Genome sequence analysis of H7N3 subtype avian influenza virus originated from wild birds and its potential infectivity in mice.

In 2021, an H7N3 avian influenza virus (AIV) was isolated from a mallard in Tianhewan Yellow River National Wetland Park, Ningxia Hui Autonomous Region, China. Sequences analysis showed that this strain received its genes from H7, H6, H5, H3, and H1 AIVs of domestic poultry and wild birds in Asia and Europe. It was mild pathogenicity in mice. These results suggest the importance of continued surveillance of the H7N3 virus to better understand the ecology and evolution of the AIVs in poultry and wild birds and the potential threat to humans.

Comparative pathogenic potential of avian influenza H7N3 viruses isolated from wild birds in Egypt and their sensitivity to commercial antiviral drugs.

Active surveillance and studying the virological features of avian-origin influenza viruses are essential for early warning and preparedness for the next potential pandemic. During our active surveillance of avian influenza viruses in wild birds in Egypt in the period 2014-2017, multiple reassortant low-pathogenic avian influenza H7N3 viruses were isolated. In this study, we investigated and compared the infectivity, pathogenicity, and transmission of four different constellation forms of Egyptian H7N3 viruses in chickens and mice and assessed the sensitivity of these viruses to different commercial antiviral drugs in vitro. Considerable variation in virus pathogenicity was observed in mice infected with different H7N3 viruses. The mortality rate ranged from 20 to 100% in infected mice. Infected chickens showed only ocular clinical signs at three days postinfection as well as systemic viral infection in different organs. Efficient virus replication and transmission in chickens was observed within each group, indicating that these subtypes can spread easily from wild birds to poultry without prior adaptation. Mutations in the viral proteins associated with antiviral drug resistance were not detected, and all strains were sensitive to the antiviral drugs tested. In conclusion, all of the viruses studied had the ability to infect mice and chickens. H7N3 viruses circulating among wild birds in Egypt could threaten poultry production and public health.

Increased virulence of a novel reassortant H1N3 avian influenza virus in mice as a result of adaptive amino acid substitutions.

In this study, a novel multiple-gene reassortant H1N3 subtype avian influenza virus (AIV) (A/chicken/Zhejiang/81213/2017, CK81213) was isolated in Eastern China, whose genes were derived from H1 (H1N3), H7 (H7N3 and H7N9), and H10 (H10N3 and H10N8) AIVs. This AIV belongs to the avian Eurasian-lineage and exhibits low pathogenicity. Serial lung-to-lung passages of CK81213 in mice was performed to study the amino acid substitutions potentially related to the adaptation of H1 AIVs in mammals. And the mouse-adapted H1N3 virus showed greater virulence than wild-type H1N3 AIV in mice and the genomic analysis revealed a total of two amino acid substitutions in the PB2 (E627K) and HA (L67V) proteins. Additionally, the results of the animal study indicate that CK81213 could infect mice without prior adaption and become highly pathogenic to mice after continuous passage. Our findings show that routine surveillance of H1 AIVs is important for the prediction of influenza epidemics.

Low Pathogenicity H7N3 Avian Influenza Viruses Have Higher Within-Host Genetic Diversity Than a Closely Related High Pathogenicity H7N3 Virus in Infected Turkeys and Chickens.

Within-host viral diversity offers a view into the early stages of viral evolution occurring after a virus infects a host. In recent years, advances in deep sequencing have allowed for routine identification of low-frequency variants, which are important sources of viral genetic diversity and can potentially emerge as a major virus population under certain conditions. We examined within-host viral diversity in turkeys and chickens experimentally infected with closely related H7N3 avian influenza viruses (AIVs), specifically one high pathogenicity AIV (HPAIV) and two low pathogenicity AIV (LPAIVs) with different neuraminidase protein stalk lengths. Consistent with the high mutation rates of AIVs, an abundance of intra-host single nucleotide variants (iSNVs) at low frequencies of 2-10% was observed in all samples collected. Furthermore, a small number of common iSNVs were observed between turkeys and chickens, and between directly inoculated and contact-exposed birds. Notably, the LPAIVs have significantly higher iSNV diversities and frequencies of nonsynonymous changes than the HPAIV in both turkeys and chickens. These findings highlight the dynamics of AIV populations within hosts and the potential impact of genetic changes, including mutations in the hemagglutinin gene that confers the high pathogenicity pathotype, on AIV virus populations and evolution.

Genetic Characterization of Novel H7Nx Low Pathogenic Avian Influenza Viruses from Wild Birds in South Korea during the Winter of 2020-2021.

Zoonotic infection with avian influenza viruses (AIVs) of subtype H7, such as H7N9 and H7N4, has raised concerns worldwide. During the winter of 2020-2021, five novel H7 low pathogenic AIVs (LPAIVs) containing different neuraminidase (NA) subtypes, including two H7N3, an H7N8, and two H7N9, were detected in wild bird feces in South Korea. Complete genome sequencing and phylogenetic analysis showed that the novel H7Nx AIVs were reassortants containing two gene segments (hemagglutinin (HA) and matrix) that were related to the zoonotic Jiangsu-Cambodian H7 viruses causing zoonotic infection and six gene segments originating from LPAIVs circulating in migratory birds in Eurasia. A genomic constellation analysis demonstrated that all H7 isolates contained a mix of gene segments from different viruses, indicating that multiple reassortment occurred. The well-known mammalian adaptive substitution (E627K and D701N) in PB2 was not detected in any of these isolates. The detection of multiple reassortant H7Nx AIVs in wild birds highlights the need for intensive surveillance in both wild birds and poultry in Eurasia.

The Pathobiology of H7N3 Low and High Pathogenicity Avian Influenza Viruses from the United States Outbreak in 2020 Differs between Turkeys and Chickens.

An outbreak caused by H7N3 low pathogenicity avian influenza virus (LPAIV) occurred in commercial turkey farms in the states of North Carolina (NC) and South Carolina (SC), United States in March of 2020. Subsequently, H7N3 high pathogenicity avian influenza virus (HPAIV) was detected on a turkey farm in SC. The infectivity, transmissibility, and pathogenicity of the H7N3 HPAIV and two LPAIV isolates, including one with a deletion in the neuraminidase (NA) protein stalk, were studied in turkeys and chickens. High infectivity [<2 log10 50% bird infectious dose (BID50)] and transmission to birds exposed by direct contact were observed with the HPAIV in turkeys. In contrast, the HPAIV dose to infect chickens was higher than for turkeys (3.7 log10 BID50), and no transmission was observed. Similarly, higher infectivity (<2-2.5 log10 BID50) and transmissibility were observed with the H7N3 LPAIVs in turkeys compared to chickens, which required higher virus doses to become infected (5.4-5.7 log10 BID50). The LPAIV with the NA stalk deletion was more infectious in turkeys but did not have enhanced infectivity in chickens. These results show clear differences in the pathobiology of AIVs in turkeys and chickens and corroborate the high susceptibility of turkeys to both LPAIV and HPAIV infections.

Genetic Characterization and Pathogenesis of Avian Influenza Virus H7N3 Isolated from Spot-Billed Ducks in South Korea, Early 2019.

Low-pathogenicity avian influenza viruses (LPAIV) introduced by migratory birds circulate in wild birds and can be transmitted to poultry. These viruses can mutate to become highly pathogenic avian influenza viruses causing severe disease and death in poultry. In March 2019, an H7N3 avian influenza virus-A/Spot-billed duck/South Korea/WKU2019-1/2019 (H7N3)-was isolated from spot-billed ducks in South Korea. This study aimed to evaluate the phylogenetic and mutational analysis of this isolate. Molecular analysis revealed that the genes for HA (hemagglutinin) and NA (neuraminidase) of this strain belonged to the Central Asian lineage, whereas genes for other internal proteins such as polymerase basic protein 1 (PB1), PB2, nucleoprotein, polymerase acidic protein, matrix protein, and non-structural protein belonged to that of the Korean lineage. In addition, a monobasic amino acid (PQIEPR/GLF) at the HA cleavage site, and the non-deletion of the stalk region in the NA gene indicated that this isolate was a typical LPAIV. Nucleotide sequence similarity analysis of HA revealed that the highest homology (99.51%) of this isolate is to that of A/common teal/Shanghai/CM1216/2017 (H7N7), and amino acid sequence of NA (99.48%) was closely related to that of A/teal/Egypt/MB-D-487OP/2016 (H7N3). An in vitro propagation of the A/Spot-billed duck/South Korea/WKU2019-1/2019 (H7N3) virus showed highest (7.38 Log10 TCID50/mL) virus titer at 60 h post-infection, and in experimental mouse lungs, the virus was detected at six days' post-infection. Our study characterizes genetic mutations, as well as pathogenesis in both in vitro and in vivo model of a new Korea H7N3 viruses in 2019, carrying multiple potential mutations to become highly pathogenic and develop an ability to infect humans; thus, emphasizing the need for routine surveillance of avian influenza viruses in wild birds.

Dominant subtype switch in avian influenza viruses during 2016-2019 in China.

We have surveyed avian influenza virus (AIV) genomes from live poultry markets within China since 2014. Here we present a total of 16,091 samples that were collected from May 2016 to February 2019 in 23 provinces and municipalities in China. We identify 2048 AIV-positive samples and perform next generation sequencing. AIV-positive rates (12.73%) from samples had decreased substantially since 2016, compared to that during 2014-2016 (26.90%). Additionally, H9N2 has replaced H5N6 and H7N9 as the dominant AIV subtype in both chickens and ducks. Notably, novel reassortants and variants continually emerged and disseminated in avian populations, including H7N3, H9N9, H9N6 and H5N6 variants. Importantly, almost all of the H9 AIVs and many H7N9 and H6N2 strains prefer human-type receptors, posing an increased risk for human infections. In summary, our nation-wide surveillance highlights substantial changes in the circulation of AIVs since 2016, which greatly impacts the prevention and control of AIVs in China and worldwide.

Emergence and Selection of a Highly Pathogenic Avian Influenza H7N3 Virus.

Low-pathogenicity avian influenza (LPAI) viruses of subtypes H5 and H7 have the ability to spontaneously mutate to highly pathogenic (HPAI) virus variants, causing high mortality in poultry. The highly pathogenic phenotype is caused by mutation of the hemagglutinin (HA) cleavage site, but additional mutations may play a role. Evidence from the field for the switch to high pathogenicity remains scarce. This study provides direct evidence for LPAI-to-HPAI virus mutation during H7N3 infection of a turkey farm in the Netherlands. No severe clinical symptoms were reported at the farm, but deep sequencing of isolates from the infected turkeys revealed a minority of HPAI virus sequences (0.06%) in the virus population. The HPAI virus contained a 12-nucleotide insertion in the HA cleavage site that was likely introduced by a single event as no intermediates with shorter inserts were identified. This suggests nonhomologous recombination as the mechanism of insertion. Analysis of different organs of the infected turkeys showed the largest amount of HPAI virus in the lung (4.4%). The HPAI virus was rapidly selected in experimentally infected chickens after both intravenous and intranasal/intratracheal inoculation with a mixed virus preparation. Full-genome sequencing revealed that both pathotypes contained a deletion in the stalk region of the neuraminidase protein. We identified additional mutations in HA and polymerase basic protein 1 (PB1) in the HPAI virus, which were already present as minority variants in the LPAI virus population. Our findings provide more insight into the molecular changes and mechanisms involved in the emergence and selection of HPAI viruses.IMPORTANCE Low-pathogenicity avian influenza (LPAI) viruses circulate in wild birds and can be transmitted to poultry. LPAI viruses can mutate to become highly pathogenic avian influenza (HPAI) viruses causing severe disease and death in poultry. Little is known about this switch to high pathogenicity. We isolated an LPAI H7N3 virus from an infected turkey farm and showed that this contains small amounts of HPAI virus. The HPAI virus rapidly outcompeted the LPAI virus in chickens that were experimentally infected with this mixture of viruses. We analyzed the genome sequences of the LPAI and HPAI viruses and identified several changes that may be important for a virus to become highly pathogenic. This knowledge may be used for timely identification of LPAI viruses that pose a risk of becoming highly pathogenic in the field.

Genetic and antigenic characterization of the first H7N7 low pathogenic avian influenza viruses isolated in Vietnam.

During the annual surveillance of avian influenza viruses (AIVs) in Vietnam in 2018, three H7N7 AIV isolates were identified in domestic ducks in a single flock in Vinh Long province. The present study is the first documented report of H7N7 virus isolates in Vietnam and aimed to characterize these viruses, both genetically and antigenically. Deduced amino acid sequences for the hemagglutinins (HAs) indicated a low pathogenicity of these viruses in chickens. Phylogenetic analysis revealed that the H7 HA genes of these isolates were closely related to each other and belonged to the European-Asian sublineage, together with those of H7N3 viruses isolated from ducks in Cambodia during 2017. They were not genetically related to those of Chinese H7N9 or H7N1 viruses that were previously detected in Vietnam during 2012. Interestingly, the M genes of the two H7N7 virus isolates were phylogenetically classified into distinct groups, suggesting an ongoing reassortment event in domestic ducks because they were isolated from the same flock. These H7N7 viruses exhibited somewhat different antigenic characteristics compared with other representative H7 low pathogenic AIVs. Surprisingly, the antigenicity of Vietnamese H7N7 viruses is similar to Chinese H7N9 highly pathogenic AIV. The findings of this study suggest that H7N7 viruses may be undergoing reassortment and antigenic diversification in poultry flocks in Vietnam. The silent spread of Vietnamese H7N7 viruses in chickens may lead to acquire high pathogenicity in chickens although the zoonotic potential of the viruses seems to be low since these viruses retain typical avian-specific motifs in the receptor-binding site in the HA and there is no mutation related to mammalian adaptation in PB2 gene. Thus, these results highlight the need for continuous and intensive surveillance of avian influenza in Vietnam, targeting not only highly pathogenic AIVs but also low pathogenic viruses.

Rapid evolution of Mexican H7N3 highly pathogenic avian influenza viruses in poultry.

Highly pathogenic avian influenza (HPAI) virus subtype H7N3 has been circulating in poultry in Mexico since 2012 and vaccination has been used to control the disease. In this study, eight Mexican H7N3 HPAI viruses from 2015-2017 were isolated and fully sequenced. No evidence of reassortment was detected with other avian influenza (AI) viruses, but phylogenetic analyses show divergence of all eight gene segments into three genetic clusters by 2015, with 94.94 to 98.78 percent nucleotide homology of the HA genes when compared to the index virus from 2012. The HA protein of viruses from each cluster showed a different number of basic amino acids (n = 5-7) in the cleavage site, and six different patterns at the predicted N-glycosylation sites. Comparison of the sequences of the Mexican lineage H7N3 HPAI viruses and American ancestral wild bird AI viruses to characterize the virus evolutionary dynamics showed that the nucleotide substitution rates in PB2, PB1, PA, HA, NP, and NS genes greatly increased once the virus was introduced into poultry. The global nonsynonymous and synonymous ratios imply strong purifying selection driving the evolution of the virus. Forty-nine positively selected sites out of 171 nonsynonymous mutations were identified in the Mexican H7N3 HPAI viruses, including 7 amino acid changes observed in higher proportion in North American poultry origin AI viruses isolates than in wild bird-origin viruses. Continuous monitoring and molecular characterization of the H7N3 HPAI virus is important for better understanding of the virus evolutionary dynamics and further improving control measures including vaccination.

A novel H7N3 reassortant originating from the zoonotic H7N9 highly pathogenic avian influenza viruses that has adapted to ducks.

The first human case of zoonotic H7N9 avian influenza virus (AIV) infection was reported in March 2013 in China. This virus continues to circulate in poultry in China while mutating to highly pathogenic AIVs (HPAIVs). Through monitoring at airports in Japan, a novel H7N3 reassortant of the zoonotic H7N9 HPAIVs, A/duck/Japan/AQ-HE30-1/2018 (HE30-1), was detected in a poultry meat product illegally brought by a passenger from China into Japan. We analysed the genetic, pathogenic and antigenic characteristics of HE30-1 by comparing it with previous zoonotic H7N9 AIVs and their reassortants. Phylogenetic analysis of the entire HE30-1 genomic sequence revealed that it comprised at least three different sources; the HA (H7), PB1, PA, NP, M and NS segments of HE30-1 were directly derived from H7N9 AIVs, whereas the NA (N3) and PB2 segments of HE30-1 were unrelated to zoonotic H7N9. Experimental infection revealed that HE30-1 was lethal in chickens but not in domestic or mallard ducks. HE30-1 was shed from and replicated in domestic and mallard ducks and chickens, whereas previous zoonotic H7N9 AIVs have not adapted well to ducks. This finding suggests the possibility that HE30-1 may disseminate to remote area by wild bird migration once it establishes in wild bird population. A haemagglutination-inhibition assay indicated that antigenic drift has occurred among the reassortants of zoonotic H7N9 AIVs; HE30-1 showed similar antigenicity to some of those H7N9 AIVs, suggesting it might be prevented by the H5/H7 inactivated vaccine that was introduced in China in 2017. Our study reports the emergence of a new reassortant of zoonotic H7N9 AIVs with novel viral characteristics and warns of the challenge we still face to control the zoonotic H7N9 AIVs and their reassortants.

Co-subsistence of avian influenza virus subtypes of low and high pathogenicity in Bangladesh: Challenges for diagnosis, risk assessment and control.

Endemic co-circulation of potentially zoonotic avian influenza viruses (AIV) of subtypes H5N1 and H9N2 (G1 lineage) in poultry in Bangladesh accelerated diversifying evolution. Two clinical samples from poultry obtained in 2016 yielded five different subtypes (highly pathogenic [HP] H5N1, HP H5N2, HP H7N1, HP H7N2, H9N2) and eight genotypes of AIV by plaque purification. H5 sequences grouped with clade 2.3.2.1a viruses while N1 was related to an older, preceding clade, 2.2.2. The internal genome segments of the plaque-purified viruses originated from clade 2.2.2 of H5N1 or from G1/H9N2 viruses. H9 and N2 segments clustered with contemporary H9N2 strains. In addition, HP H7 sequences were detected for the first time in samples and linked to Pakistani HP H7N3 viruses of 2003. The unexpected findings of mixtures of reassorted HP H5N1 and G1-like H9N2 viruses, which carry genome segments of older clades in association with the detection of HP H7 HA segments calls for confirmation of these results by targeted surveillance in the area of origin of the investigated samples. Hidden niches and obscured transmission pathways may exist that retain or re-introduce genome segments of older viruses or reassortants thereof which causes additional challenges for diagnosis, risk assessment and disease control.

Efficacy of novel recombinant fowlpox vaccine against recent Mexican H7N3 highly pathogenic avian influenza virus.

Since 2012, H7N3 highly pathogenic avian influenza (HPAI) has produced negative economic and animal welfare impacts on poultry in central Mexico. In the present study, chickens were vaccinated with two different recombinant fowlpox virus vaccines (rFPV-H7/3002 with 2015 H7 hemagglutinin [HA] gene insert, and rFPV-H7/2155 with 2002 H7 HA gene insert), and were then challenged three weeks later with H7N3 HPAI virus (A/chicken/Jalisco/CPA-37905/2015). The rFPV-H7/3002 vaccine conferred 100% protection against mortality and morbidity, and significantly reduced virus shed titers from the respiratory and gastrointestinal tracts. In contrast, 100% of sham and rFPV-H7/2155 vaccinated birds shed virus at higher titers and died within 4 days. Pre- (15/20) and post- (20/20) challenge serum of birds vaccinated with rFPV-H7/3002 had antibodies detectable by hemagglutination inhibition (HI) assay using challenge virus antigen. However, only a few birds (3/20) in the rFPV-H7/2155 vaccinated group had antibodies that reacted against the challenge strain but all birds had antibodies that reacted against the homologous vaccine antigen (A/turkey/Virginia/SEP-66/2002) (20/20). One possible explanation for differences in vaccines efficacy is the antigenic drift between circulating viruses and vaccines. Molecular analysis demonstrated that the Mexican H7N3 strains have continued to rapidly evolve since 2012. In addition, we identified in silico three potential new N-glycosylation sites on the globular head of the H7 HA of A/chicken/Jalisco/CPA-37905/2015 challenge virus, which were absent in 2012 H7N3 outbreak virus. Our results suggested that mutations in the HA antigenic sites including increased glycosylation sites, accumulated in the new circulating Mexican H7 HPAIV strains, altered the recognition of neutralizing antibodies from the older vaccine strain rFPV-H7/2155. Therefore, the protective efficacy of novel rFPV-H7/3002 against recent outbreak Mexican H7N3 HPAIV confirms the importance of frequent updating of vaccines seed strains for long-term effective control of H7 HPAI virus.

Characterization of a novel reassortant H7N3 highly pathogenic avian influenza virus isolated from a poultry meat product taken on a passenger flight to Japan.

A new reassortant H7N3 avian influenza virus (AIV) was isolated from a duck meat product that was illegally taken on board a passenger flight from China to Japan in March 2018. Sequencing analysis revealed that the H7N3 isolate, A/duck/Japan/AQ-HE30-1/2018 (Dk/HE30-1) (H7N3), was a reassortant highly pathogenic avian influenza virus (HPAIV) that contained the haemagglutinin (HA) gene of Chinese H7N9 HPAIV. Dk/HE30-1 (H7N3) possessed a novel polybasic sequence motif PEVPKRRRTAR/GLF at the HA cleavage site that has never previously been reported in H7 HPAIVs. The HA antigenicity of Dk/HE30-1 (H7N3) slightly differed from that of H7N9 HPAIVs previously reported. These findings will help further our knowledge of the circulation and genetic evolution of emerging AIVs in endemic areas.

Virus-like particles displaying H5, H7, H9 hemagglutinins and N1 neuraminidase elicit protective immunity to heterologous avian influenza viruses in chickens.

Avian influenza (AI) viruses circulating in wild birds pose a serious threat to public health. Human and veterinary vaccines against AI subtypes are needed. Here we prepared triple-subtype VLPs that co-localized H5, H7 and H9 antigens derived from H5N1, H7N3 and H9N2 viruses. VLPs also contained influenza N1 neuraminidase and retroviral gag protein. The H5/H7/H9/N1/gag VLPs were prepared using baculovirus expression. Biochemical, functional and antigenic characteristics were determined including hemagglutination and neuraminidase enzyme activities. VLPs were further evaluated in a chicken AI challenge model for safety, immunogenicity and protective efficacy against heterologous AI viruses including H5N2, H7N3 and H9N2 subtypes. All vaccinated birds survived challenges with H5N2 and H7N3 highly pathogenic AI (HPAI) viruses, while all controls died. Immune response was also detectable after challenge with low pathogenicity AI (LPAI) H9N2 virus suggesting that H5/H7/H9/N1/gag VLPs represent a promising approach for the development of broadly protective AI vaccine.

An engineered avian-origin influenza A virus for pancreatic ductal adenocarcinoma virotherapy.

Pancreatic ductal adenocarcinoma (PDA) is one of the leading causes of cancer-related deaths worldwide and the development of new treatment strategies for PDA patients is of crucial importance. Virotherapy uses natural or engineered oncolytic viruses (OVs) to selectively kill tumour cells. Due to their genetic heterogeneity, PDA cells are highly variable in their permissiveness to various OVs. The avian influenza A virus (IAV) H7N3 A/turkey/Italy/2962/03 is a potent inducer of apoptosis in PDA cells previously shown to be resistant to other OVs (Kasloff et al., 2014), suggesting that it might be effective against specific subclasses of pancreatic cancer. To improve the selectivity of the avian influenza isolate for PDA cells, here confirmed deficient for IFN response, we engineered a truncation in the NS1 gene that is the major virus-encoded IFN antagonist. The recombinant virus (NS1-77) replicated efficiently in PDA cells, but was attenuated in non-malignant pancreatic ductal cells, in which it induced a potent IFN response that acted upon bystander uninfected cancer cells, triggering their death. The engineered virus displayed an enhanced ability to debulk a PDA-derived tumour in xenograft mouse model. Our results highlight the possibility of selecting an IAV strain from the diverse natural avian reservoir on the basis of its inherent oncolytic potency in specific PDA subclasses and, through engineering, improve its safety, selectivity and debulking activity for cancer treatment.

Evidence for wild waterfowl origin of H7N3 influenza A virus detected in captive-reared New Jersey pheasants.

In August 2014, a low-pathogenic H7N3 influenza A virus was isolated from pheasants at a New Jersey gamebird farm and hunting preserve. In this study, we use phylogenetic analyses and calculations of genetic similarity to gain inference into the genetic ancestry of this virus and to identify potential routes of transmission. Results of maximum-likelihood (ML) and maximum-clade-credibility (MCC) phylogenetic analyses provide evidence that A/pheasant/New Jersey/26996-2/2014 (H7N3) had closely related H7 hemagglutinin (HA) and N3 neuraminidase (NA) gene segments as compared to influenza A viruses circulating among wild waterfowl in the central and eastern USA. The estimated time of the most recent common ancestry (TMRCA) between the pheasant virus and those most closely related from wild waterfowl was early 2013 for both the H7 HA and N3 NA gene segments. None of the viruses from waterfowl identified as being most closely related to A/pheasant/New Jersey/26996-2/2014 at the HA and NA gene segments in ML and MCC phylogenetic analyses shared ≥99 % nucleotide sequence identity for internal gene segment sequences. This result indicates that specific viral strains identified in this study as being closely related to the HA and NA gene segments of A/pheasant/New Jersey/26996-2/2014 were not the direct predecessors of the etiological agent identified during the New Jersey outbreak. However, the recent common ancestry of the H7 and N3 gene segments of waterfowl-origin viruses and the virus isolated from pheasants suggests that viral diversity maintained in wild waterfowl likely played an important role in the emergence of A/pheasant/New Jersey/26996-2/2014.

Effect of Infection with a Mesogenic Strain of Newcastle Disease Virus on Infection with Highly Pathogenic Avian Influenza Virus in Chickens.

Little is known on the interactions between avian influenza virus (AIV) and Newcastle disease virus (NDV) when coinfecting the same poultry host. In a previous study we found that infection of chickens with a mesogenic strain of NDV (mNDV) can reduce highly pathogenic AIV (HPAIV) replication, clinical disease, and mortality. This interaction depended on the titer of the viruses used and the timing of the infections. To further explore the effect of mNDV infectious dose in protecting chickens against HPAIV infection, 2-wk-old birds were inoculated with different doses of mNDV (10(4), 10(6), or 10(7) 50% embryo infective dose [EID50]) 3 days before inoculation with a HPAIV (10(5) or 10(6) EID50). Although birds coinfected with the higher mNDV doses (10(6) or 10(7)) survived for longer than birds inoculated only with HPAIV (10(5)), we did not observe the same protection with the lower dose of mNDV (10(4)) or when given the higher dose of HPAIV (10(6)), indicating that the relation between the titer of the two coinfecting viruses is determinant in the outcome. In a similar experiment, a higher number of 4-wk-old birds survived, and for longer, even when given higher HPAIV doses (10(6.3) and 10(7.3) EID50). In addition, we also examined the duration of protection provided by mNDV (10(7) EID50) on a HPAIV infection. Five-week-old chickens were inoculated with mNDV followed by inoculation with 10(6) EID50 of an HPAIV given at 2, 4, 6, or 9 days after the mNDV. HPAIV replication was affected and an increase in survival was found in all coinfected groups when compared to the HPAIV single-inoculated group, but the mortality in coinfected groups was high. In conclusion, previous inoculation with mNDV can affect HPAIV replication in chickens for at least 9 days, but this viral interference is titer dependent.

Residues in the PB2 and PA genes contribute to the pathogenicity of avian H7N3 influenza A virus in DBA/2 mice.

Replication and transmission of avian influenza virus in humans poses a pandemic threat. The molecular determinants that facilitate this process are not well understood. We used DBA/2 mice to identify viral factors that mediate the difference in pathogenesis between a virulent (H7N3) and a non-virulent (H7N9) avian influenza virus from North America. In vitro and in vivo characterization of reassortant viruses identified the PB2 and PA polymerase genes as major determinants of H7N3 pathogenesis. Analysis of individual residues in the PB2 and PA genes identified position 358 (E358V) in PB2 and positions 190 (P190S) and 400 (Q400P) in PA that reduced the virulence of H7N3 virus. The E358V and P190S substitutions also caused reduced inflammation after infection. Our results suggest that specific residues in the polymerase proteins PB2 and PA are important for replication and virulence of avian influenza viruses in a mammalian host.