Comparative evolution of influenza A virus H1 and H3 head and stalk domains across host species.

IMPORTANCE: For decades, researchers have studied the rapid evolution of influenza A viruses for vaccine design and as a useful model system for the study of host/parasite evolution. By performing an exhaustive analysis of hemagglutinin protein (HA) sequences from 49 lineages independently evolving in birds, swine, canines, equines, and humans over the last century, our work uncovers surprising features of HA evolution. In particular, the canine H3 stalk, unlike human H3 and H1 stalk domains, is not evolving slowly, suggesting that evolution in the stalk domain is not universally constrained across all host species. Therefore, a broader multi-host perspective on HA evolution may be useful during the evaluation and design of stalk-targeted vaccine candidates.

Cross-species transmission and PB2 mammalian adaptations of highly pathogenic avian influenza A/H5N1 viruses in Chile.

H5N1 highly pathogenic avian influenza viruses (HPAIV) emerged in wild birds in Chile in December 2022 and spilled over into poultry, marine mammals, and one human. Between December 9, 2022 - March 14, 2023, a coordinated government/academic response detected HPAIV by real-time RT-PCR in 8.5% (412/4735) of samples from 23 avian and 3 mammal orders. Whole-genome sequences obtained from 77 birds and 8 marine mammals revealed that all Chilean H5N1 viruses belong to lineage 2.3.4.4b and cluster monophyletically with viruses from Peru, indicating a single introduction from North America into Peru/Chile. Mammalian adaptations were identified in the PB2 segment: D701N in two sea lions, one human, and one shorebird, and Q591K in the human and one sea lion. Minor variant analysis revealed that D701N was present in 52.9 - 70.9% of sequence reads, indicating the presence of both genotypes within hosts. Further surveillance of spillover events is warranted to assess the emergence and potential onward transmission of mammalian adapted H5N1 HPAIV in South America.

Highly pathogenic avian influenza A (H5N1) in marine mammals and seabirds in Peru.

Highly pathogenic avian influenza (HPAI) A/H5N1 viruses (lineage 2.3.4.4b) are rapidly invading the Americas, threatening wildlife, poultry, and potentially evolving into the next global pandemic. In November 2022 HPAI arrived in Peru, triggering massive pelican and sea lion die-offs. We report genomic characterization of HPAI/H5N1 in five species of marine mammals and seabirds (dolphins, sea lions, sanderlings, pelicans and cormorants). Peruvian viruses belong to lineage 2.3.4.4b, but they are 4:4 reassortants where 4 genomic segments (PA, HA, NA and MP) position within the Eurasian lineage that initially entered North America from Eurasia, while the other 4 genomic segments (PB2, PB1, NP and NS) position within the American lineage (clade C) that circulated in North America. These viruses are rapidly accruing mutations, including mutations of concern, that warrant further examination and highlight an urgent need for active local surveillance to manage outbreaks and limit spillover into other species, including humans.

Accelerated evolution of SARS-CoV-2 in free-ranging white-tailed deer.

The zoonotic origin of the COVID-19 pandemic virus highlights the need to fill the vast gaps in our knowledge of SARS-CoV-2 ecology and evolution in non-human hosts. Here, we detected that SARS-CoV-2 was introduced from humans into white-tailed deer more than 30 times in Ohio, USA during November 2021-March 2022. Subsequently, deer-to-deer transmission persisted for 2-8 months, disseminating across hundreds of kilometers. Newly developed Bayesian phylogenetic methods quantified how SARS-CoV-2 evolution is not only three-times faster in white-tailed deer compared to the rate observed in humans but also driven by different mutational biases and selection pressures. The long-term effect of this accelerated evolutionary rate remains to be seen as no critical phenotypic changes were observed in our animal models using white-tailed deer origin viruses. Still, SARS-CoV-2 has transmitted in white-tailed deer populations for a relatively short duration, and the risk of future changes may have serious consequences for humans and livestock.

Influenza A viruses in gulls in landfills and freshwater habitats in Minnesota, United States.

Introduction: The unpredictable evolution of avian influenza viruses (AIVs) presents an ongoing threat to agricultural production and public and wildlife health. Severe outbreaks of highly pathogenic H5N1 viruses in US poultry and wild birds since 2022 highlight the urgent need to understand the changing ecology of AIV. Surveillance of gulls in marine coastal environments has intensified in recent years to learn how their long-range pelagic movements potentially facilitate inter-hemispheric AIV movements. In contrast, little is known about inland gulls and their role in AIV spillover, maintenance, and long-range dissemination. Methods: To address this gap, we conducted active AIV surveillance in ring-billed gulls (Larus delawarensis) and Franklin's gulls (Leucophaeus pipixcan) in Minnesota's natural freshwater lakes during the summer breeding season and in landfills during fall migration (1,686 samples). Results: Whole-genome AIV sequences obtained from 40 individuals revealed three-lineage reassortants with a mix of genome segments from the avian Americas lineage, avian Eurasian lineage, and a global "Gull" lineage that diverged more than 50 years ago from the rest of the AIV global gene pool. No poultry viruses contained gull-adapted H13, NP, or NS genes, pointing to limited spillover. Geolocators traced gull migration routes across multiple North American flyways, explaining how inland gulls imported diverse AIV lineages from distant locations. Migration patterns were highly varied and deviated far from assumed "textbook" routes. Discussion: Viruses circulating in Minnesota gulls during the summer breeding season in freshwater environments reappeared in autumn landfills, evidence of AIV persistence in gulls between seasons and transmission between habitats. Going forward, wider adoption of technological advances in animal tracking devices and genetic sequencing is needed to expand AIV surveillance in understudied hosts and habitats.

Tracking the emergence of antigenic variants in influenza A virus epidemics in Brazil.

Influenza A virus (IAV) circulation patterns differ in North America and South America, with influenza seasons often characterized by different subtypes and strains. However, South America is relatively undersampled considering the size of its population. To address this gap, we sequenced the complete genomes of 220 IAVs collected between 2009 and 2016 from hospitalized patients in southern Brazil. New genetic drift variants were introduced into southern Brazil each season from a global gene pool, including four H3N2 clades (3c, 3c2, 3c3, and 3c2a) and five H1N1pdm clades (clades 6, 7, 6b, 6c, and 6b1). In 2016, H1N1pdm viruses belonging to a new 6b1 clade caused a severe influenza epidemic in southern Brazil that arrived early and spread rapidly, peaking mid-autumn. Inhibition assays showed that the A/California/07/2009(H1N1) vaccine strain did not protect well against 6b1 viruses. Phylogenetically, most 6b1 sequences that circulated in southern Brazil belong to a single transmission cluster that rapidly diffused across susceptible populations, leading to the highest levels of influenza hospitalization and mortality seen since the 2009 pandemic. Continuous genomic surveillance is needed to monitor rapidly evolving IAVs for vaccine strain selection and understand their epidemiological impact in understudied regions.

Blue-Winged Teals in Guatemala and Their Potential Role in the Ecology of H14 Subtype Influenza a Viruses.

Wild aquatic birds are considered the natural hosts of 16 HA (H1-H16) and 9 NA (N1-N9) subtypes of influenza A viruses (FLUAV) found in different combinations. H14 FLUAVs are rarely detected in nature. Since 2011, H14 FLUAVs have been consistently detected in Guatemala, leading to the largest collection of this subtype from a single country. All H14 FLUAVs in Guatemala were detected from blue-winged teal samples. In this report, 17 new full-length H14 FLUAV genome sequences detected from 2014 until 2019 were analyzed and compared to all published H14 sequences, including Guatemala, North America, and Eurasia. The H14 FLUAVs identified in Guatemala were mostly associated with the N3 subtype (n = 25), whereas the rest were paired with either N4 (n = 7), N5 (n = 4), N6 (n = 1), and two mixed infections (N3/N5 n = 2, and N2/N3 n = 1). H14 FLUAVs in Guatemala belong to a distinct H14 lineage in the Americas that is evolving independently from the Eurasian H14 lineage. Of note, the ORF of the H14 HA segments showed three distinct motifs at the cleavage site, two of these containing arginine instead of lysine in the first and fourth positions, not previously described in other countries. The effects of these mutations on virus replication, virulence, and/or transmission remain unknown and warrant further studies.

Origins and Evolution of Seasonal Human Coronaviruses.

Four seasonal human coronaviruses (sHCoVs) are endemic globally (229E, NL63, OC43, and HKU1), accounting for 5-30% of human respiratory infections. However, the epidemiology and evolution of these CoVs remain understudied due to their association with mild symptomatology. Using a multigene and complete genome analysis approach, we find the evolutionary histories of sHCoVs to be highly complex, owing to frequent recombination of CoVs including within and between sHCoVs, and uncertain, due to the under sampling of non-human viruses. The recombination rate was highest for 229E and OC43 whereas substitutions per recombination event were highest in NL63 and HKU1. Depending on the gene studied, OC43 may have ungulate, canine, or rabbit CoV ancestors. 229E may have origins in a bat, camel, or an unsampled intermediate host. HKU1 had the earliest common ancestor (1809-1899) but fell into two distinct clades (genotypes A and B), possibly representing two independent transmission events from murine-origin CoVs that appear to be a single introduction due to large gaps in the sampling of CoVs in animals. In fact, genotype B was genetically more diverse than all the other sHCoVs. Finally, we found shared amino acid substitutions in multiple proteins along the non-human to sHCoV host-jump branches. The complex evolution of CoVs and their frequent host switches could benefit from continued surveillance of CoVs across non-human hosts.

Homotypic protection against influenza in a pediatric cohort in Managua, Nicaragua.

The period of protection from repeat infection following symptomatic influenza is not well established due to limited availability of longitudinal data. Using data from a pediatric cohort in Managua, Nicaragua, we examine the effects of natural influenza virus infection on subsequent infection with the same influenza virus subtype/lineage across multiple seasons, totaling 2,170 RT-PCR-confirmed symptomatic influenza infections. Logistic regression models assessed whether infection in the prior influenza season protected against homologous reinfection. We sequenced viruses from 2011-2019 identifying dominant clades and measuring antigenic distances between hemagglutinin clades. We observe homotypic protection from repeat infection in children infected with influenza A/H1N1pdm (OR 0.12, CI 0.02-0.88), A/H3N2 (OR 0.41, CI 0.24-0.73), and B/Victoria (OR 0.00, CI 0.00-0.14), but not with B/Yamagata viruses (OR 0.60, CI 0.09-2.10). Overall, protection wanes as time or antigenic distance increases. Individuals infected with one subtype or lineage of influenza virus have significantly lower odds of homologous reinfection for the following one to two years; after two years this protection wanes. This protection is demonstrated across multiple seasons, subtypes, and lineages among children.

Birth cohort relative to an influenza A virus's antigenic cluster introduction drives patterns of children's antibody titers.

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.

Evolution of influenza A virus in intensive and free-range swine farms in Spain.

Swine harbor genetically diverse influenza A viruses (IAVs) with the capacity to host-switch to humans, causing global pandemics. Spain is the largest swine producer in Europe and has a mixed production system that includes 'white coat' pigs raised intensively in modern buildings and free-range Iberian pigs that interface differently with humans, wildlife, and other swine. Through active longitudinal IAV surveillance in nine Spanish provinces during 2015-9, we generated forty-seven complete or near-complete genome sequences from IAVs collected from swine in both systems. Genetically diverse IAVs were identified in intensively raised white pigs and free-range Iberian pigs, including new H3N1 reassortants. Both systems are dynamic environments for IAV evolution, but driven by different processes. IAVs in white pigs were genetically related to viruses found in swine raised intensively in other European countries, reflecting high rates of viral introduction following European trade routes. In contrast, IAVs in Iberian pigs have a genetic makeup shaped by frequent introductions of human IAVs, reflecting rearing practices with high rates of human contact. Transmission between white and Iberian pigs also occurred. In conclusion, Iberian swine with high rates of human contact harbor genetically diverse IAVs and potentially serve as intermediary hosts between white pigs and humans, presenting an understudied zoonotic risk that requires further investigation.

SARS-CoV-2 infection in free-ranging white-tailed deer.

Humans have infected a wide range of animals with SARS-CoV-21-5, but the establishment of a new natural animal reservoir has not been observed. Here we document that free-ranging white-tailed deer (Odocoileus virginianus) are highly susceptible to infection with SARS-CoV-2, are exposed to multiple SARS-CoV-2 variants from humans and are capable of sustaining transmission in nature. Using real-time PCR with reverse transcription, we detected SARS-CoV-2 in more than one-third (129 out of 360, 35.8%) of nasal swabs obtained from O. virginianus in northeast Ohio in the USA during January to March 2021. Deer in six locations were infected with three SARS-CoV-2 lineages (B.1.2, B.1.582 and B.1.596). The B.1.2 viruses, dominant in humans in Ohio at the time, infected deer in four locations. We detected probable deer-to-deer transmission of B.1.2, B.1.582 and B.1.596 viruses, enabling the virus to acquire amino acid substitutions in the spike protein (including the receptor-binding domain) and ORF1 that are observed infrequently in humans. No spillback to humans was observed, but these findings demonstrate that SARS-CoV-2 viruses have been transmitted in wildlife in the USA, potentially opening new pathways for evolution. There is an urgent need to establish comprehensive 'One Health' programmes to monitor the environment, deer and other wildlife hosts globally.

SARS-CoV-2 infection in free-ranging white-tailed deer ( Odocoileus virginianus ).

Human-to-animal spillover of SARS-CoV-2 virus has occurred in a wide range of animals, but thus far, the establishment of a new natural animal reservoir has not been detected. Here, we detected SARS-CoV-2 virus using rRT-PCR in 129 out of 360 (35.8%) free-ranging white-tailed deer ( Odocoileus virginianus ) from northeast Ohio (USA) sampled between January-March 2021. Deer in 6 locations were infected with at least 3 lineages of SARS-CoV-2 (B.1.2, B.1.596, B.1.582). The B.1.2 viruses, dominant in Ohio at the time, spilled over multiple times into deer populations in different locations. Deer-to-deer transmission may have occurred in three locations. The establishment of a natural reservoir of SARS-CoV-2 in white-tailed deer could facilitate divergent evolutionary trajectories and future spillback to humans, further complicating long-term COVID-19 control strategies. ONE-SENTENCE SUMMARY: A significant proportion of SARS-CoV-2 infection in free-ranging US white-tailed deer reveals a potential new reservoir.

Tracing the Source of Influenza A Virus Zoonoses in Interconnected Circuits of Swine Exhibitions.

BACKGROUND: Since 2011, influenza A viruses circulating in US swine exhibited at county fairs are associated with >460 zoonotic infections, presenting an ongoing pandemic risk. Swine "jackpot shows" that occur before county fairs each summer intermix large numbers of exhibition swine from diverse geographic locations. We investigated the role of jackpot shows in influenza zoonoses. METHODS: We collected snout wipe or nasal swab samples from 17 009 pigs attending 350 national, state, and local swine exhibitions across 8 states during 2016-2018. RESULTS: Influenza was detected in 13.9% of swine sampled at jackpot shows, and 76.3% of jackpot shows had at least 1 pig test positive. Jackpot shows had 4.3-fold higher odds of detecting at least 1 influenza-positive pig compared to county fairs. When influenza was detected at a county fair, almost half of pigs tested positive, clarifying why zoonotic infections occur primarily at county fairs. CONCLUSIONS: The earlier timing of jackpot shows and long-distance travel for repeated showing of individual pigs provide a pathway for the introduction of influenza into county fairs. Mitigation strategies aimed at curtailing influenza at jackpot shows are likely to have downstream effects on disease transmission at county fairs and zoonoses.

Molecular characterization of respiratory syncytial viruses circulating in a paediatric cohort in Amman, Jordan.

Respiratory syncytial viruses (RSVs) are an important cause of mortality worldwide and a major cause of respiratory tract infections in children, driving development of vaccine candidates. However, there are large gaps in our knowledge of the local evolutionary and transmission dynamics of RSVs, particularly in understudied regions such as the Middle East. To address this gap, we sequenced the complete genomes of 58 RSVA and 27 RSVB samples collected in a paediatric cohort in Amman, Jordan, between 2010 and 2013. RSVA and RSVB co-circulated during each winter epidemic of RSV in Amman, and each epidemic comprised multiple independent viral introductions of RSVA and RSVB. However, RSVA and RSVB alternated in dominance across years, potential evidence of immunological interactions. Children infected with RSVA tended to be older than RSVB-infected children [30 months versus 22.4 months, respectively (P value = 0.02)], and tended to developed bronchopneumonia less frequently than those with RSVB, although the difference was not statistically significant (P value = 0.06). Differences in spatial patterns were investigated, and RSVA lineages were often identified in multiple regions in Amman, whereas RSVB introductions did not spread beyond a single region of the city, although these findings were based on small sample sizes. Multiple RSVA genotypes were identified in Amman, including GA2 viruses as well as three viruses from the ON1 sub-genotype that emerged in 2009 and are now the dominant genotype circulating worldwide. As vaccine development advances, further sequencing of RSV is needed to understand viral ecology and transmission, particularly in under-studied locations.

The Evolutionary Dynamics of Influenza A Viruses Circulating in Mallards in Duck Hunting Preserves in Maryland, USA.

Duck hunting preserves (DHP) have resident populations of farm-raised mallard ducks, which create potential foci for the evolution of novel influenza A viruses (IAVs). Through an eleven-year (2003-2013) IAV surveillance project in seven DHPs in Maryland, USA, we frequently identified IAVs in the resident, free-flying mallard ducks (5.8% of cloacal samples were IAV-positive). The IAV population had high genetic diversity, including 12 HA subtypes and 9 NA subtypes. By sequencing the complete genomes of 290 viruses, we determined that genetically diverse IAVs were introduced annually into DHP ducks, predominantly from wild birds in the Anatidae family that inhabit the Atlantic and Mississippi flyways. The relatively low viral gene flow observed out of DHPs suggests that raised mallards do not sustain long-term viral persistence nor do they serve as important sources of new viruses in wild birds. Overall, our findings indicate that DHPs offer reliable samples of the diversity of IAV subtypes, and could serve as regional sentinel sites that mimic the viral diversity found in local wild duck populations, which would provide a cost-efficient strategy for long-term IAV monitoring. Such monitoring could allow for early identification and characterization of viruses that threaten bird species of high economic and environmental interest.

Epidemiological hypothesis testing using a phylogeographic and phylodynamic framework.

Computational analyses of pathogen genomes are increasingly used to unravel the dispersal history and transmission dynamics of epidemics. Here, we show how to go beyond historical reconstructions and use spatially-explicit phylogeographic and phylodynamic approaches to formally test epidemiological hypotheses. We illustrate our approach by focusing on the West Nile virus (WNV) spread in North America that has substantially impacted public, veterinary, and wildlife health. We apply an analytical workflow to a comprehensive WNV genome collection to test the impact of environmental factors on the dispersal of viral lineages and on viral population genetic diversity through time. We find that WNV lineages tend to disperse faster in areas with higher temperatures and we identify temporal variation in temperature as a main predictor of viral genetic diversity through time. By contrasting inference with simulation, we find no evidence for viral lineages to preferentially circulate within the same migratory bird flyway, suggesting a substantial role for non-migratory birds or mosquito dispersal along the longitudinal gradient.