Emergence Of Influenza Viruses Research Articles

Nonreplicating influenza A virus vaccines confer broad protection against lethal challenge.

ABSTRACTNew vaccine technologies are being investigated for their ability to elicit broadly cross-protective immunity against a range of influenza viruses. We compared the efficacies of two intranasally delivered nonreplicating influenza virus vaccines (H1 and H5 S-FLU) that are based on the suppression of the hemagglutinin signal sequence, with the corresponding H1N1 and H5N1 cold-adapted (ca) live attenuated influenza virus vaccines in mice and ferrets. Administration of two doses of H1 or H5 S-FLU vaccines protected mice and ferrets from lethal challenge with homologous, heterologous, and heterosubtypic influenza viruses, and two doses of S-FLU and ca vaccines yielded comparable effects. Importantly, when ferrets immunized with one dose of H1 S-FLU or ca vaccine were challenged with the homologous H1N1 virus, the challenge virus failed to transmit to naive ferrets by the airborne route. S-FLU technology can be rapidly applied to any emerging influenza virus, and the promising preclinical data support further evaluation in humans.

Detection and Isolation of Swine Influenza A Virus in Spiked Oral Fluid and Samples from Individually Housed, Experimentally Infected Pigs: Potential Role of Porcine Oral Fluid in Active Influenza A Virus Surveillance in Swine.

BackgroundThe lack of seasonality of swine influenza A virus (swIAV) in combination with the capacity of swine to harbor a large number of co-circulating IAV lineages, resulting in the risk for the emergence of influenza viruses with pandemic potential, stress the importance of swIAV surveillance. To date, active surveillance of swIAV worldwide is barely done because of the short detection period in nasal swab samples. Therefore, more sensitive diagnostic methods to monitor circulating virus strains are requisite.MethodsqRT-PCR and virus isolations were performed on oral fluid and nasal swabs collected from individually housed pigs that were infected sequentially with H1N1 and H3N2 swIAV strains. The same methods were also applied to oral fluid samples spiked with H1N1 to study the influence of conservation time and temperature on swIAV infectivity and detectability in porcine oral fluid.ResultsAll swIAV infected animals were found qRT-PCR positive in both nasal swabs and oral fluid. However, swIAV could be detected for a longer period in oral fluid than in nasal swabs. Despite the high detectability of swIAV in oral fluid, virus isolation from oral fluid collected from infected pigs was rare. These results are supported by laboratory studies showing that the PCR detectability of swIAV remains unaltered during a 24 h incubation period in oral fluid, while swIAV infectivity drops dramatically immediately upon contact with oral fluid (3 log titer reduction) and gets lost after 24 h conservation in oral fluid at ambient temperature.ConclusionsOur data indicate that porcine oral fluid has the potential to replace nasal swabs for molecular diagnostic purposes. The difficulty to isolate swIAV from oral fluid could pose a drawback for its use in active surveillance programs.

Replication and Transmission of the Novel Bovine Influenza D Virus in a Guinea Pig Model.

Influenza D virus (FLUDV) is a novel influenza virus that infects cattle and swine. The goal of this study was to investigate the replication and transmission of bovine FLUDV in guinea pigs. Following direct intranasal inoculation of animals, the virus was detected in nasal washes of infected animals during the first 7 days postinfection. High viral titers were obtained from nasal turbinates and lung tissues of directly inoculated animals. Further, bovine FLUDV was able to transmit from the infected guinea pigs to sentinel animals by means of contact and not by aerosol dissemination under the experimental conditions tested in this study. Despite exhibiting no clinical signs, infected guinea pigs developed seroconversion and the viral antigen was detected in lungs of animals by immunohistochemistry. The observation that bovine FLUDV replicated in the respiratory tract of guinea pigs was similar to observations described previously in studies of gnotobiotic calves and pigs experimentally infected with bovine FLUDV but different from those described previously in experimental infections in ferrets and swine with a swine FLUDV, which supported virus replication only in the upper respiratory tract and not in the lower respiratory tract, including lung. Our study established that guinea pigs could be used as an animal model for studying this newly emerging influenza virus. Influenza D virus (FLUDV) is a novel emerging pathogen with bovine as its primary host. The epidemiology and pathogenicity of the virus are not yet known. FLUDV also spreads to swine, and the presence of FLUDV-specific antibodies in humans could indicate that there is a potential for zoonosis. Our results showed that bovine FLUDV replicated in the nasal turbinate and lungs of guinea pigs at high titers and was also able to transmit from an infected animal to sentinel animals by contact. The fact that bovine FLUDV replicated productively in both the upper and lower respiratory tracts of guinea pigs, similarly to virus infection in its native host, demonstrates that guinea pigs would be a suitable model host to study the replication and transmission potential of bovine FLUDV.

Serological evidence for the presence of influenza D virus in small ruminants

Influenza D virus (FLUDV) was isolated from diseased pigs with respiratory disease symptoms in 2011, and since then the new virus has also been spread to cattle. Little is known about the susceptibility of other agricultural animals and poultry to FLUDV. This study was designed to determine if other farm animals such as goats, sheep, chickens, and turkey are possible hosts to this newly emerging influenza virus. 648 goat and sheep serum samples and 250 chicken and turkey serum samples were collected from 141 small ruminant and 25 poultry farms from different geographical locations in the United States and Canada. Serum samples were examined using the hemagglutination inhibition (HI) assay and the sheep and goat samples were further analyzed using the serum neutralization assay. Results of this study showed FLUDV antibodies were detected in 13.5% (17/126) of the sampled sheep farms, and 5.2% (29/557) of tested sheep serum samples were positive for FLUDV antibodies. For the goat results, the FLUDV antibodies were detected in 13.3% (2/15) of the sampled farms, and 8.8% (8/91) of the tested goat serum samples were positive for FLUDV antibodies. Furthermore, all tested poultry serum samples were negative for FLUDV antibodies. Our data demonstrated that sheep and goat are susceptible to FLUDV virus and multiple states in U.S. have this virus infection already in these two species. This new finding highlights a need for future surveillance of FLUDV virus in small ruminants toward better understanding both the origin and natural reservoir of this new virus.

Herd immunity and fatal cases of influenza among the population exposed to poultry and wild birds in Russian Asia in 2013-2014.

In total 1,525 blood serum samples were collected in October, 2013 in Russian Asia from people who reside in territories that are at high risk for emergence of influenza viruses with pandemic potential. Presence of antibodies to influenza viruses in the sera was tested in hemagglutination inhibition test. None of the samples produced positive results with the antigens A/H5 and A/H7. Twelve strains of influenza A(H1N1pdm09) virus were isolated from people who died presumably from influenza during 2013-2014 epidemic season. All strains were similar to vaccine strain A/California/07/09 according to their antigenic properties and sensitivity to anti-neuraminidase drugs (oseltamivir and zanamivir). Genetic analysis revealed that all strains belong to group 6, subgroup 6B of influenza A(H1N1)pdm09 virus. Substitutions in HA1: S164F add E235K as well as E47G, A86V, K331R, N386K, N397K in NA, and K131E, N29S in NS1, and N29S, R34Q in NEP separate investigated strains into two groups: 1st group-A/Chita/1114/2014, A/Chita/1115/2014, A/Chita/853/2014, A/Barnaul/269/2014 and 2nd group-A/Chita/655/2014, A/Chita/656/2014, A/Chita/709/2014, A/Chita/873/2014. Mutation D222G in HA1, which is often associated with high morbidity of the illness, was present in strain A/Novosibirsk/114/2014. Substitution N386K in NA removes a potential N-glycosylation site in neuraminidases of A/Chita/1114/2014, A/Chita/1115/2014, A/Chita/853/2014, A/Barnaul/269/2014, A/Novosibirsk/114/2014, and A/Blagoveshensk/252/2014.

A drug-disease model describing the effect of oseltamivir neuraminidase inhibition on influenza virus progression.

A population drug-disease model was developed to describe the time course of influenza virus with and without oseltamivir treatment and to investigate opportunities for antiviral combination therapy. Data included viral titers from 208 subjects, across 4 studies, receiving placebo and oseltamivir at 20 to 200 mg twice daily for 5 days. A 3-compartment mathematical model, comprising target cells infected at rate β, free virus produced at rate p and cleared at rate c, and infected cells cleared at rate δ, was implemented in NONMEM with an inhibitory Hill function on virus production (p), accounting for the oseltamivir effect. In congruence with clinical data, the model predicts that the standard 75-mg regimen initiated 2 days after infection decreased viral shedding duration by 1.5 days versus placebo; the 150-mg regimen decreased shedding by an additional average 0.25 day. The model also predicts that initiation of oseltamivir sooner postinfection, specifically at day 0.5 or 1, results in proportionally greater decreases in viral shedding duration of 5 and 3.5 days, respectively. Furthermore, the model suggests that combining oseltamivir (acting to subdue virus production rate) with an antiviral whose activity decreases viral infectivity (β) results in a moderate additive effect dependent on therapy initiation time. In contrast, the combination of oseltamivir with an antiviral whose activity increases viral clearance (c) shows significant additive effects independent of therapy initiation time. The utility of the model for investigating the pharmacodynamic effects of novel antivirals alone or in combination on emergent influenza virus strains warrants further investigation.

Cats as a potential source of emerging influenza virus infections

Based on the findings of the present study, we conclude that cats can be infected with human influenza viruses as well as avian influenza viruses. Actually, the recent study has shown that both human-type(α2, 6-linked sialic acid) and avian-type(α2, 3-linked sialic acid)influenza virus receptors were extensively detected in the respiratory organs such as trachea, bronchus, and lung of the domestic cats(Wang et al., 2013). Therefore, cats may act as a vector for human influenza virus transmission within households, posing a potential public health concern. Furthermore, we detected both H5N1 and human virus-seropositive cats in neighboring areas at similar sampling times, suggesting that cats can be simultaneously infected with both avian and human viruses in H5N1 virus-endemic areas. Thus, cats, like pigs, may act as an intermediate host for the emergence of new, potentially p and emic viruses.

Emerging influenza viruses and the prospect of a universal influenza virus vaccine.

Influenza viruses cause annual seasonal epidemics and pandemics at irregular intervals. Several cases of human infections with avian and swine influenza viruses have been detected recently, warranting enhanced surveillance and the development of more effective countermeasures to address the pandemic potential of these viruses. The most effective countermeasure against influenza virus infection is the use of prophylactic vaccines. However, vaccines that are currently in use for seasonal influenza viruses have to be re-formulated and re-administered in a cumbersome process every year due to the antigenic drift of the virus. Furthermore, current seasonal vaccines are ineffective against novel pandemic strains. This paper reviews zoonotic influenza viruses with pandemic potential and technological advances towards better vaccines that induce broad and long lasting protection from influenza virus infection. Recent efforts have focused on the development of broadly protective/universal influenza virus vaccines that can provide immunity against drifted seasonal influenza virus strains but also against potential pandemic viruses.

The first lack of evidence of H7N9 avian influenza virus infections among pigs in Eastern China

In this study, we sought to examine whether evidence existed suggesting that pigs were being infected with the novel H7N9 avian influenza virus. From November 2012 to November 2013, blood was drawn from 1560 pigs from 100 large farms in 4 provinces of eastern China. Many of these pigs were in close proximity to wild birds or poultry. Swine sera were studied using hemagglutinin inhibition (HI) assays and enzyme-linked immunosorbent assays (ELISAs) against the H7 antigen derived from the emergent H7N9 avian influenza virus (AIV). Only 29 of the 1560 samples had HI titers of 1:20 when using the H7N9 AIV antigens, and none of the 29 (H7N9 AIV) HI-positive samples were positive when using ELISA, indicating that no samples were positive for H7N9. The negative results were also verified using a novel competitive HA-ELISA. As pigs have been shown to be infected with other avian influenza viruses and as the prevalence of novel influenza A viruses (e.g., H7N9 AIV) may be increasing among poultry in China, similar seroepidemiological studies of pigs should be periodically conducted in the future.

The development of a four-way linking framework in Egypt: an example of the FAO, OIE and WHO joint activities to facilitate national risk assessment.

Cross-sectoral assessment of health risks arising or existing at the human-animal interface is crucial to identifying and implementing effective national disease control measures. This requires availability of information from 4 functional information 'streams' - epidemiological, laboratory, animal, and human health. The Food and Agriculture Organization of the United Nations (FAO)/ World Organisation for Animal Health (OIE)/ World Health Organization (WHO) Four-Way Linking (4WL) project promotes the establishing of a national-level joint framework for data sharing, risk assessment, and risk communication, in order to both improve communications within and among governmental public health and animal health influenza laboratories, epidemiology offices, national partners, with the aim of strengthening the national capacity to detect, report and assess risks arising from emerging influenza viruses. The project is currently being implemented in countries where H5N1 avian influenza is endemic and where human cases have been reported. The project is comprised of two main activities at country level: a 'review mission', which is the project launch in the country and has the objective to assess the existing situation; and a 'scenario based workshop', with the scope to bring together key national partners and build relationships among people working in the 4 information streams and to improve understanding of national strengths and gaps. During the workshop the delegates engaged in interactive sessions on basic risk assessment and devoted to specify the needs and roles of the 4 different streams. The participants work through a mock influenza outbreak scenario, which practically illustrates how risk assessment and communication of an emergency at the animal-human interface is more effective when there is linking of the 4 streams, collaboration, communication, and coordinated action. In 2010, Egypt was the first country where the project was successfully implemented, followed by Vietnam and Indonesia.

Cross-protection against influenza virus infection by intranasal administration of nucleoprotein-based vaccine with compound 48/80 adjuvant

The nucleoprotein (NP) of influenza viruses is highly conserved and therefore has become one of the major targets of current universal influenza vaccine (UIV) studies. In this study, the recombinant nucleoprotein (NP) of the A/PR/8/34 (H1N1) influenza virus strain was expressed using an Escherichia coli (E. coli) expression system and then purified as a candidate UIV. The NP protein was administered intranasally or intraperitoneally twice at 3-week intervals to female BALB/c mice in combination with C48/80 adjuvant. Then, the mice were challenged with homologous or heterologous influenza viruses at a lethal dose 3 weeks after the last immunization. The results showed that the serum IgG titers of all of the mice immunized with NP reached a higher level and the protection provided by NP vaccine against the homologous virus depended on the administered dosage and adjuvant. In addition, immunization with 100 μg NP in combination with C48/80 adjuvant could provide good cross-protection against heterologous H9N2 avian influenza viruses. This study indicated that NP as a candidate antigen of UIV immunized intranasally could effectively induce mucosal and cell-mediated immunity, with the potential to control epidemics caused by the appearance of new emerging influenza viruses.

Challenges and Strategies of Laboratory Diagnosis for Newly Emerging Influenza Viruses in Taiwan: A Decade after SARS

Since the first case of severe acute respiratory syndrome (SARS) in Taiwan was identified in March 2003, viral respiratory infections, in particular the influenza virus, have become a national public health concern. Taiwan would face a serious threat of public health problems if another SARS epidemic overlapped with a flu outbreak. After SARS, the Taiwan Centers for Disease Control accelerated and strengthened domestic research on influenza and expanded the exchange of information with international counterparts. The capacity of influenza A to cross species barriers presents a potential threat to human health. Given the mutations of avian flu viruses such as H7N9, H6N1, and H10N8, all countries, including Taiwan, must equip themselves to face a possible epidemic or pandemic. Such preparedness requires global collaboration.

Different arms of the adaptive immune system induced by a combination vaccine work in concert to provide enhanced clearance of influenza.

Current split influenza virus vaccines that induce strain-specific neutralising antibodies provide some degree of protection against influenza infection but there is a clear need to improve their effectiveness. The constant antigenic drift of influenza viruses means that vaccines are often not an exact match to the circulating strain and so levels of relevant antibodies may not be sufficiently high to afford protection. In the situation where the emergent influenza virus is completely novel, as is the case with pandemic strains, existing vaccines may provide no benefit. In this study we tested the concept of a combination vaccine consisting of sub-optimal doses of split influenza virus vaccine mixed with a cross-protective T-cell inducing lipopeptide containing the TLR2 ligand Pam2Cys. Mice immunised with combination vaccines showed superior levels of lung viral clearance after challenge compared to either split virus or lipopeptide alone, mediated through activation of enhanced humoral and/or additional cellular responses. The mechanism of action of these vaccines was dependent on the route of administration, with intranasal administration being superior to subcutaneous and intramuscular routes, potentially through the induction of memory CD8+ T cells in the lungs. This immunisation strategy not only provides a mechanism for minimising the dose of split virus antigen but also, through the induction of cross-protective CD8+ T cells, proves a breadth of immunity to provide potential benefit upon encounter with serologically diverse influenza isolates.

Influenza surveillance in animals: what is our capacity to detect emerging influenza viruses with zoonotic potential?

A survey of national animal influenza surveillance programmes was conducted to assess the current capacity to detect influenza viruses with zoonotic potential in animals (i.e. those influenza viruses that can be naturally transmitted between animals and humans) at regional and global levels. Information on 587 animal influenza surveillance system components was collected for 99 countries from Chief Veterinary Officers (CVOs) (n=94) and published literature. Less than 1% (n=4) of these components were specifically aimed at detecting influenza viruses with pandemic potential in animals (i.e. those influenza viruses that are capable of causing epidemic spread in human populations over large geographical regions or worldwide), which would have zoonotic potential as a prerequisite. Those countries that sought to detect influenza viruses with pandemic potential searched for such viruses exclusively in domestic pigs. This work shows the global need for increasing surveillance that targets potentially zoonotic influenza viruses in relevant animal species.

Influenza: environmental remodeling, population dynamics, and the need to understand networks.

NEW CHALLENGES FOR PUBLIC HEALTH Emergence of new pathogens has been the reality of the 21st century; the role of animal reservoirs and changes in human behaviors may be the main key factors for disease dynamics. Human population growth and territory expansion have lead to habitat sharing between human beings, domestic animals, wild animals, and their pathogens; bringing new opportunities for spill over. Risk assessment regarding the main factors associated with potential reassortment and transmission between species should get to a stage where the analysis of wildlife networks and interactions with domestic animals and human beings is well mapped. This last will allow an accurate prevention and control of hot spots of influenza transmission (1, 2). The challenge for all public health professionals lies upon the integration of the analysis of environment, animal reservoirs, and human population as a whole and develop action plans accordingly to their interactions and not each of them separately (3–5). New animal production systems and human population dynamics have lead to different sources of infection that are not well understood. Although surveillance systems have improved considerably, the need for better communications and relations between environmental, health, and agricultural sectors is essential for a precise prevention of disease appearance and dispersal. Interdisciplinary and Intersectorial help becomes imperative when assessing the risk factors; such as human interactions with animals, wildlife contact with animal production systems, and environmental remodeling that contribute to disease emergence (6). In addition, the assessment of hot spots of influenza transmission should be the tool to map animal habitats that are at most risk of encounters with domestic animals that might serve as a mixing vessel and as the source of infection for humans (7, 8). The emergence of influenza viruses is just one example of many diseases that have social and environmental factors that enhance their appearance and dispersal. The new strains that have emerged have social and environmental issues in common, which contribute to the appearance of new viruses, or at least, to the spillover between species; and it is here where the efforts should focus (9, 10).

Modified vaccinia virus Ankara encoding influenza virus hemagglutinin induces heterosubtypic immunity in macaques.

Current influenza virus vaccines primarily aim to induce neutralizing antibodies (NAbs). Modified vaccinia virus Ankara (MVA) is a safe and well-characterized vector for inducing both antibody and cellular immunity. We evaluated the immunogenicity and protective efficacy of MVA encoding influenza virus hemagglutinin (HA) and/or nucleoprotein (NP) in cynomolgus macaques. Animals were given 2 doses of MVA-based vaccines 4 weeks apart and were challenged with a 2009 pandemic H1N1 isolate (H1N1pdm) 8 weeks after the last vaccination. MVA-based vaccines encoding HA induced potent serum antibody responses against homologous H1 or H5 HAs but did not stimulate strong T cell responses prior to challenge. However, animals that received MVA encoding influenza virus HA and/or NP had high frequencies of virus-specific CD4(+) and CD8(+) T cell responses within the first 7 days of H1N1pdm infection, while animals vaccinated with MVA encoding irrelevant antigens did not. We detected little or no H1N1pdm replication in animals that received vaccines encoding H1 (homologous) HA, while a vaccine encoding NP from an H5N1 isolate afforded no protection. Surprisingly, H1N1pdm viral shedding was reduced in animals vaccinated with MVA encoding HA and NP from an H5N1 isolate. This reduced shedding was associated with cross-reactive antibodies capable of mediating antibody-dependent cellular cytotoxicity (ADCC) effector functions. Our results suggest that ADCC plays a role in cross-protective immunity against influenza. Vaccines optimized to stimulate cross-reactive antibodies with ADCC function may provide an important measure of protection against emerging influenza viruses when NAbs are ineffective. Current influenza vaccines are designed to elicit neutralizing antibodies (NAbs). Vaccine-induced NAbs typically are effective but highly specific for particular virus strains. Consequently, current vaccines are poorly suited for preventing the spread of newly emerging pandemic viruses. Therefore, we evaluated a vaccine strategy designed to induce both antibody and T cell responses, which may provide more broadly cross-protective immunity against influenza. Here, we show in a translational primate model that vaccination with a modified vaccinia virus Ankara encoding hemagglutinin from a heterosubtypic H5N1 virus was associated with reduced shedding of a pandemic H1N1 virus challenge, while vaccination with MVA encoding nucleoprotein, an internal viral protein, was not. Unexpectedly, this reduced shedding was associated with nonneutralizing antibodies that bound H1 hemagglutinin and activated natural killer cells. Therefore, antibody-dependent cellular cytotoxicity (ADCC) may play a role in cross-protective immunity to influenza virus. Vaccines that stimulate ADCC antibodies may enhance protection against pandemic influenza virus.

Cellular and humoral cross-immunity against two H3N2v influenza strains in presumably unexposed healthy and HIV-infected subjects.

Human cases of infection due to a novel swine-origin variant of influenza A virus subtype H3N2 (H3N2v) have recently been identified in the United States. Pre-existing humoral and cellular immunity has been recognized as one of the key factors in limiting the infection burden of an emerging influenza virus strain, contributing to restrict its circulation and to mitigate clinical presentation. Aim of this study was to assess humoral and cell-mediated cross immune responses to H3N2v in immuno-competent (healthy donors, n = 45) and immuno-compromised hosts (HIV-infected subjects, n = 46) never exposed to H3N2v influenza strain. Humoral response against i) H3N2v (A/H3N2/Ind/08/11), ii) animal vaccine H3N2 strain (A/H3N2/Min/11/10), and iii) pandemic H1N1 virus (A/H1N1/Cal/07/09) was analysed by hemagglutination inhibition assay; cell-mediated response against the same influenza strains was analysed by ELISpot assay. A large proportion of healthy and HIV subjects displayed cross-reacting humoral and cellular immune responses against two H3N2v strains, suggesting the presence of B- and T-cell clones able to recognize epitopes from emerging viral strains in both groups. Specifically, humoral response was lower in HIV subjects than in HD, and a specific age-related pattern of antibody response against different influenza strains was observed both in HD and in HIV. Cellular immune response was similar between HD and HIV groups and no relationship with age was reported. Finally, no correlation between humoral and cellular immune response was observed. Overall, a high prevalence of HD and HIV patients showing cross reactive immunity against two H3N2v strains was observed, with a slightly lower proportion in HIV persons. Other studies focused on HIV subjects at different stages of diseases are needed in order to define how cross immunity can be affected by advanced immunosuppression.

Immunogenicity and protective efficacy of recombinant M2e.Hsp70c (Hsp70359–610) fusion protein against influenza virus infection in mice

New strategies in vaccine development are urgently needed to combat emerging influenza viruses and to reduce the risk of pandemic disease surfacing. Being conserved, the M2e protein, is a potential candidate for universal vaccine development against influenza A viruses. Mycobacterium tuberculosis Hsp70 (mHsp70) is known to cultivate the function of immunogenic antigenpresenting cells, stimulate a strong cytotoxic T lymphocyte (CTL) response, and stop the induction of tolerance. Thus, in this study, a recombinant protein from the extracellular domain of influenza A virus matrix protein 2 (M2e), was fused to the C-terminus of Mycobacterium tuberculosis Hsp70 (Hsp70c), to generate a vaccine candidate. Humoral immune responses, IFN-γ-producing lymphocyte, and strong CTL activity were all induced to confirm the immunogenicity of M2e.Hsp70c (Hsp70(359-610)). And challenge tests showed protection against H1N1 and H9N2 strains in vaccinated groups. Finally these results demonstrates M2e.Hsp70c fusion protein can be a candidate for a universal influenza A vaccine.

Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2012–2013

Emergence of influenza viruses with reduced susceptibility to neuraminidase inhibitors (NAIs) is sporadic, often follows exposure to NAIs, but occasionally occurs in the absence of NAI pressure. The emergence and global spread in 2007/2008 of A(H1N1) influenza viruses showing clinical resistance to oseltamivir due to neuraminidase (NA) H275Y substitution, in the absence of drug pressure, warrants continued vigilance and monitoring for similar viruses. Four World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance, Epidemiology and Control of Influenza (WHO CCs) tested 11,387 viruses collected by WHO-recognized National Influenza Centres (NIC) between May 2012 and May 2013 to determine 50% inhibitory concentration (IC50) data for oseltamivir, zanamivir, peramivir and laninamivir. The data were evaluated using normalized IC50 fold-changes rather than raw IC50 data. Nearly 90% of the 11,387 viruses were from three WHO regions: Western Pacific, the Americas and Europe. Only 0.2% (n=27) showed highly reduced inhibition (HRI) against at least one of the four NAIs, usually oseltamivir, while 0.3% (n=39) showed reduced inhibition (RI). NA sequence data, available from the WHO CCs and from sequence databases (n=3661), were screened for amino acid substitutions associated with reduced NAI susceptibility. Those showing HRI were A(H1N1)pdm09 with NA H275Y (n=18), A(H3N2) with NA E119V (n=3) or NA R292K (n=1) and B/Victoria-lineage with NA H273Y (n=2); amino acid position numbering is A subtype and B type specific. Overall, approximately 99% of circulating viruses tested during the 2012–2013 period were sensitive to all four NAIs. Consequently, these drugs remain an appropriate choice for the treatment and prophylaxis of influenza virus infections.

Infection and pathogenesis of canine, equine, and human influenza viruses in canine tracheas.

Influenza A viruses (IAVs) can jump species barriers and occasionally cause epidemics, epizootics, pandemics, and panzootics. Characterizing the infection dynamics at the target tissues of natural hosts is central to understanding the mechanisms that control host range, tropism, and virulence. Canine influenza virus (CIV; H3N8) originated after the transfer of an equine influenza virus (EIV) into dogs. Thus, comparing CIV and EIV isolates provides an opportunity to study the determinants of influenza virus emergence. Here we characterize the replication of canine, equine, and human IAVs in the trachea of the dog, a species to which humans are heavily exposed. We define a phenotype of infection for CIV, which is characterized by high levels of virus replication and extensive tissue damage. CIV was compared to evolutionarily distinct EIVs, and the early EIV isolates showed an impaired ability to infect dog tracheas, while EIVs that circulated near the time of CIV emergence exhibited a CIV-like infection phenotype. Inoculating dog tracheas with various human IAVs (hIAVs) showed that they infected the tracheal epithelium with various efficiencies depending on the virus tested. Finally, we show that reassortant viruses carrying gene segments of CIV and hIAV are viable and that addition of the hemagglutinin (HA) and neuraminidase (NA) of CIV to the 2009 human pandemic virus results in a virus that replicates at high levels and causes significant lesions. This provides important insights into the role of evolution on viral emergence and on the role of HA and NA as determinants of pathogenicity. Influenza A viruses (IAVs) have entered new host species in recent history, sometimes with devastating consequences. Canine influenza virus (CIV) H3N8 originated from a direct transfer of an equine influenza virus (EIV) in the early 2000s. We studied the infection patterns of IAVs that circulate in dogs or to which dogs are commonly exposed and showed that CIV emergence was likely caused by an adaptive driver, as evolutionarily distinct EIVs display distinct infection phenotypes. We also showed that many human viruses can infect dog tracheas and that reassortment with CIV results in viable viruses. Finally, we showed that the hemagglutinin and neuraminidase of CIV act as virulence factors. Our findings have significant implications because they show that dogs might act as "mixing vessels" in which novel viruses with pandemic potential could emerge and also provide experimental evidence supporting the role of viral evolution in influenza virus emergence.