Quantitative Description of Glycan-Receptor Binding of Influenza A Virus H7 Hemagglutinin

In the context of recently emerged novel influenza strains through reassortment, avian influenza subtypes such as H5N1, H7N7, H7N2, H7N3 and H9N2 pose a constant threat in terms of their adaptation to the human host. Among these subtypes, it was recently demonstrated that mutations in H5 and H9 hemagglutinin (HA) in the context of lab-generated reassorted viruses conferred aerosol transmissibility in ferrets (a property shared by human adapted viruses). We previously demonstrated that the quantitative binding affinity of HA to α2→6 sialylated glycans (human receptors) is one of the important factors governing human adaptation of HA. Although the H7 subtype has infected humans causing varied clinical outcomes from mild conjunctivitis to severe respiratory illnesses, it is not clear where the HA of these subtypes stand in regard to human adaptation since its binding affinity to glycan receptors has not yet been quantified. In this study, we have quantitatively characterized the glycan receptor-binding specificity of HAs from representative strains of Eurasian (H7N7) and North American (H7N2) lineages that have caused human infection. Furthermore, we have demonstrated for the first time that two specific mutations; Gln226→Leu and Gly228→Ser in glycan receptor-binding site of H7 HA substantially increase its binding affinity to human receptor. Our findings contribute to a framework for monitoring the evolution of H7 HA to be able to adapt to human host.National Institutes of Health (U.S.) (GM R37 GM057073-13)Singapore-MIT Alliance for Research and Technolog

Variations in the Hemagglutinin of the 2009 H1N1 Pandemic Virus: Potential for Strains with Altered Virulence Phenotype?

A novel, swine-origin influenza H1N1 virus (H1N1pdm) caused the first pandemic of the 21st century. This pandemic, although efficient in transmission, is mild in virulence. This atypical mild pandemic season has raised concerns regarding the potential of this virus to acquire additional virulence markers either through further adaptation or possibly by immune pressure in the human host. Using the mouse model we generated, within a single round of infection with A/California/04/09/H1N1 (Ca/04), a virus lethal in mice—herein referred to as mouse-adapted Ca/04 (ma-Ca/04). Five amino acid substitutions were found in the genome of ma-Ca/04: 3 in HA (D131E, S186P and A198E), 1 in PA (E298K) and 1 in NP (D101G). Reverse genetics analyses of these mutations indicate that all five mutations from ma-Ca/04 contributed to the lethal phenotype; however, the D131E and S186P mutations—which are also found in the 1918 and seasonal H1N1 viruses—in HA alone were sufficient to confer virulence of Ca/04 in mice. HI assays against H1N1pdm demonstrate that the D131E and S186P mutations caused minor antigenic changes and, likely, affected receptor binding. The rapid selection of ma-Ca/04 in mice suggests that a virus containing this constellation of amino acids might have already been present in Ca/04, likely as minor quasispecies

Monkeypox Disease Transmission in an Experimental Setting: Prairie Dog Animal Model

Monkeypox virus (MPXV) is considered the most significant human public health threat in the genus Orthopoxvirus since the eradication of variola virus (the causative agent of smallpox). MPXV is a zoonotic agent endemic to forested areas of Central and Western Africa. In 2003, MPXV caused an outbreak in the United States due to the importation of infected African rodents, and subsequent sequential infection of North American prairie dogs (Cynomys ludovicianus) and humans. In previous studies, the prairie dog MPXV model has successfully shown to be very useful for understanding MPXV since the model emulates key characteristics of human monkeypox disease. In humans, percutaneous exposure to animals has been documented but the primary method of human-to-human MPXV transmission is postulated to be by respiratory route. Only a few animal model studies of MPXV transmission have been reported. Herein, we show that MPXV infected prairie dogs are able to transmit the virus to naive animals through multiple transmission routes. All secondarily exposed animals were infected with MPXV during the course of the study. Notably, animals secondarily exposed appeared to manifest more severe disease; however, the disease course was very similar to those of experimentally challenged animals including inappetence leading to weight loss, development of lesions, production of orthopoxvirus antibodies and shedding of similar levels or in some instances higher levels of MPXV from the oral cavity. Disease was transmitted via exposure to contaminated bedding, co-housing, or respiratory secretions/nasal mucous (we could not definitively say that transmission occurred via respiratory route exclusively). Future use of the model will allow us to evaluate infection control measures, vaccines and antiviral strategies to decrease disease transmission

Milk Lacking α-Casein Leads to Permanent Reduction in Body Size in Mice

The major physiological function of milk is the transport of amino acids, carbohydrates, lipids and minerals to mammalian offspring. Caseins, the major milk proteins, are secreted in the form of a micelle consisting of protein and calcium-phosphate

A Single E627K Mutation in the PB2 Protein of H9N2 Avian Influenza Virus Increases Virulence by Inducing Higher Glucocorticoids (GCs) Level

While repeated infection of humans and enhanced replication and transmission in mice has attracted more attention to it, the pathogenesis of H9N2 virus was less known in mice. PB2 residue 627 as the virulent determinant of H5N1 virus is associated with systemic infection and impaired TCR activation, but the impact of this position in H9N2 virus on the host immune response has not been evaluated. In this study, we quantified the cellular immune response to infection in the mouse lung and demonstrate that VK627 and rTsE627K infection caused a significant reduction in the numbers of T cells and inflammatory cells (Macrophage, Neutrophils, Dendritic cells) compared to mice infected with rVK627E and TsE627. Further, we discovered (i) a high level of thymocyte apoptosis resulted in impaired T cell development, which led to the reduced amount of mature T cells into lung, and (ii) the reduced inflammatory cells entering into lung was attributed to the diminished levels in pro-inflammatory cytokines and chemokines. Thereafter, we recognized that higher GCs level in plasma induced by VK627 and rTsE627K infection was associated with the increased apoptosis in thymus and the reduced pro-inflammatory cytokines and chemokines levels in lung. These data demonstrated that VK627 and rTsE627K infection contributing to higher GCs level would decrease the magnitude of antiviral response in lung, which may be offered as a novel mechanism of enhanced pathogenicity for H9N2 AIV

Evidence of Expanded Host Range and Mammalian-Associated Genetic Changes in a Duck H9N2 Influenza Virus Following Adaptation in Quail and Chickens

H9N2 avian influenza viruses continue to circulate worldwide; in Asia, H9N2 viruses have caused disease outbreaks and established lineages in land-based poultry. Some H9N2 strains are considered potentially pandemic because they have infected humans causing mild respiratory disease. In addition, some of these H9N2 strains replicate efficiently in mice without prior adaptation suggesting that H9N2 strains are expanding their host range. In order to understand the molecular basis of the interspecies transmission of H9N2 viruses, we adapted in the laboratory a wildtype duck H9N2 virus, influenza A/duck/Hong Kong/702/79 (WT702) virus, in quail and chickens through serial lung passages. We carried out comparative analysis of the replication and transmission in quail and chickens of WT702 and the viruses obtained after 23 serial passages in quail (QA23) followed by 10 serial passages in chickens (QA23CkA10). Although the WT702 virus can replicate and transmit in quail, it replicates poorly and does not transmit in chickens. In contrast, the QA23CkA10 virus was very efficient at replicating and transmitting in quail and chickens. Nucleotide sequence analysis of the QA23 and QA23CkA10 viruses compared to the WT702 virus indicated several nucleotide substitutions resulting in amino acid changes within the surface and internal proteins. In addition, a 21-amino acid deletion was found in the stalk of the NA protein of the QA23 virus and was maintained without further modification in the QA23CkA10 adapted virus. More importantly, both the QA23 and the QA23CkA10 viruses, unlike the WT702 virus, were able to readily infect mice, produce a large-plaque phenotype, showed faster replication kinetics in tissue culture, and resulted in the quick selection of the K627 amino acid mammalian-associated signature in PB2. These results are in agreement with the notion that adaptation of H9 viruses to land-based birds can lead to strains with expanded host range

Multivalent HA DNA Vaccination Protects against Highly Pathogenic H5N1 Avian Influenza Infection in Chickens and Mice

Sustained outbreaks of highly pathogenic avian influenza (HPAI) H5N1 in avian species increase the risk of reassortment and adaptation to humans. The ability to contain its spread in chickens would reduce this threat and help maintain the capacity for egg-based vaccine production. While vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses.The ability of DNA vaccines encoding hemagglutinin (HA) proteins from different HPAI H5N1 serotypes was evaluated for its ability to elicit neutralizing antibodies and to protect against homologous and heterologous HPAI H5N1 strain challenge in mice and chickens after DNA immunization by needle and syringe or with a pressure injection device. These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs. The response was dose-dependent, and breadth was determined by the choice of the influenza virus HA in the vaccine. Monovalent and trivalent HA vaccines were tested first in mice and conferred protection against lethal H5N1 A/Vietnam/1203/2004 challenge 68 weeks after vaccination. In chickens, protection was observed against heterologous strains of HPAI H5N1 after vaccination with a trivalent H5 serotype DNA vaccine with doses as low as 5 microg DNA given twice either by intramuscular needle injection or with a needle-free device.DNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. In addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates

Characterization of an Artificial Swine-Origin Influenza Virus with the Same Gene Combination as H1N1/2009 Virus: A Genesis Clue of Pandemic Strain

Pandemic H1N1/2009 influenza virus, derived from a reassortment of avian, human, and swine influenza viruses, possesses a unique gene segment combination that had not been detected previously in animal and human populations. Whether such a gene combination could result in the pathogenicity and transmission as H1N1/2009 virus remains unclear. In the present study, we used reverse genetics to construct a reassortant virus (rH1N1) with the same gene combination as H1N1/2009 virus (NA and M genes from a Eurasian avian-like H1N1 swine virus and another six genes from a North American triple-reassortant H1N2 swine virus). Characterization of rH1N1 in mice showed that this virus had higher replicability and pathogenicity than those of the seasonal human H1N1 and Eurasian avian-like swine H1N1 viruses, but was similar to the H1N1/2009 and triple-reassortant H1N2 viruses. Experiments performed on guinea pigs showed that rH1N1 was not transmissible, whereas pandemic H1N1/2009 displayed efficient transmissibility. To further determine which gene segment played a key role in transmissibility, we constructed a series of reassortants derived from rH1N1 and H1N1/2009 viruses. Direct contact transmission studies demonstrated that the HA and NS genes contributed to the transmission of H1N1/2009 virus. Second, the HA gene of H1N1/2009 virus, when combined with the H1N1/2009 NA gene, conferred efficient contact transmission among guinea pigs. The present results reveal that not only gene segment reassortment but also amino acid mutation were needed for the generation of the pandemic influenza virus

Eurasian-Origin Gene Segments Contribute to the Transmissibility, Aerosol Release, and Morphology of the 2009 Pandemic H1N1 Influenza Virus

The epidemiological success of pandemic and epidemic influenza A viruses relies on the ability to transmit efficiently from person-to-person via respiratory droplets. Respiratory droplet (RD) transmission of influenza viruses requires efficient replication and release of infectious influenza particles into the air. The 2009 pandemic H1N1 (pH1N1) virus originated by reassortment of a North American triple reassortant swine (TRS) virus with a Eurasian swine virus that contributed the neuraminidase (NA) and M gene segments. Both the TRS and Eurasian swine viruses caused sporadic infections in humans, but failed to spread from person-to-person, unlike the pH1N1 virus. We evaluated the pH1N1 and its precursor viruses in a ferret model to determine the contribution of different viral gene segments on the release of influenza virus particles into the air and on the transmissibility of the pH1N1 virus. We found that the Eurasian-origin gene segments contributed to efficient RD transmission of the pH1N1 virus likely by modulating the release of influenza viral RNA-containing particles into the air. All viruses replicated well in the upper respiratory tract of infected ferrets, suggesting that factors other than viral replication are important for the release of influenza virus particles and transmission. Our studies demonstrate that the release of influenza viral RNA-containing particles into the air correlates with increased NA activity. Additionally, the pleomorphic phenotype of the pH1N1 virus is dependent upon the Eurasian-origin gene segments, suggesting a link between transmission and virus morphology. We have demonstrated that the viruses are released into exhaled air to varying degrees and a constellation of genes influences the transmissibility of the pH1N1 virus

Exhaled Aerosol Transmission of Pandemic and Seasonal H1N1 Influenza Viruses in the Ferret

Person-to-person transmission of influenza viruses occurs by contact (direct and fomites) and non-contact (droplet and small particle aerosol) routes, but the quantitative dynamics and relative contributions of these routes are incompletely understood. The transmissibility of influenza strains estimated from secondary attack rates in closed human populations is confounded by large variations in population susceptibilities. An experimental method to phenotype strains for transmissibility in an animal model could provide relative efficiencies of transmission. We developed an experimental method to detect exhaled viral aerosol transmission between unanesthetized infected and susceptible ferrets, measured aerosol particle size and number, and quantified the viral genomic RNA in the exhaled aerosol. During brief 3-hour exposures to exhaled viral aerosols in airflow-controlled chambers, three strains of pandemic 2009 H1N1 strains were frequently transmitted to susceptible ferrets. In contrast one seasonal H1N1 strain was not transmitted in spite of higher levels of viral RNA in the exhaled aerosol. Among three pandemic strains, the two strains causing weight loss and illness in the intranasally infected ‘donor’ ferrets were transmitted less efficiently from the donor than the strain causing no detectable illness, suggesting that the mucosal inflammatory response may attenuate viable exhaled virus. Although exhaled viral RNA remained constant, transmission efficiency diminished from day 1 to day 5 after donor infection. Thus, aerosol transmission between ferrets may be dependent on at least four characteristics of virus-host relationships including the level of exhaled virus, infectious particle size, mucosal inflammation, and viral replication efficiency in susceptible mucosa