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Although mRNA vaccine efficacy against severe coronavirus disease 2019 remains high, variant emergence has prompted booster immunizations. However, the effects of repeated exposures to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens on memory T cells are poorly understood. Here, we utilize major histocompatibility complex multimers with single-cell RNA sequencing to profile SARS-CoV-2-responsive T cells ex vivo from humans with one, two or three antigen exposures, including vaccination, primary infection and breakthrough infection. Exposure order determined the distribution between spike-specific and non-spike-specific responses, with vaccination after infection leading to expansion of spike-specific T cells and differentiation to CCR7

SummaryBacterial cell wall, a universal pathogen-associated molecular pattern (PAMP), crosses the placenta into the fetal brain. We determined that PAMPs interact with TLR2/6 on murine fetal neural progenitor cells (NPCs) to induce overexpansion of all neocortical layers leading to a larger, folded cortex and abnormal postnatal behavior. The NPC overexpansion originated at E10 and targeted ventricular radial glia (vRG), the primary NPC, by shortening cell cycle and increasing self-renewal. The mechanism involved two novel signaling pathways in NPCs mediated by recognition of bacterial PAMPs by TLR2/6 including: a) loss of primary cilia, activation of hedgehog signaling, and increased FOXG1 and b) increased PI3K/AKT activity. These findings reveal PAMP/TLR2/6 acts as a morphogen in fetal neurodevelopment. In addition, the loss ofTlr2orTlr6without pathogenic challenge, increased the number of neurons, establishing the requirement for an endogenous TLR2 signal for normal neurodevelopment in the embryo.

In late 2017, increased mortality was detected in chicken farms in Algeria undergoing A(H9N2) influenza outbreaks. Analysis of viruses isolated from affected farms showed that they were monophyletic, were of the G1 hemagglutinin (HA) lineage, and were antigenically and genetically similar to viruses detected contemporaneously in other countries in Northern Africa and the Middle East. The virus was able to spread via contact transmission between ferrets but did not cause disease in intravenously inoculated chickens.

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.

Highly pathogenic avian influenza H5N1 viruses were first isolated in Bangladesh in February 2007. Subsequently, clades 2.2.2, 2.3.4.2 and 2.3.2.1a were identified in Bangladesh, and our previous surveillance data revealed that by the end of 2014, the circulating viruses exclusively comprised clade 2.3.2.1a. We recently determined the status of circulating avian influenza viruses in Bangladesh by conducting surveillance of live poultry markets and waterfowl in wetland areas from February 2015 through February 2016. Until April 2015, clade 2.3.2.1a persisted without any change in genotype. However, in June 2015, we identified a new genotype of H5N1 viruses, clade 2.3.2.1a, which quickly became predominant. These newly emerged H5N1 viruses contained the hemagglutinin, neuraminidase and matrix genes of circulating 2.3.2.1a Bangladeshi H5N1 viruses and five other genes of low pathogenic Eurasian-lineage avian influenza A viruses. Some of these internal genes were closely related to those of low pathogenic viruses isolated from ducks in free-range farms and wild birds in a wetland region of northeastern Bangladesh, where commercially raised domestic ducks have frequent contact with migratory birds. These findings indicate that migratory birds of the Central Asian flyway and domestic ducks in the free-range farms in Tanguar haor-like wetlands played an important role in the emergence of this novel genotype of highly pathogenic H5N1 viruses.

Objectives Ducks can shed and spread influenza A viruses (IAVs) while showing no disease signs. Our objective was to clarify the role of ‘foie gras’ ducks in the circulation of IAVs in Bulgaria. Methods Monthly avian influenza surveillance was conducted on 63 ‘foie gras’ duck farms, 52 of which were surveyed throughout the study between November 2008 and April 2012. Virologic and serologic samples were collected and tested. During this time, wild bird samples were collected at major wild bird‐resting areas near the Black Sea coast and Danube River. Results The study showed high isolation frequency of low‐pathogenicity avian influenza viruses. In the raising population (

Antiviral drug susceptibility is one of the evaluation criteria of pandemic potential posed by an influenza virus. Influenza A viruses of swine (IAV-S) can play an important role in generating novel variants, yet limited information is available on the drug resistance profiles of IAV-S circulating in the U.S. Phenotypic analysis of the IAV-S isolated in the U.S. (2009-2011) (n=105) revealed normal inhibition by the neuraminidase (NA) inhibitors (NAIs) oseltamivir, zanamivir, and peramivir. Screening NA sequences from IAV-S collected in the U.S. (1930-2014) showed 0.03% (1/3396) sequences with clinically relevant H274Y-NA substitution. Phenotypic analysis of IAV-S isolated in the U.S. (2009-2011) confirmed amantadine resistance caused by the S31N-M2 and revealed an intermediate level of resistance caused by the I27T-M2. The majority (96.7%, 589/609) of IAV-S with the I27T-M2 in the influenza database were isolated from pigs in the U.S. The frequency of amantadine-resistant markers among IAV-S in the U.S. was high (71%), and their distribution was M-lineage dependent. All IAV-S of the Eurasian avian M lineage were amantadine-resistant and possessed either a single S31N-M2 substitution (78%, 585/747) or its combination with the V27A-M2 (22%, 162/747). The I27T-M2 substitution accounted for 43% (429/993) of amantadine resistance in classic swine M lineage. Phylogenetic analysis showed that both S31N-M2 and I27T-M2 emerged stochastically but appeared to be fixed in the U.S. IAV-S population. This study defines a drug-susceptibility profile, identifies the frequency of drug-resistant markers, and establishes a phylogenetic approach for continued antiviral-susceptibility monitoring of IAV-S in the U.S.

Highly pathogenic H5N1 avian influenza viruses are associated with severe disease in humans and continue to be a pandemic threat. While vaccines are available, other approaches are required for patients that typically respond poorly to vaccination, such as the elderly and the immunocompromised. To produce a therapeutic agent that is highly efficacious at low doses and is broadly specific against antigenically drifted H5N1 influenza viruses, we developed two neutralizing monoclonal antibodies and combined them into a single bispecific Fc fusion protein (the Fc dual-affinity retargeting [FcDART] molecule). In mice, a single therapeutic or prophylactic dose of either monoclonal antibody at 2.5 mg/kg of body weight provided 100% protection against challenge with A/Vietnam/1203/04 (H5N1) or the antigenically drifted strain A/Whooper swan/Mongolia/244/05 (H5N1). In ferrets, a single 1-mg/kg prophylactic dose provided 100% protection against A/Vietnam/1203/04 challenge. FcDART was also effective, as a single 2.5-mg/kg therapeutic or prophylactic dose in mice provided 100% protection against A/Vietnam/1203/04 challenge. Antibodies bound to conformational epitopes in antigenic sites on the globular head of the hemagglutinin protein, on the basis of analysis of mutants with antibody escape mutations. While it was possible to generate escape mutants in vitro , they were neutralized by the antibodies in vivo , as mice infected with escape mutants were 100% protected after only a single therapeutic dose of the antibody used to generate the escape mutant in vitro . In summary, we have combined the antigen specificities of two highly efficacious anti-H5N1 influenza virus antibodies into a bispecific FcDART molecule, which represents a strategy to produce broadly neutralizing antibodies that are effective against antigenically diverse influenza viruses. IMPORTANCE Highly pathogenic H5N1 avian influenza viruses are associated with severe disease in humans and are a pandemic threat. A vaccine is available, but other approaches are required for patients that typically respond poorly to vaccination, such as the elderly and the immunocompromised. The variability of the virus means that such an approach must be broad spectrum. To achieve this, we developed two antibodies that neutralize H5N1 influenza viruses. In mice, these antibodies provided complete protection against a spectrum of H5N1 influenza viruses at a single low dose. We then combined the two antibodies into a single molecule, FcDART, which combined the broad-spectrum activity and protective efficacy of both antibodies. This treatment provides a novel and effective therapeutic agent or prophylactic with activity against highly pathogenic H5N1 avian influenza viruses.

To clarify the epidemiology of influenza A viruses in coordinated swine production systems to which no animals from outside the system are introduced, we conducted virologic surveillance during September 2012–September 2013. Animal age, geographic location, and farm type were found to affect the prevalence of these viruses.

Avian influenza viruses (H5N1) of clades 2.3.4.1, 2.3.4.2, and 2.3.2.1 were introduced into Laos in 2009-2010. To investigate these viruses, we conducted active surveillance of poultry during March 2010. We detected viruses throughout Laos, including several interclade reassortants and 2 subgroups of clade 2.3.4, one of which caused an outbreak in May 2010.

Detection of this highly pathogenic clade in Europe poses a health threat to poultry and humans, On March 15, 2010, a highly pathogenic avian influenza virus was isolated from the carcass of a common buzzard (Buteo buteo) in Bulgaria. Phylogenetic analyses of the virus showed a close genetic relationship with influenza virus A (H5N1) clade 2.3.2.1 viruses isolated from wild birds in the Tyva Republic and Mongolia during 2009–2010. Designated A/common buzzard/Bulgaria/38WB/2010, this strain was highly pathogenic in chickens but had low pathogenicity in mice and ferrets and no molecular markers of increased pathogenicity in mammals. The establishment of clade 2.3.2.1 highly pathogenic avian influenza viruses of the H5N1 subtype in wild birds in Europe would increase the likelihood of health threats to humans and poultry in the region.

TOC Summary: Viruses belonging to these novel genotypes are indistinguishable phenotypically from endemic swine viruses., As a result of human-to-pig transmission, pandemic influenza A (H1N1) 2009 virus was detected in pigs soon after it emerged in humans. In the United States, this transmission was quickly followed by multiple reassortment between the pandemic virus and endemic swine viruses. Nine reassortant viruses representing 7 genotypes were detected in commercial pig farms in the United States. Field observations suggested that the newly described reassortant viruses did not differ substantially from pandemic (H1N1) 2009 or endemic strains in their ability to cause disease. Comparable growth properties of reassortant and endemic viruses in vitro supported these observations; similarly, a representative reassortant virus replicated in ferrets to the same extent as did pandemic (H1N1) 2009 and endemic swine virus. These novel reassortant viruses highlight the increasing complexity of influenza viruses within pig populations and the frequency at which viral diversification occurs in this ecologically important viral reservoir.

Swine influenza virus (SIV) H3N2 with triple reassorted internal genes (TRIG) has been enzootic in Unites States since 1998. Transmission of the 2009 pandemic H1N1 (pH1N1) virus to pigs in the United States was followed by reassortment with endemic SIV, resulting in reassorted viruses that include novel H3N2 genotypes (rH3N2p). Between July and December 2011, 12 cases of human infections with swine-lineage H3N2 viruses containing the pandemic matrix (pM) gene [A(H3N2)v] were detected. Whole-genome analysis of H3N2 viruses isolated from pigs from 2009 to 2011 sequenced in this study and other available H3N2 sequences showed six different rH3N2p genotypes present in the U.S. swine population since 2009. The presence of the pM gene was a common feature among all rH3N2p genotypes, but no specific genotype appeared to predominate in the swine population. We compared the pathogenic, transmission, genetic, and antigenic properties of a human A(H3N2)v isolate and two swine H3N2 isolates, H3N2-TRIG and rH3N2p. Our in vivo study detected no increased virulence in A(H3N2)v or rH3N2p viruses compared to endemic H3N2-TRIG virus. Antibodies to cluster IV H3N2-TRIG and rH3N2p viruses had reduced cross-reactivity to A(H3N2)v compared to other cluster IV H3N2-TRIG and rH3N2p viruses. Genetic analysis of the hemagglutinin gene indicated that although rH3N2p and A(H3N2)v are related to cluster IV of H3N2-TRIG, some recent rH3N2p isolates appeared to be forming a separate cluster along with the human isolates of A(H3N2)v. Continued monitoring of these H3N2 viruses is necessary to evaluate the evolution and potential loss of population immunity in swine and humans.

The increasing availability of complete influenza virus genomes is deepening our understanding of influenza evolutionary dynamics and facilitating the selection of vaccine strains. However, only one complete African influenza virus sequence is available in the public domain. Here we present a complete genome analysis of 59 influenza A/H3N2 viruses isolated from humans in Uganda during the 2008 and 2009 season. Isolates were recovered from hospital-based sentinel surveillance for influenza-like illnesses and their whole genome sequenced. The viruses circulating during these two seasons clearly differed from each other phylogenetically. They showed a slow evolution away from the 2009/10 recommended vaccine strain (A/Brisbane/10/07), instead clustering with the 2010/11 recommended vaccine strain (A/Perth/16/09) in the A/Victoria/208/09 clade, as observed in other global regions. All of the isolates carried the adamantane resistance marker S31N in the M2 gene and carried several markers of enhanced transmission; as expected, none carried any marker of neuraminidase inhibitor resistance. The hemagglutinin gene of the 2009 isolates differed from that of the 2008 isolates in antigenic sites A, B, D, and to a lesser extent, C and E indicating evidence of an early phylogenetic shift from the 2008 to 2009 viruses. The internal genes of the 2009 isolates were similar to those of one 2008 isolate, A/Uganda/MUWRP-050/2008. Another 2008 isolate had a truncated PB1-F2 protein. Whole genome sequencing can enhance surveillance of future seasonal changes in the viral genome which is crucial to ensure that selected vaccine strains are protective against the strains circulating in Eastern Africa. This data provides an important baseline for this surveillance. Overall the influenza virus activity in Uganda appears to mirror that observed in other regions of the southern hemisphere.