Biology subjects

Webby, R. J.

Publications and source records attributed to Webby, R. J..

10 recordsLinked to original sources

bioRxiv2023

Innate immune control of influenza virus interspecies adaptation

Influenza virus pandemics are caused by viruses from animal reservoirs that adapt to efficiently infect and replicate in human hosts. Here, we investigated whether Interferon-Induced Transmembrane Protein 3 (IFITM3), a host antiviral factor with known human deficiencies, plays a role in interspecies virus infection and adaptation. We found that IFITM3-deficient mice and human cells could be infected with low doses of avian influenza viruses that failed to infect WT counterparts, identifying a new role for IFITM3 in controlling the minimum infectious viral dose threshold. Remarkably, influenza viruses passaged through Ifitm3-/- mice exhibited enhanced host adaptation, a result that was distinct from passaging in mice deficient for interferon signaling, which caused virus attenuation. Our data demonstrate that IFITM3 deficiency uniquely facilitates zoonotic influenza virus infections and subsequent adaptation, implicating IFITM3 deficiencies in the human population as a vulnerability for emergence of new pandemic viruses.

microbiology↗

bioRxiv2023

Mucosal and systemic immune dynamics associated with COVID-19 outcomes: a longitudinal prospective clinical study

RationaleCOVID-19 severity varies widely; children and African Americans have low and high risk, respectively. Mechanistic data from these groups and the mucosa is lacking. ObjectivesTo quantify mucosal and systemic viral and immune dynamics in a diverse cohort to identify mechanisms underpinning COVID-19 severity and outcome predictors. MethodsIn this prospective study of unvaccinated children and adults COVID-19 outcome was based on an ordinal clinical severity scale. We quantified viral RNA, antigens, antibodies, and cytokines by PCR, ELISA, and Luminex from 579 longitudinally collected blood and nasal specimens from 78 subjects including 45 women and used modeling to determine functional relationships between these data. Measurements and Main ResultsCOVID-19 induced unique immune responses in African Americans (n=26) and children (n=20). Mild outcome was associated with more effective coordinated responses whereas moderate and severe outcomes had rapid seroconversion, significantly higher antigen, mucosal sCD40L, MCP-3, MCP-1, MIP-1, and MIP-1{beta}, and systemic IgA, IgM, IL-6, IL-8, IL-10, IL-15, IL-1RA, and IP-10, and uncoordinated early immune responses that went unresolved. Mucosal IL-8, IL-1{beta}, and IFN-{gamma} with systemic IL-1RA and IgA predicted COVID-19 outcomes. ConclusionsWe present novel mucosal data, biomarkers, and therapeutic targets from a diverse cohort. Based on our findings, children and African Americans with COVID-19 have significantly lower IL-6 and IL-17 levels which may reduce responsiveness to drugs targeting IL-6 and IL-17. Unregulated immune responses persisted indicating moderate to severe COVID-19 cases may require prolonged treatments. Reliance on slower acting adaptive responses may cause immune crisis for some adults who encounter a novel virus. At a Glance CommentaryO_ST_ABSScientific Knowledge on the SubjectC_ST_ABSDespite the disparate outcomes for African Americans and children with COVID-19 and the vital role of mucosal immunity, the majority of mechanistic clinical studies lack these groups and mucosal assessments. To date, mucosal immune responses to SARS-CoV-2 has not been adequately described and we lack data from these understudied groups. What This Study Adds to the FieldThis was a prospective cohort study of children and adults with confirmed COVID-19. Mortality was low (2.5%). Severity outcomes were associated with African American Race, shortness of breath, fever, respiratory disease, high blood pressure, and diabetes. We systematically characterized viral and immune factors in the mucosa and periphery and observed that moderate and severe COVID-19 were associated with longer duration, impaired clearance, early overexuberant antibody and cytokine production that was sustained. This study demonstrates that African Americans are at high risk of severe COVID-19 and display unique mucosal and peripheral immune responses. Children with COVID-19 also had distinct immune responses. This illustrates the importance of vaccination and careful clinical oversight of these populations (e.g., lower IL-6 and IL-17 levels may diminish tocilizumab, siltuximab, secukinumab, and brodalumab efficacy). This study identified generalizable outcomes predictors, systemic IL-1RA with mucosal IL-1{beta} and IL-8, and demonstrated the utility of mucosal sampling from diverse cohorts.

immunology↗

bioRxiv2023

Development of a nucleoside-modified mRNA vaccine against clade 2.3.4.4b H5 highly pathogenic avian influenza virus

Highly pathogenic avian influenza viruses from H5 clade 2.3.4.4b are circulating at unprecedently high levels in wild and domestic birds and have the potential to adapt to humans. We generated an mRNA lipid nanoparticle (LNP) vaccine encoding the hemagglutinin (HA) glycoprotein from a clade 2.3.4.4b H5 isolate. We show that the vaccine is immunogenic in mice and ferrets and prevents morbidity and mortality of ferrets following 2.3.4.4b H5N1 challenge.

microbiology↗

bioRxiv2022

The episodic resurgence of highly pathogenic avian influenza H5 virus

Highly pathogenic avian influenza (HPAI) H5N1 activity has intensified globally since 2021, replacing the dominant clade 2.3.4.4 H5N8 virus. H5N1 viruses have spread rapidly to four continents, causing increasing reports of mass mortality in wild birds and poultry. The ecological and virological properties required for future mitigation strategies are unclear. Using epidemiological, spatial and genomic approaches, we demonstrate changes in the source of resurgent H5 HPAI and reveal significant shifts in virus ecology and evolution. Outbreak data indicates key resurgent events in 2016/17 and 2020/21 that contributed to the panzootic spread of H5N1 in 2021/22, including an increase in virus diffusion velocity and persistence in wild birds. Genomic analysis reveals that the 2016/17 epizootics originated in Asia, where HPAI H5 reservoirs are documented as persistent. However, in 2020/21, 2.3.4.4b H5N8 viruses emerged in domestic poultry in Africa, featuring several novel mutations altering the HA structure, receptor binding, and antigenicity. The new H5N1 virus emerged from H5N8 through reassortment in wild birds along the Adriatic flyway around the Mediterranean Sea. It was characterized by extensive reassortment with low pathogenic avian influenza in domestic and wild birds as it spread globally. In contrast, earlier outbreaks of H5N8 were caused by a more stable genetic constellation, highlighting dynamic changes in HPAI H5 genomic evolution. These results suggest a shift in the epicenter of HPAI H5 beyond Asia to new regions in Africa, the Middle East, Europe, and North and South America. The persistence of HPAI H5 with resurgence potential in domestic birds indicates that elimination strategies remain a high priority.

microbiology↗

bioRxiv2022

Chelerythrine as an anti-Zika virus agent: therapeutic potential and mode of action

Zika virus (ZIKV) is a mosquito-borne virus that has been associated with adult and neonatal neurological conditions. So far, there is no approved drug or vaccine against ZIKV infection; thus, ZIKV remains a global health threat. Here, we explored the effects of chelerythrine (CTC), a known protein kinase C (PKC) inhibitor, against ZIKV infection in cell culture models to determine its potential as a therapeutic agent for ZIKV infection. We found that CTC protected Vero cells from ZIKV-induced cytopathic effects in a dose-dependent manner. It also reduced the production of ZIKV in Vero and A549 cells. In contrast, other PKC inhibitors failed to protect Vero cells from ZIKV-induced cytopathic effects, indicating PKC-independent mechanisms underlying the effects of CTC on ZIKV. Further investigation suggested that CTC inhibited ZIKV attachment/binding rather than internalization in the host cells. Pretreatment of cell-free ZIKV particles rather than pretreatment of cells with CTC resulted in reduced ZIKV infectivity in vitro, indicating that CTC blocked the attachment/binding of the ZIKV particles to host factors. In silico analyses suggested that these effects are potentially due to the binding of CTC to the ZIKV E protein, which may occlude the interaction of the E protein with attachment factors or receptors on the host cell surface. Overall, our findings suggest that CTC reduces the infectivity of ZIKV particles through PKC- and cell-independent mechanisms. Our findings also support further exploration of CTC as an anti-ZIKV agent.

microbiology↗

bioRxiv2022

Robustness of the ferret model for influenza risk assessment studies: a cross-laboratory exercise

Ferrets represent the preferred animal model for assessing the transmission potential of newly emerged zoonotic influenza viruses. However, heterogeneity among established experimental protocols and facilities across different laboratories may lead to variable results, complicating interpretation of transmission experimental data. Between 2018-2020, a global exercise was conducted by 11 participating laboratories to assess the range of variation in ferret transmission experiments using two common stock H1N1 influenza viruses that possess different transmission characteristics in ferrets. Inoculation route, dose, and volume were standardized, and all participating laboratories followed the same experimental conditions for respiratory droplet transmission, including a strict 1:1 donor:contact ratio. Additional host and environmental parameters likely to affect influenza transmission kinetics were monitored throughout. Overall transmission outcomes for both viruses across 11 laboratories were concordant, suggesting the robustness of the ferret model for zoonotic influenza risk assessment. To attain high confidence in identifying zoonotic influenza viruses with moderate-to-high or low transmissibility, our analyses support that as few as three but as many as five laboratories, respectively, would need to independently perform viral transmission experiments with concordant results. This exercise facilitates the development of a more homogenous protocol for ferret transmission experiments that are employed for the purposes of risk assessment.

microbiology↗

bioRxiv2021

Host diversity and behavior determine patterns of interspecies transmission and geographic diffusion of avian Influenza A subtypes among North American wild reservoir species

Wild birds can carry avian influenza viruses (AIV), including those with pandemic or panzootic potential, long distances. Even though AIV has a broad host range, few studies account for host diversity when estimating AIV spread. We analyzed AIV genomic sequences from North American wild birds, including 303 newly sequenced isolates, to estimate interspecies transmission and geographic diffusion patterns among multiple co-circulating subtypes. Our results show high transition rates within Anseriformes and Charadriiformes, but limited transitions between these orders. Patterns of interspecies transmission were positively associated with breeding habitat range overlap, and negatively associated with host genetic distance. Distance between regions (negative correlation) and summer temperature at origin (positive correlation) were strong predictors of diffusion. Taken together, this study demonstrates that host diversity and ecology can determine evolutionary processes that underlie AIV natural history and spread. Understanding these processes can provide important insights for effective control of AIV.

evolutionary biology↗

bioRxiv2021

Minor variants of swine H1N1 influenza viruses with enhanced polymerase activity and HA stability promote airborne transmission in ferrets

Understanding how animal influenza A viruses (IAVs) acquire airborne transmissibility in humans and ferrets is needed to prepare for and respond to pandemics. Here, we investigated in ferrets the replication and transmission of swine H1N1 isolates P4 and G15, whose majority population had decreased polymerase activity and poor HA stability, respectively. For both isolates, a minor variant was selected and transmitted in ferrets. Polymerase-enhancing variant PA-S321 airborne-transmitted and propagated in one ferret. HA-stabilizing variant HA1-S210 was selected in all G15-inoculated ferrets and was transmitted by contact and airborne routes. With an efficient polymerase and a stable HA, the purified minor variant G15-HA1-S210 had earlier and higher peak titers in inoculated ferrets and was recovered at a higher frequency after airborne transmission than P4 and G15. Overall, HA stabilization played a more prominent role than polymerase enhancement in the replication and transmission of these viruses in ferrets. The results suggest pandemic risk-assessment studies may benefit from deep sequencing to identify minor variants with human-adapted traits. IMPORTANCEDiverse IAVs circulate in animals, yet few acquire the viral traits needed to start a human pandemic. A stabilized HA and mammalian-adapted polymerase have been shown to promote the adaptation of IAVs to humans and ferrets (the gold-standard model for IAV replication, pathogenicity, and transmissibility). Here, we used swine IAV isolates of the gamma lineage a model to investigate the importance of HA stability and polymerase activity in promoting replication and transmission in ferrets. These are emerging viruses that bind to both -2,6- and -2,3-linked receptors. Using isolates containing mixed populations, a stabilized HA was selected within days in inoculated ferrets. An enhanced polymerase was also selected and propagated after airborne transmission to a ferret. Thus, HA stabilization was a stricter requirement, yet both traits promoted transmissibility. Knowing the viral traits needed for pandemic potential, and the relative importance of each, will help identify emerging viruses of greatest concern.

microbiology↗

bioRxiv2021

Tropism, susceptibility, infectivity, and cytokine releases of differentiated human tonsillar epithelial cells by different Influenza viruses

Human tonsil epithelium cells (HTEC) are a heterogeneous group of actively differentiating cells comprising stratified squamous epithelial and reticulated crypt cells with abundant keratin expression. We hypothesized that the tonsils are a primary site for influenza infection and sustained viral replication. Primary HTEC were grown using an air-liquid culture and infected apically with different influenza viruses (IVs) to measure viral growth kinetics. These cultures were highly differentiated, with subpopulations of heterogenous surface stratified squamous cells rich with both cilia and microvilli; these cells contained more 2,6-linked sialic acids, those preferentially bound by human IVs, than 2,3-linked avian like sialic acids. The stratified squamous cells were interrupted by patches of reticular epithelial cells rich in 2,3-linked sialic acids. The HTEC were permissive for influenza A and B virus replication. Following infection, a subset of cells, mostly ciliated cells, underwent apoptosis while others remained intact despite being positive for IV nucleoprotein. H3N2 virus antigen colocalized with non-ciliated cells while H1N1 virus antigen was mostly associated with ciliated cells. Exposure of HTECs to IVs triggers an early proinflammatory response that fluctuates between viruses. The H3N2 IV induces an early response that persists, whereas pH1N1 induces a primarily late response in HTECs. Our results implicated HTEC as a site for IV replication. The HTEC differentiated system provides a valuable in vitro model for studying cellular tropism, infectivity, cytokine responses and the pathogenesis of IVs. IMPORTANCETo develop an effective intervention against influenza, it is important to identify host factors affecting transmission, pathogenesis, and immune response. Tonsils are lymphoepithelial organs characterized by infiltration of B and T lymphocytes into the squamous epithelium of tonsillar crypts, beneath which germinal centers play key roles in antigen processing and immune response. The heterogenicity of HTECs as well as the sialic acid distributions supports the replication of IVs and may play a role in IV adaptation. Furthermore, Tonsillectomy is a surgical procedure in which tonsils are fully removed from the human throat and may contribute to the diverse outcomes among infected individuals.

immunology↗

bioRxiv2021

Time dependent proinflammatory responses shape virus interference during coinfections of influenza A virus and influenza D virus

Both influenza A virus (IAV) and influenza D virus (IDV) are enzootic in pigs. IAV causes approximately 100% morbidity with low mortality, whereas IDV leads to only mild respiratory diseases in pigs. In this study, we performed a series of coinfection experiments in vitro and in vivo to understand how IAV and IDV interact and cause pathogenesis during coinfection. Results showed that IAV inhibited IDV replication when infecting swine tracheal epithelial cells (STEC) with IAV 24- or 48-hours prior to IDV inoculation, and that IDV suppressed IAV replication when IDV preceded IAV inoculation by 48 hours. Virus interference was not identified during simultaneous IAV/IDV infections or with 6 hours between the two viral infections, regardless of their order. The interference pattern at 24- and 48-hours correlated with proinflammatory responses induced by the first infection, which was about 24-hours slower for IDV than IAV. The viruses did not interfere with each other if both infected the cells before proinflammatory responses were induced. Coinfection in pigs further demonstrated that IAV interfered both viral shedding and virus replication of IDV, especially in the upper respiratory tract. Clinically, coinfection of IDV and IAV did not show significant enhancement of disease pathogenesis, compared with the pigs infected with IAV alone. In summary, this study suggests that interference during coinfection of IAV and IDV is primarily due to the proinflammatory response and is therefore dependent on the time between infection, and the order of infection. ImportanceBoth IAV and IDV are enzootic in pigs, and feral pigs have a higher risk for both IAV and IDV exposures than IDV exposure alone. This study suggests that in coinfection with IAV and IDV either virus can interfere with the replication of the other virus by stimulating proinflammatory responses; however, the proinflammatory response was 24 hours slower for IDV than IAV. In vitro there was no interference during simultaneous coinfection, regardless of infection order. Coinfection of IDV and IAV in pigs did not show enhanced pathogenesis, compared with those infected only with IAV. This study can facilitate our understanding of virus epidemiology and pathogenesis associated with IAV and IDV coinfection.

microbiology↗