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The emergence of a pandemic influenza virus may be better anticipated if we better understand the evolutionary steps taken by avian influenza viruses as they adapt to mammals. We used ancestral sequence reconstruction to resurrect viruses representing initial adaptive stages of the European avian-like H1N1 virus as it transitioned from avian to swine hosts. We demonstrate that efficient transmissibility in pigs was gained through stepwise adaptation after 1983. These time-dependent adaptations resulted in changes in hemagglutinin receptor binding specificity and increased viral polymerase activity. An NP-R351K mutation under strong positive selection increased the transmissibility of a reconstructed virus. The stepwise-adaptation of a wholly avian influenza virus to a mammalian host suggests a window where targeted intervention may have highest impact. Successful intervention will, however, require strategic coordination of surveillance and risk assessment activities to identify these adapting viruses and guide pandemic preparedness resources.

Understanding the evolutionary adaptations that enable avian influenza viruses to transmit in mammalian hosts could allow better detection of zoonotic viruses with pandemic potential. We applied ancestral sequence reconstruction to gain viruses representing different adaptive stages of the European avian-like (EA) H1N1 swine influenza virus as it transitioned from avian to swine hosts since 1979. Ancestral viruses representing the avian-like precursor virus and EA swine influenza viruses from 1979-1983, 1984-1987 and 1988-1992 were reconstructed and characterized. Glycan-binding analyses showed stepwise changes in the haemagglutinin receptor-binding specificity of the EA swine influenza viruses-that is, from recognition of both α2,3- and α2,6-linked sialosides to recognition of α2,6-linked sialosides only; however, efficient transmission in piglets was enabled by adaptive changes in the viral polymerase protein and nucleoprotein, which have been fixed since 1983. PB1-Q621R and NP-R351K increased viral replication and transmission in piglets when introduced into the 1979-1983 ancestral virus that lacked efficient transmissibility. The stepwise adaptation of an avian influenza virus to a mammalian host suggests that there may be opportunities to intervene and prevent interspecies jumps through strategic coordination of surveillance and risk assessment activities.

Summary The failure to mount an antibody response following viral infection or seroconversion failure is a largely underappreciated and poorly understood phenomenon. Here, we identified immunologic markers associated with robust antibody responses after influenza virus infection in two independent human cohorts, SHIVERS and FLU09, based in Auckland, New Zealand and Memphis, Tennessee, USA, respectively. In the SHIVERS cohort, seroconversion significantly associates with (1) hospitalization, (2) greater numbers of proliferating, activated CD4+ T cells, but not CD8+ T cells, in the periphery during the acute phase of illness, and (3) fewer inflammatory monocytes (CD14hiCD16+) by convalescence. In the FLU09 cohort, fewer CD14hiCD16+ monocytes during early illness in the nasal mucosa were also associated with the generation of influenza-specific mucosal immunoglobulin A (IgA) and IgG antibodies. Our study demonstrates that seroconversion failure after infection is a definable immunological phenomenon, associated with quantifiable cellular markers that can be used to improve diagnostics, vaccine efficacy, and epidemiologic efforts.

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.

During influenza A virus (IAV) entry, the hemagglutinin (HA) protein is triggered by endosomal low pH to undergo irreversible structural changes that mediate membrane fusion. HA proteins from different isolates vary in the pH at which they become activated in endosomes or become irreversible inactivated if exposed to extracellular acid. Little is known about extracellular pH in the upper respiratory tracts of mammals, how pH may shift during IAV infection, and its impact on replication of viruses that vary in HA activation pH. Here, we inoculated DBA/2J mice intranasally with A/TN/1-560/2009 (H1N1) (activation pH 5.5) or a mutant containing the destabilizing mutation HA1-Y17H (pH 6.0). We measured the kinetics of extracellular pH during infection using an optical pH-sensitive microsensor probe placed in the naris, nasal sinus, soft palate, and trachea. We also measured intracellular pH of single-cell suspensions of live, primary lung epithelial cells with various wavelength pH-sensitive dyes localized to cell membranes, cytosol, endosomes, secretory vesicles, microtubules, and lysosomes. Infection with either virus decreased extracellular pH and increased intracellular pH. Peak host immune responses were observed at 2 days post infection (DPI) and peak pH changes at 5 DPI. Extracellular and intracellular pH returned to baseline by 7 DPI in mice infected with HA1-Y17H and was restored later in wildtype-infected. Overall, IAV infection altered respiratory tract pH, which in turn modulated replication efficiency. This suggests a virus-host pH feedback loop that may select for IAV strains containing HA proteins of optimal pH stability, which may be approximately pH 5.5 in mice but may differ in other species.

Since the first report of SARS-CoV-2 in December 2019, genetic variants have continued to emerge, complicating strategies for mitigating the disease burden of COVID-19. Positive SARS-CoV-2 nasopharyngeal swabs (n=8,735) were collected from Missouri, USA, from March-October 2020, and viral genomes (n=178) were sequenced. Hospitalization status and length of stay were extracted from medical charts of 1,335 patients and integrated with emerging genetic variants and viral shedding analyses for assessment of clinical impacts. Multiple introductions of SARS-CoV-2 into Missouri, primarily from Australia, Europe, and domestic states, were observed. Four local lineages rapidly emerged and spread across urban and rural regions in Missouri. While the majority of Missouri viruses harbored Spike-D614G mutations, a large number of unreported mutations were identified among Missouri viruses, including seven in the RNA-dependent RNA polymerase complex and Spike protein that were positively selected. A 15.6-fold increase in viral RNA levels in swab samples occurred from March to May and remained elevated. Accounting for other comorbidities, individuals test-positive for COVID-19 with high viral loads were less likely to be hospitalized (odds ratio=0.39, 95% confidence interval [CI]=0.20, 0.77) and had shorter hospital stays (hazard ratio=0.34, p=0.003) than those with low viral loads. Overall, the first eight months of the pandemic in Missouri saw multiple locally acquired mutants emerge and dominate in urban and rural locations. Although we were unable to find associations between specific variants and greater disease severity, Missouri COVID-positive individuals that presented with increased viral shedding had less severe disease by several measures.

Background Human spillovers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to dogs and the emergence of a highly contagious avian-origin H3N2 canine influenza virus have raised concerns on the role of dogs in the spread of SARS-CoV-2 and their susceptibility to existing human and avian influenza viruses, which might result in further reassortment. Methods We systematically studied the replication kinetics of SARS-CoV-2, SARS-CoV, influenza A viruses of H1, H3, H5, H7, and H9 subtypes, and influenza B viruses of Yamagata-like and Victoria-like lineages in ex vivo canine nasal cavity, soft palate, trachea, and lung tissue explant cultures and examined ACE2 and sialic acid (SA) receptor distribution in these tissues. Results There was limited productive replication of SARS-CoV-2 in canine nasal cavity and SARS-CoV in canine nasal cavity, soft palate, and lung, with unexpectedly high ACE2 levels in canine nasal cavity and soft palate. Canine tissues were susceptible to a wide range of human and avian influenza viruses, which matched with the abundance of both human and avian SA receptors. Conclusions Existence of suitable receptors and tropism for the same tissue foster virus adaptation and reassortment. Continuous surveillance in dog populations should be conducted given the many chances for spillover during outbreaks.

Laninamivir (LAN) is a long-acting neuraminidase (NA) inhibitor (NAI) with a similar binding profile in the influenza NA enzyme active site as those of other NAIs, oseltamivir (OS), zanamivir (ZAN), and peramivir, and may share common resistance markers with these NAIs. We screened viruses with NA substitutions previously found during OS and ZAN selection in avian influenza viruses (AIVs) of the N3 to N9 subtypes for LAN susceptibility. Of the 72 NA substitutions, 19 conferred resistance to LAN, which ranged from 11.2- to 549.8-fold-decreased inhibitory activity over that of their parental viruses. Ten NA substitutions reduced the susceptibility to all four NAIs, whereas the remaining 26 substitutions yielded susceptibility to one or more NAIs. To determine whether the in vitro susceptibility of multi-NAI-resistant AIVs is associated with in vivo susceptibility, we infected BALB/c mice with recombinant AIVs with R292K (ma81K-N3R292K) or Q136K (ma81K-N8Q136K) NA substitutions, which impart in vitro susceptibility only to LAN or OS, respectively. Both ma81K-N3R292K and ma81K-N8Q136K virus-infected mice exhibited reduced weight loss, mortality, and lung viral titers when treated with their susceptible NAIs, confirming the in vitro susceptibility of these substitutions. Together, LAN resistance profiling of AIVs of a range of NA subtypes improves the understanding of NAI resistance mechanisms. Furthermore, the association of in vitro and in vivo NAI susceptibility indicates that our models are useful tools for monitoring NAI susceptibility of AIVs. IMPORTANCE The chemical structures of neuraminidase inhibitors (NAIs) possess similarities, but slight differences can result in variable susceptibility of avian influenza viruses (AIVs) carrying resistance-associated NA substitutions. Therefore, comprehensive susceptibility profiling of these substitutions in AIVs is critical for understanding the mechanism of antiviral resistance. In this study, we profiled resistance to the anti-influenza drug laninamivir in AIVs with substitutions known to impart resistance to other NAIs. We found 10 substitutions that conferred resistance to all four NAIs tested. On the other hand, we found that the remaining 26 NA substitutions were susceptible to at least one or more NAIs and showed for a small selection that in vitro data predicted in vivo behavior. Therefore, our findings highlight the usefulness of screening resistance markers in NA enzyme inhibition assays and animal models of AIV infections.

COVID-19 remains a challenge worldwide, and testing of asymptomatic individuals remains critical to pandemic control measures. Starting March 2020, a total of 7497 hospital employees were tested at least weekly for SARS CoV-2; the cumulative incidence of asymptomatic infections was 5.64%. Consistently over a 14-month period half of COVID-19 infections (414 of 820, total) were detected through the asymptomatic screening program, a third of whom never developed any symptoms during follow-up. Prompt detection and isolation of these cases substantially reduced the risk of potential workplace and outside of workplace transmission. COVID-19 vaccinations of the workforce were initiated in December 2020. Twenty-one individuals tested positive after being fully vaccinated (3.9 per 1000 vaccinated). Most (61.9%) remained asymptomatic and in majority (75%) the virus could not be sequenced due to low template RNA levels in swab samples. Further routine testing of vaccinated asymptomatic employees was stopped and will be redeployed if needed; routine testing for those not vaccinated continues. Asymptomatic SARS-CoV-2 testing, as a part of enhanced screening, monitors local dynamics of the COVID-19 pandemic and can provide valuable data to assess the ongoing impact of COVID-19 vaccination and SARS-CoV-2 variants, inform risk mitigation, and guide adaptive, operational planning including titration of screening strategies over time, based on infection risk modifiers such as vaccination.

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 and geographic viral transition patterns among multiple co-circulating subtypes. Our results show high transition rates within Anseriformes and Charadriiformes, but limited transitions between these orders. Patterns of transition between species 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 transition between locations. 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.

Stringent nonpharmaceutical interventions (NPIs) such as lockdowns and border closures are not currently recommended for pandemic influenza control. New Zealand used these NPIs to eliminate coronavirus disease 2019 during its first wave. Using multiple surveillance systems, we observed a parallel and unprecedented reduction of influenza and other respiratory viral infections in 2020. This finding supports the use of these NPIs for controlling pandemic influenza and other severe respiratory viral threats., New Zealand has been relatively successful in controlling COVID-19 due to implementation of strict non-pharmaceutical interventions. Here, the authors demonstrate a striking decline in reports of influenza and other non-influenza respiratory pathogens over winter months in which the interventions have been in place.

The improvement of influenza virus vaccines and the development of a universal product have been long-standing goals in pre-clinical and clinical research. To meet these goals and to understand the strengths and weaknesses of current vaccine strategies, scientists routinely study human responses toward seasonal influenza vaccines. This research is frequently performed with clinical samples taken throughout an influenza season, often without strict attention to the month of inoculation for each study participant. Here, we ask how the timing of vaccination affects outcomes. Results demonstrate significant influences of inoculation month on the immune response. During the progression from fall to winter months, there are changes in host lifestyles and in the frequencies of clinical/sub-clinical viral infections that can significantly alter vaccine immunogenicity. We now recommend routine assessment of inoculation month during clinical studies to inform data interpretation and expedite the development of successful vaccines. This recommendation is pertinent to numerous vaccine development efforts within and outside the influenza virus field.

To optimize the public health response to coronavirus disease 2019 (COVID-19), we must first understand the antibody response to individual proteins on the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and the antibody&rsquo, s cross reactivity to other coronaviruses. Using a panel of 37 convalescent COVID-19 human serum samples, we showed that the magnitude and specificity of responses varied across individuals, independent of their reactivity to seasonal human coronaviruses (HCoVs). These data suggest that COVID-19 vaccines will elicit primary humoral immune responses in na&iuml, ve individuals and variable responses in those previously exposed to SARS-CoV-2. Unlike the limited cross-coronavirus reactivities in humans, serum samples from 96 dogs and 10 cats showed SARS-CoV-2 protein-specific responses focused on non&ndash, S1 proteins. The correlation of this response with those to other coronaviruses suggests that the antibodies are cross-reactive and generated to endemic viruses within these hosts, which must be considered in seroepidemiologic studies. We conclude that substantial variation in antibody generation against coronavirus proteins will influence interpretations of serologic data in the clinical and veterinary settings.

Summary The failure to mount an antibody response following viral infection or seroconversion failure is a largely underappreciated and poorly understood phenomenon. Here, we identified immunologic markers associated with robust antibody responses after influenza virus infection in two independent human cohorts, SHIVERS and FLU09, based in Auckland, New Zealand and Memphis, Tennessee, USA, respectively. In the SHIVERS cohort, seroconversion significantly associates with (1) hospitalization, (2) greater numbers of proliferating, activated CD4+ T cells, but not CD8+ T cells, in the periphery during the acute phase of illness, and (3) fewer inflammatory monocytes (CD14hiCD16+) by convalescence. In the FLU09 cohort, fewer CD14hiCD16+ monocytes during early illness in the nasal mucosa were also associated with the generation of influenza-specific mucosal immunoglobulin A (IgA) and IgG antibodies. Our study demonstrates that seroconversion failure after infection is a definable immunological phenomenon, associated with quantifiable cellular markers that can be used to improve diagnostics, vaccine efficacy, and epidemiologic efforts., Graphical abstract, Highlights Post-infection seroconversion is associated with severity of influenza virus infection Seroconverters have early proliferation and activation of CD4+ T cells CD8+ T cells are unaffected CD14hiCD16+ monocytes in the blood and nasal mucosa is associated with antibody response, Wong et al. show that antibody responsiveness after influenza virus infection is associated with CD4+ T cells and CD14hiCD16+ monocytes. CD14hiCD16+ monocytes are also important in the mucosal antibody response. This demonstrates that seroconversion failure after infection is a definable immunological phenomenon, an important consideration for diagnostics and epidemiological studies.

International audience; Since its detection in swine, influenza D virus (IDV) has been shown to be present in multiple animal hosts, and bovines have been identified as its natural reservoir. However, it remains unclear how IDVs emerge, evolve, spread, and maintain in bovine populations. Through multiple years of virological and serological surveillance in a single order-buyer cattle facility in Mississippi, we showed consistently high seroprevalence of IDVs in cattle and recovered a total of 32 IDV isolates from both healthy and sick animals, including those with antibodies against IDV. Genomic analyses of these isolates along with those isolated from other areas showed that active genetic reassortment occurred in IDV and that five reassortants were identified in the Mississippian facility. Two antigenic groups were identified through antigenic cartography analyses for these 32 isolates and representative IDVs from other areas. Remarkably, existing antibodies could not protect cattle from experimental reinfection with IDV. Additional phenotypic analyses demonstrated variations in growth dynamics and pathogenesis in mice between viruses independent of genomic constellation. In summary, this study suggests that, in addition to epidemiological factors, the ineffectiveness of preexisting immunity and cocirculation of a diverse viral genetic pool could facilitate its high prevalence in animal populations.IMPORTANCE Influenza D viruses (IDVs) are panzootic in multiple animal hosts, but the underlying mechanism is unclear. Through multiple years of surveillance in the same order-buyer cattle facility, 32 IDV isolates were recovered from both healthy and sick animals, including those with evident antibodies against IDV. Active reassortment occurred in the cattle within this facility and in those across other areas, and multiple reassortants cocirculated in animals. These isolates are shown with a large extent of phenotypic diversity in replication efficiency and pathogenesis but little in antigenic properties. Animal experiments demonstrated that existing antibodies could not protect cattle from experimental reinfection with IDV. This study suggests that, in addition to epidemiological factors, limited protection from preex isting immunity against IDVs in cattle herds and cocirculation of a diverse viral genetic pool likely facilitate the high prevalence of IDVs in animal populations.

International audience; Influenza A/H5N1 has circulated in Asia since 2003 and is now enzootic in many countries in that region. In Cambodia, the virus has circulated since 2004 and has intermittently infected humans. During this period, we have noted differences in the rate of infections in humans, potentially associated with the circulation of different viral clades. In particular, a reassortant clade 1.1.2 virus emerged in early 2013 and was associated with a dramatic increase in infections of humans (34 cases) until it was replaced by a clade 2.3.2.1c virus in early 2014. In contrast, only one infection of a human has been reported in the 6 years since the clade 2.3.2.1c virus became the dominant circulating virus. We selected three viruses to represent the main viral clades that have circulated in Cambodia (clade 1.1.2, clade 1.1.2 reassortant, and clade 2.3.2.1c), and we conducted experiments to assess the virulence and transmissibility of these viruses in avian (chicken, duck) and mammalian (ferret) models. Our results suggest that the clade 2.3.2.1c virus is more "avian-like," with high virulence in both ducks and chickens, but there is no evidence of aerosol transmission of the virus from ducks to ferrets. In contrast, the two clade 1 viruses were less virulent in experimentally infected and contact ducks. However, evidence of chicken-to-ferret aerosol transmission was observed for both clade 1 viruses. The transmission experiments provide insights into clade-level differences that might explain the variation in A/H5N1 infections of humans observed in Cambodia and other settings.

The host innate defence against influenza virus infection is an intricate system with a plethora of antiviral factors involved. We have identified host histone deacetylase 6 (HDAC6) as an anti-influenza virus factor in cultured cells. Consistent with this, we report herein that HDAC6 knockout (KO) mice are more susceptible to influenza virus A/PR/8/1934 (H1N1) infection than their wild type (WT) counterparts. The KO mice lost weight faster than the WT mice and, unlike WT mice, could not recover their original body weight. Consequently, more KO mice succumbed to infection, which corresponded with higher lung viral loads. Conversely, the expression of the critical innate antiviral response genes interferon alpha/beta, CD80, CXCL10 and IL15 was significantly downregulated in KO mouse lungs compared to WT mouse lungs. These data are consistent with the known function of HDAC6 of de-acetylating the retinoic acid inducible gene-I (RIG-I) and activating the host innate antiviral response cascade. Loss of HDAC6 thus leads to a blunted innate response and increased susceptibility of mice to influenza A virus infection.

Pandemic influenza A viruses can emerge from swine, an intermediate host that supports adaptation of human-preferred receptor-binding specificity by the hemagglutinin (HA) surface antigen. Other HA traits necessary for pandemic potential are poorly understood. For swine influenza viruses isolated in 2009–2016, gamma-clade viruses had less stable HA proteins (activation pH 5.5–5.9) than pandemic clade (pH 5.0–5.5). Gamma-clade viruses replicated to higher levels in mammalian cells than pandemic clade. In ferrets, a model for human adaptation, a relatively stable HA protein (pH 5.5–5.6) was necessary for efficient replication and airborne transmission. The overall airborne transmission frequency in ferrets for four isolates tested was 42%, and isolate G15 airborne transmitted 100% after selection of a variant with a stabilized HA. The results suggest swine influenza viruses containing both a stabilized HA and alpha-2,6 receptor binding in tandem pose greater pandemic risk. Increasing evidence supports adding HA stability to pre-pandemic risk assessment algorithms.

The genesis of novel influenza viruses through reassortment poses a continuing risk to public health. This is of particular concern in Bangladesh, where highly pathogenic avian influenza viruses of the A(H5N1) subtype are endemic and cocirculate with other influenza viruses. Active surveillance of avian influenza viruses in Bangladeshi live poultry markets detected three A(H5) genotypes, designated H5N1-R1, H5N1-R2, and H5N2-R3, that arose from reassortment of A(H5N1) clade 2.3.2.1a viruses. The H5N1-R1 and H5N1-R2 viruses contained HA, NA, and M genes from the A(H5N1) clade 2.3.2.1a viruses and PB2, PB1, PA, NP, and NS genes from other Eurasian influenza viruses. H5N2-R3 viruses contained the HA gene from circulating A(H5N1) clade 2.3.2.1a viruses, NA and M genes from concurrently circulating A(H9N2) influenza viruses, and PB2, PB1, PA, NP, and NS genes from other Eurasian influenza viruses. Representative viruses of all three genotypes and a parental clade 2.3.2.1a strain (H5N1-R0) infected and replicated in mice without prior adaptation; the H5N2-R3 virus replicated to highest titers in the lung. All viruses efficiently infected and killed chickens. All viruses replicated in inoculated ferrets, but no airborne transmission was detected, and only H5N2-R3 showed limited direct-contact transmission. Our findings demonstrate that although the A(H5N1) viruses circulating in Bangladesh have the capacity to infect and replicate in mammals, they show very limited capacity for transmission. However, reassortment does generate viruses of distinct phenotypes. IMPORTANCE Highly pathogenic avian influenza A(H5N1) viruses have circulated continuously in Bangladesh since 2007, and active surveillance has detected viral evolution driven by mutation and reassortment. Recently, three genetically distinct A(H5N1) reassortant viruses were detected in live poultry markets in Bangladesh. Currently, we cannot assign pandemic risk by only sequencing viruses; it must be conducted empirically. We found that the H5Nx highly pathogenic avian influenza viruses exhibited high virulence in mice and chickens, and one virus had limited capacity to transmit between ferrets, a property considered to be consistent with a higher zoonotic risk.

Background The Centers of Excellence for Influenza Research and Surveillance (CEIRS) network, funded by the US National Institutes of Health, has been operational since 2007 and is tasked with conducting research to improve understanding of influenza viruses. Recently, CEIRS developed an Influenza Response Plan (IRP) to improve science preparedness for the network. Methods Development of the IRP involved a collaborative process between project staff, CEIRS center directors or their designees, and NIAID CEIRS leadership (referred to as the Pandemic Planning Advisory Committee [PPAC]). Project staff identified and summarized the response capabilities of each center and then worked with the PPAC to identify and rank research priorities for an emergency response using a modified Delphi method. Results Key elements of the response plan include tables of response capabilities for each CEIRS center, a framework that outlines and ranks research priorities for CEIRS during an emergency situation, and an operational strategy for executing the research priorities. Conclusions The CEIRS IRP highlights the importance of enhancing science preparedness in advance of an influenza pandemic or other influenza‐related zoonotic incident to ensure that research can be carried out expeditiously and effectively in emergency situations and to improve global health security.

Influenza A virus infection in swine impacts the agricultural industry in addition to its zoonotic potential. Here, we utilize epigraph, a computational algorithm, to design a universal swine H3 influenza vaccine. The epigraph hemagglutinin proteins are delivered using an Adenovirus type 5 vector and are compared to a wild type hemagglutinin and the commercial inactivated vaccine, FluSure. In mice, epigraph vaccination leads to significant cross-reactive antibody and T-cell responses against a diverse panel of swH3 isolates. Epigraph vaccination also reduces weight loss and lung viral titers in mice after challenge with three divergent swH3 viruses. Vaccination studies in swine, the target species for this vaccine, show stronger levels of cross-reactive antibodies and T-cell responses after immunization with the epigraph vaccine compared to the wild type and FluSure vaccines. In both murine and swine models, epigraph vaccination shows superior cross-reactive immunity that should be further investigated as a universal swH3 vaccine., A range of Influenza vaccines have been linked to induction of adaptive immunity in a number of animal models. Here, the authors utilize a computational design strategy and produce a swine H3 influenza vaccine which shows enhanced efficacy in both murine and porcine infectious disease models.

Data on the immunologic responses to neuraminidase (NA) is lacking compared to what is available on hemagglutinin (HA) responses, despite growing evidence that NA immunity can be protective and broadly cross-reactive. Understanding these NA responses during natural infection is key to exploiting these properties for improving influenza vaccines. Using two community-acquired influenza cohorts, we showed that the induction of both HA and NA antibodies after infection is influenced by age and subtypes. Such response dynamics suggest the influence of immunological memory, and understanding how this process is regulated will be critical to any vaccine effort targeting NA immunity., Despite evidence that antibodies targeting the influenza virus neuraminidase (NA) protein can be protective and are broadly cross-reactive, the immune response to NA during infection is poorly understood compared to the response to hemagglutinin (HA) protein. As such, we compared the antibody profile to HA and NA in two naturally infected human cohorts in Auckland, New Zealand: (i) a serosurvey cohort, consisting of pre- and post-influenza season sera from PCR-confirmed influenza cases (n = 50), and (ii) an immunology cohort, consisting of paired sera collected after PCR-confirmation of infection (n = 94). The induction of both HA and NA antibodies in these cohorts was influenced by age and subtype. Seroconversion to HA was more frequent in those

The panzootic caused by A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) A(H5) viruses has occurred in multiple waves since 1996. From 2013 onwards, clade 2.3.4.4 viruses of subtypes A(H5N2), A(H5N6), and A(H5N8) emerged to cause panzootic waves of unprecedented magnitude among avian species accompanied by severe losses to the poultry industry around the world. Clade 2.3.4.4 A(H5) viruses have expanded in distinct geographical and evolutionary pathways likely via long distance migratory bird dispersal onto several continents and by poultry trade among neighboring countries. Coupled with regional circulation, the viruses have evolved further by reassorting with local viruses. As of February 2019, there have been 23 cases of humans infected with clade 2.3.4.4 H5N6 viruses, 16 (70%) of which had fatal outcomes. To date, no HPAI A(H5) virus has caused sustainable human-to-human transmission. However, due to the lack of population immunity in humans and ongoing evolution of the virus, there is a continuing risk that clade 2.3.4.4 A(H5) viruses could cause an influenza pandemic if the ability to transmit efficiently among humans was gained. Therefore, multisectoral collaborations among the animal, environmental, and public health sectors are essential to conduct risk assessments and develop countermeasures to prevent disease and to control spread. In this article, we describe an assessment of the likelihood of clade 2.3.4.4 A(H5) viruses gaining human-to-human transmissibility and impact on human health should such human-to-human transmission occur. This structured analysis assessed properties of the virus, attributes of the human population, and ecology and epidemiology of these viruses in animal hosts.

Anecdotal evidence showed a negative correlation between Bacille Calmette-Guerin (BCG) vaccination and incidence of COVID-19. Incidence of the disease in children is much lower than in adults. It is hypothesized that BCG and other childhood vaccinations may provide some protection against SARS-CoV-2 infection through trained or adaptive immune responses. Here, we tested whether BCG, Pneumococcal, Rotavirus, Diphtheria, Tetanus, Pertussis, Hepatitis B, Haemophilus influenzae, Hepatitis B, Meningococcal, Measles, Mumps, and Rubella vaccines provide cross-reactive neutralizing antibodies against SARS-CoV-2 in BALB/c mice. Results indicated that none of these vaccines provided antibodies capable of neutralizing SARS-CoV-2 up to seven weeks post vaccination. We conclude that if such vaccines have any role in COVID-19 immunity, this role is not antibody-mediated.

Ducks are a natural reservoir of influenza A viruses (IAVs) and can act as a reassortment vessel. Wetlands, such as Hakaluki and Tanguar haor in Bangladesh, have unique ecosystems including domestic duck (Anas platyrhynchos domesticus) rearing, especially household and free-range ducks. A cross-sectional study was, therefore, conducted to explore avian influenza status and its distribution and risk factors in the wetland areas. During the three consecutive winters of 2015&ndash, 2017, specifically in December of these years, we collected a total of 947 samples including blood, oropharyngeal and cloacal swabs from domestic ducks (free-range duck (n = 312 samples) and household ducks (n = 635 samples) in wetlands. We screened serum samples using a nucleoprotein competitive enzyme-linked immunosorbent assay (c-ELISA) to estimate seroprevalence of IAV antibodies and swab samples by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) to detect IA viral M gene. Eleven (11) M gene positive samples were subjected to sequencing and phylogenetic analysis. Serological and viral prevalence rates of IAVs were 63.8% (95% CI: 60.6&ndash, 66.8) and 10.7% (8.8&ndash, 12.8), respectively. Serological and viral RNA prevalence rates were 51.8% (95% CI: 47.2&ndash, 56.4) and 10.2% (7.6&ndash, 13.3) in Hakaluki haor, 75.6% (71.5&ndash, 79.4) and 11.1% (8.5&ndash, 14.3) in Tanguar haor, 66.3% (62.5&ndash, 69.9) and 11.2% (8.8&ndash, 13.9) in household ducks and 58.7% (52.9&ndash, 64.2) and 9.6% (6.5&ndash, 13.4) in free-range ducks, respectively. The risk factors identified for higher odds of AI seropositive ducks were location (OR = 2.9, 95% CI: 2.2&ndash, 3.8, p &lt, 0.001, Tanguar haor vs. Hakaluki haor), duck-rearing system (OR = 1.4, 1.1&ndash, 1.8, household vs. free-range), farmer&rsquo, s education status (OR = 1.5, 1.2&ndash, 2.0, p &lt, 0.05 illiterate vs. literate) and contact type (OR = 3.0, 2.1&ndash, 4.3, p &lt, contact with chicken vs. no contact with chicken). The risk factors identified for higher odds of AI RNA positive ducks were farmer&rsquo, s education status (OR = 1.5, 1.0&ndash, 2.3, p &lt, 0.05 for illiterate vs literate), contact type (OR = 2.7, 1.7&ndash, 4.2, p &lt, ducks having contact with chicken vs. ducks having contact with waterfowl). The phylogenetic analysis of 11 partial M gene sequences suggested that the M gene sequences detected in free-range duck were very similar to each other and were closely related to the M gene sequences of previously reported highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) subtypes in waterfowl in Bangladesh and Southeast Asian countries. Results of the current study will help provide significant information for future surveillance programs and model IAV infection to predict the spread of the viruses among migratory waterfowl, free-range ducks and domestic poultry in Bangladesh.

The continual emergence of novel influenza A strains from non-human hosts requires constant vigilance and the need for ongoing research to identify strains that may pose a human public health risk. Since 1999, canine H3 influenza A viruses (CIVs) have caused many thousands or millions of respiratory infections in dogs in the United States. While no human infections with CIVs have been reported to date, these viruses could pose a zoonotic risk. In these studies, the National Institutes of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) network collaboratively demonstrated that CIVs replicated in some primary human cells and transmitted effectively in mammalian models. While people born after 1970 had little or no pre-existing humoral immunity against CIVs, the viruses were sensitive to existing antivirals and we identified a panel of H3 cross-reactive human monoclonal antibodies (hmAbs) that could have prophylactic and/or therapeutic value. Our data predict these CIVs posed a low risk to humans. Importantly, we showed that the CEIRS network could work together to provide basic research information important for characterizing emerging influenza viruses, although there were valuable lessons learned., Author summary The 2009 influenza pandemic was a stark reminder that ongoing vigilance is critical to protect the public from an influenza pandemic. The continual evolution of influenza viruses and emergence from animal reservoirs, leads to the need to quickly identify strains that pose a public health risk. In these studies, members of the National Institutes of Allergy and Infectious Diseases (NIAID) Centers for Excellence in Influenza Research and Surveillance (CEIRS) network worked together to demonstrate that the emerging canine H3 influenza viruses posed a low risk to public health and identified several therapeutic options in the event of an emergence. In addition to providing important new basic research, many lessons were learned that may be important in dealing with any emerging disease outbreak.

An individual’s antibody titers to influenza A strains are a result of the complicated interplay between infection history, cross-reactivity, immune waning, and other factors. It has been challenging to disentangle how population-level patterns of humoral immunity change as a function of age, calendar year, and birth cohort from cross-sectional data alone. We analyzed 1,589 longitudinal sera samples from 260 children across three studies in Nicaragua, 2006–16. Hemagglutination inhibition (HAI) titers were determined against four H3N2 strains, one H1N1 strain, and two H1N1pdm strains. We assessed temporal patterns of HAI titers using an age–period–cohort modeling framework. We found that titers against a given virus depended on calendar year of serum collection and birth cohort but not on age. Titer cohort patterns were better described by participants’ ages relative to year of likely introduction of the virus’s antigenic cluster than by age relative to year of strain introduction or by year of birth. These cohort effects may be driven by a decreasing likelihood of early-life infection after cluster introduction and by more broadly reactive antibodies at a young age. H3N2 and H1N1 viruses had qualitatively distinct cohort patterns, with cohort patterns of titers to specific H3N2 strains reaching their peak in children born 3 years prior to that virus’s antigenic cluster introduction and with titers to H1N1 and H1N1pdm strains peaking for children born 1–2 years prior to cluster introduction but not being dramatically lower for older children. Ultimately, specific patterns of strain circulation and antigenic cluster introduction may drive population-level antibody titer patterns in children.

SARS-CoV-2 infection causes diverse outcomes ranging from asymptomatic infection to respiratory distress and death. A major unresolved question is whether prior immunity to endemic, human common cold coronaviruses (hCCCoV) impacts susceptibility to SARS-CoV-2 infection or immunity following infection and vaccination. Therefore, we analyzed samples from the same individuals before and after SARS-CoV-2 infection or vaccination. We found hCCCoV antibody levels increase after SARS-CoV-2 exposure, demonstrating cross-reactivity. However, a case-control study indicates baseline hCCCoV antibody levels are not associated with protection against SARS-CoV-2 infection. Rather, higher magnitudes of pre-existing betacoronavirus antibodies correlate with more SARS-CoV-2 antibodies following infection, an indicator of greater disease severity. Additionally, immunization with hCCCoV spike proteins before SARS-CoV-2 immunization impedes generation of SARS-CoV-2 neutralizing antibodies in mice. Together, these data suggest pre-existing hCCCoV antibodies hinder SARS-CoV-2 antibody-based immunity following infection and provide insight on how pre-existing coronavirus immunity impacts SARS-CoV-2 infection, which is critical considering emerging variants., Graphical Abstract, A major unresolved question is whether prior immunity to endemic, human common cold coronaviruses (hCCCoV) impacts susceptibility to SARS-CoV-2 infection. Lin et al. analyze hCCCoV antibodies in the same individuals before and after SARS-CoV-2 infection, finding pre-existing betacoronavirus antibodies may hinder SARS-CoV-2 effective immunity following infection.

The H9N2 influenza viruses that have become established in Bangladeshi live poultry markets possess five gene segments of the highly pathogenic H7N3 avian influenza virus. We assessed the replication, transmission, and disease potential of three H9N2 viruses in chickens and New World quail. Each virus replicated to high titers and transmitted by the airborne route to contacts in both species. Infected chickens showed no disease signs, and the viruses differed in their disease potential in New World quail. New World quail were more susceptible than chickens to H9N2 viruses and shed virus after airborne transmission for 10 days. Consequently, New World quail are a potential threat in the maintenance and spread of influenza virus in live poultry markets.

Among 16 haemagglutinin (HA) subtypes of avian influenza viruses (AIVs), H13 AIVs have rarely been isolated in wild waterfowl. H13 AIVs cause asymptomatic infection and are maintained mainly in gull and tern populations; however, the recorded antigenic information relating to the viruses has been limited. In this study, 2 H13 AIVs, A/duck/Hokkaido/W345/2012 (H13N2) and A/duck/Hokkaido/WZ68/2012 (H13N2), isolated from the same area in the same year in our surveillance, were genetically and antigenically analyzed with 10 representative H13 strains including a prototype strain, A/gull/Maryland/704/1977 (H13N6). The HA genes of H13 AIVs were phylogenetically divided into 3 groups (I, II, and III). A/duck/Hokkaido/W345/2012 (H13N2) was genetically classified into Group III. This virus was distinct from a prototype strain, A/gull/Maryland/704/1977 (H13N6), and the virus, A/duck/Hokkaido/WZ68/2012 (H13N2), both belonging to Group I. Antigenic analysis indicated that the viruses of Group I were antigenically closely related to those of Group II, but distinct from those of Group III, including A/duck/Hokkaido/W345/2012 (H13N2). In summary, our study indicates that H13 AIVs have undergone antigenic diversification in nature.

Influenza viruses undergo rapid antigenic evolution and reassortment, resulting in annual epidemics and the occasional pandemics. Exposure to influenza virus hemagglutinin (HA) and neuraminidase (NA) antigen, either through vaccination or infection, induces an antibody response able to recognize only the homologous antigenic subtype. However, atypical antibody responses recognizing non-homologous influenza subtypes have been reported during infection and vaccination. Here, we review the incidence of these phenomena in published literature and discuss the potential mechanisms underlying them.

Influenza D virus (IDV) has been identified in domestic cattle, swine, camelid, and small ruminant populations across North America, Europe, Asia, South America, and Africa. Our study investigated seroprevalence and transmissibility of IDV in feral swine. During 2012-2013, we evaluated feral swine populations in 4 US states; of 256 swine tested, 57 (19.1%) were IDV seropositive. Among 96 archived influenza A virus-seropositive feral swine samples collected from 16 US states during 2010-2013, 41 (42.7%) were IDV seropositive. Infection studies demonstrated that IDV-inoculated feral swine shed virus 3-5 days postinoculation and seroconverted at 21 days postinoculation; 50% of in-contact naive feral swine shed virus, seroconverted, or both. Immunohistochemical staining showed viral antigen within epithelial cells of the respiratory tract, including trachea, soft palate, and lungs. Our findings suggest that feral swine might serve an important role in the ecology of IDV.

Surveillance of wild aquatic birds and free-range domestic ducks in the Tanguar Haor wetlands in Bangladesh has identified influenza virus subtypes H3N6, H7N1, H7N5, H7N9, and H15N9. Molecular characterization of these viruses indicates their contribution to the genesis of new genotypes of H5N1 influenza viruses from clade 2.3.2.1a that are dominant in poultry markets in Bangladesh as well as to the genesis of the highly pathogenic H5N8 virus currently causing disease outbreaks in domestic poultry in Europe and the Middle East. Therefore, we studied the antigenicity, replication, and pathogenicity of influenza viruses isolated from Tanguar Haor in the DBA/2J mouse model. All viruses replicated in the lung without prior mammalian adaptation, and H7N1 and H7N9 viruses caused 100% and 60% mortality, respectively. H7N5 viruses replicated only in the lungs, whereas H7N1 and H7N9 viruses also replicated in the heart, liver, and brain. Replication and transmission studies in mallard ducks showed that H7N1 and H7N9 viruses replicated in ducks without clinical signs of disease and shed at high titers from the cloaca of infected and contact ducks, which could facilitate virus transmission and spread. Our results indicate that H7 avian influenza viruses from free-range ducks can replicate in mammals, cause severe disease, and be efficiently transmitted to contact ducks. Our study highlights the role of free-range ducks in the spread of influenza viruses to other species in live poultry markets and the potential for these viruses to infect and cause disease in mammals.

In 2015, highly pathogenic avian influenza A(H5N1) viruses reemerged in poultry in West Africa. We describe the introduction of a reassortant clade 2.3.2.1c virus into Togo in April 2018. Our findings signal further local spread and evolution of these viruses, which could affect animal and human health.Keywords

Background There is insufficient knowledge about the relation of avian influenza virus (AIV) to migratory birds in South America. Accordingly, we studied samples obtained over a 4-year period (2009–2012) from wild birds at a major wintering site in Southern Brazil. Methods We obtained 1212 oropharyngeal/cloacal samples from wild birds at Lagoa do Peixe National Park and screened them for influenza A virus by RT-PCR amplification of the matrix gene. Virus isolates were subjected to genomic sequencing and antigenic characterization. Results Forty-eight samples of 1212 (3.96%) contained detectable influenza virus RNA. Partial viral sequences were obtained from 12 of these samples, showing the presence of H2N2 (1), H6Nx (1), H6N1 (8), H9N2 (1), and H12N5 (1) viruses. As H6 viruses predominated, we generated complete genomes from all nine H6 viruses. Phylogenetic analyses showed that they were most similar to viruses of South American lineage. The H6N1 viruses caused no disease signs in infected ferrets and, despite genetic differences, were antigenically similar to North American isolates. Conclusions Lagoa do Peixe National Park is a source of multiple AIV subtypes, with the levels of influenza virus in birds being highest at the end of their wintering period in this region. H6N1 viruses were the predominant subtype identified. These viruses were more similar to viruses of South American lineage than to those of North American lineage. This article is protected by copyright. All rights reserved.

Immunocompromised patients are highly susceptible to influenza virus infections. Although neuraminidase inhibitor (NAI) therapy has proved effective in these patients, the treatment regimens require optimization, which can be partly addressed via animal models. Here, we describe a pharmacologically immunosuppressed mouse model for studying the pathogenesis of influenza B viruses and evaluating the efficacy of antiviral treatment. We modeled clinical regimens for dexamethasone and cyclophosphamide to immunosuppress BALB/c mice that were then inoculated with B/Phuket/3073/2013 (Yamagata lineage) or B/Brisbane/60/2008 (BR/08, Victoria lineage) virus. Although both viruses caused morbidity and mortality in immunosuppressed mice, BR/08 was more virulent, consistently inducing greater morbidity and 100% lethality in mice inoculated with at least 10(3) TCID(50)/mouse. The replication of both viruses was prolonged in the lungs of immunosuppressed mice, but the extent of pulmonary inflammation in these mice was markedly less than that in immunocompetent animals. Most of the examined cytokines, including IFN-γ, IL-1β, and RANTES, were significantly decreased in the lungs of immunosuppressed mice, as compared to immunocompetent animals, until at least 10 days post-infection. Treatment with the NAI oseltamivir for 8 or 16 days increased the mean survival time and reduced virus spread in the lungs of immunosuppressed mice challenged with a lethal dose of BR/08 but did not completely provide protection or decrease the virus titers. Our data suggests that the synergy of the viral load and aberrant immune responses is a key contributor to the severity of infection, as well as the limited efficacy of oseltamivir, which in immunosuppressed mice curtails virus release without clearing infected cells.

Stable, effective, easy-to-manufacture vaccines are critical to stopping the COVID-19 pandemic resulting from the coronavirus SARS-CoV-2. We constructed a vaccine candidate CoV-RBD121-NP, which is comprised of the SARS-CoV-2 receptor-binding domain (RBD) of the spike glycoprotein (S) fused to a human IgG1 Fc domain (CoV-RBD121) and conjugated to a modified tobacco mosaic virus (TMV) nanoparticle. In vitro, CoV-RBD121 bound to the host virus receptor ACE2 and to the monoclonal antibody CR3022, a neutralizing antibody that blocks S binding to ACE2. The CoV-RBD121-NP vaccine candidate retained key SARS-CoV-2 spike protein epitopes, had consistent manufacturing release properties of safety, identity, and strength, and displayed stable potency when stored for 12 months at 2–8 °C or 22–28 °C. Immunogenicity studies revealed strong antibody responses in C57BL/6 mice with non-adjuvanted or adjuvanted (7909 CpG) formulations. The non-adjuvanted vaccine induced a balanced Th1/Th2 response and antibodies that recognized both the S1 domain and full S protein from SARS2-CoV-2, whereas the adjuvanted vaccine induced a Th1-biased response. Both adjuvanted and non-adjuvanted vaccines induced virus neutralizing titers as measured by three different assays. Collectively, these data showed the production of a stable candidate vaccine for COVID-19 through the association of the SARS-CoV-2 RBD with the TMV-like nanoparticle.

Background Aloe vera is a functional food with various pharmacological functions, including an immune-modulating effect. Until now, A. vera has never been studied as an adjuvant in influenza vaccine, and its effects on upper respiratory tract infection (URI) are unknown. Purpose The objective of our study was to investigate the effect of processed A. vera gel (PAG) on immunogenicity of quadrivalent inactivated influenza vaccine and URI in healthy adults. Study design A randomized, double-blind, placebo-controlled clinical trial was performed. Methods This study was conducted in 100 healthy adults at a single center from September 2017 to May 2018. Subjects were randomly divided into a PAG group (n = 50) and a placebo group (n = 50). The enrolled subjects were instructed to ingest the study drug for 8 weeks. The participants received a single dose of quadrivalent inactivated influenza vaccine after taking the study drug for the first 4 weeks of the study. The primary endpoint was seroprotection rate against at least one viral strain at 4 weeks post-vaccination. Other outcomes were seroprotection rate at 24 weeks post-vaccination, seroconversion rate, geometric mean fold increase (GMFI) at 4 and 24 weeks post-vaccination, seroprotection rate ratio and geometric mean titer ratio (GMTR) at 4 weeks post-vaccination between PAG and placebo groups, and incidence, severity, and duration of URI. Results The European Committee for proprietary medicinal products (CPMP) evaluation criteria were met at least one in the PAG and placebo groups for all strains. However, there was no significant difference in the seroprotection rate at 4 weeks post-vaccination against all strains in both PAG and placebo groups. Among secondary endpoints, the GMFI at 4 weeks post-vaccination for the A/H3N2 was significantly higher in the PAG than in placebo group. The GMTR as adjuvant effect was 1.382 (95% CI, 1.014-1.1883). Kaplan–Meier curve analysis showed a reduction in incidence of URI (p = 0.035), and a generalized estimating equation model identified a decrease in repeated URI events (odds ratio 0.57; 95% CI, 0.39-0.83; p = 0.003) in the PAG group. Conclusions Oral intake of PAG did not show a significant increase in seroprotection rate from an immunogenicity perspective. However, it reduced the number of URI episodes. A well-designed further study is needed on the effect of PAG's antibody response against A/H3N2 in the future.

This study was approved by the St. Jude Children’s Research Hospital Institutional Animal Care and Use Committee (Amended IACUC Approval # 546-100491-07/17; expires 4/5/23).; International audience; Influenza D virus (IDV) was first isolated in 2011 in Oklahoma, USA from pigs presenting with influenza-like symptoms. IDV is known to mainly circulate in ruminants, especially cattle. In Africa, there is limited information on the epidemiology of IDV, although the virus has likely circulated in the region since 2012. In the present study, we investigated the seropositivity of IDV among domestic ruminants and swine in West and East Africa from 2017 to 2020. Serum samples were analyzed using the hemagglutination inhibition (HI) assay. Our study demonstrated that IDV is still circulating in Africa, with variations in seropositivity among countries and species. The highest seropositivity was detected in cattle (3.9 to 20.9%). Our data highlights a need for extensive surveillance of IDV in Africa in order to better understand the epidemiology of the virus in the region.

The emergence of SARS-CoV-2 antigenic variants with increased transmissibility is a public health threat. Some variants show substantial resistance to neutralization by SARS-CoV-2 infection- or vaccination-induced antibodies. Here, we analyzed receptor binding domain-binding monoclonal antibodies derived from SARS-CoV-2 mRNA vaccine-elicited germinal center B cells for neutralizing activity against the WA1/2020 D614G SARS-CoV-2 strain and variants of concern. Of five monoclonal antibodies that potently neutralized the WA1/2020 D614G strain, all retained neutralizing capacity against the B.1.617.2 variant, four also neutralized the B.1.1.7 variant, and only one, 2C08, also neutralized the B.1.351 and B.1.1.28 variants. 2C08 reduced lung viral load and morbidity in hamsters challenged with the WA1/2020 D614G, B.1.351, or B.1.617.2 strains. Clonal analysis identified 2C08-like public clonotypes among B cells responding to SARS-CoV-2 infection or vaccination in 41 out of 181 individuals. Thus, 2C08-like antibodies can be induced by SARS-CoV-2 vaccines and mitigate resistance by circulating variants of concern.

Introduction Influenza viruses cause significant socioeconomic impact due to annual outbreaks and pandemic risks. Human tonsil epithelium cells (HTEC) are a heterogeneous group of actively differentiating epithelia comprising stratified squamous epithelium and reticulated crypt cells with abundant keratin expression. Hypothesis We hypothesized that the tonsils are a primary site for influenza infection and sustained viral replication. Methods and Results Primary HTEC (ScienCell Research Laboratories) were grown using an air-liquid interface and infected apically with different influenza viruses at various MOIs to measure viral growth kinetics. These cells were highly differentiated, with subpopulations of cells including ciliated, non-ciliated cells and specialized cells with secretory functions. There was a heterogenous distribution of both human-like (α2,6-linked) and avian-like (α2,3-linked) sialic acid receptors. The HTEC surface and crypts were lined with pseudostratified columnar ciliated cells possessing both α2,6-linked and α2,3-linked sialic acid receptors that were interrupted by patches of reticular epithelial cells. The HTEC epithelial cells were permissive for growth of influenza A and B viruses. A subset of cells, mostly ciliated cells, underwent apoptosis while others including non-ciliated cells remained intact despite being positive for influenza virus nucleoprotein. Interestingly, differences were seen between subtypes with colocalization of H3N2 virus and non-ciliated cells while H1N1 virus mostly associated with ciliated cells. Conclusion Our results implicated human tonsillar crypt epithelium as a site for influenza virus replication. The tonsil epithelium cell culture differentiated system provides a valuable in vitro model for studying cellular tropism, infectivity, cytokines immune response and the pathogenesis of influenza viruses for better development of effective universal vaccine and therapies against different strains of influenza viruses.

The majority of the Egyptian swine population was culled in the aftermath of the 2009 H1N1 pandemic, but small-scale growing remains. We sampled pigs from piggeries and an abattoir in Cairo. We found virological evidence of infection with avian H9N2 and H5N1 viruses as well as human pandemic H1N1 influenza virus. Serological evidence suggested previous exposure to avian H5N1 and H9N2, human pandemic H1N1, and swine avian-like and human-like viruses. This raises concern about potential reassortment of influenza viruses in pigs and highlights the need for better control and prevention of influenza virus infection in pigs.

North American wild birds are an important reservoir of influenza A viruses, yet the potential of viruses in this reservoir to transmit and cause disease in mammals is not well understood. Our surveillance of avian influenza viruses (AIVs) at Delaware Bay, USA, revealed a group of similar H1N1 AIVs isolated in 2009, some of which were airborne-transmissible in the ferret model without prior adaptation. Comparison of the genomes of these viruses revealed genetic markers of airborne transmissibility in the Polymerase Basic 2 (PB2), PB1, PB1-F2, Polymerase Acidic-X (PA-X), Nonstructural Protein 1 (NS1), and Nuclear Export Protein (NEP) genes. We studied the role of NS1 in airborne transmission and found that NS1 mutants that were not airborne-transmissible caused limited tissue pathology in the upper respiratory tract (URT). Viral maturation was also delayed, evident as strong intranuclear staining and little virus at the mucosa. Our study of this naturally occurring constellation of genetic markers has provided insights into the poorly understood phenomenon of AIV airborne transmissibility by revealing a role for NS1 and characteristics of viral replication in the URT that were associated with airborne transmission. The transmissibility of these viruses further highlights the pandemic potential of AIVs in the wild bird reservoir and the need to maintain surveillance.

Background In the fall of 2014, highly pathogenic avian influenza (HPAI) subtype H5N8 clade 2.3.4.4 was introduced into North America by migrating waterfowl from Asia where, through reassortment, novel HPAI H5N2 and H5N1 viruses emerged. Objectives Assess the susceptibility of pigs to HPAI H5N1, H5N2, and H5N8 clade 2.3.3.3 from North America. Methods Pigs and trachea explants were inoculated with a representative panel of H5NX clade 2.3.4.4 HPAI viruses from North America. Nasal swabs, BALF, and sera were collected to assess replication and transmission in challenged and direct contact pigs by RRT-PCR, virus isolation, hemagglutination inhibition, and ELISA. Results Limited virus replication was restricted to the lower respiratory tract of challenged pigs, though absent in the nasal passages and trachea cultures, as determined by RRT-PCR in all samples. Seroconversion of inoculated pigs was detected by NP ELISA but was not reliably detected by antigen-specific hemagglutination inhibition. Boost with adjuvanted virus was required for the production of neutralizing antibodies to assess cross-reactivity between wild-type avian strains. All RRT-PCR and serology tests were negative for contact animals indicating a failure of transmission from primary inoculated pigs. Conclusions H5NX clade 2.3.4.4 strains can replicate in the lower respiratory tract of swine upon high titer inoculation, though appear to be incapable of replication in swine nasal epithelium in vivo or ex vivo in trachea explants in culture. Infected pigs did not produce high levels of serum antibodies following infection. Collectively, our data show HPAI H5NX clade 2.3.4.4 viruses to be poorly adapted for replication and transmission in swine.

Highly pathogenic avian influenza A(H5N8) clade 2.3.4.4 virus emerged in 2016 and spread to Russia, Europe, and Africa. Our analysis of viruses from domestic ducks at Tanguar haor, Bangladesh, showed genetic similarities with other viruses from wild birds in central Asia, suggesting their potential role in the genesis of A(H5N8).

The endemicity of avian influenza viruses (AIVs) among Egyptian poultry represents a public health risk. Co-circulation of low pathogenic AIV H9N2 subtype with highly pathogenic AIV H5N1 subtype in Egyptian farms provides a possibility to generate novel reassortant viruses. Here, the genetic characteristics of surface glycoproteins of 59 Egyptian H9N2 viruses, isolated between 2013 and 2015, were analysed. To elucidate the potential of genetic reassortment, 10 H9N2 isolates were selected based on different avian hosts (chickens, ducks, pigeons and quails) and phylogenetic analyses of their full genome sequences were conducted. Additionally, we performed antigenic analysis to further investigate the antigenic evolution of H9N2 viruses isolated during 2011-2015. Different viral characteristics including receptor-binding affinity and drug resistance of representative Egyptian H9N2 viruses were further investigated. The surface glycoproteins of current Egyptian H9N2 viruses were closely related to viruses of the G1-like lineage isolated from Egypt. Several genetic markers that enhance virulence in poultry and transmission to humans were detected. Analysis of the full genome of 10 H9N2 isolates indicated that two pigeon isolates inherited five internal genes from Eurasian AIVs circulating in wild birds. Antigenic conservation of different Egyptian H9N2 isolates from chickens, pigeons and ducks was observed, whereas quail isolates showed antigenic drift. The Egyptian H9N2 viruses preferentially bound to the human-like receptor rather than to the avian-like receptor. Our results suggest that the endemic H9N2 viruses in Egypt contain elements that may favour avian-to-human transmission and thus represent a public health risk.

In 2013, a novel avian-origin H7N9 influenza A virus causing severe lower respiratory tract disease in humans emerged in China, with continued sporadic cases. An effective vaccine is needed for this virus in case it acquires transmissibility among humans; however, PR8-based A/Anhui/1/2013 (Anhui/1, H7N9), a WHO-recommended H7N9 candidate vaccine virus (CVV) for vaccine production, does not replicate well in chicken eggs, posing an obstacle to egg-based vaccine production. To address this issue, we explored the possibility that PR8's hemagglutinin (HA) and neuraminidase (NA) packaging signals mediate improvement of Anhui/1 CVV yield in eggs. We constructed chimeric HA and NA genes having the coding region of Anhui/1 HA and NA flanked by the 5' and 3' packaging signals of PR8's HA and NA, respectively. The growth of CVVs containing the chimeric HA was not affected, but that of those containing the chimeric NA gene grew in embryonated chicken eggs with a more than 2-fold higher titer than that of WT CVV. Upon 6 passages in eggs further yield increase was achieved although this was not associated with any changes in the chimeric NA gene. The HA of the passaged CVV, did, however, exhibit egg-adaptive mutations and one of them (HA-G218E) improved CVV growth in eggs without significantly changing antigenicity. The HA-G218E substitution and a chimeric NA, thus, combine to provide an Anhui/1 CVV with properties more favorable for vaccine manufacture.

Summary Equine coronaviruses (ECoV) are the only coronavirus known to infect horses. So far, data on ECoV infection in horses remain limited to the USA, France and Japan and its geographic distribution is not well understood. We carried out RT‐PCR on 306 nasal and 315 rectal swabs and tested 243 sera for antibodies to detect coronavirus infections in apparently healthy horses in Saudi Arabia and Oman. We document evidence of infection with ECoV and HKU23 coronavirus by RT‐PCR. There was no conclusive evidence of Middle East respiratory syndrome coronavirus infection in horses. Serological data suggest that lineage A betacoronavirus infections are commonly infecting horses in Saudi Arabia and Oman but antibody cross‐reactivities between these viruses do not permit us to use serological data alone to identify which coronaviruses are causing these infections.