Patterns of within-host influenza A virus (IAV) diversity and evolution have been described in natural human infections, but these patterns remain poorly characterized in non-human hosts. Elucidating these dynamics is important to better understand IAV biology and the evolutionary processes that govern spillover into humans. Here, we sampled an IAV outbreak in pigs during a week-long county fair to characterize viral diversity and evolution in this important reservoir host. Nasal wipes were collected on a daily basis from all pigs present at the fair, yielding up to 421 samples per day. Subtyping of PCR-positive samples revealed the co-circulation of H1N1 and H3N2 subtype swine IAVs. PCR-positive samples with robust Ct values were deep-sequenced, yielding 506 sequenced samples from a total of 253 pigs. Based on higher-depth re-sequenced data from a subset of these initially sequenced samples (260 samples from 168 pigs), we characterized patterns of within-host IAV genetic diversity and evolution. We find that IAV genetic diversity in single-subtype infected pigs is low, with the majority of intrahost Single Nucleotide Variants (iSNVs) present at frequencies of <10%. The ratio of the number of nonsynonymous to the number of synonymous iSNVs is significantly lower than under the neutral expectation, indicating that purifying selection shapes patterns of within-host viral diversity in swine. The dynamic turnover of iSNVs and their pronounced frequency changes further indicate that genetic drift also plays an important role in shaping IAV populations within pigs. Taken together, our results highlight similarities in patterns of IAV genetic diversity and evolution between humans and swine, including the role of stochastic processes in shaping within-host IAV dynamics.

For the first time, a complete genome of an RNA virus has been sequenced in its original form. Previously, RNA was sequenced by the chemical degradation of radiolabelled RNA, a difficult method that produced only short sequences. Instead, RNA has usually been sequenced indirectly by copying it into cDNA, which is often amplified to dsDNA by PCR and subsequently analyzed using a variety of DNA sequencing methods. We designed an adapter to short highly conserved termini of the influenza virus genome to target the (-) sense RNA into a protein nanopore on the Oxford Nanopore MinION sequencing platform. Utilizing this method and total RNA extracted from the allantoic fluid of infected chicken eggs, we demonstrate successful sequencing of the complete influenza virus genome with 100% nucleotide coverage, 99% consensus identity, and 99% of reads mapped to influenza. By utilizing the same methodology we can redesign the adapter in order to expand the targets to include viral mRNA and (+) sense cRNA, which are essential to the viral life cycle. This has the potential to identify and quantify splice variants and base modifications, which are not practically measurable with current methods.

While primarily considered a respiratory pathogen, influenza A virus (IAV) is nonetheless capable of spreading to, and replicating in, numerous extrapulmonary tissues in humans. However, within-host assessments of genetic diversity during multicycle replication have been largely limited to respiratory tract tissues and specimens. As selective pressures can vary greatly between anatomical sites, there is a need to examine how measures of viral diversity may vary between influenza viruses exhibiting different tropisms in humans, as well as following influenza virus infection of cells derived from different organ systems. Here, we employed human primary tissue constructs emulative of the human airway or corneal surface, and we infected both with a panel of human- and avian-origin IAV, inclusive of H1 and H3 subtype human viruses and highly pathogenic H5 and H7 subtype viruses, which are associated with both respiratory disease and conjunctivitis following human infection. While both cell types supported productive replication of all viruses, airway-derived tissue constructs elicited greater induction of genes associated with antiviral responses than did corneal-derived constructs. We used next-generation sequencing to examine viral mutations and population diversity, utilizing several metrics. With few exceptions, generally comparable measures of viral diversity and mutational frequency were detected following homologous virus infection of both respiratory-origin and ocular-origin tissue constructs. Expansion of within-host assessments of genetic diversity to include IAV with atypical clinical presentations in humans or in extrapulmonary cell types can provide greater insight into understanding those features most prone to modulation in the context of viral tropism. IMPORTANCE Influenza A virus (IAV) can infect tissues both within and beyond the respiratory tract, leading to extrapulmonary complications, such as conjunctivitis or gastrointestinal disease. Selective pressures governing virus replication and induction of host responses can vary based on the anatomical site of infection, yet studies examining within-host assessments of genetic diversity are typically only conducted in cells derived from the respiratory tract. We examined the contribution of influenza virus tropism on these properties two different ways: by using IAV associated with different tropisms in humans, and by infecting human cell types from two different organ systems susceptible to IAV infection. Despite the diversity of cell types and viruses employed, we observed generally similar measures of viral diversity postinfection across all conditions tested; these findings nonetheless contribute to a greater understanding of the role tissue type contributes to the dynamics of virus evolution within a human host.

During the evolution of SARS-CoV-2 in humans a D614G substitution in the spike (S) protein emerged and became the predominant circulating variant (S-614G) of the COVID-19 pandemic (1) . However, whether the increasing prevalence of the S-614G variant represents a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains elusive. Here, we generated isogenic SARS-CoV-2 variants and demonstrate that the S-614G variant has (i) enhanced binding to human ACE2, (ii) increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a novel human ACE2 knock-in mouse model, and (iii) markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Collectively, our data show that while the S-614G substitution results in subtle increases in binding and replication in vitro , it provides a real competitive advantage in vivo , particularly during the transmission bottle neck, providing an explanation for the global predominance of S-614G variant among the SARS-CoV-2 viruses currently circulating.

The report “Genomic Surveillance for SARS-CoV-2 Variants: Predominance of the Delta (B.1.617.2) and Omicron (B.1.1.529) Variants — United States, June 2021–January 2022” contained several errors.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

In the United States, annual vaccination against seasonal influenza is recommended for all persons aged ≥6 months except when contraindicated (1). Currently available influenza vaccines are designed to protect against four influenza viruses: A(H1N1)pdm09 (the 2009 pandemic virus), A(H3N2), B/Victoria lineage, and B/Yamagata lineage. Most influenza viruses detected this season have been A(H3N2) (2). With the exception of the 2020-21 season, when data were insufficient to generate an estimate, CDC has estimated the effectiveness of seasonal influenza vaccine at preventing laboratory-confirmed, mild/moderate (outpatient) medically attended acute respiratory infection (ARI) each season since 2004-05. This interim report uses data from 3,636 children and adults with ARI enrolled in the U.S. Influenza Vaccine Effectiveness Network during October 4, 2021-February 12, 2022. Overall, vaccine effectiveness (VE) against medically attended outpatient ARI associated with influenza A(H3N2) virus was 16% (95% CI = -16% to 39%), which is considered not statistically significant. This analysis indicates that influenza vaccination did not reduce the risk for outpatient medically attended illness with influenza A(H3N2) viruses that predominated so far this season. Enrollment was insufficient to generate reliable VE estimates by age group or by type of influenza vaccine product (1). CDC recommends influenza antiviral medications as an adjunct to vaccination; the potential public health benefit of antiviral medications is magnified in the context of reduced influenza VE. CDC routinely recommends that health care providers continue to administer influenza vaccine to persons aged ≥6 months as long as influenza viruses are circulating, even when VE against one virus is reduced, because vaccine can prevent serious outcomes (e.g., hospitalization, intensive care unit (ICU) admission, or death) that are associated with influenza A(H3N2) virus infection and might protect against other influenza viruses that could circulate later in the season.

Genomic surveillance is a critical tool for tracking emerging variants of SARS-CoV-2 (the virus that causes COVID-19), which can exhibit characteristics that potentially affect public health and clinical interventions, including increased transmissibility, illness severity, and capacity for immune escape. During June 2021-January 2022, CDC expanded genomic surveillance data sources to incorporate sequence data from public repositories to produce weighted estimates of variant proportions at the jurisdiction level and refined analytic methods to enhance the timeliness and accuracy of national and regional variant proportion estimates. These changes also allowed for more comprehensive variant proportion estimation at the jurisdictional level (i.e., U.S. state, district, territory, and freely associated state). The data in this report are a summary of findings of recent proportions of circulating variants that are updated weekly on CDC's COVID Data Tracker website to enable timely public health action.(†) The SARS-CoV-2 Delta (B.1.617.2 and AY sublineages) variant rose from 1% to >50% of viral lineages circulating nationally during 8 weeks, from May 1-June 26, 2021. Delta-associated infections remained predominant until being rapidly overtaken by infections associated with the Omicron (B.1.1.529 and BA sublineages) variant in December 2021, when Omicron increased from 1% to >50% of circulating viral lineages during a 2-week period. As of the week ending January 22, 2022, Omicron was estimated to account for 99.2% (95% CI = 99.0%-99.5%) of SARS-CoV-2 infections nationwide, and Delta for 0.7% (95% CI = 0.5%-1.0%). The dynamic landscape of SARS-CoV-2 variants in 2021, including Delta- and Omicron-driven resurgences of SARS-CoV-2 transmission across the United States, underscores the importance of robust genomic surveillance efforts to inform public health planning and practice.

During the evolution of SARS-CoV-2 in humans a D614G substitution in the spike (S) protein emerged and became the predominant circulating variant (S-614G) of the COVID-19 pandemic(1). However, whether the increasing prevalence of the S-614G variant represents a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains elusive. Here, we generated isogenic SARS-CoV-2 variants and demonstrate that the S-614G variant has (i) enhanced binding to human host cell surface receptor angiotensin-converting enzyme 2 (ACE2), (ii) increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a novel human ACE2 knock-in mouse model, and (iii) markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Collectively, our data show that while the S-614G substitution results in subtle increases in binding and replication in vitro, it provides a real competitive advantage in vivo, particularly during the transmission bottle neck, providing an explanation for the global predominance of S-614G variant among the SARS-CoV-2 viruses currently circulating.

BACKGROUND: At the start of the 2019-2020 influenza season, concern arose that circulating B/Victoria viruses of the globally emerging clade V1A.3 were antigenically drifted from the strain included in the vaccine. Intense B/Victoria activity was followed by circulation of genetically diverse A(H1N1)pdm09 viruses, that were also antigenically drifted. We measured vaccine effectiveness (VE) in the United States against illness from these emerging viruses. METHODS: We enrolled outpatients aged ≥6 months with acute respiratory illness at five sites. Respiratory specimens were tested for influenza by reverse-transcriptase polymerase chain reaction (RT-PCR). Using the test-negative design, we determined influenza VE by virus sub-type/lineage and genetic subclades by comparing odds of vaccination in influenza cases versus test-negative controls. RESULTS: Among 8,845 enrollees, 2,722 (31%) tested positive for influenza, including 1,209 (44%) for B/Victoria and 1,405 (51%) for A(H1N1)pdm09. Effectiveness against any influenza illness was 39% (95% confidence interval [CI]: 32-44), 45% (95%CI: 37-52) against B/Victoria and 30% (95%CI: 21-39) against A(H1N1)pdm09 associated illness. Vaccination offered no protection against A(H1N1)pdm09 viruses with antigenically drifted clade 6B.1A 183P-5A+156K HA genes (VE 7%; 95%CI: -14 to 23%) which predominated after January. CONCLUSIONS: Vaccination provided protection against influenza illness, mainly due to infections from B/Victoria viruses. Vaccine protection against illness from A(H1N1)pdm09 was lower than historically observed effectiveness of 40-60%, due to late-season vaccine mismatch following emergence of antigenically drifted viruses. The effect of drift on vaccine protection is not easy to predict and, even in drifted years, significant protection can be observed.

BACKGROUND: Increased illness due to antigenically drifted A(H3N2) clade 3C.3a influenza viruses prompted concerns about vaccine effectiveness and vaccine strain selection. We used U.S. virologic surveillance and Influenza Vaccine Effectiveness (VE) Network data to evaluate consequences of this clade. METHODS: Distribution of influenza viruses was described using virologic surveillance data. The VE Network enrolled ambulatory patients aged >/=6 months with acute respiratory illness at five sites. Respiratory specimens were tested by RT-PCR for influenza and sequenced. Using a test-negative design, we estimated VE comparing odds of influenza among vaccinated versus unvaccinated participants. RESULTS: During the 2018-2019 influenza season, A(H3N2) clade 3C.3a viruses caused an increasing proportion of influenza cases. Among 2,763 VE Network case patients, 1,325 (48%) were infected with A(H1N1)pdm09 and 1,350 (49%) with A(H3N2); clade 3C.3a accounted for 977 (93%) of 1,054 sequenced A(H3N2) viruses. VE was 44% (95% confidence interval [CI], 37 to 51%) against A(H1N1)pdm09 and 9% (95% CI, -4 to 20%) against A(H3N2); effectiveness was 5% (95% CI, -10 to 19%) against A(H3N2) clade 3C.3a viruses. CONCLUSIONS: Predominance of A(H3N2) clade 3C.3a viruses during the latter part of the 2018-2019 season was associated with decreased vaccine effectiveness, supporting the A(H3N2) vaccine component update for 2019-2020 northern hemisphere influenza vaccines.

Bangladesh has reported repeated outbreaks of highly pathogenic avian influenza (HPAI) A(H5) viruses in poultry since 2007. Because of the large number of live poultry markets (LPM) relative to the population density of poultry throughout the country, these markets can serve as sentinel sites for HPAI A(H5) detection. Through active LPM surveillance during June 2016-June 2017, HPAI A(H5N6) viruses along with 14 other subtypes of influenza A viruses were detected. The HPAI A(H5N6) viruses belonged to clade 2.3.4.4 and were likely introduced into Bangladesh around March 2016. Human infections with influenza clade 2.3.4.4 viruses in Bangladesh have not been identified, but the viruses had several molecular markers associated with potential human infection. Vigilant surveillance at the animal-human interface is essential to identify emerging avian influenza viruses with the potential to threaten public and animal health.

The fifth-wave of the H7N9 influenza epidemic in China was distinguished by a sudden increase in human infections, an extended geographic distribution, and the emergence of highly pathogenic avian influenza (HPAI) viruses. Genetically, some H7N9 viruses from the fifth-wave have acquired novel amino acid changes at positions involved in mammalian adaptation, antigenicity, and HA cleavability. Here, several low pathogenic avian influenza (LPAI) and HPAI H7N9 human isolates from the fifth epidemic wave were assessed for their pathogenicity and transmissibility in mammalian models, as well as their ability to replicate in human airway epithelial cells. We found that a LPAI virus exhibited a similar capacity to replicate and cause disease in two animal species as viruses from previous waves. In contrast, HPAI H7N9 viruses possessed enhanced virulence, causing greater lethargy and mortality, with an extended tropism for brain tissues in both ferret and mouse models. These HPAI viruses also showed signs of adaptation to mammalian hosts by acquiring the ability to fuse at a lower pH threshold compared with other H7N9 viruses. All of the fifth-wave H7N9 viruses were able to transmit among cohoused ferrets, but exhibited a limited capacity to transmit by respiratory droplets and deep sequencing analysis revealed that the H7N9 viruses sampled after transmission showed a reduced amount of minor variants. Taken together, we conclude that the fifth-wave HPAI H7N9 viruses have gained the ability to cause enhanced disease in mammalian models, and with further adaptation may acquire the ability to cause an H7N9 pandemic.ImportanceThe potential pandemic risk posed by avian influenza H7N9 viruses was heightened during the fifth epidemic wave in China due to the sudden increased number of human infections and the emergence of antigenically distinct LPAI and HPAI H7N9 viruses. In this study, a group of fifth-wave HPAI and LPAI viruses were evaluated for their ability to infect, cause disease, and transmit in small animal models. The ability of HPAI H7N9 viruses to cause more severe disease and to replicate in brain tissues in animal models as well as their ability to fuse at a lower pH threshold compared to LPAI H7N9 viruses suggest that the fifth-wave H7N9 viruses have evolved to acquire novel traits with the potential to pose a higher risk to humans. Although the fifth-wave H7N9 viruses have not yet gained the ability to transmit efficiently by air, continuous surveillance and risk assessment remain essential parts of our pandemic preparedness efforts.

For the first time, a coding complete genome of an RNA virus has been sequenced in its original form. Previously, RNA was sequenced by the chemical degradation of radiolabeled RNA, a difficult method that produced only short sequences. Instead, RNA has usually been sequenced indirectly by copying it into cDNA, which is often amplified to dsDNA by PCR and subsequently analyzed using a variety of DNA sequencing methods. We designed an adapter to short highly conserved termini of the influenza A virus genome to target the (-) sense RNA into a protein nanopore on the Oxford Nanopore MinION sequencing platform. Utilizing this method with total RNA extracted from the allantoic fluid of influenza rA/Puerto Rico/8/1934 (H1N1) virus infected chicken eggs (EID50 6.8 x 10(9)), we demonstrate successful sequencing of the coding complete influenza A virus genome with 100% nucleotide coverage, 99% consensus identity, and 99% of reads mapped to influenza A virus. By utilizing the same methodology one can redesign the adapter in order to expand the targets to include viral mRNA and (+) sense cRNA, which are essential to the viral life cycle, or other pathogens. This approach also has the potential to identify and quantify splice variants and base modifications, which are not practically measurable with current methods.

Influenza A(H1) viruses circulating in swine represent an emerging virus threat as zoonotic infections occur sporadically following exposure to swine. A fatal infection caused by an H1N1 variant (H1N1v) virus was detected in a patient with reported exposure to swine and who presented with pneumonia, respiratory failure, and cardiac arrest. To understand the genetic and phenotypic characteristics of the virus, genome sequence analysis, antigenic characterization, and ferret pathogenesis and transmissibility experiments were performed. Antigenic analysis of the virus isolated from the fatal case, A/Ohio/09/2015, demonstrated significant antigenic drift away from classical swine H1N1 variant viruses and H1N1 pandemic 2009 viruses. A substitution in the H1 hemagglutinin (G155E) was identified that likely impacted antigenicity, and reverse genetics was employed to understand the molecular mechanism of antibody escape. Reversion of the substitution to 155G, in a reverse genetics A/Ohio/09/2015 virus, showed that this residue was central to the loss of hemagglutination inhibition by ferret antisera raised against a prototypical H1N1 pandemic 2009 virus (A/California/07/2009), as well as gamma lineage classical swine H1N1 viruses, demonstrating the importance of this residue for antibody recognition of this H1 lineage. When analyzed in the ferret model, A/Ohio/09/2015 and another H1N1v virus (A/Iowa/39/2015), as well as A/California/07/2009, replicated efficiently in the respiratory tract of ferrets. The two H1N1v viruses transmitted efficiently among cohoused ferrets, but respiratory droplet transmission studies showed that A/California/07/2009 transmitted through the air more efficiently. Pre-existing immunity to A/California/07/2009 did not fully protect ferrets from challenge with A/Ohio/09/2015.IMPORTANCE Human infections with classical swine influenza A(H1N1) viruses that circulate in pigs continue to occur in the United States following exposure to swine. To understand the genetic and virologic characteristics of a virus (A/Ohio/09/2015) associated with a fatal infection and a virus associated with a non-fatal infection (A/Iowa/39/2015), we performed genome sequence analysis, antigenic testing, and pathogenicity and transmission studies in a ferret model. Reverse genetics was employed to identify a single antigenic site substitution (HA G155E) responsible for antigenic variation of A/Ohio/09/2015 compared to related classical swine influenza A(H1N1) viruses. Ferrets with pre-existing immunity to the pandemic A(H1N1) virus were challenged with A/Ohio/09/2015 demonstrating decreased protection. This data illustrates the potential for currently circulating swine influenza viruses to infect and cause illness in humans with pre-existing immunity to H1N1 pandemic 2009 viruses and a need for ongoing risk assessment and development of candidate vaccine viruses for improved pandemic preparedness.

Influenza A and B viruses are the causative agents of annual influenza epidemics that can be severe; influenza A viruses intermittently cause pandemics. Sequence information from influenza genomes is instrumental in determining mechanisms underpinning antigenic evolution and antiviral resistance. However, due to sequence diversity and the dynamics of influenza evolution, rapid and high-throughput sequencing of influenza viruses remains a challenge. We developed a single-reaction FluA/B Multiplex RT-PCR method that amplifies the most critical genomic segments (HA, NA, and M) of seasonal influenza A and B viruses for next-generation sequencing, regardless of viral types, subtypes, or lineages. Herein we demonstrate that the strategy is highly sensitive and robust. The strategy was validated on thousands of seasonal influenza A and B virus positive specimens using multiple next-generation sequencing platforms.

The emergence of A(H7N9) virus strains with resistance to neuraminidase (NA) inhibitors highlights a critical need to discover new countermeasures for treatment of A(H7N9) virus-infected patients. We previously described an anti-NA mAb (3c10-3) that has prophylactic and therapeutic efficacy in mice lethally challenged with A(H7N9) virus when delivered intraperitoneally (i.p.). Here we show that intrananasal (i.n.) administration of 3c10-3 protects 100% of mice from mortality when treated 24h post-challenge and further characterize the protective efficacy of 3c10-3 using a nonlethal A(H7N9) challenge model. Administration of 3c10-3 i.p. 24h prior to challenge resulted in a significant decrease in viral lung titers and deep sequencing analysis indicated that treatment did not consistently select for viral variants in NA. Furthermore, prophylactic administration of 3c10-3 did not inhibit the development of protective immunity to subsequent homologous virus re-challenge. Taken together, 3c10-3 highlights the potential use of anti-NA mAb to mitigate influenza virus infection.