Global Interactomics Uncovers Extensive Organellar Targeting by Zika Virus.

Zika virus (ZIKV) is a membrane enveloped Flavivirus with a positive strand RNA genome, transmitted by Aedes mosquitoes. The geographical range of ZIKV has dramatically expanded in recent decades resulting in increasing numbers of infected individuals, and the spike in ZIKV infections has been linked to significant increases in both Guillain-Barré syndrome and microcephaly. Although a large number of host proteins have been physically and/or functionally linked to other Flaviviruses, very little is known about the virus-host protein interactions established by ZIKV. Here we map host cell protein interaction profiles for each of the ten polypeptides encoded in the ZIKV genome, generating a protein topology network comprising 3033 interactions among 1224 unique human polypeptides. The interactome is enriched in proteins with roles in polypeptide processing and quality control, vesicle trafficking, RNA processing and lipid metabolism. >60% of the network components have been previously implicated in other types of viral infections; the remaining interactors comprise hundreds of new putative ZIKV functional partners. Mining this rich data set, we highlight several examples of how ZIKV may usurp or disrupt the function of host cell organelles, and uncover an important role for peroxisomes in ZIKV infection.

Evaluation of 5 Commercially Available Zika Virus Immunoassays.

Because of the global spread of Zika virus, accurate and high-throughput diagnostic immunoassays are needed. We compared the sensitivity and specificity of 5 commercially available Zika virus serologic assays to the recommended protocol of Zika virus IgM-capture ELISA and plaque-reduction neutralization tests. Most commercial immunoassays showed low sensitivity, which can be increased.

Reduction of Neuraminidase Activity Exacerbates Disease in 2009 Pandemic Influenza Virus-Infected Mice.

During the first wave of the 2009 pandemic, caused by a H1N1 influenza virus (pH1N1) of swine origin, antivirals were the only form of therapeutic available to control the proliferation of disease until the conventional strain-matched vaccine was produced. Oseltamivir is an antiviral that inhibits the sialidase activity of the viral neuraminidase (NA) protein and was shown to be effective against pH1N1 viruses in ferrets. Furthermore, it was used in humans to treat infections during the pandemic and is still used for current infections without reported complication or exacerbation of illness. However, in an evaluation of the effectiveness of oseltamivir against pH1N1 infection, we unexpectedly observed an exacerbation of disease in virus-infected mice treated with oseltamivir, transforming an otherwise mild illness into one with high morbidity and mortality. In contrast, an identical treatment regime alleviated all signs of illness in mice infected with the pathogenic mouse-adapted virus A/WSN/33 (H1N1). The worsened clinical outcome with pH1N1 viruses occurred over a range of oseltamivir doses and treatment schedules and was directly linked to a reduction in NA enzymatic activity. Our results suggest that the suppression of NA activity with antivirals may exacerbate disease in a host-dependent manner by increasing replicative fitness in viruses that are not optimally adapted for replication in that host. IMPORTANCE: Here, we report that treatment of pH1N1-infected mice with oseltamivir enhanced disease progression, transforming a mild illness into a lethal infection. This raises a potential pitfall of using the mouse model for evaluation of the therapeutic efficacy of neuraminidase inhibitors. We show that antiviral efficacy determined in a single animal species may not represent treatment in humans and that caution should be used when interpreting the outcome. Furthermore, increased virulence due to oseltamivir treatment was the effect of a shift in the hemagglutinin (HA) and neuraminidase (NA) activity balance. This is the first study that has demonstrated that altering the HA/NA activity balance by reduction in NA activity can result in an increase in virulence in any animal model from nonpathogenic to lethal and the first to demonstrate a situation in which treatment with a NA activity inhibitor has an effect opposite to the intended therapeutic effect of ameliorating the infection.

Hemagglutinin-Neuraminidase Balance Influences the Virulence Phenotype of a Recombinant H5N3 Influenza A Virus Possessing a Polybasic HA0 Cleavage Site.

UNLABELLED: Although a polybasic HA0 cleavage site is considered the dominant virulence determinant for highly pathogenic avian influenza (HPAI) H5 and H7 viruses, naturally occurring virus isolates possessing a polybasic HA0 cleavage site have been identified that are low pathogenic in chickens. In this study, we generated a reassortant H5N3 virus that possessed the hemagglutinin (HA) gene from H5N1 HPAI A/swan/Germany/R65/2006 and the remaining gene segments from low pathogenic A/chicken/British Columbia/CN0006/2004 (H7N3). Despite possessing the HA0 cleavage site GERRRKKR/GLF, this rH5N3 virus exhibited a low pathogenic phenotype in chickens. Although rH5N3-inoculated birds replicated and shed virus and seroconverted, transmission to naive contacts did not occur. To determine whether this virus could evolve into a HPAI form, it underwent six serial passages in chickens. A progressive increase in virulence was observed with the virus from passage number six being highly transmissible. Whole-genome sequencing demonstrated the fixation of 12 nonsynonymous mutations involving all eight gene segments during passaging. One of these involved the catalytic site of the neuraminidase (NA; R293K) and is associated with decreased neuraminidase activity and resistance to oseltamivir. Although introducing the R293K mutation into the original low-pathogenicity rH5N3 increased its virulence, transmission to naive contact birds was inefficient, suggesting that one or more of the remaining changes that had accumulated in the passage number six virus also play an important role in transmissibility. Our findings show that the functional linkage and balance between HA and NA proteins contributes to expression of the HPAI phenotype. IMPORTANCE: To date, the contribution that hemagglutinin-neuraminidase balance can have on the expression of a highly pathogenic avian influenza virus phenotype has not been thoroughly examined. Reassortment, which can result in new hemagglutinin-neuraminidase combinations, may have unpredictable effects on virulence and transmission characteristics of a virus. Our data show the importance of the neuraminidase in complementing a polybasic HA0 cleavage site. Furthermore, it demonstrates that adaptive changes selected for during the course of virus evolution can result in unexpected traits such as antiviral drug resistance.

A (H1N1) pdm09 HA D222 variants associated with severity and mortality in patients during a second wave in Mexico.

BACKGROUND: Pandemic type A (H1N1) influenza arose in early 2009, probably in Mexico and the United States, and reappeared in North America in September for seven more months. An amino acid substitution in the hemagglutinin (HA), D222G, has been reported in a significant proportion of patients with a severe and fatal outcome. We studied the prevalence of HA222 substitutions in patients in Mexico during the second wave and its association with clinical outcome and pathogenicity in a mouse model. METHODS: The nucleotide sequences of hemagglutinin (HA) from viruses collected from 77 patients were determined including 50 severe and fatal cases and 27 ambulatory cases. Deep sequencing was done on 5 samples from severe or fatal cases in order to determine the quasispecies proportion. Weight loss and mortality due to infection with cultured influenza viruses were analyzed in a mouse model. RESULTS: Viruses from 14 out of 50 hospitalized patients (28%) had a non aspartic acid residue at the HA 222 position (nD222), while all 27 ambulatory patients had D222 (p=0.0014). G222 was detected as sole species or in coexistence with N222 and D222 in 12 patients with severe disease including 8 who died. N222 in coexistence with D222 was detected in 1 patient who died and co-occurrence of A222 and V222, together with D222, was detected in another patient who died. The patients with a nD222 residue had higher mortality (71.4%), compared to the group with only D222 (22.2%, p=0.0008). Four of the 14 viruses from hospitalized patients were cultured and intranasally infected into mice. Two viruses with G222 were lethal while a third virus, with G222, caused only mild illness in mice similar to the fourth virus that contained D222. CONCLUSIONS: We confirm the elevated incidence of HA222 (H1N1)pdm09 variants in severe disease and mortality. Both clinical and mouse infection data support the idea that nD222 mutations contribute to increased severity of disease but additional determinants in disease outcome may be present.

Discovery and synthesis of novel benzofurazan derivatives as inhibitors of influenza A virus.

The identification of a novel hit compound inhibitor of the protein-protein interaction between the influenza RNA-polymerase PA and PB1 subunits has been accomplished by means of high-throughput screening. A small family of structurally related molecules has been synthesized and biologically evaluated with most of the compounds showing micromolar potency of inhibition against viral replication.

Comparison of fifteen SARS-CoV-2 nucleic acid amplification test assays used during the Canadian Laboratory Response Network's National SARS-CoV-2 Proficiency Program, May 2020 to June 2021.

Background: On March 11, 2020, the World Health Organization declared a pandemic caused by the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This led to increased clinical testing and decentralizing of this testing from provincial health laboratories to regional and private facilities. Leveraging the results from the Canadian Laboratory Response Network's National SARS-CoV-2 Proficiency Test (PT) Program, this study compares multiple commercial and laboratory-developed nucleic acid amplification tests, assessing both sensitivity and specificity across multiple users. Methods: Each panel consisted of six blinded, contrived-clinical samples. Panels were distributed to international, provincial and territorial laboratories and subsequently to partner facilities. Participating laboratories were asked to run these sample through their respective extraction/PCR workflows and submit results to the National Microbiology Laboratory, outlining the nucleic acid extraction platform and nucleic acid amplification test employed, as well as the viral gene target and Ct values or equivalent obtained. Data were compiled for each molecular platform and gene target used. Results: The PT schemes were deployed in May 2020, November 2020 and June 2021, resulting in 683 data sets using 37 different nucleic acid amplification tests. Over the course of three PT schemes, the average score obtained was 99.3% by participants demonstrating consistent testing between laboratories and testing platforms. Conclusion: This study confirmed the rapid and successful implementation of a Canadian PT Program and provided comparative analysis of the various emergency use authorized and laboratory developed tests employed for the detection of SARS-CoV-2 and demonstrated an overall 99.3% test concordance nationwide.

Evolution of nucleic acid amplification testing across Canada as observed through the Canadian Laboratory Response Network's SARS-CoV-2 Proficiency Test Program, May 2020 to June 2021.

To help accommodate the surge in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) clinical testing due to the coronavirus disease 2019 pandemic, the decentralization of testing from provincial public health laboratories to regional laboratories and private facilities was necessary. To further support the growing number of test sites in Canada, the National Microbiology Laboratory developed a proficiency test program for the detection of SARS-CoV-2 using nucleic acid amplification tests and administered it under an arm of the Canadian Laboratory Response Network (CLRN). Since its conception in May 2020, CLRN has conducted three proficiency test schemes, from May 2020 to June 2021, and has observed an increase in participation of more than 400%. This article will explore the evolution of CLRN's SARS-CoV-2 Proficiency Test Program and its support of the Canadian pandemic response.

A four specimen-pooling scheme reliably detects SARS-CoV-2 and influenza viruses using the BioFire FilmArray Respiratory Panel 2.1.

The COVID-19 pandemic required increased testing capacity, enabling rapid case identification and effective contract tracing to reduce transmission of disease. The BioFire FilmArray is a fully automated nucleic acid amplification test system providing specificity and sensitivity associated with gold standard molecular methods. The FilmArray Respiratory Panel 2.1 targets 22 viral and bacterial pathogens, including SARS-CoV-2 and influenza virus. While each panel provides a robust output of information regarding pathogen detection, the specimen throughput is low. This study evaluates the FilmArray Respiratory Panel 2.1 using 33 pools of contrived nasal samples and 22 pools of clinical nasopharyngeal specimens to determine the feasibility of increasing testing capacity, while maintaining detection of both SARS-CoV-2 and influenza virus. We observed 100% detection and 90% positive agreement for SARS-CoV-2 and 98% detection and 95% positive agreement for influenza viruses with pools of contrived or clinical specimens, respectively. While discordant results were mainly attributed to loss in sensitivity, the sensitivity of the pooling assay was well within accepted limits of detection for a nucleic acid amplification test. Overall, this study provides evidence supporting the use of pooling patient specimens, one in four with the FilmArray Respiratory Panel 2.1 for the detection of SARS-CoV-2 and influenza virus.

Identification of high-affinity PB1-derived peptides with enhanced affinity to the PA protein of influenza A virus polymerase.

The influenza A virus polymerase complex, consisting of the subunits PB1, PB2, and PA, represents a promising target for the development of new antiviral drugs. We have previously demonstrated the feasibility of targeting the protein-protein interaction domain between PA and PB1 using peptides derived from the extreme N terminus of PB1 (amino acids [aa] 1 to 15), comprising the PA-binding domain of PB1. To increase the binding affinity of these peptides, we performed a systematic structure-affinity relationship analysis. Alanine and aspartic acid scans revealed that almost all amino acids in the core binding region (aa 5 to 11) are indispensable for PA binding. Using a library of immobilized peptides representing all possible single amino acid substitutions, we were able to identify amino acid positions outside the core PA-binding region (aa 1, 3, 12, 14, and 15) that are variable and can be replaced by affinity-enhancing residues. Surface plasmon resonance binding studies revealed that combination of several affinity-enhancing mutations led to an additive effect. Thus, the feasibility to enhance the PA-binding affinity presents an intriguing possibility to increase antiviral activity of the PB1-derived peptide and one step forward in the development of an antiviral drug against influenza A viruses.

Clinical evaluation of the GeneXpert® Xpert® Xpress SARS-CoV-2/Flu/RSV combination test.

The Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV combination test received emergency use authorization approval by the United States Food and Drug Administration in December 2020, and Health Canada approval in January 2021. The performance characteristics of the GeneXpert Xpert Xpress SARS-CoV-2/Flu/RSV combination test were assessed at Lakeridge Health Oshawa and the National Microbiology Laboratory of Canada. The combination test was compared to the Xpert SARS-CoV-2 and Xpert Flu/RSV assays, and the BioFire FilmArray Respiratory Panel 2.1 (RP2.1) test kit. Materials evaluated were serial dilutions of chemically-inactivated SARS-CoV-2 and remnant clinical specimens (nasal or nasopharyngeal swabs) collected from patients. The limit of detection (LOD) for the SARS-CoV-2 component of the Xpert SARS-CoV-2/Flu/RSV combination test was determined to be <100 viral copies/mL when using chemically-inactivated SARS-CoV-2. In total, 86 clinical positive and 51 clinical negative samples were used for this study, with mixtures of clinical positives being used to mimic coinfection and screen for competitive inhibition. The combination test showed a high percent agreement with the Xpert SARS-CoV-2 and Xpert Flu/RSV tests, as well as the BioFire FilmArray RP2.1. Based on the findings from this study and a growing body of research, the Xpert SARS-CoV-2/Flu/RSV combination test will serve as an effective replacement for the Xpert SARS-CoV-2 and Xpert Flu/RSV assays.

Characterization of Ebola Virus Risk to Bedside Providers in an Intensive Care Environment.

BACKGROUND: The 2014-2016 Ebola outbreak in West Africa recapitulated that nosocomial spread of Ebola virus could occur and that health care workers were at particular risk including notable cases in Europe and North America. These instances highlighted the need for centers to better prepare for potential Ebola virus cases; including understanding how the virus spreads and which interventions pose the greatest risk. METHODS: We created a fully equipped intensive care unit (ICU), within a Biosafety Level 4 (BSL4) laboratory, and infected multiple sedated non-human primates (NHPs) with Ebola virus. While providing bedside care, we sampled blood, urine, and gastric residuals; as well as buccal, ocular, nasal, rectal, and skin swabs, to assess the risks associated with routine care. We also assessed the physical environment at end-point. RESULTS: Although viral RNA was detectable in blood as early as three days post-infection, it was not detectable in the urine, gastric fluid, or swabs until late-stage disease. While droplet spread and fomite contamination were present on a few of the surfaces that were routinely touched while providing care in the ICU for the infected animal, these may have been abrogated through good routine hygiene practices. CONCLUSIONS: Overall this study has helped further our understanding of which procedures may pose the highest risk to healthcare providers and provides temporal evidence of this over the clinical course of disease.

Characterization of a novel STAT 2 knock-out hamster model of Crimean-Congo hemorrhagic fever virus pathogenesis.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne pathogen causing a febrile illness in humans, which can progress to hemorrhagic manifestations, multi-organ failure, and death. Current mouse models of CCHFV infection reliably succumb to virus challenge but vary in their ability to reflect signs of disease similar to humans. In this study, we established a signal transducer and activator of transcription 2 (STAT2) knockout hamster model to expand the repertoire of animal models of CCHFV pathogenesis that can be used for therapeutic development. These hamsters demonstrated a systemic and lethal disease in response to infection. Hallmarks of human disease were observed including petechial rash, blood coagulation dysfunction, and various biochemistry and blood cell count abnormalities. Furthermore, we also demonstrated the utility of this model for anti-CCHFV therapeutic evaluation. The STAT2 knock-out hamster model of CCHFV infection may provide some further insights into clinical disease, viral pathogenesis, and pave the way for testing of potential drug and vaccine candidates.

Establishment of an RNA polymerase II-driven reverse genetics system for Nipah virus strains from Malaysia and Bangladesh.

Nipah virus (NiV) has emerged as a highly lethal zoonotic paramyxovirus that is capable of causing a febrile encephalitis and/or respiratory disease in humans for which no vaccines or licensed treatments are currently available. There are two genetically and geographically distinct lineages of NiV: NiV-Malaysia (NiV-M), the strain that caused the initial outbreak in Malaysia, and NiV-Bangladesh (NiV-B), the strain that has been implicated in subsequent outbreaks in India and Bangladesh. NiV-B appears to be both more lethal and have a greater propensity for person-to-person transmission than NiV-M. Here we describe the generation and characterization of stable RNA polymerase II-driven infectious cDNA clones of NiV-M and NiV-B. In vitro, reverse genetics-derived NiV-M and NiV-B were indistinguishable from a wildtype isolate of NiV-M, and both viruses were pathogenic in the Syrian hamster model of NiV infection. We also describe recombinant NiV-M and NiV-B with enhanced green fluorescent protein (EGFP) inserted between the G and L genes that enable rapid and sensitive detection of NiV infection in vitro. This panel of molecular clones will enable studies to investigate the virologic determinants of henipavirus pathogenesis, including the pathogenic differences between NiV-M and NiV-B, and the high-throughput screening of candidate therapeutics.

Establishment of a comprehensive and high throughput serological algorithm for Zika virus diagnostic testing.

The previous serological algorithm for Zika virus (ZIKV) comprised screening by anti-ZIKV IgM capture ELISA (MAC-ELISA) for samples collected within 3 months postexposure or onset (MPEO). Samples positive by MAC-ELISA and samples collected beyond 3 MPEO were tested by the confirmatory plaque reduction neutralization test (PRNT), which proved laborious and time-consuming during the 2015 outbreak. Thus, we evaluated several ZIKV ELISAs to establish an anti-IgM and anti-IgG combination for use as a screening tool for all samples prior to PRNT confirmation. The MAC-ELISA or InBios-M in combination with the Euroimmun-G demonstrated sensitivities of 99.1% and 97.2%, respectively, and nonflavivirus specificity of 96.0%. Their cross-reactivities were 71.4% and 50.0%, respectively, for sera positive for Dengue virus antibodies. Due to near-perfect interrater agreement with PRNT and excellent detection of samples collected beyond 3 MPEO, these combinations were recommended as a screening protocol in a new high-throughput algorithm with special considerations for ZIKV diagnostics.

Impact of intensive care unit supportive care on the physiology of Ebola virus disease in a universally lethal non-human primate model.

BACKGROUND: There are currently limited data for the use of specific antiviral therapies for the treatment of Ebola virus disease (EVD). While there is anecdotal evidence that supportive care may be effective, there is a paucity of direct experimental data to demonstrate a role for supportive care in EVD. We studied the impact of ICU-level supportive care interventions including fluid resuscitation, vasoactive medications, blood transfusion, hydrocortisone, and ventilator support on the pathophysiology of EVD in rhesus macaques infected with a universally lethal dose of Ebola virus strain Makona C07. METHODS: Four NHPs were infected with a universally lethal dose Ebola virus strain Makona, in accordance with the gold standard lethal Ebola NHP challenge model. Following infection, the following therapeutic interventions were employed: continuous bedside supportive care, ventilator support, judicious fluid resuscitation, vasoactive medications, blood transfusion, and hydrocortisone as needed to treat cardiovascular compromise. A range of physiological parameters were continuously monitored to gage any response to the interventions. RESULTS: All four NHPs developed EVD and demonstrated a similar clinical course. All animals reached a terminal endpoint, which occurred at an average time of 166.5 ± 14.8 h post-infection. Fluid administration may have temporarily blunted a rise in lactate, but the effect was short lived. Vasoactive medications resulted in short-lived improvements in mean arterial pressure. Blood transfusion and hydrocortisone did not appear to have a significant positive impact on the course of the disease. CONCLUSIONS: The model employed for this study is reflective of an intramuscular infection in humans (e.g., needle stick) and is highly lethal to NHPs. Using this model, we found that the animals developed progressive severe organ dysfunction and profound shock preceding death. While the overall impact of supportive care on the observed pathophysiology was limited, we did observe some time-dependent positive responses. Since this model is highly lethal, it does not reflect the full spectrum of human EVD. Our findings support the need for continued development of animal models that replicate the spectrum of human disease as well as ongoing development of anti-Ebola therapies to complement supportive care.

Correction to: Impact of intensive care unit supportive care on the physiology of Ebola virus disease in a universally lethal non-human primate model.

Please note that four authors (Logan Banadyga, Alixandra Albietz, Brad Pickering, and Gary Wong) have been erroneously omitted from the author list in the published original article [1].

Comprehending a Killer: The Akt/mTOR Signaling Pathways Are Temporally High-Jacked by the Highly Pathogenic 1918 Influenza Virus.

Previous transcriptomic analyses suggested that the 1918 influenza A virus (IAV1918), one of the most devastating pandemic viruses of the 20th century, induces a dysfunctional cytokine storm and affects other innate immune response patterns. Because all viruses are obligate parasites that require host cells for replication, we globally assessed how IAV1918 induces host protein dysregulation. We performed quantitative mass spectrometry of IAV1918-infected cells to measure host protein dysregulation. Selected proteins were validated by immunoblotting and phosphorylation levels of members of the PI3K/AKT/mTOR pathway were assessed. Compared to mock-infected controls, >170 proteins in the IAV1918-infected cells were dysregulated. Proteins mapped to amino sugar metabolism, purine metabolism, steroid biosynthesis, transmembrane receptors, phosphatases and transcription regulation. Immunoblotting demonstrated that IAV1918 induced a slight up-regulation of the lamin B receptor whereas all other tested virus strains induced a significant down-regulation. IAV1918 also strongly induced Rab5b expression whereas all other tested viruses induced minor up-regulation or down-regulation. IAV1918 showed early reduced phosphorylation of PI3K/AKT/mTOR pathway members and was especially sensitive to rapamycin. These results suggest the 1918 strain requires mTORC1 activity in early replication events, and may explain the unique pathogenicity of this virus.

The interaction between the Nipah virus nucleocapsid protein and phosphoprotein regulates virus replication.

Continued outbreaks of Henipaviruses in South Asia and Australia cause severe and lethal disease in both humans and animals. Together, with evidence of human to human transmission for Nipah virus and the lack of preventative or therapeutic measures, its threat to cause a widespread outbreak and its potential for weaponization has increased. In this study we demonstrate how overexpression of the Nipah virus nucleocapsid protein regulates viral polymerase activity and viral RNA production. By overexpressing the Nipah virus nucleocapsid protein in trans viral transcription was inhibited; however, an increase in viral genome synthesis was observed. Together, the bias of polymerase activity towards genome production led to the severe inhibition of viral progeny. We identified two domains within the nucleocapsid protein, which were each independently capable of binding the viral phosphoprotein. Evident by our data, we propose that the nucleocapsid protein's ability to interact with the phosphoprotein of the polymerase complex causes a change in polymerase activity and subsequent deficiency in viral replication. This study not only provides insights into the dynamics of Henipavirus RNA synthesis and replication, but also provides insight into potential targets for antiviral drug development.

Evaluation of the Diasorin Liaison® XL Zika Capture IgM CMIA for Zika virus serological testing.

Due to the increase of Zika virus (ZIKV) transmission throughout the world, many commercial kits have recently become available to aid in laboratory diagnosis of ZIKV infections in clinical samples. Here, we analyze the fully automated Liaison® XL Zika Capture immunoglobulin M (IgM) assay against the recommended IgM-capture enzyme-linked immunosorbent assay.