H5N1 influenza: Urgent questions and directions.

H5N1 is an avian influenza virus that causes respiratory disease in birds and several land and sea mammals. The recent outbreak in the United States, including infection of dairy workers, has increased the concern around potential transmission and spread. We asked virologists, epidemiologists, and public health experts what the most urgent questions and action points are at this stage of the outbreak.

The origins of viral infection sleuth.

The events of the past year have underscored the serious and rapid threat that emerging viruses pose to global health. However, much of the rapid progress in understanding and combating SARS-CoV-2 was made possible because of the decades of important groundwork laid from researchers studying other emergent infectious diseases. The 2021 John Dirks Canada Gairdner Global Health award recognizes the contributions of Joseph Sriyal Malik Peiris and Yi Guan toward understanding the origins and options for control of newly emerging infectious disease outbreaks in Asia, notably zoonotic influenza and severe acute respiratory syndrome (SARS). Cell's Nicole Neuman corresponded with Yi Guan about his path to becoming a viral infection sleuth and the challenges of understanding emerging pathogens and their origins. Excerpts of their exchange are included here.

Pathogenicity and transmissibility of bovine H5N1 influenza virus.

Highly pathogenic H5N1 avian influenza (HPAI H5N1) viruses occasionally infect, but typically do not transmit, in mammals. In the spring of 2024, an unprecedented outbreak of HPAI H5N1 in bovine herds occurred in the USA, with virus spread within and between herds, infections in poultry and cats, and spillover into humans, collectively indicating an increased public health risk1-4. Here we characterize an HPAI H5N1 virus isolated from infected cow milk in mice and ferrets. Like other HPAI H5N1 viruses, the bovine H5N1 virus spread systemically, including to the mammary glands of both species, however, this tropism was also observed for an older HPAI H5N1 virus isolate. Bovine HPAI H5N1 virus bound to sialic acids expressed in human upper airways and inefficiently transmitted to exposed ferrets (one of four exposed ferrets seroconverted without virus detection). Bovine HPAI H5N1 virus thus possesses features that may facilitate infection and transmission in mammals.

Identification, characterization, and natural selection of mutations driving airborne transmission of A/H5N1 virus.

Recently, A/H5N1 influenza viruses were shown to acquire airborne transmissibility between ferrets upon targeted mutagenesis and virus passage. The critical genetic changes in airborne A/Indonesia/5/05 were not yet identified. Here, five substitutions proved to be sufficient to determine this airborne transmission phenotype. Substitutions in PB1 and PB2 collectively caused enhanced transcription and virus replication. One substitution increased HA thermostability and lowered the pH of membrane fusion. Two substitutions independently changed HA binding preference from α2,3-linked to α2,6-linked sialic acid receptors. The loss of a glycosylation site in HA enhanced overall binding to receptors. The acquired substitutions emerged early during ferret passage as minor variants and became dominant rapidly. Identification of substitutions that are essential for airborne transmission of avian influenza viruses between ferrets and their associated phenotypes advances our fundamental understanding of virus transmission and will increase the value of future surveillance programs and public health risk assessments.

BTN3A3 evasion promotes the zoonotic potential of influenza A viruses.

Spillover events of avian influenza A viruses (IAVs) to humans could represent the first step in a future pandemic1. Several factors that limit the transmission and replication of avian IAVs in mammals have been identified. There are several gaps in our understanding to predict which virus lineages are more likely to cross the species barrier and cause disease in humans1. Here, we identified human BTN3A3 (butyrophilin subfamily 3 member A3)2 as a potent inhibitor of avian IAVs but not human IAVs. We determined that BTN3A3 is expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts primarily at the early stages of the virus life cycle by inhibiting avian IAV RNA replication. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F or, rarely, 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, avian IAV serotypes, such as H7 and H9, that spilled over into humans also evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure3. Thus, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.

Structural determinants for naturally evolving H5N1 hemagglutinin to switch its receptor specificity.

Of the factors governing human-to-human transmission of the highly pathogenic avian-adapted H5N1 virus, the most critical is the acquisition of mutations on the viral hemagglutinin (HA) to "quantitatively switch" its binding from avian to human glycan receptors. Here, we describe a structural framework that outlines a necessary set of H5 HA receptor-binding site (RBS) features required for the H5 HA to quantitatively switch its preference to human receptors. We show here that the same RBS HA mutations that lead to aerosol transmission of A/Vietnam/1203/04 and A/Indonesia/5/05 viruses, when introduced in currently circulating H5N1, do not lead to a quantitative switch in receptor preference. We demonstrate that HAs from circulating clades require as few as a single base pair mutation to quantitatively switch their binding to human receptors. The mutations identified by this study can be used to monitor the emergence of strains having human-to-human transmission potential.

Ventilation does not affect close-range transmission of influenza virus in a ferret playpen setup.

Sustained community spread of influenza viruses relies on efficient person-to-person transmission. Current experimental transmission systems do not mimic environmental conditions (e.g., air exchange rates, flow patterns), host behaviors, or exposure durations relevant to real-world settings. Therefore, results from these traditional systems may not be representative of influenza virus transmission in humans. To address this pitfall, we developed a close-range transmission setup that implements a play-based scenario and used it to investigate the impact of ventilation rates on transmission. In this setup, four immunologically naive recipient ferrets were exposed to a donor ferret infected with a genetically barcoded 2009 H1N1 virus (H1N1pdm09) for 4 h. The ferrets interacted in a shared space that included toys, similar to a childcare setting. Transmission efficiency was assessed under low and high ventilation, with air exchange rates of ~1.3 h-1 and 23 h-1, respectively. Transmission efficiencies observed in three independent replicate studies were similar between ventilation conditions. The presence of infectious virus or viral RNA on surfaces and in air throughout the exposure area was also not impacted by the ventilation rate. While high viral genetic diversity in donor ferret nasal washes was maintained during infection, recipient ferret nasal washes displayed low diversity, revealing a narrow transmission bottleneck regardless of ventilation rate. Examining the frequency and duration of ferret physical touches revealed no link between these interactions and a successful transmission event. Our findings indicate that exposures characterized by frequent, close-range interactions and the presence of fomites can overcome the benefits of increased ventilation.

Stability of influenza A virus in droplets and aerosols is heightened by the presence of commensal respiratory bacteria.

Aerosol transmission remains a major challenge for control of respiratory viruses, particularly those causing recurrent epidemics, like influenza A virus (IAV). These viruses are rarely expelled alone, but instead are embedded in a consortium of microorganisms that populate the respiratory tract. The impact of microbial communities and inter-pathogen interactions upon stability of transmitted viruses is well-characterized for enteric pathogens, but is under-studied in the respiratory niche. Here, we assessed whether the presence of five different species of commensal respiratory bacteria could influence the persistence of IAV within phosphate-buffered saline and artificial saliva droplets deposited on surfaces at typical indoor air humidity, and within airborne aerosol particles. In droplets, presence of individual species or a mixed bacterial community resulted in 10- to 100-fold more infectious IAV remaining after 1 h, due to bacterial-mediated flattening of drying droplets and early efflorescence. Even when no efflorescence occurred at high humidity or the bacteria-induced changes in droplet morphology were abolished by aerosolization instead of deposition on a well plate, the bacteria remained protective. Staphylococcus aureus and Streptococcus pneumoniae were the most stabilizing compared to other commensals at equivalent density, indicating the composition of an individual's respiratory microbiota is a previously unconsidered factor influencing expelled virus persistence.IMPORTANCEIt is known that respiratory infections such as coronavirus disease 2019 and influenza are transmitted by release of virus-containing aerosols and larger droplets by an infected host. The survival time of viruses expelled into the environment can vary depending on temperature, room air humidity, UV exposure, air composition, and suspending fluid. However, few studies consider the fact that respiratory viruses are not alone in the respiratory tract-we are constantly colonized by a plethora of bacteria in our noses, mouth, and lower respiratory system. In the gut, enteric viruses are known to be stabilized against inactivation and environmental decay by gut bacteria. Despite the presence of a similarly complex bacterial microbiota in the respiratory tract, few studies have investigated whether viral stabilization could occur in this niche. Here, we address this question by investigating influenza A virus stabilization by a range of commensal bacteria in systems representing respiratory aerosols and droplets.

Forecasting of influenza activity and associated hospital admission burden and estimating the impact of COVID-19 pandemic on 2019/20 winter season in Hong Kong.

Like other tropical and subtropical regions, influenza viruses can circulate year-round in Hong Kong. However, during the COVID-19 pandemic, there was a significant decrease in influenza activity. The objective of this study was to retrospectively forecast influenza activity during the year 2020 and assess the impact of COVID-19 public health social measures (PHSMs) on influenza activity and hospital admissions in Hong Kong. Using weekly surveillance data on influenza virus activity in Hong Kong from 2010 to 2019, we developed a statistical modeling framework to forecast influenza virus activity and associated hospital admissions. We conducted short-term forecasts (1-4 weeks ahead) and medium-term forecasts (1-13 weeks ahead) for the year 2020, assuming no PHSMs were implemented against COVID-19. We estimated the reduction in transmissibility, peak magnitude, attack rates, and influenza-associated hospitalization rate resulting from these PHSMs. For short-term forecasts, mean ambient ozone concentration and school holidays were found to contribute to better prediction performance, while absolute humidity and ozone concentration improved the accuracy of medium-term forecasts. We observed a maximum reduction of 44.6% (95% CI: 38.6% - 51.9%) in transmissibility, 75.5% (95% CI: 73.0% - 77.6%) in attack rate, 41.5% (95% CI: 13.9% - 55.7%) in peak magnitude, and 63.1% (95% CI: 59.3% - 66.3%) in cumulative influenza-associated hospitalizations during the winter-spring period of the 2019/2020 season in Hong Kong. The implementation of PHSMs to control COVID-19 had a substantial impact on influenza transmission and associated burden in Hong Kong. Incorporating information on factors influencing influenza transmission improved the accuracy of our predictions.

Simulating respiratory disease transmission within and between classrooms to assess pandemic management strategies at schools.

The global spread of coronavirus disease 2019 (COVID-19) has emphasized the need for evidence-based strategies for the safe operation of schools during pandemics that balance infection risk with the society's responsibility of allowing children to attend school. Due to limited empirical data, existing analyses assessing school-based interventions in pandemic situations often impose strong assumptions, for example, on the relationship between class size and transmission risk, which could bias the estimated effect of interventions, such as split classes and staggered attendance. To fill this gap in school outbreak studies, we parameterized an individual-based model that accounts for heterogeneous contact rates within and between classes and grades to a multischool outbreak data of influenza. We then simulated school outbreaks of respiratory infectious diseases of ongoing threat (i.e., COVID-19) and potential threat (i.e., pandemic influenza) under a variety of interventions (changing class structures, symptom screening, regular testing, cohorting, and responsive class closures). Our results suggest that interventions changing class structures (e.g., reduced class sizes) may not be effective in reducing the risk of major school outbreaks upon introduction of a case and that other precautionary measures (e.g., screening and isolation) need to be employed. Class-level closures in response to detection of a case were also suggested to be effective in reducing the size of an outbreak.

Persistence of Influenza H5N1 and H1N1 Viruses in Unpasteurized Milk on Milking Unit Surfaces.

Examining the persistence of highly pathogenic avian influenza A(H5N1) from cattle and human influenza A(H1N1)pdm09 pandemic viruses in unpasteurized milk revealed that both remain infectious on milking equipment materials for several hours. Those findings highlight the risk for H5N1 virus transmission to humans from contaminated surfaces during the milking process.

Evaluating the epizootic and zoonotic threat of an H7N9 low-pathogenicity avian influenza virus (LPAIV) variant associated with enhanced pathogenicity in turkeys.

Between 2013 and 2017, the A/Anhui/1/13-lineage (H7N9) low-pathogenicity avian influenza virus (LPAIV) was epizootic in chickens in China, causing mild disease, with 616 fatal human cases. Despite poultry vaccination, H7N9 has not been eradicated. Previously, we demonstrated increased pathogenesis in turkeys infected with H7N9, correlating with the emergence of the L217Q (L226Q H3 numbering) polymorphism in the haemagglutinin (HA) protein. A Q217-containing virus also arose and is now dominant in China following vaccination. We compared infection and transmission of this Q217-containing 'turkey-adapted' (ty-ad) isolate alongside the H7N9 (L217) wild-type (wt) virus in different poultry species and investigated the zoonotic potential in the ferret model. Both wt and ty-ad viruses demonstrated similar shedding and transmission in turkeys and chickens. However, the ty-ad virus was significantly more pathogenic than the wt virus in turkeys but not in chickens, causing 100 and 33% mortality in turkeys respectively. Expanded tissue tropism was seen for the ty-ad virus in turkeys but not in chickens, yet the viral cell receptor distribution was broadly similar in the visceral organs of both species. The ty-ad virus required exogenous trypsin for in vitro replication yet had increased replication in primary avian cells. Replication was comparable in mammalian cells, and the ty-ad virus replicated successfully in ferrets. The L217Q polymorphism also affected antigenicity. Therefore, H7N9 infection in turkeys can generate novel variants with increased risk through altered pathogenicity and potential HA antigenic escape. These findings emphasize the requirement for enhanced surveillance and understanding of A/Anhui/1/13-lineage viruses and their risk to different species.

H5 and H9 avian influenza - potential re-emergent zoonotic threats to humans.

PURPOSE OF REVIEW: This review aims to discuss the current state of human infections with Avian Influenza A (H5) and (H9) viruses, to support awareness of the global epidemiology among clinicians and public health professionals interested in emerging respiratory infections. RECENT FINDINGS: Among increasing numbers of detections in avian species of Avian Influenza A(H5N1) clade 2.3.4.4b globally, reported human cases of severe infection have been rare.Enhanced surveillance of persons exposed to avian species infected with Influenza A (H5N1) clade 2.3.4.4b in different countries has identified small numbers of asymptomatic individuals with Avian Influenza A (H5N1) detected by PCR from the upper respiratory tract; some of these instances have been considered to represent contamination rather than infection.There have also been recent sporadic human cases of Avian Influenza A(H9N2) internationally, including in China and Cambodia. SUMMARY: Human infections with Avian Influenza A(H5) and (H9) viruses remain of interest as an emerging infection both to clinicians and public health professionals. While maintaining effective surveillance is essential, one health strategies to control infection in avian species will be key to mitigating these risks.

The resurgence of influenza A/H3N2 virus in Australia after the relaxation of COVID-19 restrictions during the 2022 season.

This study retrospectively analyzed the genetic characteristics of influenza A H3N2 (A/H3N2) viruses circulating in New South Wales (NSW), the Australian state with the highest number of influenza cases in 2022, and explored the phylodynamics of A/H3N2 transmission within Australia during this period. Sequencing was performed on 217 archived specimens, and A/H3N2 evolution and spread within Australia were analyzed using phylogenetic and phylodynamic methods. Hemagglutinin genes of all analyzed NSW viruses belonged to subclade 3C.2a1b.2a.2 and clustered together with the 2022 vaccine strain. Complete genome analysis of NSW viruses revealed highly frequent interclade reassortments between subclades 3C.2a1b.2a.2 and 3C.2a1b.1a. The estimated earliest introduction time of the dominant subgroup 3C.2a1b.2a.2a.1 in Australia was February 22, 2022 (95% highest posterior density: December 19, 2021-March 13, 2022), following the easing of Australian travel restrictions, suggesting a possible international source. Phylogeographic analysis revealed that Victoria drove the transmission of A/H3N2 viruses across the country during this season, while NSW did not have a dominant role in viral dissemination to other regions. This study highlights the importance of continuous surveillance and genomic characterization of influenza viruses in the postpandemic era, which can inform public health decision-making and enable early detection of novel strains with pandemic potential.

Long-term effects of non-pharmaceutical interventions on total disease burden in parsimonious epidemiological models.

The recent global COVID-19 pandemic resulted in governments enacting non-pharmaceutical interventions (NPIs) targeted at reducing transmission of SARS-CoV-2. But the NPIs also affected the transmission of viruses causing non-target seasonal respiratory diseases, including influenza and respiratory syncytial virus (RSV). In many countries, the NPIs were found to reduce cases of such seasonal respiratory diseases, but there is also evidence that subsequent relaxation of NPIs led to outbreaks of these diseases that were larger than pre-pandemic ones, due to the accumulation of susceptible individuals prior to relaxation. Therefore, the net long-term effects of NPIs on the total disease burden of non-target diseases remain unclear. Knowledge of this is important for infectious disease management and maintenance of public health. In this study, we shed light on this issue for the simplified scenario of a set of NPIs that prevent or reduce transmission of a seasonal respiratory disease for about a year and are then removed, using mathematical analyses and numerical simulations of a suite of four epidemiological models with varying complexity and generality. The model parameters were estimated using empirical data pertaining to seasonal respiratory diseases and covered a wide range. Our results showed that NPIs reduced the total disease burden of a non-target seasonal respiratory disease in the long-term. Expressed as a percentage of population size, the reduction was greater for larger values of the basic reproduction number and the immunity loss rate, reflecting larger outbreaks and hence more infections averted by imposition of NPIs. Our study provides a foundation for exploring the effects of NPIs on total disease burden in more-complex scenarios.

The influenza virus enigma.

Both seasonal and pandemic influenza continue to challenge both scientists and clinicians. Drug-resistant H1N1 influenza viruses have dominated the 2009 flu season, and the H5N1 avian influenza virus continues to kill both people and poultry in Eurasia. Here, we discuss the pathogenesis and transmissibility of influenza viruses and we emphasize the need to find better predictors of both seasonal and potentially pandemic influenza.

Markovian Approach for Exploring Competitive Diseases with Heterogeneity-Evidence from COVID-19 and Influenza in China.

Due to the complex interactions between multiple infectious diseases, the spreading of diseases in human bodies can vary when people are exposed to multiple sources of infection at the same time. Typically, there is heterogeneity in individuals' responses to diseases, and the transmission routes of different diseases also vary. Therefore, this paper proposes an SIS disease spreading model with individual heterogeneity and transmission route heterogeneity under the simultaneous action of two competitive infectious diseases. We derive the theoretical epidemic spreading threshold using quenched mean-field theory and perform numerical analysis under the Markovian method. Numerical results confirm the reliability of the theoretical threshold and show the inhibitory effect of the proportion of fully competitive individuals on epidemic spreading. The results also show that the diversity of disease transmission routes promotes disease spreading, and this effect gradually weakens when the epidemic spreading rate is high enough. Finally, we find a negative correlation between the theoretical spreading threshold and the average degree of the network. We demonstrate the practical application of the model by comparing simulation outputs to temporal trends of two competitive infectious diseases, COVID-19 and seasonal influenza in China.