Biology subjects

Lakdawala, S.

Publications and source records attributed to Lakdawala, S..

15 recordsLinked to original sources

bioRxiv2026

A lethal human H5N5 influenza virus isolate exhibits low pandemic risk traits

In fall of 2025, a fatal infection of highly pathogenic avian influenza (HPAI) virus H5N5 occurred. To define the risk of this emerging virus to humans, we performed a comprehensive analysis based on our established triage. Serological analysis revealed that humans across all birth years had no detectable neutralizing antibodies to this H5N5 isolate. Further characterization revealed a lack of phenotypic signatures associated with epidemiologically successful influenza viruses in humans, including reduced replication in human airway cells and an avian-like pH of inactivation. Additionally, assessment of H5N5 in ferrets revealed a lack of direct contact transmission and moderate disease severity. H5N5 infection in ferrets with prior immunity against the 2009 H1N1 pandemic strain resulted in fewer clinical signs and reduced viral shedding. Together our data suggest that the current H5N5 HPAI lineage poses a low pandemic risk. ImportanceHPAI H5N5 viruses have caused widespread infection and death in avian species, and characterizing their pandemic risk traits is critical to understanding the threat posed to humans. In this work we analyzed an isolate that resulted in a human fatality in 2025. We found that this strain lacks many key features of influenza viruses with epidemiological success in humans including reduced growth in human lung cultures, a pH of inactivation less than 5.0, and lack of transmission to cohoused recipient ferrets. Prior immunity with seasonal H1N1 strain also reduced the viral load and disease burden of the virus. Taken together, these data suggest that currently circulating H5N5 poses a low risk to humans but highlights the importance of phenotypic characterizations for future risk assessments as the virus evolves in wild birds.

microbiology↗

bioRxiv2026

Within-host adaptive evolution is limited by genetic drift in experimental human influenza A virus infections

Selection of advantageous mutations drives the emergence of dominant variants during seasonal influenza epidemics. However, within-host detection of such variants remains rare, limiting our understanding of how selection operates at the scale of individual hosts. In this study, we used a controlled human infection model to examine the within-host evolutionary dynamics in thirteen participants intranasally infected with a seasonal H3N2 influenza A virus. Although this clinical trial is ongoing, our work represents a pre-planned, interim, exploratory analysis. Results in this system were contrasted with those observed in a ferret model of infection. The inoculum, used in both humans and ferrets, carried standing diversity that enabled evaluation of variant trajectories during infection. Although the dynamics were variable among participants, in humans, the minor variants in the PA and NP gene segments tended to increase in frequency as infection progressed. Variant dynamics were more consistent among ferrets but showed differences from humans in the fate of the minor NP allele. Based on these observations, we fit a population genetic model to longitudinal measurements of variant frequencies. Estimates of variant selection coefficients and effective viral population sizes indicated that in humans the two minor variants had a selective advantage over the major variants, but genetic drift was strong, limiting the efficiency of selection. In ferrets, the PA minor variant also was estimated to have a selective advantage, while the NP minor variant was estimated to have a selective disadvantage. Moreover, effective viral population sizes were estimated to be considerably higher in ferrets than in humans, indicating that genetic drift was weaker in ferrets. Our analyses reveal differing selective environments acting on influenza viruses in human and ferret hosts and indicate that selection at the within-host level is weakened by genetic drift.

microbiology↗

bioRxiv2025

Fatal Human H3N8 Influenza Virus has a Moderate Pandemic Risk

In China, low pathogenic avian influenza (LPAI) H3N8 virus is widespread among chickens and has recently caused three zoonotic infections, with the last one in 2023 being fatal. Here we evaluated the relative pandemic risk of this 2023 zoonotic H3N8 influenza virus, utilizing our previously published decision tree. Serological analysis indicated that a large proportion of the human population does not have any cross-neutralizing antibodies against this H3N8 strain. LPAI H3N8 displayed a dual affinity for 2-3 and 2-6 sialic acids and replicated efficiently in human bronchial epithelial cells. Furthermore, we observed H3N8 transmission via direct contact but not aerosols to ferrets with pre-existing H3N2 immunity. Although pre-existing H3N2 immunity resulted in a shortened disease course in ferrets, it did not reduce disease severity or replication in the respiratory tract. This study suggests that this zoonotic H3N8 strain has moderate pandemic potential and emphasizes the continued need for avian influenza surveillance.

microbiology↗

bioRxiv2024

Pre-existing H1N1 immunity reduces severe disease with bovine H5N1 influenza virus

The emergence of highly pathogenic H5N1 avian influenza in dairy cattle herds across the United States has caused multiple mild human infections. There is an urgent need to understand the risk of spillover into humans. Here, we show that pre-existing immunity from the 2009 H1N1 pandemic influenza virus provided protection from mortality and severe clinical disease to ferrets intranasally infected with bovine H5N1. H1N1 immune ferrets exhibited a differential tissue tropism with little bovine H5N1 viral dissemination to organs outside the respiratory tract and significantly less H5N1 virus found in nasal secretions and the respiratory tract. Additionally, ferrets with H1N1 prior immunity produced antibodies that cross-reacted with H5N1 neuraminidase protein. Taken together, these results suggest that mild disease in humans may be linked to prior immunity to human seasonal influenza viruses.

microbiology↗

bioRxiv2023

Ventilation does not affect influenza virus transmission efficiency 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 modified, more realistic transmission setup and used it to investigate the impact of ventilation rates on transmission in a close-range, play-based scenario. In this setup, four immunologically naive recipient ferrets were exposed to a donor ferret infected with a genetically barcoded 2009 H1N1 virus (H1N1pdm09) for four hours. The ferrets interacted in a shared space that included toys, similar to a child care setting. The transmission efficiency was determined under conditions of low and high ventilation rates; air exchange rates of [~] 1.3 hr-1 and 23 hr-1, respectively. Despite the large difference in ventilation rate, transmission efficiency was the same, 50% in two independent replicate studies. The presence of infectious virus or viral RNA on surfaces and in air throughout the exposure area was similar regardless of 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. Our findings indicate that in exposures characterized by frequent close-range, play-based interactions and the presence of fomites, ventilation does not significantly impact transmission efficiency. SignificanceImproved ventilation in building has the potential to reduce transmission of respiratory viruses, but its effect in different settings is not well understood. We developed a novel system to study influenza virus transmission in the ferret animal model in an environment that mimics a child care center. We demonstrate that increased ventilation is not effective at disrupting transmission in this setting, suggesting that transmission occurs mainly at close-range or via fomites. Multiple interventions are needed to reduce the spread of influenza virus in this type of setting.

microbiology↗

bioRxiv2023

Influenza infected macrophages release viral ribonucleoproteins that shape the local host response

Airway epithelial cells and macrophages (M{Phi}) represent cellular targets of infection by influenza A virus (IAV). Epithelial cells support IAV infection and replication by enabling the generation and release of new viral particles (productive replication). In contrast, M{Phi} are susceptible to initial IAV infection but the release of infectious viral particles is inhibited through abortive replication. Despite the lack of infectious virions released from infected M{Phi}, we detected newly synthesised viral RNA and nucleoprotein (NP) in M{Phi} supernatants. We show that viral RNA is released from infected M{Phi} as viral ribonucleoprotein (vRNP) complexes which elicits potent inflammatory responses when exposed to uninfected cells. These vRNPs specifically induced IL-1{beta}, CXCL13, IL-32, CCL4 and CXCL10 in uninfected cells and were at least partially sensed through the RIG-I/MDA5 pathway. While M{Phi} represent a dead-end for IAV infection through abortive replication, the release of vRNPs shapes immune repsonses of uninfected cells in the local microenvironment.

microbiology↗

bioRxiv2023

Seasonal influenza viruses decay more rapidly at intermediate humidity in droplets containing saliva compared to respiratory mucus

Expulsions of virus-laden aerosols or droplets are an important source of onward respiratory virus transmission and can originate from both the oral and nasal cavity of an infected host. However, the presence of infectious influenza virus in the oral cavity during infection has not been widely considered, and thus little work has explored the environmental persistence of influenza virus in oral cavity expulsions that may facilitate transmission. Using the ferret model, we detected infectious virus in the nasal and oral cavities, suggesting that virus can be expelled into the environment from either anatomical site. We also assessed the stability of two influenza A viruses (H1N1 and H3N2) in droplets of human saliva or respiratory mucus over a range of relative humidities. We observed that influenza virus infectivity decays rapidly in saliva droplets at intermediate relative humidity, while viruses in airway surface liquid droplets retain infectivity. Virus inactivation was not associated with bulk protein content, salt content, or droplet drying time. Instead, we found that saliva droplets exhibited distinct inactivation kinetics during the wet and dry phases at intermediate relative humidity and that droplet residue morphology may lead to the elevated first-order inactivation rate observed during the dry phase. Additionally, distinct differences in crystalline structure and nanobead localization were observed between saliva and airway surface liquid droplets. Together, our work demonstrates that different respiratory fluids exhibit unique virus persistence profiles and suggests that influenza viruses expelled from the oral cavity may contribute to virus transmission in low and high humidity environments. ImportanceDetermining how viruses persist in the environment is important for mitigating transmission risk. Expelled infectious droplets and aerosols are composed of respiratory fluids, including saliva and complex mucus mixtures, but how influenza viruses survive in such fluids is largely unknown. Here, we find that infectious influenza virus is present in the oral cavity of infected ferrets, suggesting that saliva-containing expulsions can play a role in onward transmission. Additionally, influenza virus in droplets composed of saliva degrades more rapidly than virus within respiratory mucus. Droplet composition impacts the crystalline structure and virus localization in dried droplets. These results suggest that viruses from distinct sites in the respiratory tract could have variable persistence in the environment, which will impact viral transmission fitness.

microbiology↗

bioRxiv2023

Variability in donor lung culture and relative humidity impact the stability of 2009 pandemic H1N1 influenza virus on nonporous surfaces

Respiratory viruses can transmit by multiple modes, including contaminated surfaces, commonly referred to as fomites. Efficient fomite transmission requires that a virus remain infectious on a given surface material over a wide range of environmental conditions, including different relative humidities. Prior work examining the stability of influenza viruses on surfaces has relied upon virus grown in media or eggs, which does not mimic the composition of virus-containing droplets expelled from the human respiratory tract. In this study, we examined the stability of the 2009 pandemic H1N1 (H1N1pdm09) virus on a variety of nonporous surface materials at four different humidity conditions. Importantly, we used virus grown in primary human bronchial epithelial (HBE) cultures from different donors to recapitulate the physiological microenvironment of expelled viruses. We observed rapid inactivation of H1N1pdm09 on copper under all experimental conditions. In contrast to copper, viruses were stable on polystyrene plastic, stainless steel, aluminum, and glass, at multiple relative humidity conditions, but greater decay on ABS plastic was observed at short time points. However, the half-lives of viruses at 23% relative humidity were similar among non-copper surfaces and ranged from 4.5 to 5.9 hours. Assessment of H1N1pdm09 longevity on nonporous surfaces revealed that virus persistence was governed more by differences among HBE culture donors than by surface material. Our findings highlight the potential role of an individuals respiratory fluid on viral persistence and could help explain heterogeneity in transmission dynamics. IMPORTANCESeasonal epidemics and sporadic pandemics of influenza cause a large public health burden. Although influenza viruses disseminate through the environment in respiratory secretions expelled from infected individuals, they can also be transmitted by contaminated surfaces where virus-laden expulsions can deposit. Understanding virus stability on surfaces within the indoor environment is critical to assessing influenza transmission risk. We found that influenza virus stability is affected by the host respiratory secretion in which the virus is expelled, the surface material on which the droplet lands, and the ambient relative humidity of the environment. Influenza viruses can remain infectious on many common surfaces for prolonged periods, with half-lives of 4.5-5.9 hours. These data imply that influenza viruses are highly persistent in indoor environments in biologically-relevant matrices. Decontamination and engineering controls should be used to mitigate influenza virus transmission.

microbiology↗

bioRxiv2023

Detection of Influenza virus and Streptococcus pneumoniae in air sampled from co-infected ferrets and analysis of their influence on pathogen stability

Secondary infection with Streptococcus pneumoniae has contributed significantly to morbidity and mortality during multiple influenza virus pandemics and remains a common threat today. During a concurrent infection, both pathogens can influence the transmission of each other, but the mechanisms behind this are unclear. In this study, condensation air sampling and cyclone bioaerosol sampling were performed using ferrets first infected with the 2009 H1N1 pandemic influenza virus (H1N1pdm09) and secondarily infected with S. pneumoniae strain D39 (Spn). We detected viable pathogens and microbial nucleic acid in expelled aerosols from co-infected ferrets, suggesting that these microbes could be present in the same respiratory expulsions. To assess whether microbial communities impact pathogen stability within an expelled droplet, we performed experiments measuring viral and bacterial persistence in 1 L droplets. We observed that H1N1pdm09 stability was unchanged in the presence of Spn. Further, Spn stability was moderately increased in the presence of H1N1pdm09, although the degree of stabilization differed between airways surface liquid collected from individual patient cultures. These findings are the first to collect both pathogens from the air and in doing so, they provide insight into the interplay between these pathogens and their hosts. ImportanceThe impact of microbial communities on transmission fitness and environmental persistence is under-studied. Environmental stability of microbes is crucial to identifying transmission risks and mitigation strategies, such as removal of contaminated aerosols and decontamination of surfaces. Co-infection with S. pneumoniae is very common during influenza virus infection, but little work has been done to understand whether S. pneumoniae alters stability of influenza virus, or vice versa, in a relevant system. Here, we demonstrate that influenza virus and S. pneumoniae are expelled by co-infected hosts. Our stability assays did not reveal any impact of S. pneumoniae on influenza virus stability, and a trend towards increased stability of S. pneumoniae in the presence of influenza viruses. Future work characterizing environmental persistence of viruses and bacteria should include microbially-complex solutions to better mimic physiologically relevant conditions.

microbiology↗

bioRxiv2022

Environmental Stability of Enveloped Viruses is Impacted by the Initial Volume and Evaporation Kinetics of Droplets

Efficient spread of respiratory viruses requires the virus to maintain infectivity in the environment. Environmental stability of viruses can be influenced by many factors, including temperature and humidity. Our study measured the impact of initial droplet volume (50, 5, and 1 {micro}L) and relative humidity (RH: 40%, 65%, and 85%) on the stability of influenza A virus, bacteriophage, Phi6, a common surrogate for enveloped viruses, and SARS-CoV-2 under a limited set of conditions. Our data suggest that the drying time required for the droplets to reach quasi-equilibrium (i.e. a plateau in mass) varied with RH and initial droplet volume. The macroscale physical characteristics of the droplets at quasi-equilibrium varied with RH but not with initial droplet volume. We observed more rapid virus decay when the droplets were still wet and undergoing evaporation, and slower decay after the droplets had dried. Initial droplet volume had a major effect on virus viability over the first few hours; whereby the decay rate of influenza virus was faster in smaller droplets. In general, influenza virus and SARS-CoV-2 decayed similarly. Overall, this study suggests that virus decay in media is closely correlated with the extent of droplet evaporation, which is controlled by RH. Taken together, these data suggest that decay of different viruses is more similar at higher RH and in smaller droplets and is distinct at lower RH and in larger droplets. Importantly, accurate assessment of transmission risk requires use of physiologically relevant droplet volumes and careful consideration of the use of surrogates. FundingNational Institute of Allergy and Infectious Diseases, National Institute of Neurological Disorders and Stroke, National Institutes of Health; Department of Health and Human Services; Flu Lab. ImportanceDuring the COVID-19 pandemic, policy decisions were being driven by virus stability experiments involving SARS-CoV-2 applied to surfaces in large droplets at various humidity conditions. The results of our study indicate that determination of half-lives for emerging pathogens in large droplets likely over-estimates transmission risk for contaminated surfaces, as occurred during the COVID-19 pandemic. Our study implicates the need for the use of physiologically relevant droplet sizes with use of relevant surrogates in addition to what is already known about the importance of physiologically relevant media for risk assessment of future emerging pathogens.

microbiology↗

bioRxiv2022

Host origin is a determinant of parallel evolution between influenza virus gene segments.

Several emerging influenza viruses, including H7N9 and H5N6 viruses, trace their origins to reassortment with H9N2 viruses that contributed internal gene segments. However, the evolutionary constraints governing reassortment of H9N2 viruses remain unknown. In seasonal human influenza A viruses, gene segments evolve in parallel at both the gene and protein levels. Here, we demonstrate that parallel evolution in human H3N2 viruses differs from avian H9 viruses, with both genes and proteins of avian H9 viruses characterized by high phylogenetic divergence. Strikingly, protein trees corresponding to avian H9 polymerase subunits diverge despite known functional constraints on polymerase evolution. Gene divergence was consistent across avian H9 isolates from different continents, suggesting that parallel evolution between H9 gene segments is not dependent on regionally defined lineages. Instead, parallel evolution in H9 viruses was dependent upon host origin. Our study reveals the role of the host in parallel evolution of influenza gene segments and suggests that high reassortment potential in avian species may be a consequence of evolutionary flexibility between gene segments.

evolutionary biology↗

bioRxiv2021

Parallel evolution between genomic segments of seasonal human influenza viruses reveals RNA-RNA relationships

The influenza A virus (IAV) genome consists of eight negative-sense viral RNA (vRNA) segments that are selectively assembled into progeny virus particles through RNA-RNA interactions. To identify relationships between vRNA segments, we examined parallel evolution between vRNA segments of seasonal human IAV, finding that evolutionary relationships between vRNA segments differ between subtypes and antigenically-shifted strains. Intersegmental relationships were distinct between H3N2 and H1N1 viruses, but largely conserved over time in H3N2 viruses. However, parallel evolution of vRNA segments diverged between H1N1 strains isolated before and after the 2009 pandemic. Surprisingly, intersegmental relationships were not driven solely by protein sequence, which is potentially indicative of RNA-RNA driven coevolution. Colocalization of highly coevolved vRNA segments was enriched over other pairs at the nuclear periphery during a productive viral infection. This study illustrates how phylogenetics can be applied to interrogate putative RNA interactions underlying selective assembly of IAV.

microbiology↗

bioRxiv2020

Pre-existing immunity provides a barrier to airborne transmission of influenza viruses

Human-to-human transmission of influenza viruses is a serious public health threat, yet the precise role of immunity from previous infections on the susceptibility to airborne viruses is still unknown. Using human seasonal influenza viruses in a ferret model, we examined the roles of exposure duration and heterosubtypic immunity on influenza transmission. We found that airborne transmission of seasonal influenza strains is abrogated in recipient animals with pre-existing non-neutralizing immunity, indicating that transmissibility of a given influenza virus strain should be examined in the context of ferrets that are not immunologically naive.

microbiology↗

bioRxiv2020

The Viral Polymerase Complex Mediates the Interaction of vRNPs with Recycling Endosomes During SeV Assembly

Paramyxoviruses are negative sense single-stranded RNA viruses that comprise many important human and animal pathogens, including human parainfluenza viruses. These viruses bud from the plasma membrane of infected cells after the viral ribonucleoprotein complex (vRNP) is transported from the cytoplasm to the cell membrane via Rab11a-marked recycling endosomes. The viral proteins that are critical for mediating this important initial step in viral assembly are unknown. Here we use the model paramyxovirus, murine parainfluenza virus 1, or Sendai virus (SeV), to investigate the roles of viral proteins in Rab11a-driven virion assembly. We previously reported that infection with SeV containing high levels of copy-back defective viral genomes (DVGs) generates heterogenous populations of cells. Cells enriched in full-length virus produce viral particles containing standard or defective viral genomes, while cells enriched in DVGs do not, despite high levels of defective viral genome replication. Here we take advantage of this heterogenous cell phenotype to identify proteins that mediate interaction of vRNPs with Rab11a. We examine the role of matrix protein and nucleoprotein and determine that they are not sufficient to drive interaction of vRNPs with recycling endosomes. Using a combination of mass spectrometry and comparative protein abundance and localization in DVG- and FL-high cells, we identify viral polymerase complex components L and, specifically, its cofactor C proteins as interactors with Rab11a. We find that accumulation of these proteins within the cell is the defining feature that differentiates cells that proceed to viral egress from cells which remain in replication phases. IMPORTANCEParamyxoviruses are a family of viruses that include a number of pathogens with significant burdens on human health. Particularly, human parainfluenza viruses are an important cause of pneumonia and bronchiolitis in children for which there are no vaccines or direct acting antivirals. These cytoplasmic replicating viruses bud from the plasma membrane and coopt cellular endosomal recycling pathways to traffic viral ribonucleoprotein complexes from the cytoplasm to the membrane of infected cells. The viral proteins required for viral engagement with the recycling endosome pathway are still not known. Here we use the model paramyxovirus Sendai virus, or murine parainfluenza virus 1, to investigate the role of viral proteins in this initial step of viral assembly. We find that viral polymerase components large protein L and accessory C proteins are necessary for engagement with recycling endosomes. These findings are important in identifying viral proteins as potential targets for development of antivirals.

microbiology↗

bioRxiv2018

Learning the sequence of influenza A genome assembly during viral replication using point process models and fluorescence in situ hybridization

Within influenza virus infected cells, viral genomic RNA are selectively packed into progeny virions, which predominantly contain a single copy of 8 viral RNA segments. Intersegmental RNA-RNA interactions are thought to mediate selective packaging of each viral ribonucleoprotein complex (vRNP). Clear evidence of a specific interaction network culminating in the full genomic set has yet to be identified. Using multi-color fluorescence in situ hybridization to visualize four vRNP segments within a single cell, we developed image-based models of vRNP-vRNP spatial dependence. These models were used to construct likely sequences of vRNP associations resulting in the full genomic set. Our results support the notion that selective packaging occurs during cytoplasmic transport and identifies the formation of multiple distinct vRNP sub-complexes that likely form as intermediate steps toward full genomic inclusion into a progeny virion. The methods employed demonstrate a statistically driven, model based approach applicable to other interaction and assembly problems. Author SummaryInfluenza virus consists of eight viral ribonucleoproteins (vRNPs) that are assembled by infected cells to produce new virions. The process by which all eight vRNPs are assembled is not yet understood. We therefore used images from a previous study in which up to four vRNPs had been visualized in the same cell to construct spatial point process models that measure how well the subcellular distribution of one vRNP can be predicted from one or more other vRNPs. We used the likelihood of these models as an estimate of the extent of association between vRNPs and thereby constructed likely sequences of vRNP assembly that would produce full virions. Our work identifies the formation of multiple distinct vRNP sub-complexes that likely form as intermediate steps toward production of a virion. The results may be of use in designing strategies to interfere with virus assembly. We also anticipate that the approach may be useful for studying other assembly processes, especially for complexes with modest affinities and more components than can be visualized simultaneously.

bioinformatics↗