Kateri Bertran

Lack of chicken adaptation of newly emergent Eurasian H5N8 and reassortant H5N2 high pathogenicity avian influenza viruses in the U.S. is consistent with restricted poultry outbreaks in the Pacific flyway during 2014–2015

In 2014-2015, the U.S. experienced an unprecedented outbreak of Eurasian clade 2.3.4.4 H5 highly pathogenic avian influenza (HPAI) virus, initially affecting mainly wild birds and few backyard and commercial poultry premises. To better model the outbreak, the pathogenesis and transmission dynamics of representative Eurasian H5N8 and reassortant H5N2 clade 2.3.4.4 HPAI viruses detected early in the North American outbreak were investigated in chickens. High mean chicken infectious doses and lack of seroconversion in survivors indicated the viruses were poorly chicken adapted. Pathobiological features were consistent with HPAI virus infection, although the delayed appearance of lesions, longer mean death times, and reduced replication in endothelial cells differed from features of most other Eurasian H5N1 HPAI viruses. Although these initial U.S. H5 HPAI viruses had reduced adaptation and transmissibility in chickens, multi-generational passage in poultry could generate poultry adapted viruses with higher infectivity and transmissibility.

Evolution, global spread, and pathogenicity of highly pathogenic avian influenza H5Nx clade 2.3.4.4

Novel subtypes of Asian-origin (Goose/Guangdong lineage) H5 highly pathogenic avian influenza (HPAI) viruses belonging to clade 2.3.4, such as H5N2, H5N5, H5N6, and H5N8, have been identified in China since 2008 and have since evolved into four genetically distinct clade 2.3.4.4 groups (A–D). Since 2014, HPAI clade 2.3.4.4 viruses have spread rapidly via migratory wild aquatic birds and have evolved through reassortment with prevailing local low pathogenicity avian influenza viruses. Group A H5N8 viruses and its reassortant viruses caused outbreaks in wide geographic regions (Asia, Europe, and North America) during 2014–2015. Novel reassortant Group B H5N8 viruses caused outbreaks in Asia, Europe, and Africa during 2016–2017. Novel reassortant Group C H5N6 viruses caused outbreaks in Korea and Japan during the 2016–2017 winter season. Group D H5N6 viruses caused outbreaks in China and Vietnam. A wide range of avian species, including wild and domestic waterfowl, domestic poultry, and even zoo birds, seem to be permissive for infection by and/or transmission of clade 2.3.4.4 HPAI viruses. Further, compared to previous H5N1 HPAI viruses, these reassortant viruses show altered pathogenicity in birds. In this review, we discuss the evolution, global spread, and pathogenicity of H5 clade 2.3.4.4 HPAI viruses.

Infectivity, transmission and pathogenicity of H5 highly pathogenic avian influenza clade 2.3.4.4 (H5N8 and H5N2) United States index viruses in Pekin ducks and Chinese geese

In late 2014, a H5N8 highly pathogenic avian influenza (HPAI) virus, clade 2.3.4.4, spread by migratory waterfowl into North America reassorting with low pathogenicity AI viruses to produce a H5N2 HPAI virus. Since domestic waterfowl are common backyard poultry frequently in contact with wild waterfowl, the infectivity, transmissibility, and pathogenicity of the United States H5 HPAI index viruses (H5N8 and H5N2) was investigated in domestic ducks and geese. Ducks infected with the viruses had an increase in body temperature but no or mild clinical signs. Infected geese did not show increase in body temperature and most only had mild clinical signs; however, some geese presented severe neurological signs. Ducks became infected and transmitted the viruses to contacts when inoculated with high virus doses [(104 and 106 50% embryo infective dose (EID50)], but not with a lower dose (102 EID50). Geese inoculated with the H5N8 virus became infected regardless of the virus dose given, and transmitted the virus to direct contacts. Only geese inoculated with the higher doses of the H5N2 and their contacts became infected, indicating differences in infectivity between the two viruses and the two waterfowl species. Geese shed higher titers of virus and for a longer period of time than ducks. In conclusion, the H5 HPAI viruses can infect domestic waterfowl and easily transmit to contact birds, with geese being more susceptible to infection and disease than ducks. The disease is mostly asymptomatic, but infected birds shed virus for several days representing a risk to other poultry species.

The pathogenesis of H7N8 low and highly pathogenic avian influenza viruses from the United States 2016 outbreak in chickens, turkeys and mallards

In January 2016, a combined outbreak of highly pathogenic (HP) avian influenza virus (AIV) and low pathogenicity (LP) AIV occurred in commercial turkeys in the state of Indiana, United States. Genetically, the viruses were highly similar, belonged to the North American wild bird lineage, and had not been previously detected in poultry. In order to understand the pathobiology of the H7N8 LPAIV and HPAIV, infectivity, transmission and pathogenicity studies were conducted in chickens, turkeys, and mallards. Among the three species the lowest mean infectious dose for both the LP and HP phenotype was for turkeys, and also disease from the LPAIV was only observed with turkeys. Furthermore, although the HPAIV was lethal for both chickens and turkeys, clinical signs caused by the HPAIV isolate differed between the two species; neurological signs were only observed in turkeys. Mallards could be infected with and transmit both viruses to contacts, but neither caused clinical disease. Interestingly, with all three species, the mean infectious dose of the HP isolate was at least ten times lower than that of the LP isolate. This study corroborates the high susceptibility of turkeys to AIV as well as a pathobiology that is different from chickens. Further, this study demonstrates that mallards can be asymptomatically infected with HP and LP AIV from gallinaceous poultry and may not just be involved in transmitting AIV to them.

Pathobiology of Clade 2.3.4.4 H5Nx High-Pathogenicity Avian Influenza Virus Infections in Minor Gallinaceous Poultry Supports Early Backyard Flock Introductions in the Western United States in 2014-2015

ABSTRACT In 2014 and 2015, the United States experienced an unprecedented outbreak of Eurasian clade 2.3.4.4 H5 highly pathogenic avian influenza (HPAI) virus. Initial cases affected mainly wild birds and mixed backyard poultry species, while later outbreaks affected mostly commercial chickens and turkeys. The pathogenesis, transmission, and intrahost evolutionary dynamics of initial Eurasian H5N8 and reassortant H5N2 clade 2.3.4.4 HPAI viruses in the United States were investigated in minor gallinaceous poultry species (i.e., species for which the U.S. commercial industries are small), namely, Japanese quail, bobwhite quail, pearl guinea fowl, chukar partridges, and ring-necked pheasants. Low mean bird infectious doses (<2 to 3.7 log10) support direct introduction and infection of these species as observed in mixed backyard poultry during the early outbreaks. Pathobiological features and systemic virus replication in all species tested were consistent with HPAI virus infection. Sustained virus shedding with transmission to contact-exposed birds, alongside long incubation periods, may enable unrecognized dissemination and adaptation to other gallinaceous species, such as chickens and turkeys. Genome sequencing of excreted viruses revealed numerous low-frequency polymorphisms and 20 consensus-level substitutions in all genes and species, but especially in Japanese quail and pearl guinea fowl and in internal proteins PB1 and PB2. This genomic flexibility after only one passage indicates that influenza viruses can continue to evolve in galliform species, increasing their opportunity to adapt to other species. Our findings suggest that these gallinaceous poultry are permissive for infection and sustainable transmissibility with the 2014 initial wild bird-adapted clade 2.3.4.4 virus, with potential acquisition of mutations leading to host range adaptation. IMPORTANCE The outbreak of clade 2.3.4.4 H5 highly pathogenic avian influenza (HPAI) virus that occurred in the United States in 2014 and 2015 represents the worst livestock disease event in the country, with unprecedented socioeconomic and commercial consequences. Epidemiological and molecular investigations can identify transmission pathways of the HPAI virus. However, understanding the pathogenesis, transmission, and intrahost evolutionary dynamics of new HPAI viruses in different avian species is paramount. The significance of our research is in examining the susceptibility of minor gallinaceous species to HPAI virus, as this poultry sector also suffers from HPAI epizootics, and identifying the biological potential of these species as an epidemiological link between the waterfowl reservoir and the commercial chicken and turkey populations, with the ultimate goal of refining surveillance in these populations to enhance early detection, management, and control in future HPAI virus outbreaks.

Protection of White Leghorn chickens by U.S. emergency H5 vaccination against clade 2.3.4.4 H5N2 high pathogenicity avian influenza virus

During December 2014-June 2015, the U.S. experienced a high pathogenicity avian influenza (HPAI) outbreak caused by clade 2.3.4.4 H5Nx Goose/Guangdong lineage viruses with devastating consequences for the poultry industry. Three vaccines, developed based on updating existing registered vaccines or currently licensed technologies, were evaluated for possible use: an inactivated reverse genetics H5N1 vaccine (rgH5N1) and an RNA particle vaccine (RP-H5), both containing the hemagglutinin gene of clade 2.3.4.4 strain, and a recombinant herpesvirus turkey vectored vaccine (rHVT-H5) containing the hemagglutinin gene of clade 2.2 strain. The efficacy of the three vaccines, alone or in combination, was assessed in White Leghorn chickens against clade 2.3.4.4 H5N2 HPAI virus challenge. In Study 1, single (rHVT-H5) and prime-boost (rHVT-H5+rgH5N1 or rHVT-H5+RP-H5) vaccination strategies protected chickens with high levels of protective immunity and significantly reduced virus shedding. In Study 2, single vaccination with either rgH5N1 or RP-H5 vaccines provided clinical protection in adult chickens and significantly reduced virus shedding. In Study 3, double rgH5N1 vaccination protected adult chickens from clinical signs and mortality when challenged 20weeks post-boost, with high levels of long-lasting protective immunity and significantly reduced virus shedding. These studies support the use of genetically related vaccines, possibly in combination with a broad protective priming vaccine, for emergency vaccination programs against clade 2.3.4.4 H5Nx HPAI virus in young and adult layer chickens.

Airborne Transmission of Highly Pathogenic Influenza Virus during Processing of Infected Poultry

Exposure to infected poultry is a suspected cause of avian influenza (H5N1) virus infections in humans. We detected infectious droplets and aerosols during laboratory-simulated processing of asymptomatic chickens infected with human- (clades 1 and 2.2.1) and avian- (clades 1.1, 2.2, and 2.1) origin H5N1 viruses. We detected fewer airborne infectious particles in simulated processing of infected ducks. Influenza virus–naive chickens and ferrets exposed to the air space in which virus-infected chickens were processed became infected and died, suggesting that the slaughter of infected chickens is an efficient source of airborne virus that can infect birds and mammals. We did not detect consistent infections in ducks and ferrets exposed to the air space in which virus-infected ducks were processed. Our results support the hypothesis that airborne transmission of HPAI viruses can occur among poultry and from poultry to humans during home or live-poultry market slaughter of infected poultry.

The efficacy of recombinant turkey herpesvirus vaccines targeting the H5 of highly pathogenic avian influenza virus from the 2014–2015 North American outbreak

The outbreak of highly pathogenic avian influenza virus in North American poultry during 2014 and 2015 demonstrated the devastating effects of the disease and highlighted the need for effective emergency vaccine prevention and control strategies targeted at currently circulating strains. This study evaluated the efficacy of experimental recombinant turkey herpesvirus vector vaccines with three different inserts targeting the hemagglutinin gene of an isolate from the recent North American influenza outbreak. White leghorn chickens were vaccinated at one day of age and challenged with A/Turkey/Minnesota/12582/2015 H5N2 at 4 weeks of age. Birds were analyzed for survival, viral shedding at two and four days after infection, and specific antibody prior to challenge and from surviving birds. The three experimental vaccines demonstrated 100%, 45% and 15% survival with the most effective vaccine significantly reducing oral and cloacal viral shedding compared to all other groups and generated specific antibody prior to challenge with highly pathogenic avian influenza virus. More studies are needed using diverse H5Nx highly pathogenic virus isolates to fully determine the breadth of coverage against possible exposure strains, as well as possible impact of maternally derived antibody on protection and vaccine efficacy.

Infection with Some Infectious Bursal Disease Virus Pathotypes Produces Virus in Chicken Muscle Tissue and the Role of Humoral Immunity as a Mitigation Strategy.

SUMMARY Infectious bursal disease virus (IBDV) causes important economic losses and negatively affects global trade in poultry and poultry products. This study determined the presence of IBDV in primary lymphoid tissues and muscle tissue of infected broilers and the role of vaccination as a mitigation strategy. In the first study, specific-pathogen-free (SPF) broiler chickens were challenged with STC (classical [cIBDV]), Indiana (variant [varIBDV]), rA (very virulent [vvIBDV]), or Ohio (serotype 2, avirulent) IBDV. Infection was confirmed in all groups, but only the cIBDV group experienced morbidity or mortality. Virus was only isolated in low titers from a few breast and/or thigh muscle tissue samples from cIBDV and vvIBDV-infected chickens. For the second study, SPF broilers from three different treatment groups were challenged with IBDV viruses that currently circulate in the United States, varIBDV or vvIBDV: 1) maternal antibody–positive (MAb+), vaccinated with recombinant HVT-IBDV vaccine (Vaxxitek®, Merial; MAb+/Vax); 2) MAb+, not-vaccinated (MAb+/Unvax); and 3) maternal antibody–negative, not-vaccinated chickens (MAb−/Unvax). MAb+/Vax and MAb+/Unvax chickens had significantly lower virus titers in primary lymphoid tissues compared to MAb−/Unvax chickens. No virus was detected in muscle tissues from any of the groups challenged with varIBDV, confirming the results of the first experiment. Only 1 of 36 (MAb+/Vax) and 2 of 36 (MAb+/Unvax) muscle samples were positive at minimal amounts (101.97 EID50/ml) in vvIBDV challenge, compared to the 9 of 36 muscle samples that were positive in the MAb−/Unvax group. This study indicates that only cIBDV and vvIBDV strains can be found in muscle at low titers of SPF meat chickens and that the breeder vaccination with MAb transfer to progeny with or without accompanying progeny vaccination, as practiced in the United States, was an effective mitigation strategy for vvIBDV-challenged birds.

Pathobiology of Tennessee 2017 H7N9 low and high pathogenicity avian influenza viruses in commercial broiler breeders and specific pathogen free layer chickens

In March 2017, H7N9 highly pathogenic avian influenza (HPAI) virus was detected in 2 broiler breeder farms in the state of Tennessee, USA. Subsequent surveillance detected the low pathogenicity avian influenza (LPAI) virus precursor in multiple broiler breeder farms and backyard poultry in Tennessee and neighboring states. The pathogenesis of the H7N9 LPAI virus was investigated in commercial broiler breeders, the bird type mostly affected in this outbreak. Infectivity, transmissibility, and pathogenesis of the H7N9 HPAI and LPAI viruses were also studied in 4-week-old specific pathogen free (SPF) leghorn chickens. The mean bird infectious doses (BID50) for the LPAI isolate was 5.6 log10 mean egg infectious dose (EID50) for broiler breeders and 4.3 log10 EID50 for SPF layer chickens, and no transmission to contact-exposed birds was observed. In both bird types, virus shedding was almost exclusively from the oropharyngeal route. These findings suggest sub-optimal adaptation for sustained transmission with the H7N9 LPAI isolate, indicating that factors other than the birds genetic background may explain the epidemiology of the outbreak. The BID50 for the HPAI isolate in SPF layer chickens was more than 2 logs lower (<2 log10 EID50) than the LPAI isolate. Also, the HPAI virus was shed by both the oropharyngeal and cloacal routes and transmitted to contacts. Greater susceptibility and easier transmission of the H7N9 HPAI virus are features of the HP phenotype that could favor the spread of HPAI over LPAI viruses during outbreaks.