Evolution of the North American Lineage H7 Avian Influenza Viruses in Association with H7 Virus's Introduction to Poultry.

The incursions of H7 subtype low-pathogenicity avian influenza virus (LPAIV) from wild birds into poultry and its mutations to highly pathogenic avian influenza virus (HPAIV) have been an ongoing concern in North America. Since 2000, 10 phylogenetically distinct H7 virus outbreaks from wild birds have been detected in poultry, six of which mutated to HPAIV. To study the molecular evolution of the H7 viruses that occurs when changing hosts from wild birds to poultry, we performed analyses of the North American H7 hemagglutinin (HA) genes to identify amino acid changes as the virus circulated in wild birds from 2000 to 2019. Then, we analyzed recurring HA amino acid changes and gene constellations of the viruses that spread from wild birds to poultry. We found six HA amino acid changes occurring during wild bird circulation and 10 recurring changes after the spread to poultry. Eight of the changes were in and around the HA antigenic sites, three of which were supported by positive selection. Viruses from each H7 outbreak had a unique genotype, with no specific genetic group associated with poultry outbreaks or mutation to HPAIV. However, the genotypes of the H7 viruses in poultry outbreaks tended to contain minor genetic groups less observed in wild bird H7 viruses, suggesting either a biased sampling of wild bird AIVs or a tendency of having reassortment with minor genetic groups prior to the virus's introduction to poultry. IMPORTANCE Wild bird-origin H7 subtype avian influenza viruses are a constant threat to commercial poultry, both directly by the disease they cause and indirectly through trade restrictions that can be imposed when the virus is detected in poultry. It is important to understand the genetic basis of why the North American lineage H7 viruses have repeatedly crossed the species barrier from wild birds to poultry. We examined the amino acid changes in the H7 viruses associated with poultry outbreaks and tried to determine gene reassortment related to poultry adaptation and mutations to HPAIV. The findings in this study increase the understanding of the evolutionary pathways of wild bird AIV before infecting poultry and the HA changes associated with adaptation of the virus in poultry.

Multiple Gene Segments Are Associated with Enhanced Virulence of Clade 2.3.4.4 H5N8 Highly Pathogenic Avian Influenza Virus in Mallards.

Highly pathogenic avian influenza (HPAI) viruses from the H5Nx Goose/Guangdong/96 lineage continue to cause outbreaks in domestic and wild bird populations. Two distinct genetic groups of H5N8 HPAI viruses, hemagglutinin (HA) clades 2.3.4.4A and 2.3.4.4B, caused intercontinental outbreaks in 2014 to 2015 and 2016 to 2017, respectively. Experimental infections using viruses from these outbreaks demonstrated a marked difference in virulence in mallards, with the H5N8 virus from 2014 causing mild clinical disease and the 2016 H5N8 virus causing high mortality. To assess which gene segments are associated with enhanced virulence of H5N8 HPAI viruses in mallards, we generated reassortant viruses with 2014 and 2016 viruses. For single-segment reassortants in the genetic backbone of the 2016 virus, pathogenesis experiments in mallards revealed that morbidity and mortality were reduced for all eight single-segment reassortants compared to the parental 2016 virus, with significant reductions in mortality observed with the polymerase basic protein 2 (PB2), nucleoprotein (NP), and matrix (M) reassortants. No differences in morbidity and mortality were observed with reassortants that either have the polymerase complex segments or the HA and neuraminidase (NA) segments of the 2016 virus in the genetic backbone of the 2014 virus. In vitro assays showed that the NP and polymerase acidic (PA) segments of the 2014 virus lowered polymerase activity when combined with the polymerase complex segments of the 2016 virus. Furthermore, the M segment of the 2016 H5N8 virus was linked to filamentous virion morphology. Phylogenetic analyses demonstrated that gene segments related to the more virulent 2016 H5N8 virus have persisted in the contemporary H5Nx HPAI gene pool until 2020. IMPORTANCE Outbreaks of H5Nx HPAI viruses from the goose/Guangdong/96 lineage continue to occur in many countries and have resulted in substantial impact on wild birds and poultry. Epidemiological evidence has shown that wild waterfowl play a major role in the spread of these viruses. While HPAI virus infection in gallinaceous species causes high mortality, a wide range of disease outcomes has been observed in waterfowl species. In this study, we examined which gene segments contribute to severe disease in mallards infected with H5N8 HPAI viruses. No virus gene was solely responsible for attenuating the high virulence of a 2016 H5N8 virus, but the PB2, NP, and M segments significantly reduced mortality. The findings herein advance our knowledge on the pathobiology of avian influenza viruses in waterfowl and have potential implications on the ecology and epidemiology of H5Nx HPAI in wild bird populations.

Mutations in PB1, NP, HA, and NA Contribute to Increased Virus Fitness of H5N2 Highly Pathogenic Avian Influenza Virus Clade 2.3.4.4 in Chickens.

The H5N8 highly pathogenic avian influenza (HPAI) clade 2.3.4.4 virus spread to North America by wild birds and reassorted to generate the H5N2 HPAI virus that caused the poultry outbreak in the United States in 2015. In previous studies, we showed that H5N2 viruses isolated from poultry in the later stages of the outbreak had higher infectivity and transmissibility in chickens than the wild bird index H5N2 virus. Here, we determined the genetic changes that contributed to the difference in host virus fitness by analyzing sequence data from all of the viruses detected during the H5N2 outbreak, and studying the pathogenicity of reassortant viruses generated with the index wild bird virus and a chicken virus from later in the outbreak. Viruses with the wild bird virus backbone and either PB1, NP, or the entire polymerase complex of the chicken isolate, caused higher and earlier mortality in chickens, with three mutations (PB1 E180D, M317V, and NP I109T) identified to increase polymerase activity in chicken cells. The reassortant virus with the HA and NA from the chicken virus, where mutations in functionally known gene regions were acquired as the virus circulated in turkeys (HA S141P and NA S416G) and later in chickens (HA M66I, L322Q), showed faster virus growth, bigger plaque size and enhanced heat persistence in vitro, and increased pathogenicity and transmissibility in chickens. Collectively, these findings demonstrate an evolutionary pathway in which a HPAI virus from wild birds can accumulate genetic changes to increase fitness in poultry.IMPORTANCE H5Nx highly pathogenic avian influenza (HPAI) viruses of the A/goose/Guangdong/1/96 lineage continue to circulate widely affecting both poultry and wild birds. These viruses continue to change and reassort, which affects their fitness to different avian hosts. In this study, we defined mutations associated with increased virus fitness in chickens as the clade 2.3.4.4. H5N2 HPAI virus circulated in different avian species. We identified mutations in the PB1, NP, HA, and NA virus proteins that were highly conserved in the poultry isolates and contributed to the adaptation of this virus in chickens. This knowledge is important for understanding the epidemiology of H5Nx HPAI viruses and specifically the changes related to adaptation of these viruses in poultry.

Influenza A viruses remain infectious for more than seven months in northern wetlands of North America.

In this investigation, we used a combination of field- and laboratory-based approaches to assess if influenza A viruses (IAVs) shed by ducks could remain viable for extended periods in surface water within three wetland complexes of North America. In a field experiment, replicate filtered surface water samples inoculated with duck swabs were tested for IAVs upon collection and again after an overwintering period of approximately 6-7 months. Numerous IAVs were molecularly detected and isolated from these samples, including replicates maintained at wetland field sites in Alaska and Minnesota for 181-229 days. In a parallel laboratory experiment, we attempted to culture IAVs from filtered surface water samples inoculated with duck swabs from Minnesota each month during September 2018-April 2019 and found monthly declines in viral viability. In an experimental challenge study, we found that IAVs maintained in filtered surface water within wetlands of Alaska and Minnesota for 214 and 226 days, respectively, were infectious in a mallard model. Collectively, our results support surface waters of northern wetlands as a biologically important medium in which IAVs may be both transmitted and maintained, potentially serving as an environmental reservoir for infectious IAVs during the overwintering period of migratory birds.

Loss of Fitness of Mexican H7N3 Highly Pathogenic Avian Influenza Virus in Mallards after Circulating in Chickens.

Outbreaks of highly pathogenic avian influenza (HPAI) virus subtype H7N3 have been occurring in commercial chickens in Mexico since its first introduction in 2012. In order to determine changes in virus pathogenicity and adaptation in avian species, three H7N3 HPAI viruses from 2012, 2015, and 2016 were evaluated in chickens and mallards. All three viruses caused high mortality in chickens when given at medium to high doses and replicated similarly. No mortality or clinical signs and similar infectivity were observed in mallards inoculated with the 2012 and 2016 viruses. However, the 2012 H7N3 HPAI virus replicated well in mallards and transmitted to contacts, whereas the 2016 virus replicated poorly and did not transmit to contacts, which indicates that the 2016 virus is less adapted to mallards. In vitro, the 2016 virus grew slower and to lower titers than did the 2012 virus in duck fibroblast cells. Full-genome sequencing showed 115 amino acid differences between the 2012 and the 2016 viruses, with some of these changes previously associated with changes in replication in avian species, including hemagglutinin (HA) A125T, nucleoprotein (NP) M105V, and NP S377N. In conclusion, as the Mexican H7N3 HPAI virus has passaged through large populations of chickens in a span of several years and has retained its high pathogenicity for chickens, it has decreased in fitness in mallards, which could limit the potential spread of this HPAI virus by waterfowl.IMPORTANCE Not much is known about changes in host adaptation of avian influenza (AI) viruses in birds after long-term circulation in chickens or other terrestrial poultry. Although the origin of AI viruses affecting poultry is wild aquatic birds, the role of these birds in further dispersal of poultry-adapted AI viruses is not clear. Previously, we showed that HPAI viruses isolated early from poultry outbreaks could still infect and transmit well in mallards. In this study, we demonstrate that the Mexican H7N3 HPAI virus after four years of circulation in chickens replicates poorly and does not transmit in mallards but remains highly pathogenic in chickens. This information on changes in host adaptation is important for understanding the epidemiology of AI viruses and the role that wild waterfowl may play in disseminating viruses adapted to terrestrial poultry.

Pathogenicity in Chickens and Turkeys of a 2021 United States H5N1 Highly Pathogenic Avian Influenza Clade 2.3.4.4b Wild Bird Virus Compared to Two Previous H5N8 Clade 2.3.4.4 Viruses.

Highly pathogenic avian influenza viruses (HPAIVs) of subtype H5 of the Gs/GD/96 lineage remain a major threat to poultry due to endemicity in wild birds. H5N1 HPAIVs from this lineage were detected in 2021 in the United States (U.S.) and since then have infected many wild and domestic birds. We evaluated the pathobiology of an early U.S. H5N1 HPAIV (clade 2.3.4.4b, 2021) and two H5N8 HPAIVs from previous outbreaks in the U.S. (clade 2.3.4.4c, 2014) and Europe (clade 2.3.4.4b, 2016) in chickens and turkeys. Differences in clinical signs, mean death times (MDTs), and virus transmissibility were found between chickens and turkeys. The mean bird infective dose (BID50) of the 2021 H5N1 virus was approximately 2.6 log10 50% embryo infective dose (EID50) in chickens and 2.2 log10 EID50 in turkeys, and the virus transmitted to contact-exposed turkeys but not chickens. The BID50 for the 2016 H5N8 virus was also slightly different in chickens and turkeys (4.2 and 4.7 log10 EID50, respectively); however, the BID50 for the 2014 H5N8 virus was higher for chickens than turkeys (3.9 and ~0.9 log10 EID50, respectively). With all viruses, turkeys took longer to die (MDTs of 2.6-8.2 days for turkeys and 1-4 days for chickens), which increased the virus shedding period and facilitated transmission to contacts.

H5N1 highly pathogenic avian influenza clade 2.3.4.4b in wild and domestic birds: Introductions into the United States and reassortments, December 2021-April 2022.

Highly pathogenic avian influenza viruses (HPAIVs) of the A/goose/Guangdong/1/1996 lineage H5 clade 2.3.4.4b continue to have a devastating effect on domestic and wild birds. Full genome sequence analyses using 1369 H5N1 HPAIVs detected in the United States (U.S.) in wild birds, commercial poultry, and backyard flocks from December 2021 to April 2022, showed three phylogenetically distinct H5N1 virus introductions in the U.S. by wild birds. Unreassorted Eurasian genotypes A1 and A2 entered the Northeast Atlantic states, whereas a genetically distinct A3 genotype was detected in Alaska. The A1 genotype spread westward via wild bird migration and reassorted with North American wild bird avian influenza viruses. Reassortments of up to five internal genes generated a total of 21 distinct clusters; of these, six genotypes represented 92% of the HPAIVs examined. By phylodynamic analyses, most detections in domestic birds were shown to be point-source transmissions from wild birds, with limited farm-to-farm spread.

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.

Genome Sequence Variations of Infectious Bronchitis Virus Serotypes From Commercial Chickens in Mexico.

New variants of infectious bronchitis viruses (IBVs; Coronaviridae) continuously emerge despite routine vaccinations. Here, we report genome sequence variations of IBVs identified by random non-targeted next generation sequencing (NGS) of vaccine and field samples collected on FTA cards from commercial flocks in Mexico in 2019-2021. Paired-ended sequencing libraries prepared from rRNA-depleted RNAs were sequenced using Illumina MiSeq. IBV RNA was detected in 60.07% (n = 167) of the analyzed samples, from which 33 complete genome sequences were de novo assembled. The genomes are organized as 5'UTR-[Rep1a-Rep1b-S-3a-3b-E-M-4b-4c-5a-5b-N-6b]-3'UTR, except in eight sequences lacking non-structural protein genes (accessory genes) 4b, 4c, and 6b. Seventeen sequences have auxiliary S2' cleavage site located 153 residues downstream the canonically conserved primary furin-specific S1/S2 cleavage site. The sequences distinctly cluster into lineages GI-1 (Mass-type; n = 8), GI-3 (Holte/Iowa-97; n = 2), GI-9 (Arkansas-like; n = 8), GI-13 (793B; n = 14), and GI-17 (California variant; CAV; n = 1), with regional distribution in Mexico; this is the first report of the presence of 793B- and CAV-like strains in the country. Various point mutations, substitutions, insertions and deletions are present in the S1 hypervariable regions (HVRs I-III) across all 5 lineages, including in residues 38, 43, 56, 63, 66, and 69 that are critical in viral attachment to respiratory tract tissues. Nine intra-/inter-lineage recombination events are present in the S proteins of three Mass-type sequences, two each of Holte/Iowa-97 and Ark-like sequence, and one each of 793B-like and CAV-like sequences. This study demonstrates the feasibility of FTA cards as an attractive, adoptable low-cost sampling option for untargeted discovery of avian viral agents in field-collected clinical samples. Collectively, our data points to co-circulation of multiple distinct IBVs in Mexican commercial flocks, underscoring the need for active surveillance and a review of IBV vaccines currently used in Mexico and the larger Latin America region.

Phylogenetic analysis, molecular changes, and adaptation to chickens of Mexican lineage H5N2 low-pathogenic avian influenza viruses from 1994 to 2019.

The Mexican lineage H5N2 low pathogenic avian influenza viruses (LPAIVs) were first detected in 1994 and mutated to highly pathogenic avian influenza viruses (HPAIVs) in 1994-1995 causing widespread outbreaks in poultry. By using vaccination and other control measures, the HPAIVs were eradicated but the LPAIVs continued circulating in Mexico and spread to several other countries. To get better resolution of the phylogenetics of this virus, the full genome sequences of 44 H5N2 LPAIVs isolated from 1994 to 2011, and 6 detected in 2017 and 2019, were analysed. Phylogenetic incongruence demonstrated genetic reassortment between two separate groups of the Mexican lineage H5N2 viruses between 2005 and 2010. Moreover, the recent H5N2 viruses reassorted with previously unidentified avian influenza viruses. Bayesian phylogeographic results suggested that mechanical transmission involving human activity is the most probable cause of the virus spillover to Central American, Caribbean, and East Asian countries. Increased infectivity and transmission of a 2011 H5N2 LPAIV in chickens compared to a 1994 virus demonstrates improved adaptation to chickens, while low virus shedding, and limited contact transmission was observed in mallards with the same 2011 virus. The sporadic increase in basic amino acids in the HA cleavage site, changes in potential N-glycosylation sites in the HA, and truncations of PB1-F2 should be further examined in relation to the increased infectivity and transmission in poultry. The genetic changes that occur as this lineage of H5N2 LPAIVs continues circulating in poultry is concerning not only because of the effect of these changes on vaccination efficacy, but also because of the potential of the viruses to mutate to the highly pathogenic form. Continued vigilance and surveillance efforts, and the pathogenic and genetic characterization of circulating viruses, are required for the effective control of this virus.

Comparable outcomes from long and short read random sequencing of total RNA for detection of pathogens in chicken respiratory samples.

Co-infections of avian species with different RNA viruses and pathogenic bacteria are often misdiagnosed or incompletely characterized using targeted diagnostic methods, which could affect the accurate management of clinical disease. A non-targeted sequencing approach with rapid and precise characterization of pathogens should help respiratory disease management by providing a comprehensive view of the causes of disease. Long-read portable sequencers have significant potential advantages over established short-read sequencers due to portability, speed, and lower cost. The applicability of short reads random sequencing for direct detection of pathogens in clinical poultry samples has been previously demonstrated. Here we demonstrate the feasibility of long read random sequencing approaches to identify disease agents in clinical samples. Experimental oropharyngeal swab samples (n = 12) from chickens infected with infectious bronchitis virus (IBV), avian influenza virus (AIV) and Mycoplasma synoviae (MS) and field-collected clinical oropharyngeal swab samples (n = 11) from Kenyan live bird markets previously testing positive for Newcastle disease virus (NDV) were randomly sequenced on the MinION platform and results validated by comparing to real time PCR and short read random sequencing in the Illumina MiSeq platform. In the swabs from experimental infections, each of three agents in every RT-qPCR-positive sample (Ct range 19-34) was detectable within 1 h on the MinION platform, except for AIV one agent in one sample (Ct = 36.21). Nine of 12 IBV-positive samples were assigned genotypes within 1 h, as were five of 11 AIV-positive samples. MinION relative abundances of the test agent (AIV, IBV and MS) were highly correlated with RT-qPCR Ct values (R range-0.82 to-0.98). In field-collected clinical swab samples, NDV (Ct range 12-37) was detected in all eleven samples within 1 h of MinION sequencing, with 10 of 11 samples accurately genotyped within 1 h. All NDV-positive field samples were found to be co-infected with one or more additional respiratory agents. These results demonstrate that MinION sequencing can provide rapid, and sensitive non-targeted detection and genetic characterization of co-existing respiratory pathogens in clinical samples with similar performance to the Illumina MiSeq.

Age-Associated Changes in Recombinant H5 Highly Pathogenic and Low Pathogenic Avian Influenza Hemagglutinin Tissue Binding in Domestic Poultry Species.

The 2014 outbreak of clade 2.3.4.4A highly pathogenic avian influenza (HPAI) led to the culling of millions of commercial chickens and turkeys and death of various wild bird species. In this outbreak, older chickens and turkeys were commonly infected, and succumbed to clinical disease compared to younger aged birds such chicken broilers. Some experimental studies using waterfowl species have shown age-related differences in susceptibility to clinical disease with HPAI viruses. Here, we evaluate differences in H5 Hemagglutinin (HA) tissue binding across age groups, using recombinant H5 HA (rHA) proteins generated using gene sequences from low pathogenic (A/mallard/MN/410/2000(H5N2 (LPAIV)) and a HPAIV (A/Northern pintail/Washington/40964/2014(H5N2)) influenza A virus (IAV). Respiratory and intestinal tracts from chickens, ducks (Mallard, Pekin, Muscovy) and turkeys of different age groups were used to detect rHA binding with protein histochemistry, which was quantified as the median area of binding (MAB) used for statistical analysis. There were species and tissue specific differences in the rHA binding among the age groups; however, turkeys had significant differences in the HPAIV rHA binding in the respiratory tract, with younger turkeys having higher levels of binding in the lung compared to the older group. In addition, in the intestinal tract, younger turkeys had higher levels of binding compared to the older birds. Using LPAIV, similar species and tissues, specific differences were seen among the age groups; however, only turkeys had overall significant differences in the respiratory tract MAB, with the older birds having higher levels of binding compared to the younger group. No age-related differences were seen in the overall intestinal tract rHA binding. Age-related differences in rHA binding of the LPAIV and HPAIV demonstrated in this study may partially, but not completely, explain differences in host susceptibility to infection observed during avian influenza outbreaks and in experimental infection studies.

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.

Recombinant hemagglutinin glycoproteins provide insight into binding to host cells by H5 influenza viruses in wild and domestic birds.

Clade 2.3.4.4, H5 subtype highly pathogenic avian influenza viruses (HPAIVs) have caused devastating effects across wild and domestic bird populations. We investigated differences in the intensity and distribution of the hemagglutinin (HA) glycoprotein binding of a clade 2.3.4.4 H5 HPAIV compared to a H5 low pathogenic avian influenza virus (LPAIV). Recombinant HA from gene sequences from a HPAIV, A/Northern pintail/Washington/40964/2014(H5N2) and a LPAIV, A/mallard/MN/410/2000(H5N2) were generated and, via protein histochemistry, HA binding in respiratory, intestinal and cloacal bursal tissue was quantified as median area of binding (MAB). Poultry species, shorebirds, ducks and terrestrial birds were used. Differences in MAB were observed between the HPAIV and LPAIV H5 HAs. We demonstrate that clade 2.3.4.4 HPAIV H5 HA has a broader host cell binding across a variety of bird species compared to the LPAIV H5 HA. These findings support published results from experimental trials, and outcomes of natural disease outbreaks with these viruses.

Pathogenicity and genomic changes of a 2016 European H5N8 highly pathogenic avian influenza virus (clade 2.3.4.4) in experimentally infected mallards and chickens.

Highly pathogenic avian influenza H5N8 clade 2.3.4.4 virus caused outbreaks in poultry and unusually high mortality in wild birds in 2016-2017. The pathobiology of one of these viruses was examined in mallards and chickens. High mortality and transmission to direct contacts were observed in mallards inoculated with medium and high doses of the virus. However, in chickens, high mortality occurred only when birds are given the high virus dose and no transmission was observed, indicating that the virus was better adapted to mallards. In comparison with the virus inoculum, viral sequences obtained from the chickens had a higher number of nucleotide changes but lower intra-host genomic diversity than viral sequences obtained from the mallards. These observations are consistent with population bottlenecks occurring when viruses infect and replicate in a host that it is not well adapted to. Whether these observations apply to influenza viruses in general remains to be determined.

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.

Minimum Infectious Dose Determination of the Arkansas Delmarva Poultry Industry Infectious Bronchitis Virus Vaccine Delivered by Hatchery Spray Cabinet.

The Arkansas Delmarva Poultry Industry (ArkDPI) infectious bronchitis virus (IBV) vaccine is effective when administered by eye drop, where the vaccine virus is able to infect and replicate well in birds and is able to induce protection against homologous challenge. However, accumulating evidence indicates that the ArkDPI vaccine is ineffective when applied by hatchery spray cabinet using the same manufacturer-recommended dose per bird. For this study, we aimed to determine the minimum infectious dose for the spray-administered ArkDPI vaccine, which we designate as the dose that achieves the same level of infection and replication as the eye drop-administered ArkDPI vaccine. To this end, we used increasing doses of commercial ArkDPI vaccine to vaccinate 100 commercial broiler chicks at day of hatch, using a commercial hatchery spray cabinet. The choanal cleft of each bird was swabbed at 7 and 10 days postvaccination, and real-time reverse-transcriptase PCR was performed. We observed that the level of infection and replication with spray vaccination matches with that of eye drop vaccination when chicks received 100 times the standard dose for the commercial ArkDPI vaccine. We further examined the S1 spike gene sequence from a subset of reisolated ArkDPI vaccine virus samples and observed that certain nucleotide changes arise in vaccine viruses reisolated from chicks, as previously reported. This suggests that the ArkDPI vaccine has a certain virus subpopulation that, while successful at infecting and replicating in chicks, represents only a minor virus subpopulation in the original vaccine. Thus, the minimum infectious dose for the ArkDPI vaccine using a hatchery spray cabinet appears to be dependent on the amount of this minor subpopulation reaching the chicks.

Polymorphisms in the S1 spike glycoprotein of Arkansas-type infectious bronchitis virus (IBV) show differential binding to host tissues and altered antigenicity.

Sequencing avian infectious bronchitis virus spike genes re-isolated from vaccinated chicks revealed that many sequence changes are found on the S1 spike gene. In the ArkDPI strain, Y43H and ∆344 are the two most common changes observed. This study aims to examine the roles of Y43H and ∆344 in selection in vivo. Using recombinant ArkDPI S1 proteins, we conducted binding assays on chicken tracheas and embryonic chorioallantoic membrane (CAM). Protein histochemistry showed that the Y43H change allows for enhanced binding to trachea, whereas the ArkDPI S1 spike with H43 alone was able to bind CAM. Using Western blot under denaturing conditions, ArkDPI serotype-specific sera did not bind to S1 proteins with ∆344, suggesting that ∆344 alters antigenicity of S1. These findings are important because they propose that specific changes in S1 enhances virus fitness by more effective binding to host tissues (Y43H) and by evading a vaccine-induced antibody response (∆344).

Insights from molecular structure predictions of the infectious bronchitis virus S1 spike glycoprotein.

Infectious bronchitis virus is an important respiratory pathogen in chickens. The IBV S1 spike is a viral structural protein that is responsible for attachment to host receptors and is a major target for neutralizing antibodies. To date, there is no experimentally determined structure for the IBV S1 spike. In this study, we sought to find a predicted tertiary structure for IBV S1 using I-TASSER, which is an automated homology modeling platform. We found that the predicted structures obtained were robust and consistent with experimental data. For instance, we observed that all four residues (38, 43, 63, and 68) that have been shown to be critical for binding to host tissues, were found at the surface of the predicted structure of Massachusetts (Mass) S1 spike. Together with antigenicity index analysis, we were also able to show that Ma5 vaccine has higher antigenicity indices at residues close to the receptor-binding region than M41 vaccine, thereby providing a possible mechanism on how Ma5 achieves better protection against challenge. Examination of the predicted structure of the Arkansas IBV S1 spike also gave insights on the effect of polymorphisms at position 43 on the surface availability of receptor binding residues. This study showcases advancements in protein structure prediction and contributes useful, inexpensive tools to provide insights into the biology of IBV.