Amino acids in the polymerase complex of shorebird-isolated H1N1 influenza virus impact replication and host-virus interactions in mammalian models.

A diverse population of avian influenza A viruses (AIVs) are maintained in wild birds and ducks yet the zoonotic potential of AIVs in these environmental reservoirs and the host-virus interactions involved in mammalian infection are not well understood. In studies of a group of subtype H1N1 AIVs isolated from migratory wild birds during surveillance in North America, we previously identified eight amino acids in the polymerase genes PB2 and PB1 that were important for the transmissibility of these AIVs in a ferret model of human influenza virus transmission. In this current study we found that PB2 containing amino acids associated with transmissibility at 67, 152, 199, 508, and 649 and PB1 at 298, 642, and 667 were associated with more rapid viral replication kinetics, greater infectivity, more active polymerase complexes and greater kinetics of viral genome replication and transcription. Pathogenicity in the mouse model was also impacted, evident as greater weight loss and lung pathology associated with greater inflammatory lung cytokine expression. Further, these AIVs all contained the avian-type amino acids of PB2-E627, D701, G590, Q591 and T271. Therefore, our study provides novel insights into the role of the AIV polymerase complex in the zoonotic transmission of AIVs in mammals.

Handheld plasmonic biosensor for virus detection in field-settings.

After World Health Organization (WHO) announced COVID-19 outbreak a pandemic, we all again realized the importance of developing rapid diagnostic kits. In this article, we introduced a lightweight and field-portable biosensor employing a plasmonic chip based on nanohole arrays integrated to a lensfree-imaging framework for label-free detection of viruses in field-settings. The platform utilizes a CMOS (complementary metal-oxide-semiconductor) camera with high quantum efficiency in the spectral window of interest to monitor diffraction field patterns of nanohole arrays under the uniform illumination of an LED (light-emitting diode) source which is spectrally tuned to the plasmonic mode supported by the nanohole arrays. As an example for the applicability of our biosensor for virus detection, we could successfully demonstrate the label-free detection of H1N1 viruses, e.g., swine flu, with medically relevant concentrations. We also developed a low-cost and easy-to-use sample preparation kit to prepare the surface of the plasmonic chip for analyte binding, e.g., virus-antibody binding. In order to reveal a complete biosensor technology, we also developed a user friendly Python™ - based graphical user interface (GUI) that allows direct access to biosensor hardware, taking and processing diffraction field images, and provides virus information to the end-user. Employing highly sensitive nanohole arrays and lensfree-imaging framework, our platform could yield an LOD as low as 103 TCID50/mL. Providing accurate and rapid sensing information in a handheld platform, weighing only 70 g and 12 cm tall, without the need for bulky and expensive instrumentation, our biosensor could be a very strong candidate for diagnostic applications in resource-poor settings. As our detection scheme is based on the use of antibodies, it could quickly adapt to the detection of different viral diseases, e.g., COVID-19 or influenza, by simply coating the plasmonic chip surface with an antibody possessing affinity to the virus type of interest. Possessing this ability, our biosensor could be swiftly deployed to the field in need for rapid diagnosis, which may be an important asset to prevent the spread of diseases before turning into a pandemic by isolating patients from the population.

Molecular Characterization of Closely Related H6N2 Avian Influenza Viruses Isolated from Turkey, Egypt, and Uganda.

Genetic analysis of circulating avian influenza viruses (AIVs) in wild birds at different geographical regions during the same period could improve our knowledge about virus transmission dynamics in natural hosts, virus evolution as well as zoonotic potential. Here, we report the genetic and molecular characterization of H6N2 influenza viruses isolated from migratory birds in Turkey, Egypt, and Uganda during 2017-2018. The Egyptian and Turkish isolates were genetically closer to each other than they were to the virus isolated from Uganda. Our results also suggest that multiple reassortment events were involved in the genesis of the isolated viruses. All viruses contained molecular markers previously associated with increased replication and/or pathogenicity in mammals. The results of this study indicate that H6N2 viruses carried by migratory birds on the West Asian/East African and Mediterranean/Black Sea flyways have the potential to transmit to mammals including humans. Additionally, adaptation markers in these viruses indicate the potential risk for poultry, which also increases the possibility of human exposure to these viruses.

Potential for Low-Pathogenic Avian H7 Influenza A Viruses To Replicate and Cause Disease in a Mammalian Model.

H7 subtype influenza A viruses are widely distributed and have been responsible for human infections and numerous outbreaks in poultry with significant impact. Despite this, the disease-causing potential of the precursor low-pathogenic (LP) H7 viruses from the wild bird reservoir has not been investigated. Our objective was to assess the disease-causing potential of 30 LP H7 viruses isolated from wild avian species in the United States and Canada using the DBA/2J mouse model. Without prior mammalian adaptation, the majority of viruses, 27 (90%), caused mortality in mice. Of these, 17 (56.7%) caused 100% mortality and 24 were of pathogenicity similar to that of A/Anhui/1/2013 (H7N9), which is highly pathogenic in mice. Viruses of duck origin were more pathogenic than those of shorebird origin, as 13 of 18 (72.2%) duck origin viruses caused 100% mortality while 4 of 12 (33.3%) shorebird origin viruses caused 100% mortality, despite there being no difference in mean lung viral titers between the groups. Replication beyond the respiratory tract was also evident, particularly in the heart and brain. Of the 16 viruses studied for fecal shedding, 11 were detected in fecal samples. These viruses exhibited a strong preference for avian-type α2,3-linked sialic acids; however, binding to mammalian-type α2,6-linked sialic acids was also detected. These findings indicate that LP avian H7 influenza A viruses are able to infect and cause disease in mammals without prior adaptation and therefore pose a potential public health risk. IMPORTANCE: Low-pathogenic (LP) avian H7 influenza A viruses are widely distributed in the avian reservoir and are the precursors of numerous outbreaks of highly pathogenic avian influenza viruses in commercial poultry farms. However, unlike highly pathogenic H7 viruses, the disease-causing potential of LP H7 viruses from the wild bird reservoir has not been investigated. To address this, we studied 30 LP avian H7 viruses isolated from wild avian species in the United States and Canada using the DBA/2J mouse model. Surprisingly, the majority of these viruses, 90%, caused mortality in mice without prior mammalian adaptation, and 56.7% caused 100% mortality. There was also evidence of spread beyond the respiratory tract and fecal shedding. Therefore, the disease-causing potential of LP avian H7 influenza A viruses in mammals may be underestimated, and these viruses therefore pose a potential public health risk.

Possible basis for the emergence of H1N1 viruses with pandemic potential from avian hosts.

Influenza A viruses of the H1N1 subtype have emerged from the avian influenza gene pool in aquatic birds and caused human pandemics at least twice during the past century. Despite this fact, surprisingly little is known about the H1N1 gene pool in the aquatic bird reservoir. A preliminary study showed that an H1N1 virus from a shorebird of the Charadriiformes order was transmitted between animals through the airborne route of infection, whereas an H1N1 virus from a bird of the Anseriformes order was not. Here we show that two of the three H1N1 viruses isolated from Charadriiformes species in 2009 were transmitted between animals through the airborne route of infection, and five H1N1 isolates from Anseriformes species were not. The one H1N1 virus from a Charadriiformes species that failed to transmit through the airborne route was a reassortant possessing multiple internal gene segments from Anseriformes species. The molecular differences between the airborne-transmissible and non-airborne-transmissible H1N1 viruses were multigenic, involving the selection of virus with human-like receptor-binding specificity (α2-6 sialic acid) and multiple differences in the polymerase complex, mainly in the PB2, PB1-F2, and nonstructural genes.

The Genomic Contributions of Avian H1N1 Influenza A Viruses to the Evolution of Mammalian Strains.

Among the influenza A viruses (IAVs) in wild aquatic birds, only H1, H2, and H3 subtypes have caused epidemics in humans. H1N1 viruses of avian origin have also caused 3 of 5 pandemics. To understand the reappearance of H1N1 in the context of pandemic emergence, we investigated whether avian H1N1 IAVs have contributed to the evolution of human, swine, and 2009 pandemic H1N1 IAVs. On the basis of phylogenetic analysis, we concluded that the polymerase gene segments (especially PB2 and PA) circulating in North American avian H1N1 IAVs have been reintroduced to swine multiple times, resulting in different lineages that led to the emergence of the 2009 pandemic H1N1 IAVs. Moreover, the similar topologies of hemagglutinin and nucleoprotein and neuraminidase and matrix gene segments suggest that each surface glycoprotein coevolved with an internal gene segment within the H1N1 subtype. The genotype of avian H1N1 IAVs of Charadriiformes origin isolated in 2009 differs from that of avian H1N1 IAVs of Anseriformes origin. When the antigenic sites in the hemagglutinin of all 31 North American avian H1N1 IAVs were considered, 60%-80% of the amino acids at the antigenic sites were identical to those in 1918 and/or 2009 pandemic H1N1 viruses. Thus, although the pathogenicity of avian H1N1 IAVs could not be inferred from the phylogeny due to the small dataset, the evolutionary process within the H1N1 IAV subtype suggests that the circulation of H1N1 IAVs in wild birds poses a continuous threat for future influenza pandemics in humans.

Survival analysis of infected mice reveals pathogenic variations in the genome of avian H1N1 viruses.

Most influenza pandemics have been caused by H1N1 viruses of purely or partially avian origin. Here, using Cox proportional hazard model, we attempt to identify the genetic variations in the whole genome of wild-type North American avian H1N1 influenza A viruses that are associated with their virulence in mice by residue variations, host origins of virus (Anseriformes-ducks or Charadriiformes-shorebirds), and host-residue interactions. In addition, through structural modeling, we predicted that several polymorphic sites associated with pathogenicity were located in structurally important sites, especially in the polymerase complex and NS genes. Our study introduces a new approach to identify pathogenic variations in wild-type viruses circulating in the natural reservoirs and ultimately to understand their infectious risks to humans as part of risk assessment efforts towards the emergence of future pandemic strains.

Possible role of songbirds and parakeets in transmission of influenza A(H7N9) virus to humans.

Avian-origin influenza A(H7N9) recently emerged in China, causing severe human disease. Several subtype H7N9 isolates contain influenza genes previously identified in viruses from finch-like birds. Because wild and domestic songbirds interact with humans and poultry, we investigated the susceptibility and transmissibility of subtype H7N9 in these species. Finches, sparrows, and parakeets supported replication of a human subtype H7N9 isolate, shed high titers through the oropharyngeal route, and showed few disease signs. Virus was shed into water troughs, and several contact animals seroconverted, although they shed little virus. Our study demonstrates that a human isolate can replicate in and be shed by such songbirds and parakeets into their environment. This finding has implications for these birds' potential as intermediate hosts with the ability to facilitate transmission and dissemination of A(H7N9) virus.

Fecal influenza in mammals: selection of novel variants.

In aquatic birds, influenza A viruses mainly replicate in the intestinal tract without significantly affecting the health of the host, but in mammals, they replicate in the respiratory tract and often cause disease. Occasionally, influenza viruses have been detected in stool samples of hospitalized patients and in rectal swabs of naturally or experimentally infected mammals. In this study, we compared the biological and molecular differences among four wild-type avian H1N1 influenza viruses and their corresponding fecal and lung isolates in DBA/2J and BALB/cJ mice. All fecal and lung isolates were more pathogenic than the original wild-type viruses, when inoculated into mice of both strains. The increased virulence was associated with the acquisition of genetic mutations. Most of the novel genotypes emerged as PB2(E627K), HA(F128V), HA(F454L), or HA(H300P) variations, and double mutations frequently occurred in the same isolate. However, influenza virus strain- and host-specific differences were also observed in terms of selected variants. The avian H1N1 virus of shorebird origin appeared to be unique in its ability to rapidly adapt to BALB/cJ mice via the fecal route, compared to the adaptability of the H1N1 virus of mallard origin. Furthermore, a bimodal distribution in fecal shedding was observed in mice infected with the fecal isolates, while a normal distribution was observed after infection with the lung isolates or wild-type virus. Fecal isolates contained HA mutations that increased the activation pH of the HA protein. We conclude that influenza virus variants that emerge in fecal isolates in mammals might influence viral transmission, adaptation to mammals, and viral ecology or evolution.

Subglacial Lake Vostok (Antarctica) accretion ice contains a diverse set of sequences from aquatic, marine and sediment-inhabiting bacteria and eukarya.

Lake Vostok, the 7(th) largest (by volume) and 4(th) deepest lake on Earth, is covered by more than 3,700 m of ice, making it the largest subglacial lake known. The combination of cold, heat (from possible hydrothermal activity), pressure (from the overriding glacier), limited nutrients and complete darkness presents extreme challenges to life. Here, we report metagenomic/metatranscriptomic sequence analyses from four accretion ice sections from the Vostok 5G ice core. Two sections accreted in the vicinity of an embayment on the southwestern end of the lake, and the other two represented part of the southern main basin. We obtained 3,507 unique gene sequences from concentrates of 500 ml of 0.22 µm-filtered accretion ice meltwater. Taxonomic classifications (to genus and/or species) were possible for 1,623 of the sequences. Species determinations in combination with mRNA gene sequence results allowed deduction of the metabolic pathways represented in the accretion ice and, by extension, in the lake. Approximately 94% of the sequences were from Bacteria and 6% were from Eukarya. Only two sequences were from Archaea. In general, the taxa were similar to organisms previously described from lakes, brackish water, marine environments, soil, glaciers, ice, lake sediments, deep-sea sediments, deep-sea thermal vents, animals and plants. Sequences from aerobic, anaerobic, psychrophilic, thermophilic, halophilic, alkaliphilic, acidophilic, desiccation-resistant, autotrophic and heterotrophic organisms were present, including a number from multicellular eukaryotes.

Emergence of Influenza Viruses and Crossing the Species Barrier.

Influenza A viruses are zoonotic pathogens that infect a variety of host species including wild aquatic birds, domestic poultry, and a limited number of mammals including humans. The error-prone nature of the virus's replication machinery and its ability to transmit among multiple hosts lead to generation of novel virus variants with altered pathogenicity and virulence. Spatial, molecular, and physiological barriers inhibit cross-species infections, particularly in the case of human infection with avian viruses. Pigs are proposed as a mixing vessel that facilitates movement of avian viruses from the wild bird reservoir into humans. However, the past decade has witnessed the emergence of highly pathogenic and virulent avian H5 and H7 viruses that have breached these barriers, bypassed the pig intermediate host, and infected humans with a high mortality rate, but have not established human-to-human transmissible lineages. Because influenza viruses pose a significant risk to both human and animal health, it is becoming increasingly important to attempt to predict their identities and pathogenic potential before their widespread emergence. Surveillance of the wild bird reservoir, molecular characterization and documentation of currently circulating viruses in humans and animals, and a comprehensive risk assessment analysis of individual isolates should remain a high priority. Such efforts are critical to the pursuit of prevention and control strategies, including vaccine development and assessment of antiviral susceptibility, that will have a direct impact on the well-being of humans and animals worldwide.

Ecology of subglacial lake vostok (antarctica), based on metagenomic/metatranscriptomic analyses of accretion ice.

Lake Vostok is the largest of the nearly 400 subglacial Antarctic lakes and has been continuously buried by glacial ice for 15 million years. Extreme cold, heat (from possible hydrothermal activity), pressure (from the overriding glacier) and dissolved oxygen (delivered by melting meteoric ice), in addition to limited nutrients and complete darkness, combine to produce one of the most extreme environments on Earth. Metagenomic/metatranscriptomic analyses of ice that accreted over a shallow embayment and over the southern main lake basin indicate the presence of thousands of species of organisms (94% Bacteria, 6% Eukarya, and two Archaea). The predominant bacterial sequences were closest to those from species of Firmicutes, Proteobacteria and Actinobacteria, while the predominant eukaryotic sequences were most similar to those from species of ascomycetous and basidiomycetous Fungi. Based on the sequence data, the lake appears to contain a mixture of autotrophs and heterotrophs capable of performing nitrogen fixation, nitrogen cycling, carbon fixation and nutrient recycling. Sequences closest to those of psychrophiles and thermophiles indicate a cold lake with possible hydrothermal activity. Sequences most similar to those from marine and aquatic species suggest the presence of marine and freshwater regions.

The potential of avian H1N1 influenza A viruses to replicate and cause disease in mammalian models.

H1N1 viruses in which all gene segments are of avian origin are the most frequent cause of influenza pandemics in humans; therefore, we examined the disease-causing potential of 31 avian H1N1 isolates of American lineage in DBA/2J mice. Thirty of 31 isolates were very virulent, causing respiratory tract infection; 22 of 31 resulted in fecal shedding; and 10 of 31 were as pathogenic as the pandemic 2009 H1N1 viruses. Preliminary studies in BALB/cJ mice and ferrets showed that 1 of 4 isolates tested was more pathogenic than the pandemic 2009 H1N1 viruses in BALB/cJ mice, and 1 of 2 strains transmitted both by direct and respiratory-droplet contact in ferrets. Preliminary studies of other avian subtypes (H2, H3, H4, H6, H10, H12) in DBA/2J mice showed lower pathogenicity than the avian H1N1 viruses. These findings suggest that avian H1N1 influenza viruses are unique among influenza A viruses in their potential to infect mammals.