Long-term adaptation following influenza A virus host shifts results in increased within-host viral fitness due to higher replication rates, broader dissemination within the respiratory epithelium and reduced tissue damage.

The mechanisms and consequences of genome evolution on viral fitness following host shifts are poorly understood. In addition, viral fitness -the ability of an organism to reproduce and survive- is multifactorial and thus difficult to quantify. Influenza A viruses (IAVs) circulate broadly among wild birds and have jumped into and become endemic in multiple mammalian hosts, including humans, pigs, dogs, seals, and horses. H3N8 equine influenza virus (EIV) is an endemic virus of horses that originated in birds and has been circulating uninterruptedly in equine populations since the early 1960s. Here, we used EIV to quantify changes in infection phenotype associated to viral fitness due to genome-wide changes acquired during long-term adaptation. We performed experimental infections of two mammalian cell lines and equine tracheal explants using the earliest H3N8 EIV isolated (A/equine/Uruguay/63 [EIV/63]), and A/equine/Ohio/2003 (EIV/2003), a monophyletic descendant of EIV/63 isolated 40 years after the emergence of H3N8 EIV. We show that EIV/2003 exhibits increased resistance to interferon, enhanced viral replication, and a more efficient cell-to-cell spread in cells and tissues. Transcriptomics analyses revealed virus-specific responses to each virus, mainly affecting host immunity and inflammation. Image analyses of infected equine respiratory explants showed that despite replicating at higher levels and spreading over larger areas of the respiratory epithelium, EIV/2003 induced milder lesions compared to EIV/63, suggesting that adaptation led to reduced tissue pathogenicity. Our results reveal previously unknown links between virus genotype and the host response to infection, providing new insights on the relationship between virus evolution and fitness.

Antiviral Activity of Canine RIG-I against Canine Influenza Virus and Interactions between Canine RIG-I and CIV.

RIG-I functions as a virus sensor that induces a cellular antiviral response. Although it has been investigated in other species, there have been no further studies to date on canine RIG-I against canine influenza virus (CIV). In the present study, we cloned the RIG-I gene of beagle dogs and characterized its expression, subcellular localization, antiviral response, and interactions with CIV proteins. RIG-I was highly expressed and mainly localized in the cytoplasm, with low levels detected in the nucleus. The results revealed that overexpression of the CARD domain of RIG-I and knockdown of RIG-I showed its ability to activate the RLR pathway and induced the expression of downstream interferon-stimulated genes. Moreover, overexpression of canine RIG-I suppressed the replication of CIV. The association between RIG-I and CIV was evaluated with the luciferase assay and by indirect immunofluorescence and bimolecular fluorescence complementation analyses. The results showed that CIV nonstructural protein 1 (NS1) can strongly suppress the RIG-I-mediated innate immune response, and the novel interactions between CIV matrix proteins (M1 and M2) and canine RIG-I were disclosed. These findings provide a basis for investigating the antiviral mechanism of canine RIG-I against CIV, which can lead to effective strategies for preventing CIV infection in dogs.

PB1 S524G mutation of wild bird-origin H3N8 influenza A virus enhances virulence and fitness for transmission in mammals.

Influenza H3N8 viruses have been recovered frequently from wild bird species, including Anseriformes (primarily from migratory ducks) and Charadriiformes (primarily from shorebirds). However, little attention has been given to the transmission ability of H3N8 avian influenza viruses among mammals. Here, we study the potential human health threat and the molecular basis of mammalian transmissibility of H3N8 avian influenza viruses isolated from wild bird reservoirs. We classified eight H3N8 viruses into seven different genotypes based on genomic diversity. Six of eight H3N8 viruses isolated naturally from wild birds have acquired the ability to bind to the human-type receptor. However, the affinity for α-2,6-linked SAs was lower than that for α-2,3-linked SAs. Experiments on guinea pigs demonstrated that three viruses transmitted efficiently to direct-contact guinea pigs without prior adaptation. Notably, one virus transmitted efficiently via respiratory droplets in guinea pigs but not in ferrets. We further found that the PB1 S524G mutation conferred T222 virus airborne transmissibility between ferrets. We also determined that the 524G mutant increased viral pathogenicity slightly in mice compared with the WT (wild type). Based on these results, we elucidated the potential human health threat and molecular basis of mammalian transmissibility of H3N8 influenza viruses. We emphasized the need for continued surveillance of the H3N8 influenza viruses circulating in birds.

Fatal multiple outbreaks of equine influenza H3N8 in Nigeria, 2019: The first introduction of Florida clade 1 to West Africa.

In December 2018, suspected outbreaks of equine influenza (EI) were observed in donkeys in Sokoto State, in the extreme northwest of Nigeria bordering the Republic of the Niger. Equine influenza virus (EIV) subtype H3N8 was the etiologic agent identified in the outbreaks using real-time RT-qPCR and sequencing of both the partial haemagglutinin (HA) gene and the complete genome. Since then the H3N8 virus spread to 7 of the 19 northern states of Nigeria, where it affected both donkeys and horses. Phylogenetic analysis of the partial and complete HA gene revealed the closest nucleotide similarity (99.7%) with EIVs belonging to the Florida clade 1 (Fc-1) of the American lineage isolated in 2018 from Argentina and Chile. In total, 80 amino acid substitutions were observed in the viral proteins when compared to the OIE-recommended Fc-1 vaccine strains. The HA and neuraminidase proteins respectively had 13 and 16 amino acid substitutions. This study represents the first reported outbreak of EI caused by an Fc-1 virus in Nigeria and in the West Africa sub-region. Based on this report, extensive disease surveillance in equids is required to establish the circulating lineages and design an effective control strategy to protect the considerable population of horses and donkeys in the country.

Truncation of PA-X Contributes to Virulence and Transmission of H3N8 and H3N2 Canine Influenza Viruses in Dogs.

Equine-origin H3N8 and avian-origin H3N2 canine influenza viruses (CIVs) prevalent in dogs are thought to pose a public health threat arising from intimate contact between dogs and humans. However, our understanding of CIV virulence is still limited. Influenza A virus PA-X is a fusion protein encoded in part by a +1 frameshifted open reading frame (X-ORF) in segment 3. The X-ORF can be translated in full-length (61-amino-acid) or truncated (41-amino-acid) form. Genetic analysis indicated that the X-ORFs of equine H3N8 and avian H3N2 influenza viruses encoded 61 amino acids but were truncated after introduction into dogs. To determine the effect of PA-X truncation on the biological characteristics of CIVs, we constructed four recombinant viruses on H3N8 and H3N2 CIV backgrounds bearing truncated or full-length PA-Xs. We observed that truncation of PA-X increased growth of both H3N8 and H3N2 CIVs in MDCK cells and suppressed expression from cotransfected plasmids in MDCK cells. Furthermore, truncation of PA-X enhanced viral pathogenicity in dogs, as shown by aggravated clinical symptoms and histopathological changes, increased viral replication in the respiratory system, and prolonged virus shedding. Additionally, CIVs with truncated PA-Xs were transmitted more efficiently in dogs. Global gene expression profiling of the lungs of infected dogs revealed that differentially expressed genes were mainly associated with inflammatory responses, which might contribute to the pathogenicity of PA-X-truncated CIVs. Our findings revealed that truncation of PA-X might be important for the adaptation of influenza viruses to dogs.IMPORTANCE Epidemics of equine-origin H3N8 and avian-origin H3N2 influenza viruses in canine populations are examples of successful cross-species transmission of influenza A viruses. Genetic analysis showed that the PA-X genes of equine H3N8 or avian H3N2 influenza viruses were full-length, with X-ORFs encoding 61 amino acids; however, those of equine-origin H3N8 or avian-origin H3N2 CIVs were truncated, suggesting that PA-X truncation occurred after transmission to dogs. In this study, we extended the PA-X genes of H3N8 and H3N2 CIVs and compared the biological characteristics of CIVs bearing different lengths of PA-X. We demonstrated that for both H3N8 and H3N2 viruses, truncation of PA-X increased virus yields in MDCK cells and enhanced viral replication, pathogenicity, and transmission in dogs. These results might reflect enhanced suppression of host gene expression and upregulation of genes related to inflammatory responses. Collectively, our data partially explain the conservation of truncated PA-X in CIVs.

Bearing the brunt: Mongolian khulan (Equus hemionus hemionus) are exposed to multiple influenza A strains.

The majority of influenza A virus strains are hosted in nature by avian species in the orders of Anseriformes and Charadriformes. A minority of strains have been able to cross species boundaries and establish themselves in novel non-avian hosts. Influenza viruses of horses, donkeys, and mules represent such successful events of avian to mammal influenza virus adaptation. Mongolia has over 3 million domestic horses and is home to two wild equids, the Asiatic wild ass or khulan (Equus hemionus hemionus), and Przewalski's horse (Equus ferus przewalskii). Domestic and wild equids are sympatric across most of their range in Mongolia. Epizootic influenza A virus outbreaks among Mongolian domestic horses have been frequently recorded. However, the exposure, circulation and relation to domestic horse influenza A virus outbreaks among wild equids is unknown. We evaluated serum samples of Asiatic wild asses in Mongolia for antibodies against influenza A viruses, using modified protein microarray technique. We detected antibodies against hemagglutinin (H) H1, H3, H5, H7, H8 and H10 influenza A viruses. Asiatic wild asses may represent a previously unidentified influenza A virus reservoir in an ecosystem shared with populations of domestic horses in which influenza strains circulate.

Attenuation of highly pathogenic avian influenza A(H5N1) viruses in Indonesia following the reassortment and acquisition of genes from low pathogenicity avian influenza A virus progenitors.

The highly pathogenic avian influenza (HPAI) A(H5N1) virus is endemic in Indonesian poultry and has caused sporadic human infection in Indonesia since 2005. Surveillance of H5N1 viruses in live bird markets (LBMs) during 2012 and 2013 was carried out to provide epidemiologic and virologic information regarding viral circulation and the risk of human exposure. Real-time RT-PCR of avian cloacal swabs and environmental samples revealed influenza A-positive specimens, which were then subjected to virus isolation and genomic sequencing. Genetic analysis of specimens collected at multiple LBMs in Indonesia identified both low pathogenicity avian influenza (LPAI) A(H3N8) and HPAI A(H5N1) viruses belonging to clade 2.1.3.2a. Comparison of internal gene segments among the LPAI and HPAI viruses revealed that the latter had acquired the PB2, PB1, and NS genes from LPAI progenitors and other viruses containing a wild type (wt) genomic constellation. Comparison of murine infectivity of the LPAI A(H3N8), wt HPAI A(H5N1) and reassortant HPAI A(H5N1) viruses showed that the acquisition of LPAI internal genes attenuated the reassortant HPAI virus, producing a mouse infectivity/virulence phenotype comparable to that of the LPAI virus. Comparison of molecular markers in each viral gene segment suggested that mutations in PB2 and NS1 may facilitate attenuation. The discovery of an attenuated HPAI A(H5N1) virus in mice that resulted from reassortment may have implications for the capability of these viruses to transmit and cause disease. In addition, surveillance suggests that LBMs in Indonesia may play a role in the generation of reassortant A(H5) viruses and should be monitored.

RETRACTED: An epidemiological investigation of associated risk factors with equine influenza (H3N8) epidemic 2015-16 in Pakistan.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editors-in-Chief. The article duplicates significant parts of a paper that had already appeared in Preventative Veterinary Medicine, 149, 132-139; https://doi.org/10.1016/j.prevetmed.2017.12.005. One of the conditions of submission of a paper for publication is that authors declare explicitly that the paper has not been previously published and is not under consideration for publication elsewhere. Re-use of any data should be appropriately cited. As such this article represents a misuse of the scientific publishing system. The scientific community takes a very strong view on this matter and apologies are offered to readers of the journal that this was not detected during the submission process.

Equine influenza: evolution of a highly infectious virus.

Fleur Whitlock, Adam Rash and Debra Elton of the equine influenza group at the Animal Health Trust provide a timely reminder of risk of equine influenza and the importance of vaccination.

Heterosubtypic immunity increases infectious dose required to infect Mallard ducks with Influenza A virus.

Previous field and experimental studies have demonstrated that heterosubtypic immunity (HSI) is a potential driver of Influenza A virus (IAV) prevalence and subtype diversity in mallards. Prior infection with IAV can reduce viral shedding during subsequent reinfection with IAV that have genetically related hemagglutinins (HA). In this experiment, we evaluated the effect of HSI conferred by an H3N8 IAV infection against increasing challenge doses of closely (H4N6) and distantly (H6N2) related IAV subtypes in mallards. Two groups of thirty 1-month-old mallards each, were inoculated with 105.9 50% embryo infectious doses (EID50) of an H3N8 virus or a mock-inoculum. One month later, groups of five birds each were challenged with increasing doses of H4N6 or H6N2 virus; age-matched, single infection control ducks were included for all challenges. Results demonstrate that naïve birds were infected after inoculation with 103 and 104 EID50 doses of the H4N6 or H6N2 virus, but not with 102 EID50 doses of either IAV. In contrast, with birds previously infected with H3N8 IAV, only one duck challenged with 104 EID50 of H4N6 IAV was shedding viral RNA at 2 days post-inoculation, and with H6N2 IAV, only birds challenged with the 104 EID50 dose were positive to virus isolation. Viral shedding in ducks infected with H6N2 IAV was reduced on days 2 and 3 post-inoculation compared to control birds. To explain the differences in the dose necessary to produce infection among H3-primed ducks challenged with H4N6 or H6N2 IAV, we mapped the amino acid sequence changes between H3 and H4 or H6 HA on predicted three-dimensional structures. Most of the sequence differences occurred between H3 and H6 at antigenic sites A, B, and D of the HA1 region. These findings demonstrate that the infectious dose necessary to infect mallards with IAV can increase as a result of HSI and that this effect is most pronounced when the HA of the viruses are genetically related.

Refinement of the equine influenza model in the natural host: A meta-analysis to determine the benefits of individual nebulisation for experimental infection and vaccine evaluation in the face of decreased strain pathogenicity.

Equine Influenza (EI) is an important respiratory disease of horses caused by H3N8 equine influenza viruses (EIV). Vaccination is a key strategy to prevent or control this disease. However, EIV undergoes continuous antigenic drift and whilst numerous EI vaccines are commercially available worldwide, an accurate evaluation of their efficacy is frequently required through clinical trials conducted in the natural host. Room nebulisation is one of the chosen methods to challenge horses during EI vaccine studies. A potential decreased pathogenicity observed with recent Florida Clade 2 (FC2) EIV isolates have increased the heterogeneity of the clinical response and virus shedding measured after infection by room nebulisation, which reduced the statistical power of studies. Our objectives were to compare clinical and virological parameters following experimental infection with several different EIV strains and to confirm that individual nebulisation is a model refinement that prevents an increase of the number of animals per group. This study is a retrospective comparison and meta-analysis of clinical and virological results collected from 9 independent EIV infection studies in the natural host. Naïve Welsh mountain ponies were experimentally infected by room or individual nebulisation with FC2 EIV strains, including A/equine/Richmond/1/07 (R/07), A/equine/East Renfrewshire/11 (ER/11), A/equine/Cambremer/1/2012 (C/12) and A/equine/Northamptonshire/1/13 (N/1/13). The retrospective meta-analysis confirmed a decreased pathogenicity of the EIV ER/11 and C/12 strains when compared with R/07. Experimental infection by individual nebulisation improved the clinical and virological parameters induced by recent FC2 strains, when compared with conventional room nebulisation. In conclusion, individual nebulisation offers a better control of the challenge dose administered and a greater homogeneity of the response measured in control animals. This in turn, helps maintain the number of animals per group to the minimum necessary required to obtain meaningful results in vaccine efficacy studies, which adheres to the 3Rs (Replacement, Reduction and Refinement) principles.

Infection, contagion and causality in Colonial Britain: the 1889-90 influenza pandemic and the British Medical Journal.

The influenza pandemic of 1889 was the first truly global flu outbreak in scope. Characterised by high morbidity and low mortality, it spread rapidly across Europe and the rest of the world along trading routes. It reached mainland Britain in December 1889. The responses of medical practitioners in Britain and the British colonies to the pandemic were heavily featured in the British Medical Journal and reveal a confusing picture around causality, contagion and infection. Cases from the colonies (Cape Town, India, Australia, Samoan Islands, Hong Kong) as presented in the journal are explored in an attempt to reconstruct the mainstream medical belief of the time. The evidence sadly shows a lack of confidence in contagionism, almost complete absence of monocausalism and a vague picture of the epidemic constitution. Original case studies from colonial medical officers as well as editorials triggered a debate in the pages of the BMJ. In this context, the journal succeeded in playing a key role in recording the first thoroughly documented attack of influenza. In a world that was only learning to be interconnected, the BMJ became the point of reference for the British medical establishment, which ranged from London to Scotland and from Africa and India to Oceania.

Attenuation of the virulence of a recombinant influenza virus expressing the naturally truncated NS gene from an H3N8 equine influenza virus in mice.

Equine influenza virus (EIV) causes a highly contagious disease in horses and other equids. Recently, we isolated an H3N8 EIV (A/equine/Kyonggi/SA1/2011) from a domestic horse in South Korea that exhibited symptoms of respiratory disease, and found that the EIV strain contained a naturally mutated NS gene segment encoding a truncated NS1 protein. In order to determine whether there was an association between the NS gene truncation and viral virulence, a reverse genetics system was applied to generate various NS gene recombinant viruses using the backbone of the H1N1 A/Puerto Rico/8/1934 (PR/8) virus. In a mouse model, the recombinant PR/8 virus containing the mutated NS gene of the Korean H3N8 EIV strain showed a dramatically reduced virulence: it induced no weight loss, no clinical signs and no histopathological lesions. However, the mice infected with the recombinant viruses with NS genes of PR/8 and H3N8 A/equine/2/Miami/1963 showed severe clinical signs including significant weight loss and 100% mortality. In addition, the levels of the pro-inflammatory cytokines; IL-6, CCL5, and IFN-γ, in the lungs of mice infected with the recombinant viruses expressing a full-length NS1 were significantly higher than those of mice infected with the virus with the NS gene from the Korean H3N8 EIV strain. In this study, our results suggest that the C-terminal moiety of NS1 contains a number of virulence determinants and might be a suitable target for the development of a vaccine candidate against equine influenza.

Phylogenetic analysis and pathogenicity of H3 subtype avian influenza viruses isolated from live poultry markets in China.

H3 subtype influenza A virus is one of the main subtypes that threats both public and animal health. However, the evolution and pathogenicity of H3 avian influenza virus (AIV) circulating in domestic birds in China remain largely unclear. In this study, seven H3 AIVs (four H3N2 and three H3N8) were isolated from poultry in live poultry market (LPM) in China. Phylogenetic analyses of full genomes showed that all viruses were clustered into Eurasian lineage, except N8 genes of two H3N8 isolates fell into North American lineage. Intriguingly, the N8 gene of one H3N8 and PB2, PB1, NP and NS of two H3N2 isolates have close relationship with those of the highly pathogenic H5N8 viruses circulating in Korea and United States, suggesting that the H3-like AIV may contribute internal genes to the highly pathogenic H5N8 viruses. Phylogenetic tree of HA gene and antigenic cross-reactivity results indicated that two antigenically different H3 viruses are circulating in LPM in China. Most of the H3 viruses replicated in mice lung and nasal turbinate without prior adaptation, and the representative H3 viruses infected chickens without causing clinical signs. The reassortment of H3 subtype influenza viruses warrants continuous surveillance in LPM in China.

Pathology of Equine Influenza virus (H3N8) in Murine Model.

Equine influenza viruses (EIV)-H3N8 continue to circulate in equine population throughout the world. They evolve by the process of antigenic drift that leads to substantial change in the antigenicity of the virus, thereby necessitating substitution of virus strain in the vaccines. This requires frequent testing of the new vaccines in the in vivo system; however, lack of an appropriate laboratory animal challenge model for testing protective efficacy of equine influenza vaccine candidates hinders the screening of new vaccines and other therapeutic approaches. In the present investigation, BALB/c mouse were explored for suitability for conducting pathogenecity studies for EIV. The BALB/c mice were inoculated intranasally @ 2×106.24 EID50 with EIV (H3N8) belonging to Clade 2 of Florida sublineage and monitored for setting up of infection and associated parameters. All mice inoculated with EIV exhibited clinical signs viz. loss in body weights, lethargy, dyspnea, etc, between 3 and 5 days which commensurate with lesions observed in the respiratory tract including rhinitis, tracheitis, bronchitis, bronchiolitis, alveolitis and diffuse interstitial pneumonia. Transmission electron microscopy, immunohistochemistry, virus quantification through titration and qRT-PCR demonstrated active viral infection in the upper and lower respiratory tract. Serology revealed rise in serum lactate dehydrogenase levels along with sero-conversion. The pattern of disease progression, pathological lesions and virus recovery from nasal washings and lungs in the present investigations in mice were comparable to natural and experimental EIV infection in equines. The findings establish BALB/c mice as small animal model for studying EIV (H3N8) infection and will have immense potential for dissecting viral pathogenesis, vaccine efficacy studies, preliminary screening of vaccine candidates and antiviral therapeutics against EIV.

Cross-Species Infectivity of H3N8 Influenza Virus in an Experimental Infection in Swine.

UNLABELLED: Avian influenza A viruses have gained increasing attention due to their ability to cross the species barrier and cause severe disease in humans and other mammal species as pigs. H3 and particularly H3N8 viruses, are highly adaptive since they are found in multiple avian and mammal hosts. H3N8 viruses have not been isolated yet from humans; however, a recent report showed that equine influenza A viruses (IAVs) can be isolated from pigs, although an established infection has not been observed thus far in this host. To gain insight into the possibility of H3N8 avian IAVs to cross the species barrier into pigs, in vitro experiments and an experimental infection in pigs with four H3N8 viruses from different origins (equine, canine, avian, and seal) were performed. As a positive control, an H3N2 swine influenza virus A was used. Although equine and canine viruses hardly replicated in the respiratory systems of pigs, avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Interestingly, antibodies against hemagglutinin could not be detected after infection by hemagglutination inhibition (HAI) test with avian and seal viruses. This phenomenon was observed not only in pigs but also in mice immunized with the same virus strains. Our data indicated that H3N8 IAVs from wild aquatic birds have the potential to cross the species barrier and establish successful infections in pigs that might spread unnoticed using the HAI test as diagnostic tool. IMPORTANCE: Although natural infection of humans with an avian H3N8 influenza A virus has not yet been reported, this influenza A virus subtype has already crossed the species barrier. Therefore, we have examined the potential of H3N8 from canine, equine, avian, and seal origin to productively infect pigs. Our results demonstrated that avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Surprisingly, we could not detect specific antibodies against hemagglutinin in any H3N8-infected pigs. Therefore, special attention should be focused toward viruses of the H3N8 subtype since they could behave as stealth viruses in pigs.

Inefficient transmissibility of NS-truncated H3N8 equine influenza virus in dogs.

H3N8 equine influenza virus (EIV) causes respiratory diseases in the horse population, and it has been demonstrated that EIV can transmit into dogs owing to its availability on receptors of canine respiratory epithelial cells. Recently, we isolated H3N8 EIV from an EIV-vaccinated horse that showed symptoms of respiratory disease, and which has a partially truncated nonstructural gene (NS). However, it is not clear that the NS-truncated EIV has an ability to cross the host species barrier from horses to dogs as well. Here, we experimentally infected the NS-truncated H3N8 EIV into dogs, and monitored their clinical signs and viral load in respiratory organs to determine the virus's transmissibility.

Respiratory transmission of an avian H3N8 influenza virus isolated from a harbour seal.

The ongoing human H7N9 influenza infections highlight the threat of emerging avian influenza viruses. In 2011, an avian H3N8 influenza virus isolated from moribund New England harbour seals was shown to have naturally acquired mutations known to increase the transmissibility of highly pathogenic H5N1 influenza viruses. To elucidate the potential human health threat, here we evaluate a panel of avian H3N8 viruses and find that the harbour seal virus displays increased affinity for mammalian receptors, transmits via respiratory droplets in ferrets and replicates in human lung cells. Analysis of a panel of human sera for H3N8 neutralizing antibodies suggests that there is no population-wide immunity to these viruses. The prevalence of H3N8 viruses in birds and multiple mammalian species including recent isolations from pigs and evidence that it was a past human pandemic virus make the need for surveillance and risk analysis of these viruses of public health importance.

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

UNLABELLED: 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. IMPORTANCE: 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.

The zoonotic potential of avian influenza viruses isolated from wild waterfowl in Zambia.

Whilst remarkable progress in elucidating the mechanisms governing interspecies transmission and pathogenicity of highly pathogenic avian influenza viruses (AIVs) has been made, similar studies focusing on low-pathogenic AIVs isolated from the wild waterfowl reservoir are limited. We previously reported that two AIV strains (subtypes H6N2 and H3N8) isolated from wild waterfowl in Zambia harbored some amino acid residues preferentially associated with human influenza virus proteins (so-called human signatures) and replicated better in the lungs of infected mice and caused more morbidity than a strain lacking such residues. To further substantiate these observations, we infected chickens and mice intranasally with AIV strains of various subtypes (H3N6, H3N8, H4N6, H6N2, H9N1 and H11N9) isolated from wild waterfowl in Zambia. Although some strains induced seroconversion, all of the tested strains replicated poorly and were nonpathogenic for chickens. In contrast, most of the strains having human signatures replicated well in the lungs of mice, and one of these strains caused severe illness in mice and induced lung injury that was characterized by a severe accumulation of polymorphonuclear leukocytes. These results suggest that some strains tested in this study may have the potential to infect mammalian hosts directly without adaptation, which might possibly be associated with the possession of human signature residues. Close monitoring and evaluation of host-associated signatures may help to elucidate the prevalence and emergence of AIVs with potential for causing zoonotic infections.