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The host response to SARS-CoV-2 infection provide insights into both viral pathogenesis and patient management. The host-encoded microRNA (miRNA) response to SARS-CoV-2 infection, however, remains poorly defined. Here we profiled circulating miRNAs from ten COVID-19 patients sampled longitudinally and ten age and gender matched healthy donors. We observed 55 miRNAs that were altered in COVID-19 patients during early-stage disease, with the inflammatory miR-31-5p the most strongly upregulated. Supervised machine learning analysis revealed that a three-miRNA signature (miR-423-5p, miR-23a-3p and miR-195-5p) independently classified COVID-19 cases with an accuracy of 99.9%. In a ferret COVID-19 model, the three-miRNA signature again detected SARS-CoV-2 infection with 99.7% accuracy, and distinguished SARS-CoV-2 infection from influenza A (H1N1) infection and healthy controls with 95% accuracy. Distinct miRNA profiles were also observed in COVID-19 patients requiring oxygenation. This study demonstrates that SARS-CoV-2 infection induces a robust host miRNA response that could improve COVID-19 detection and patient management., Author summary While it is recognized that the host response to infection plays a critical role in determining the severity and outcome of COVID-19, the host microRNA (miRNA) response to SARS-CoV-2 infection is poorly defined. Here we have used next-generation sequencing and bioinformatics to profile circulating miRNAs in 10 COVID-19 patients that were sampled longitudinally over time. COVID-19 was associated with altered expression of 55 plasma miRNAs, with miR-776-3p and miR-1275 among the most strongly down-regulated, and miR-4742-3p, miR-31-5p and miR-3215-3p the most up-regulated. An artificial intelligence methodology was used to identify a miRNA signature, consisting of miR423-5p, miR-23a-3p, miR-195-5p, which could independently classify COVID-19 patients from healthy controls with 99.9% accuracy. When applied to the ferret model of COVID-19, the same signature classified COVID-19 cases with 99.8% accuracy and could distinguish between COVID-19 and influenza A(H1N1) infection with >95% accuracy. In summary this study profiles the host miRNA response to COVID-19 and suggests that the measurement of select host molecules may have potential to independently detect disease cases.

The global COVID-19 pandemic caused by SARS-CoV-2 has resulted in over 2.2 million deaths. Disease outcomes range from asymptomatic to severe with, so far, minimal genotypic change to the virus so understanding the host response is paramount. Transcriptomics has become incredibly important in understanding host-pathogen interactions, however, post-transcriptional regulation plays an important role in infection and immunity through translation and mRNA stability, allowing tight control over potent host responses by both the host and the invading virus. Here, we apply ribosome profiling to assess post-transcriptional regulation of host genes during SARS-CoV-2 infection of a human lung epithelial cell line (Calu-3). We have identified numerous transcription factors (JUN, ZBTB20, ATF3, HIVEP2 and EGR1) as well as select antiviral cytokine genes, namely IFNB1, IFNL1,2 and 3, IL-6 and CCL5, that are restricted at the post-transcriptional level by SARS-CoV-2 infection and discuss the impact this would have on the host response to infection. This early phase restriction of antiviral transcripts in the lungs may allow high viral load and consequent immune dysregulation typically seen in SARS-CoV-2 infection.

The human protein-coding gene ILRUN (inflammation and lipid regulator with UBA-like and NBR1-like domain, previously C6orf106) is a recently-characterised inhibitor of the transcription regulators p300 and CREB-binding protein (CBP). Here we have utilised RNA-seq to define cellular pathways regulated by ILRUN in the context of severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2) infection. We find that inhibition of ILRUN expression increases cellular expression of several members of the renin-angiotensin aldosterone system (RAAS), including the SARS-CoV-2 entry receptor angiotensin converting enzyme 2 (ACE2). Furthermore, inhibition of ILRUN results in increased SARS-CoV-2 replication. These data identify ILRUN as a novel inhibitor of SARS-CoV-2 replication and represents, to our knowledge, the first report of ILRUN as a regulator of the RAAS.SIGNIFICANCE STATEMENTThere is no doubt that the current rapid global spread of COVID-19 has had significant and far-reaching impacts on our health and economy and will continue to do so. Research in emerging infectious diseases, such as severe acute respiratory syndrome-associated coronavirus (SARS-CoV-2), is growing rapidly, with new breakthroughs in the understanding of host-virus interactions and the development of innovative and exciting therapeutic strategies and new knowledge and tools to better protect against the impacts of disease. The human protein-coding gene ILRUN is a recently-characterised inhibitor of the transcription regulators p300 and CREB-binding protein (CBP). Here we present the first evidence that ILRUN modulation has implications for SARS-CoV-2 infections. Virus infectivity assays confirmed that gene silencing of ILRUN had a proviral effect and increased SARS-CoV-2 replication, whilst over-expression of ILRUN inhibited SARS-CoV-2 production. Additionally, we observed that ILRUN also regulates the expression of key elements of the RAAS. These data have important implications for the development of antiviral strategies to deal with the current SARS-CoV-2 pandemic.

Recent studies suggest that extracellular cGAMP can be taken up by macrophages to engage STING through several mechanisms. Our work demonstrates that connexin-dependent communication between epithelial cells and macrophages plays a significant role in the amplification of antiviral responses mediated by cGAMP and suggests that pharmacological strategies aimed at modulating connexins may have therapeutic applications to control antiviral responses in humans., Activation of cyclic GMP-AMP (cGAMP) synthase (cGAS) plays a critical role in antiviral responses to many DNA viruses. Sensing of cytosolic DNA by cGAS results in synthesis of the endogenous second messenger cGAMP that activates stimulator of interferon genes (STING) in infected cells. Critically, cGAMP can also propagate antiviral responses to uninfected cells through intercellular transfer, although the modalities of this transfer between epithelial and immune cells remain poorly defined. We demonstrate here that cGAMP-producing epithelial cells can transactivate STING in cocultured macrophages through direct cGAMP transfer. cGAMP transfer was reliant upon connexin expression by epithelial cells and pharmacological inhibition of connexins blunted STING-dependent transactivation of the macrophage compartment. Macrophage transactivation by cGAMP contributed to a positive-feedback loop amplifying antiviral responses, significantly protecting uninfected epithelial cells against viral infection. Collectively, our findings constitute the first direct evidence of a connexin-dependent cGAMP transfer to macrophages by epithelial cells, to amplify antiviral responses.

Highlights • Polyethylene glycol (PEG)-mediated precipitation is an established methodology for concentration of virus stocks and is applied here to SARS-COV-2 derived from VeroE6 supernatant. • PEG precipitation resulted in a 1.5log10 increase in SARS-CoV-2 infectious titres in VeroE6, Calu-3 and Caco-2 cells. • This protocol is a simple and reliable methodology for the concentration of SARS-COV-2 stocks for a range of virology applications., The development of medical countermeasures against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires robust viral assays. Here we have adapted a protocol for polyethylene glycol (PEG)-mediated precipitation of SARS-CoV-2 stocks without the need for ultracentrifugation. Virus precipitation resulted in a ∼1.5 log10 increase in SARS-CoV-2 titres of virus prepared in VeroE6 cells and enabled the infection of several immortalized human cell lines (Caco-2 and Calu-3) at a high multiplicity of infection not practically achievable without virus concentration. This protocol underscores the utility of PEG-mediated precipitation for SARS-CoV-2 and provides a resource for a range of coronavirus research areas.

Hendra virus (HeV) is an emerging zoonotic pathogen harbored by Australian mainland flying foxes. HeV infection can cause lethal disease in humans and horses, and to date all cases of human HeV disease have resulted from contact with infected horses. Currently, diagnosis of acute HeV infections in horses relies on the productive phase of infection when virus shedding may occur. An assay that identifies infected horses during the preclinical phase of infection would reduce the risk of zoonotic viral transmission during management of HeV outbreaks. Having previously shown that the host microRNA (miR)-146a is upregulated in the blood of HeV-infected horses days prior to the detection of viremia, we have profiled miRNAs at the transcriptome-wide level to comprehensively assess differences between infected and uninfected horses. Next-generation sequencing and the miRDeep2 algorithm identified 742 mature miRNA transcripts corresponding to 593 miRNAs in whole blood of six horses (three HeV-infected, three uninfected). Thirty seven miRNAs were differentially expressed in infected horses, two of which were validated by qRT-PCR. This study describes a methodology for the transcriptome-wide profiling of miRNAs in whole blood and supports the notion that measuring host miRNA expression levels may aid infectious disease diagnosis in the future.

Hendra and Nipah viruses (family Paramyxoviridae, genus Henipavirus) are bat-borne viruses that cause fatal disease in humans and a range of other mammalian species. Gaining a deeper understanding of host pathways exploited by henipaviruses for infection may identify targets for new anti-viral therapies. Here we have performed genome-wide high-throughput agonist and antagonist screens at biosafety level 4 to identify host-encoded microRNAs (miRNAs) impacting henipavirus infection in human cells. Members of the miR-181 and miR-17~93 families strongly promoted Hendra virus infection. miR-181 also promoted Nipah virus infection, but did not affect infection by paramyxoviruses from other genera, indicating specificity in the virus-host interaction. Infection promotion was primarily mediated via the ability of miR-181 to significantly enhance henipavirus-induced membrane fusion. Cell signalling receptors of ephrins, namely EphA5 and EphA7, were identified as novel negative regulators of henipavirus fusion. The expression of these receptors, as well as EphB4, were suppressed by miR-181 overexpression, suggesting that simultaneous inhibition of several Ephs by the miRNA contributes to enhanced infection and fusion. Immune-responsive miR-181 levels was also up-regulated in the biofluids of ferrets and horses infected with Hendra virus, suggesting that the host innate immune response may promote henipavirus spread and exacerbate disease severity. This study is the first genome-wide screen of miRNAs influencing infection by a clinically significant mononegavirus and nominates select miRNAs as targets for future anti-viral therapy development., Author Summary The henipaviruses Hendra and Nipah are bat-borne paramyxoviruses that are highly pathogenic in humans. Until recently the constraints of working at biosafety level 4 had hindered the large scale study of host factors associated with henipavirus infection. MicroRNAs are a class of single-stranded non-coding RNAs that regulate biological processes in eukaryotes. An emerging body of evidence suggests that host microRNAs may favour infection of vertebrate RNA viruses. We have performed high-throughput agonist and antagonist screens at biosafety level 4 to identify host-encoded microRNAs impacting henipavirus infection in human cells. Members of the miR-181 and miR-17~93 families strongly promoted Hendra virus infection and appear to suppress multiple antiviral host molecules. Infection promotion is primarily mediated via the ability of miR-181 to repress Eph receptors that negatively regulate henipavirus glycoprotein-mediated cell-cell fusion. This study is the first large-scale screen of host-encoded microRNAs influencing infection by a clinically significant mononegavirus, and of a BSL-4 virus, and supports the emerging notion that host miRNAs can play a role in supporting infection of RNA viruses.

Objectives: Pandemic H1N1/09 viruses with the neuraminidase H274Y mutation have emerged in untreated patients or following oseltamivir therapy or prophylaxis. There have been no reports yet of zanamivir-resistant H1N1/09 viruses in previously healthy patients. We wanted to determine whether we could select for neuraminidase mutations conferring high-level resistance to zanamivir by in vitro passage of the virus. We also wanted to investigate if passaging in a combination of zanamivir and oseltamivir could prevent the emergence of the H274Y mutation. Methods: An H1N1/09 virus was passaged in cell culture in increasing concentrations of either zanamivir or a combination of zanamivir and oseltamivir. Results: Passage in zanamivir selected a virus with N146S neuraminidase and G158E haemagglutinin mutations. The neuraminidase mutation only reduced drug susceptibility by 2-fold in enzyme assays. The haemagglutinin mutation conferred drug dependence and drug resistance in cells to oseltamivir and zanamivir and reduced binding to red blood cells. After four passages in zanamivir and oseltamivir, virus with a D198G neuraminidase mutation was selected with around 10-fold reduced susceptibility to oseltamivir, zanamivir and peramivir in the enzyme assay. Further passaging selected a virus with both D198G and H274Y mutations that was highly resistant to oseltamivir and peramivir, but not zanamivir. All mutations affected growth in cell culture and decreased affinities of the neuraminidases for substrate. Conclusions: We did not select a virus with a neuraminidase mutation conferring high-level resistance to zanamivir. Dual exposure to zanamivir and oseltamivir was not sufficient to prevent selection of the H274Y mutation.

Avian influenza viruses of H5 subtype can cause highly pathogenic disease in poultry. In March 2014, a new reassortant H5N6 subtype highly pathogenic avian influenza virus emerged in Lao People’s Democratic Republic. We have assessed the pathogenicity, pathobiology and immunological responses associated with this virus in chickens. Infection caused moderate to advanced disease in 6 of 6 chickens within 48 h of mucosal inoculation. High virus titers were observed in blood and tissues (kidney, spleen, liver, duodenum, heart, brain and lung) taken at euthanasia. Viral antigen was detected in endothelium, neurons, myocardium, lymphoid tissues and other cell types. Pro-inflammatory cytokines were elevated compared to non-infected birds. Our study confirmed that this new H5N6 reassortant is highly pathogenic, causing disease in chickens similar to that of Asian H5N1 viruses, and demonstrated the ability of such clade 2.3.4-origin H5 viruses to reassort with non-N1 subtype viruses while maintaining a fit and infectious phenotype. Recent detection of influenza H5N6 poultry infections in Lao PDR, China and Viet Nam, as well as six fatal human infections in China, demonstrate that these emergent highly pathogenic H5N6 viruses may be widely established in several countries and represent an emerging threat to poultry and human populations.

A recent hypothesis to explain the recurrence of bluetongue disease after winter seasonal absences of the vector has suggested a role for persistent infection of sheep. This report presents combined independent work from two laboratories investigating the possible recovery of Bluetongue virus (BTV) over a protracted period after infection of both sheep and cattle. Prior to infection with either cell-culture-adapted or non-culture-adapted BTV, sheep were subjected to a preliminary exposure to Culicoides sp. insects, which reportedly facilitates recovery of virus from infected sheep several months post-infection (p.i.). A series of skin biopsies at different intervals p.i. was used to establish skin fibroblast (SF) cultures from which attempts were made to detect virus by isolation and by molecular and immunological methods. Also examined was the effect on virus recovery of additional exposure to Culicoides sp. prior to skin biopsy during the post-inoculation period. A herd of cattle sentinels for surveillance of natural BTV infection in northern Australia was monitored prospectively for seroconversion. Evidence of infection initiated attempted virus recovery by establishing SF cultures. It was found that in both cattle and sheep there was not a protracted period over which BTV could be recovered from SF cultures. The data do not support a general hypothesis that BTV persists in either sheep or cattle.

Background Using a recently described monolayer assay amenable to high throughput screening format for the identification of potential Nipah virus and Hendra virus antivirals, we have partially screened a low molecular weight compound library (>8,000 compounds) directly against live virus infection and identified twenty eight promising lead molecules. Initial single blind screens were conducted with 10 μM compound in triplicate with a minimum efficacy of 90% required for lead selection. Lead compounds were then further characterised to determine the median efficacy (IC50), cytotoxicity (CC50) and the in vitro therapeutic index in live virus and pseudotype assay formats. Results While a number of leads were identified, the current work describes three commercially available compounds: brilliant green, gentian violet and gliotoxin, identified as having potent antiviral activity against Nipah and Hendra virus. Similar efficacy was observed against pseudotyped Nipah and Hendra virus, vesicular stomatitis virus and human parainfluenza virus type 3 while only gliotoxin inhibited an influenza A virus suggesting a non-specific, broad spectrum activity for this compound. Conclusion All three of these compounds have been used previously for various aspects of anti-bacterial and anti-fungal therapy and the current results suggest that while unsuitable for internal administration, they may be amenable to topical antiviral applications, or as disinfectants and provide excellent positive controls for future studies.

Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae) are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections., Author Summary The henipaviruses Hendra and Nipah are bat-borne paramyxoviruses that are highly pathogenic in humans. The need for high biocontainment when studying Hendra and Nipah virus biology has hindered the development of therapeutics and knowledge of the viral infection cycle. This study describes a genome-wide functional genomics screen of human host genes required for henipavirus infection, to our knowledge the first such study conducted at biosafety level 4. Our study demonstrates that henipavirus infection is critically reliant on fibrillarin, a methyltransferase enzyme residing in the cell nucleolus. Despite henipavirus genome replication occurring in the cytoplasm of infected cells, viral RNA synthesis was greatly impaired in cells lacking fibrillarin. Furthermore during the early stages of infection the Hendra virus matrix protein shuttles to the nucleolus and binds fibrillarin. Collectively these results suggest a hitherto unappreciated role for nucleolar host-virus interactions in the early replication phase of henipavirus infection. Finally, mutating the catalytic activity of fibrillarin inhibits henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections.