Real-time genomic epidemiologic investigation of a multispecies plasmid-associated hospital outbreak of NDM-5-producing Enterobacterales infections.

OBJECTIVES: New Delhi metallo-ß-lactamase (NDM) is an emergent mechanism of carbapenem resistance associated with high mortality and limited treatment options. Because the blaNDM resistance gene is often carried on plasmids, traditional infection prevention and control (IP&C) surveillance methods and reactive whole genome sequencing (WGS) may not detect plasmid transfer in multispecies outbreaks. METHODS: Initial outbreak detection of NDM-producing Enterobacterales identified at an acute care hospital occurred via traditional IP&C methods and was supplemented by real-time WGS surveillance performed weekly. To resolve NDM-encoding plasmids, we performed long-read sequencing and constructed hybrid assemblies. WGS data for suspected outbreaks was shared with the IP&C team for assessment and intervention. RESULTS: We observed a multispecies outbreak of NDM-5-producing Enterobacterales isolated from 15 patients between February 2021 and February 2023. The 19 clinical and surveillance isolates sequenced included 7 bacterial species encoding the same NDM-5 plasmid. WGS surveillance and epidemiologic investigation characterized 10 horizontal plasmid transfer events and 6 bacterial transmission events between patients in varying hospital units. CONCLUSIONS: Our investigation revealed a complex, multispecies outbreak of NDM involving multiple plasmid transfer and bacterial transmission events. We highlight the utility of combining traditional IP&C and prospective genomic methods in identifying and containing plasmid-associated outbreaks.

Genomic Epidemiologic Investigation of a Multispecies Hospital Outbreak of NDM-5-Producing Enterobacterales Infections.

Background: New Delhi metallo-ß-lactamase (NDM) represents an emergent mechanism of carbapenem resistance associated with high mortality and limited antimicrobial treatment options. Because the blaNDM resistance gene is often carried on plasmids, traditional infection prevention and control (IP&C) surveillance methods like speciation, antimicrobial resistance testing, and reactive whole genome sequencing (WGS) may not detect plasmid transfer in multispecies outbreaks. Methods: Initial outbreak detection of NDM-producing Enterobacterales identified at an acute care hospital occurred via traditional IP&C methods and was supplemented by real-time WGS surveillance, which was performed weekly using the Illumina platform. To resolve NDM-encoding plasmids, we performed long-read Oxford Nanopore sequencing and constructed hybrid assemblies using Illumina and Nanopore sequencing data. Reports of relatedness between NDM-producing organisms and reactive WGS for suspected outbreaks were shared with the IP&C team for assessment and intervention. Findings: We observed a multispecies outbreak of NDM-5-producing Enterobacterales isolated from 15 patients between February 2021 and February 2023. The 19 clinical and surveillance isolates sequenced included seven bacterial species and each encoded the same NDM-5 plasmid, which showed high homology to NDM plasmids previously observed in Asia. WGS surveillance and epidemiologic investigation characterized ten horizontal plasmid transfer events and six bacterial transmission events between patients housed in varying hospital units. Transmission prevention focused on enhanced observation and adherence to basic infection prevention measures. Interpretation: Our investigation revealed a complex, multispecies outbreak of NDM that involved multiple plasmid transfer and bacterial transmission events, increasing the complexity of outbreak identification and transmission prevention. Our investigation highlights the utility of combining traditional IP&C and prospective genomic methods in identifying and containing plasmid-associated outbreaks. Funding: This work was funded in part by the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH) (R01AI127472) (R21AI1783691).

Implementation of a Rapid Multiplex Polymerase Chain Reaction Pneumonia Panel and Subsequent Antibiotic De-escalation.

Background: Net effects of implementation of a multiplex polymerase chain reaction (PCR) pneumonia panel (PNP) on antimicrobial stewardship are thus far unknown. This retrospective study evaluated the real-world impact of the PNP on time to antibiotic de-escalation in critically ill patients treated for pneumonia at an academic medical center. Methods: This retrospective, quasi-experimental study included adult intensive care unit (ICU) patients with respiratory culture results from 1 May to 15 August 2019 (pre-PNP group) and adult ICU patients with PNP results from 1 May to 15 August 2020 (PNP group) at Nebraska Medical Center. Patients were excluded for the following reasons: any preceding positive coronavirus disease 2019 PCR test, lack of antibiotic receipt, or non-respiratory tract infection indications for antibiotics. The primary outcome was time to discontinuation of anti-methicillin-resistant Staphylococcus aureus (MRSA) therapy. Secondary outcomes included time to discontinuation of antipseudomonal therapy, frequency of early discontinuation for atypical coverage, and overall duration (in days) of antibiotic therapy for pneumonia. Results: Sixty-six patients in the pre-PNP group and 58 in the PNP group were included. There were significant differences in patient characteristics between groups. The median time to anti-MRSA agent discontinuation was 49.1 hours in the pre-PNP and 41.8 hours in the PNP group (P = .28). The median time to discontinuation of antipseudomonal agents was 134.4 hours in the pre-PNP versus 98.1 hours in the PNP group (P = .47). Other outcomes were numerically but not significantly improved in our sample. Conclusions: This early look at implementation of a multiplex PNP did not demonstrate a statistically significant difference in antibiotic use but lays the groundwork to further evaluate a significant real-world impact on antibiotic de-escalation in ICU patients treated for pneumonia.

Utility of Human In Vitro Data in Risk Assessments of Influenza A Virus Using the Ferret Model.

As influenza A viruses (IAV) continue to cross species barriers and cause human infection, the establishment of risk assessment rubrics has improved pandemic preparedness efforts. In vivo pathogenicity and transmissibility evaluations in the ferret model represent a critical component of this work. As the relative contribution of in vitro experimentation to these rubrics has not been closely examined, we sought to evaluate to what extent viral titer measurements over the course of in vitro infections are predictive or correlates of nasal wash and tissue measurements for IAV infections in vivo. We compiled data from ferrets inoculated with an extensive panel of over 50 human and zoonotic IAV (inclusive of swine-origin and high- and low-pathogenicity avian influenza viruses associated with human infection) under a consistent protocol, with all viruses concurrently tested in a human bronchial epithelial cell line (Calu-3). Viral titers in ferret nasal wash specimens and nasal turbinate tissue correlated positively with peak titer in Calu-3 cells, whereas additional phenotypic and molecular determinants of influenza virus virulence and transmissibility in ferrets varied in their association with in vitro viral titer measurements. Mathematical modeling was used to estimate more generalizable key replication kinetic parameters from raw in vitro viral titers, revealing commonalities between viral infection progression in vivo and in vitro. Meta-analyses inclusive of IAV that display a diverse range of phenotypes in ferrets, interpreted with mathematical modeling of viral kinetic parameters, can provide critical information supporting a more rigorous and appropriate contextualization of in vitro experiments toward pandemic preparedness. IMPORTANCE Both in vitro and in vivo models are employed for assessing the pandemic potential of novel and emerging influenza A viruses in laboratory settings, but systematic examinations of how well viral titer measurements obtained in vitro align with results from in vivo experimentation are not frequently performed. We show that certain viral titer measurements following infection of a human bronchial epithelial cell line are positively correlated with viral titers in specimens collected from virus-inoculated ferrets and employ mathematical modeling to identify commonalities between viral infection progression between both models. These analyses provide a necessary first step in enhanced interpretation and incorporation of in vitro-derived data in risk assessment activities and highlight the utility of employing mathematical modeling approaches to more closely examine features of virus replication not identifiable by experimental studies alone.

Inherent Heterogeneity of Influenza A Virus Stability following Aerosolization.

Efficient human-to-human transmission represents a necessary adaptation for a zoonotic influenza A virus (IAV) to cause a pandemic. As such, many emerging IAVs are characterized for transmissibility phenotypes in mammalian models, with an emphasis on elucidating viral determinants of transmission and the role host immune responses contribute to mammalian adaptation. Investigations of virus infectivity and stability in aerosols concurrent with transmission assessments have increased in recent years, enhancing our understanding of this dynamic process. Here, we employed a diverse panel of 17 human and zoonotic IAVs, inclusive of seasonally circulating H1N1 and H3N2 viruses, as well as avian and swine viruses associated with human infection, to evaluate differences in spray factor (a value that assesses efficiency of the aerosolization process), stability, and infectivity following aerosolization. While most seasonal influenza viruses did not exhibit substantial variability within these parameters, there was more heterogeneity among zoonotic influenza viruses, which possess a diverse range of transmission phenotypes. Aging of aerosols at different relative humidities identified strain-specific levels of stability with different profiles identified between zoonotic H3, H5, and H7 subtype viruses associated with human infection. As studies continue to elucidate the complex components governing virus transmissibility, notably aerosol matrices and environmental parameters, considering the relative role of subtype- and strain-specific factors to modulate these parameters will improve our understanding of the pandemic potential of zoonotic influenza A viruses. IMPORTANCE Transmission of respiratory pathogens through the air can facilitate the rapid and expansive spread of infection and disease through a susceptible population. While seasonal influenza viruses are quite capable of airborne spread, there is a lack of knowledge regarding how well influenza viruses remain viable after aerosolization and whether influenza viruses capable of jumping species barriers to cause human infection differ in this property from seasonal strains. We evaluated a diverse panel of influenza viruses associated with human infection (originating from human, avian, and swine reservoirs) for their ability to remain viable after aerosolization in the laboratory under a range of conditions. We found greater diversity among avian and swine-origin viruses compared to seasonal influenza viruses; strain-specific stability was also noted. Although influenza virus stability in aerosols is an underreported property, if molecular markers associated with enhanced stability are identified, we will be able to quickly recognize emerging strains of influenza that present the greatest pandemic threat.

Evaluation of cycle threshold values at deisolation.

The decision to discontinue isolation in hospitalized patients with persistently positive severe acute respiratory coronavirus virus 2 (SARS-CoV-2) molecular testing is nuanced. Improvement in clinical status should be evaluated with expert consultation when considering whether discontinuation of isolation is appropriate. The cycle threshold value may serve as a useful adjunct to this decision-making process.

Author Correction: Aerosol and surface contamination of SARS-CoV-2 observed in quarantine and isolation care.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetically and Antigenically Divergent Influenza A(H9N2) Viruses Exhibit Differential Replication and Transmission Phenotypes in Mammalian Models.

Low-pathogenicity avian influenza A(H9N2) viruses, enzootic in poultry populations in Asia, are associated with fewer confirmed human infections but higher rates of seropositivity compared to A(H5) or A(H7) subtype viruses. Cocirculation of A(H5) and A(H7) viruses leads to the generation of reassortant viruses bearing A(H9N2) internal genes with markers of mammalian adaptation, warranting continued surveillance in both avian and human populations. Here, we describe active surveillance efforts in live poultry markets in Vietnam in 2018 and compare representative viruses to G1 and Y280 lineage viruses that have infected humans. Receptor binding properties, pH thresholds for HA activation, in vitro replication in human respiratory tract cells, and in vivo mammalian pathogenicity and transmissibility were investigated. While A(H9N2) viruses from both poultry and humans exhibited features associated with mammalian adaptation, one human isolate from 2018, A/Anhui-Lujiang/39/2018, exhibited increased capacity for replication and transmission, demonstrating the pandemic potential of A(H9N2) viruses.IMPORTANCE A(H9N2) influenza viruses are widespread in poultry in many parts of the world and for over 20 years have sporadically jumped species barriers to cause human infection. As these viruses continue to diversify genetically and antigenically, it is critical to closely monitor viruses responsible for human infections, to ascertain if A(H9N2) viruses are acquiring properties that make them better suited to infect and spread among humans. In this study, we describe an active poultry surveillance system established in Vietnam to identify the scope of influenza viruses present in live bird markets and the threat they pose to human health. Assessment of a recent A(H9N2) virus isolated from an individual in China in 2018 is also reported, and it was found to exhibit properties of adaptation to humans and, importantly, it shows similarities to strains isolated from the live bird markets of Vietnam.

Clinical evaluation of the BioFire® Respiratory Panel 2.1 and detection of SARS-CoV-2.

We evaluated the performance of the BioFire® Respiratory Panel 2.1 (RP2.1) in the detection of SARS CoV-2 in comparison against three other SARS CoV-2 EUA assays. In these studies, the RP2.1 panel had 98 % positive percent agreement (48/49) and 100 % negative percent agreement (49/49). Since 30 % of nasopharyngeal swab specimens have a SARS CoV-2 Ct >30 and thus detection of virus in low titers is clinically relevant, a sample with a high titer was diluted and each 10 fold dilution was tested in triplicate and compared against 6 other EUA approved SARS CoV-2 assays. These data suggested that the BioFire® RP2.1 panel, along with four other SARS CoV-2 assays (Roche cobas, Cepheid Xpert Xpress, BioFire® Defense COVID19, and NECoV19), consistently detected viral RNA at the 10-7 dilution. Overall, these studies suggest that the BioFire® RP2.1 assay can be used to detect acute cases of SARS CoV2 in addition to patients with low viral titer later in disease presentation.

Aerosol and surface contamination of SARS-CoV-2 observed in quarantine and isolation care.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originated in Wuhan, China in late 2019, and its resulting coronavirus disease, COVID-19, was declared a pandemic by the World Health Organization on March 11, 2020. The rapid global spread of COVID-19 represents perhaps the most significant public health emergency in a century. As the pandemic progressed, a continued paucity of evidence on routes of SARS-CoV-2 transmission has resulted in shifting infection prevention and control guidelines between classically-defined airborne and droplet precautions. During the initial isolation of 13 individuals with COVID-19 at the University of Nebraska Medical Center, we collected air and surface samples to examine viral shedding from isolated individuals. We detected viral contamination among all samples, supporting the use of airborne isolation precautions when caring for COVID-19 patients.

Advanced Preparation Makes Research in Emergencies and Isolation Care Possible: The Case of Novel Coronavirus Disease (COVID-19).

The optimal time to initiate research on emergencies is before they occur. However, timely initiation of high-quality research may launch during an emergency under the right conditions. These include an appropriate context, clarity in scientific aims, preexisting resources, strong operational and research structures that are facile, and good governance. Here, Nebraskan rapid research efforts early during the 2020 coronavirus disease pandemic, while participating in the first use of U.S. federal quarantine in 50 years, are described from these aspects, as the global experience with this severe emerging infection grew apace. The experience has lessons in purpose, structure, function, and performance of research in any emergency, when facing any threat.

Identification of key hemagglutinin residues responsible for cleavage, acid stability, and virulence of fifth-wave highly pathogenic avian influenza A(H7N9) viruses.

We previously demonstrated that despite no airborne transmissibility increase compared to low pathogenic avian influenza viruses, select human isolates of highly pathogenic avian influenza A(H7N9) virus exhibit greater virulence in animal models and a lower threshold pH for fusion. In the current study, we utilized both in vitro and in vivo approaches to identify key residues responsible for hemagglutinin (HA) intracellular cleavage, acid stability, and virulence in mice. We found that the four amino acid insertion (-KRTA-) at the HA cleavage site of A/Taiwan/1/2017 virus is essential for HA intracellular cleavage and contributes to disease in mice. Furthermore, a lysine to glutamic acid mutation at position HA2-64 increased the threshold pH for HA activation, reduced virus stability, and replication in mice. Identification of a key residue responsible for enhanced acid stability of A(H7N9) viruses is of great significance for future surveillance activities and improvements in vaccine stability.

Risk Assessment of Fifth-Wave H7N9 Influenza A Viruses in Mammalian Models.

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 hemagglutinin cleavability. Here, several human low-pathogenic avian influenza (LPAI) and HPAI H7N9 virus 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 an 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 than 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.IMPORTANCE The potential pandemic risk posed by avian influenza H7N9 viruses was heightened during the fifth epidemic wave in China due to the sudden increase in the 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 was evaluated for its 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 than LPAI H7N9 viruses suggests 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.

Infection of Cultured Mammalian Cells with Aerosolized Influenza Virus.

Inoculation of animals via inhaled aerosols has long been used to study the infectivity and pathogenesis of both influenza virus and other respiratory pathogens in a context that mimics natural infection. In contrast, traditional in vitro studies of cellular tropism have been limited to the use of liquid inocula. We have recently shown that cultured cells can become successfully infected after exposure to aerosolized influenza virus. In this chapter, we describe the methodology employed, including the operation of aerosolization instrumentation and calculation of infectious dose, both in experimental planning and after exposure occurs.

Pathogenesis and Transmission of Genetically Diverse Swine-Origin H3N2 Variant Influenza A Viruses from Multiple Lineages Isolated in the United States, 2011-2016.

While several swine-origin influenza A H3N2 variant (H3N2v) viruses isolated from humans prior to 2011 have been previously characterized for their virulence and transmissibility in ferrets, the recent genetic and antigenic divergence of H3N2v viruses warrants an updated assessment of their pandemic potential. Here, four contemporary H3N2v viruses isolated during 2011 to 2016 were evaluated for their replicative ability in both in vitro and in vivo in mammalian models as well as their transmissibility among ferrets. We found that all four H3N2v viruses possessed similar or enhanced replication capacities in a human bronchial epithelium cell line (Calu-3) compared to a human seasonal influenza virus, suggestive of strong fitness in human respiratory tract cells. The majority of H3N2v viruses examined in our study were mildly virulent in mice and capable of replicating in mouse lungs with different degrees of efficiency. In ferrets, all four H3N2v viruses caused moderate morbidity and exhibited comparable titers in the upper respiratory tract, but only 2 of the 4 viruses replicated in the lower respiratory tract in this model. Furthermore, despite efficient transmission among cohoused ferrets, recently isolated H3N2v viruses displayed considerable variance in their ability to transmit by respiratory droplets. The lack of a full understanding of the molecular correlates of virulence and transmission underscores the need for close genotypic and phenotypic monitoring of H3N2v viruses and the importance of continued surveillance to improve pandemic preparedness.IMPORTANCE Swine-origin influenza viruses of the H3N2 subtype, with the hemagglutinin (HA) and neuraminidase (NA) derived from historic human seasonal influenza viruses, continue to cross species barriers and cause human infections, posing an indelible threat to public health. To help us better understand the potential risk associated with swine-origin H3N2v viruses that emerged in the United States during the 2011-2016 influenza seasons, we use both in vitro and in vivo models to characterize the abilities of these viruses to replicate, cause disease, and transmit in mammalian hosts. The efficient respiratory droplet transmission exhibited by some of the H3N2v viruses in the ferret model combined with the existing evidence of low immunity against such viruses in young children and older adults highlight their pandemic potential. Extensive surveillance and risk assessment of H3N2v viruses should continue to be an essential component of our pandemic preparedness strategy.

Antigenically Diverse Swine Origin H1N1 Variant Influenza Viruses Exhibit Differential Ferret Pathogenesis and Transmission Phenotypes.

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 the 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. Preexisting 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 nonfatal 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 preexisting immunity to the pandemic A(H1N1) virus were challenged with A/Ohio/09/2015, demonstrating decreased protection. These data illustrate the potential for currently circulating swine influenza viruses to infect and cause illness in humans with preexisting immunity to H1N1 pandemic 2009 viruses and a need for ongoing risk assessment and development of candidate vaccine viruses for improved pandemic preparedness.

Infection and Replication of Influenza Virus at the Ocular Surface.

Although influenza viruses typically cause respiratory tract disease, some viruses, particularly those with an H7 hemagglutinin, have been isolated from the eyes of conjunctivitis cases. Previous work has shown that isolates of multiple subtypes from both ocular and respiratory infections are capable of replication in human ex vivo ocular tissues and corneal or conjunctival cell monolayers, leaving the determinants of ocular tropism unclear. Here, we evaluated the effect of several variables on tropism for ocular cells cultured in vitro and examined the potential effect of the tear film on viral infectivity. All viruses tested were able to replicate in primary human corneal epithelial cell monolayers subjected to aerosol inoculation. The temperature at which cells were cultured postinoculation minimally affected infectivity. Replication efficiency, in contrast, was reduced at 33°C relative to that at 37°C, and this effect was slightly greater for the conjunctivitis isolates than for the respiratory ones. With the exception of a seasonal H3N2 virus, the subset of viruses studied in multilayer corneal tissue constructs also replicated productively after either aerosol or liquid inoculation. Human tears significantly inhibited the hemagglutination of both ocular and nonocular isolates, but the effect on viral infectivity was more variable, with tears reducing the infectivity of nonocular isolates more than ocular isolates. These data suggest that most influenza viruses may be capable of establishing infection if they reach the surface of ocular cells but that this is more likely for ocular-tropic viruses, as they are better able to maintain their infectivity during passage through the tear film.IMPORTANCE The potential spread of zoonotic influenza viruses to humans represents an important threat to public health. Unfortunately, despite the importance of cellular and tissue tropism to pathogenesis, determinants of influenza virus tropism have yet to be fully elucidated. Here, we sought to identify factors that limit the ability of most influenza viruses to cause ocular infection. Although ocular symptoms in humans caused by avian influenza viruses tend to be relatively mild, these infections are concerning due to the potential of the ocular surface to serve as a portal of entry for viruses that go on to establish respiratory infections. Furthermore, a better understanding of the factors that influence infection and replication in this noncanonical site may point toward novel determinants of tropism in the respiratory tract.

Pathogenesis, Transmissibility, and Tropism of a Highly Pathogenic Avian Influenza A(H7N7) Virus Associated With Human Conjunctivitis in Italy, 2013.

H7 subtype influenza viruses represent a persistent public health threat because of their continued detection in poultry and ability to cause human infection. An outbreak of highly pathogenic avian influenza H7N7 virus in Italy during 2013 resulted in 3 cases of human conjunctivitis. We determined the pathogenicity and transmissibility of influenza A/Italy/3/2013 virus in mouse and ferret models and examined the replication kinetics of this virus in several human epithelial cell types. The moderate virulence observed in mammalian models and the capacity for transmission in a direct contact model underscore the need for continued study of H7 subtype viruses.

Assessment of Molecular, Antigenic, and Pathological Features of Canine Influenza A(H3N2) Viruses That Emerged in the United States.

Background: A single subtype of canine influenza virus (CIV), A(H3N8), was circulating in the United States until a new subtype, A(H3N2), was detected in Illinois in spring 2015. Since then, this CIV has caused thousands of infections in dogs in multiple states. Methods: In this study, genetic and antigenic properties of the new CIV were evaluated. In addition, structural and glycan array binding features of the recombinant hemagglutinin were determined. Replication kinetics in human airway cells and pathogenesis and transmissibility in animal models were also assessed. Results: A(H3N2) CIVs maintained molecular and antigenic features related to low pathogenicity avian influenza A(H3N2) viruses and were distinct from A(H3N8) CIVs. The structural and glycan array binding profile confirmed these findings and revealed avian-like receptor-binding specificity. While replication kinetics in human airway epithelial cells was on par with that of seasonal influenza viruses, mild-to-moderate disease was observed in infected mice and ferrets, and the virus was inefficiently transmitted among cohoused ferrets. Conclusions: Further adaptation is needed for A(H3N2) CIVs to present a likely threat to humans. However, the potential for coinfection of dogs and possible reassortment of human and other animal influenza A viruses presents an ongoing risk to public health.