466 replicated well in the nasal turbinates of ferrets. In nonhuman primates, Anhui/1 and Dk/GM466 replicated efficiently in the upper and lower respiratory tracts, whereas the replicative ability of conventional human influenza viruses is typically restricted to the upper respiratory tract of infected primates. By contrast, Anhui/1 did not replicate well in miniature pigs after intranasal inoculation. Critically, Anhui/1 transmitted through respiratory droplets in one of three pairs of ferrets. Glycan arrays showed that Anhui/1, Shanghai/1 and A/Hangzhou/1/2013 (H7N9) (a third human A(H7N9) virus tested in this assay) bind to human virus-type receptors, a property that may be critical for virus transmissibility in ferrets. Anhui/1 was found to be less sensitive in mice to neuraminidase inhibitors than a pandemic H1N1 2009 virus, although both viruses were equally susceptible to an experimental antiviral polymerase inhibitor. The robust replicative ability in mice, ferrets and nonhuman primates and the limited transmissibility in ferrets of Anhui/1 suggest that A(H7N9) viruses have pandemic potential.

Emergence of Oseltamivir-Resistant H7N9 Influenza Viruses in Immunosuppressed Cynomolgus Macaques.

Antiviral compounds (eg, the neuraminidase inhibitor oseltamivir) are invaluable for the treatment of individuals infected with influenza A viruses of the H7N9 subtype (A[H7N9]), which have infected and killed hundreds of persons. However, oseltamivir treatment often leads to the emergence of resistant viruses in immunocompromised individuals. To better understand the emergence and properties of oseltamivir-resistant A(H7N9) viruses in immunosuppressed individuals, we infected immunosuppressed cynomolgus macaques with an A(H7N9) virus and treated them with oseltamivir. Disease severity and mortality were higher in immunosuppressed than in immunocompetent animals. Oseltamivir treatment at 2 different doses reduced A(H7N9) viral titers in infected animals, but even high-dose oseltamivir did not block viral replication sufficiently to suppress the emergence of resistant variants. Some resistant variants were not appreciably attenuated in cultured cells, but an oseltamivir-resistant A(H7N9) virus did not transmit among ferrets. These findings are useful for the control of A(H7N9) virus infections in clinical settings.

Virulence-affecting amino acid changes in the PA protein of H7N9 influenza A viruses.

UNLABELLED: Novel avian-origin influenza A(H7N9) viruses were first reported to infect humans in March 2013. To date, 143 human cases, including 45 deaths, have been recorded. By using sequence comparisons and phylogenetic and ancestral inference analyses, we identified several distinct amino acids in the A(H7N9) polymerase PA protein, some of which may be mammalian adapting. Mutant viruses possessing some of these amino acid changes, singly or in combination, were assessed for their polymerase activities and growth kinetics in mammalian and avian cells and for their virulence in mice. We identified several mutants that were slightly more virulent in mice than the wild-type A(H7N9) virus, A/Anhui/1/2013. These mutants also exhibited increased polymerase activity in human cells but not in avian cells. Our findings indicate that the PA protein of A(H7N9) viruses has several amino acid substitutions that are attenuating in mammals. IMPORTANCE: Novel avian-origin influenza A(H7N9) viruses emerged in the spring of 2013. By using computational analyses of A(H7N9) viral sequences, we identified several amino acid changes in the polymerase PA protein, which we then assessed for their effects on viral replication in cultured cells and mice. We found that the PA proteins of A(H7N9) viruses possess several amino acid substitutions that cause attenuation in mammals.

The cellular factors Vps18 and Mon2 are required for efficient production of infectious HIV-1 particles.

Like all viruses, HIV-1 requires cellular host factors for replication. The mechanisms for production of progeny virions involving these host factors, however, are not fully understood. To better understand these mechanisms, we used a yeast (Saccharomyces cerevisiae) genetic screen to identify mutant strains in which HIV-1 Gag targeting to the plasma membrane was aberrant. Of the 917 mutants identified, we selected 14 mutants whose missing genes had single orthologous counterparts in human and tested them for Gag-induced viruslike particle (VLP) release in yeast cells. We found that the Vps18 and Mon2 proteins were important for HIV-1 Gag-induced VLP release in yeast. In eukaryote cells, these host proteins are highly conserved and function in protein trafficking. Depletion of hVps18 or hMon2 reduced the efficient production of infectious HIV-1 virions in human cells. Our data suggest that these cellular factors play an important role in the efficient production of infectious HIV-1 virion particles.

Frequent occurrence of mutations in nsp3 and nsp4 of SARS-CoV-2, presumably caused by the inhaled asthma drug ciclesonide.

Mutations in nonstructural protein 3 (nsp3) and nsp4 of SARS-CoV-2, presumably induced by the asthma drug ciclesonide (which also has anti-SARS-CoV-2 activity), were counted 5,851 cases in the GISAID EpiCoV genome database. Sporadic occurrence of mutants not linked to each other in the phylogenetic tree were identified at least 88 times; of which, 58 had one or more descendants in the same branch. Five of these had spread to more than 100 cases, and one had expanded to 4,748 cases, suggesting the mutations are frequent, selected in individual patients, and transmitted to form clusters of cases. Clinical trials of ciclesonide as a treatment for COVID-19 are the presumed cause of the frequent occurrence of mutations between 2020 June and 2021 November. In addition, because ciclesonide is a common treatment for asthma, it can drive mutations in asthmatics suffering from COVID-19. Ciclesonide-resistant mutations, which have unpredictable effects in humans, are likely to continue to emerge because SARS-CoV-2 remains prevalent globally.

Detection of the ORF1 Gene Is an Indicator of the Possible Isolation of Severe Acute Respiratory Syndrome Coronavirus 2.

In the ongoing coronavirus diseases 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), real-time RT-PCR based diagnostic assays have been used for the detection of infection, but the positive signal of real-time RT-PCR does not necessarily indicate the infectivity of the patient. Due to the unique replication system of the coronavirus, primer/probe sets targeted nucleocapsid (N) and spike (S) protein detect the abundantly synthesized subgenomic RNAs as well as the virus genome, possibly making the assay unsuitable for estimation of the infectivity of the specimen, although it has an advantage for the diagnostic tests. In this study, the primer/probe set targeting the open reading frame 1a (ORF1a) gene was developed to specifically detect viral genomic RNA. Then the relation between the ORF1a signal and infectivity of the clinical specimens was validated by virus isolation using VeroE6 cells, which constitutively express transmembrane protease, serine 2, (VeroE6/TMPRSS2). The analytical sensitivity of developed ORF1a set was similar to that of previously developed N and S sets. Nevertheless, in the assay of the clinical specimen, detection rate of the ORF1a gene was lower than that of the N and S genes. These data indicated that clinical specimens contain a significant amount of subgenomic RNAs. However, as expected, the isolation-succeeded specimen always showed an RT-PCR-positive signal for the ORF1a gene, suggesting ORF1a detection in combination with N and S sets could be a more rational indicator for the possible infectivity of the clinical specimens.

Essential role of TMPRSS2 in SARS-CoV-2 infection in murine airways.

In cultured cells, SARS-CoV-2 infects cells via multiple pathways using different host proteases. Recent studies have shown that the furin and TMPRSS2 (furin/TMPRSS2)-dependent pathway plays a minor role in infection of the Omicron variant. Here, we confirm that Omicron uses the furin/TMPRSS2-dependent pathway inefficiently and enters cells mainly using the cathepsin-dependent endocytosis pathway in TMPRSS2-expressing VeroE6/TMPRSS2 and Calu-3 cells. This is the case despite efficient cleavage of the spike protein of Omicron. However, in the airways of TMPRSS2-knockout mice, Omicron infection is significantly reduced. We furthermore show that propagation of the mouse-adapted SARS-CoV-2 QHmusX strain and human clinical isolates of Beta and Gamma is reduced in TMPRSS2-knockout mice. Therefore, the Omicron variant isn't an exception in using TMPRSS2 in vivo, and analysis with TMPRSS2-knockout mice is important when evaluating SARS-CoV-2 variants. In conclusion, this study shows that TMPRSS2 is critically important for SARS-CoV-2 infection of murine airways, including the Omicron variant.

Performance Evaluation of Real-Time RT-PCR Assays for the Detection of Severe Acute Respiratory Syndrome Coronavirus-2 Developed by the National Institute of Infectious Diseases, Japan.

Soon after the 2019 outbreak of coronavirus disease 2019 in Wuhan, China, a protocol for real-time RT-PCR assay detection of severe acute respiratory syndrome coronavirus (SARS-CoV-2) was established by the National Institute of Infectious Diseases (NIID) in Japan. The protocol used Charité's nucleocapsid (Sarbeco-N) and NIID nucleocapsid (NIID-N2) assays. During the following months, SARS-CoV-2 spread and caused a global pandemic, and various SARS-CoV-2 sequences were registered in public databases, such as the Global Initiative on Sharing All Influenza Data (GISAID). In this study, we evaluated the S2 assay (NIID-S2) that was newly developed to replace the Sarbeco-N assay and the performance of the NIID-N2 and NIID-S2 assays, referring to mismatches in the primer/probe targeted region. We found that the analytical sensitivity and specificity of the NIID-S2 set were comparable to those of the NIID-N2 assay, and the detection rate for clinical specimens was identical to that of the NIID-N2 assay. Furthermore, among the available sequences (approximately 192,000), the NIID-N2 and NIID-S2 sets had 2.6% and 1.2% mismatched sequences, respectively, although most of these mismatches did not affect the amplification efficiency, except the 3' end of the NIID-N2 forward primer. These findings indicate that the previously developed NIID-N2 assay is suitable for the detection of SARS-CoV-2 with support from the newly developed NIID-S2 set.

Network-Guided Discovery of Influenza Virus Replication Host Factors.

The positions of host factors required for viral replication within a human protein-protein interaction (PPI) network can be exploited to identify drug targets that are robust to drug-mediated selective pressure. Host factors can physically interact with viral proteins, be a component of virus-regulated pathways (where proteins do not interact with viral proteins), or be required for viral replication but unregulated by viruses. Here, we demonstrate a method of combining human PPI networks with virus-host PPI data to improve antiviral drug discovery for influenza viruses by identifying target host proteins. Analysis shows that influenza virus proteins physically interact with host proteins in network positions significant for information flow, even after the removal of known abundance-degree bias within PPI data. We have isolated a subnetwork of the human PPI network that connects virus-interacting host proteins to host factors that are important for influenza virus replication without physically interacting with viral proteins. The subnetwork is enriched for signaling and immune processes distinct from those associated with virus-interacting proteins. Selecting proteins based on subnetwork topology, we performed an siRNA screen to determine whether the subnetwork was enriched for virus replication host factors and whether network position within the subnetwork offers an advantage in prioritization of drug targets to control influenza virus replication. We found that the subnetwork is highly enriched for target host proteins-more so than the set of host factors that physically interact with viral proteins. Our findings demonstrate that network positions are a powerful predictor to guide antiviral drug candidate prioritization.IMPORTANCE Integrating virus-host interactions with host protein-protein interactions, we have created a method using these established network practices to identify host factors (i.e., proteins) that are likely candidates for antiviral drug targeting. We demonstrate that interaction cascades between host proteins that directly interact with viral proteins and host factors that are important to influenza virus replication are enriched for signaling and immune processes. Additionally, we show that host proteins that interact with viral proteins are in network locations of power. Finally, we demonstrate a new network methodology to predict novel host factors and validate predictions with an siRNA screen. Our results show that integrating virus-host proteins interactions is useful in the identification of antiviral drug target candidates.

The host protein CLUH participates in the subnuclear transport of influenza virus ribonucleoprotein complexes.

The nucleus is highly compartmentalized yet dynamic. Subnuclear functions are regulated by controlling the subnuclear localization of the nuclear proteins. Influenza viral ribonucleoprotein (vRNP) is replicated in the nucleus and then exported to the cytoplasm. However, the precise subnuclear localization and transport of vRNPs remain unclear. Here, we show that CLUH, a host protein whose cellular function is not well established, plays a key role in the subnuclear transport of vRNP. Viral PB2 and M1 induced CLUH translocation to the nucleoplasm and SC35-positive speckles, respectively, even though CLUH is usually cytoplasmic. CLUH depletion inhibited the translocation of M1 to SC35-positive speckles, but did not interfere with PB2 localization to the nucleoplasm and disrupted the subnuclear transport of vRNP, abolishing vRNP nuclear export without affecting viral RNA or protein expression. Our findings suggest that CLUH plays a role in the subnuclear transport of progeny vRNP.

Amino acids substitutions in the PB2 protein of H7N9 influenza A viruses are important for virulence in mammalian hosts.

We tested the biological significance of two amino acid mutations in the PB2 protein (glutamic acid to lysine at position 627 and aspartic acid to asparagine at position 701) of A(H7N9) viruses for mammalian adaptation. Mutants were assessed for their viral polymerase activities, growth kinetics in mammalian and avian cells, and pathogenicity in mice. We found that lysine at position 627 and asparagine at position 701 in PB2 are essential for mammalian adaptation of A(H7N9) viruses.

Circulating avian influenza viruses closely related to the 1918 virus have pandemic potential.

Wild birds harbor a large gene pool of influenza A viruses that have the potential to cause influenza pandemics. Foreseeing and understanding this potential is important for effective surveillance. Our phylogenetic and geographic analyses revealed the global prevalence of avian influenza virus genes whose proteins differ only a few amino acids from the 1918 pandemic influenza virus, suggesting that 1918-like pandemic viruses may emerge in the future. To assess this risk, we generated and characterized a virus composed of avian influenza viral segments with high homology to the 1918 virus. This virus exhibited pathogenicity in mice and ferrets higher than that in an authentic avian influenza virus. Further, acquisition of seven amino acid substitutions in the viral polymerases and the hemagglutinin surface glycoprotein conferred respiratory droplet transmission to the 1918-like avian virus in ferrets, demonstrating that contemporary avian influenza viruses with 1918 virus-like proteins may have pandemic potential.

Influenza virus-host interactome screen as a platform for antiviral drug development.

Host factors required for viral replication are ideal drug targets because they are less likely than viral proteins to mutate under drug-mediated selective pressure. Although genome-wide screens have identified host proteins involved in influenza virus replication, limited mechanistic understanding of how these factors affect influenza has hindered potential drug development. We conducted a systematic analysis to identify and validate host factors that associate with influenza virus proteins and affect viral replication. After identifying over 1,000 host factors that coimmunoprecipitate with specific viral proteins, we generated a network of virus-host protein interactions based on the stage of the viral life cycle affected upon host factor downregulation. Using compounds that inhibit these host factors, we validated several proteins, notably Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) and JAK1, as potential antiviral drug targets. Thus, virus-host interactome screens are powerful strategies to identify targetable host factors and guide antiviral drug development.

Mutation of host DnaJ homolog inhibits brome mosaic virus negative-strand RNA synthesis.

The replication of positive-strand RNA viruses involves not only viral proteins but also multiple cellular proteins and intracellular membranes. In both plant cells and the yeast Saccharomyces cerevisiae, brome mosaic virus (BMV), a member of the alphavirus-like superfamily, replicates its RNA in endoplasmic reticulum (ER)-associated complexes containing viral 1a and 2a proteins. Prior to negative-strand RNA synthesis, 1a localizes to ER membranes and recruits both positive-strand BMV RNA templates and the polymerase-like 2a protein to ER membranes. Here, we show that BMV RNA replication in S. cerevisiae is markedly inhibited by a mutation in the host YDJ1 gene, which encodes a chaperone Ydj1p related to Escherichia coli DnaJ. In the ydj1 mutant, negative-strand RNA accumulation was inhibited even though 1a protein associated with membranes and the positive-strand RNA3 replication template and 2a protein were recruited to membranes as in wild-type cells. In addition, we found that in ydj1 mutant cells but not wild-type cells, a fraction of 2a protein accumulated in a membrane-free but insoluble, rapidly sedimenting form. These and other results show that Ydj1p is involved in forming BMV replication complexes active in negative-strand RNA synthesis and suggest that a chaperone system involving Ydj1p participates in 2a protein folding or assembly into the active replication complex.