Efficacy of commercial recombinant HVT vaccines against a North American clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus in chickens.

The outbreak of clade 2.3.4.4b H5 highly pathogenic avian influenza (HPAI) in North America that started in 2021 has increased interest in applying vaccination as a strategy to help control and prevent the disease in poultry. Two commercially available vaccines based on the recombinant herpes virus of turkeys (rHVT) vector were tested against a recent North American clade 2.3.4.4b H5 HPAI virus isolate: A/turkey/Indiana/22-003707-003/2022 H5N1 in specific pathogen free white leghorn (WL) chickens and commercial broiler chickens. One rHVT-H5 vaccine encodes a hemagglutinin (HA) gene designed by the computationally optimized broadly reactive antigen method (COBRA-HVT vaccine). The other encodes an HA gene of a clade 2.2 virus (2.2-HVT vaccine). There was 100% survival of both chicken types COBRA-HVT vaccinated groups and in the 2.2-HVT vaccinated groups there was 94.8% and 90% survival of the WL and broilers respectively. Compared to the 2.2-HVT vaccinated groups, WL in the COBRA-HVT vaccinated group shed significantly lower mean viral titers by the cloacal route and broilers shed significantly lower titers by the oropharyngeal route than broilers. Virus titers detected in oral and cloacal swabs were otherwise similar among both vaccine groups and chicken types. To assess antibody-based tests to identify birds that have been infected after vaccination (DIVA-VI), sera collected after the challenge were tested with enzyme-linked lectin assay-neuraminidase inhibition (ELLA-NI) for N1 neuraminidase antibody detection and by commercial ELISA for detection of antibodies to the NP protein. As early as 7 days post challenge (DPC) 100% of the chickens were positive by ELLA-NI. ELISA was less sensitive with a maximum of 75% positive at 10DPC in broilers vaccinated with 2.2-HVT. Both vaccines provided protection from challenge to both types of chickens and ELLA-NI was sensitive at identifying antibodies to the challenge virus therefore should be evaluated further for DIVA-VI.

Characterization of highly pathogenic avian influenza virus in retail dairy products in the US.

In March 2024, clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus (HPAIV) was detected in dairy cattle in the US, and it was discovered that the virus could be detected in raw milk. Although affected cow's milk is diverted from human consumption and current pasteurization requirements are expected to reduce or eliminate infectious HPAIV from the milk supply, a study was conducted to characterize whether the virus could be detected by quantitative real-time RT-PCR (qrRT-PCR) in pasteurized retail dairy products and, if detected, to determine whether the virus was viable. From 18 April to 22 April 2024, a total of 297 samples of Grade A pasteurized retail milk products (23 product types) were collected from 17 US states that represented products from 132 processors in 38 states. Viral RNA was detected in 60 samples (20.2%), with qrRT-PCR-based quantity estimates (non-infectious) of up to 5.4log1050% egg infectious doses per mL, with a mean and median of 3.0log10/mL and 2.9log10/mL, respectively. Samples that were positive for type A influenza by qrRT-PCR were confirmed to be clade 2.3.4.4 H5 HPAIV by qrRT-PCR. No infectious virus was detected in any of the qrRT-PCR-positive samples in embryonating chicken eggs. Further studies are needed to monitor the milk supply, but these results provide evidence that the infectious virus did not enter the US pasteurized milk supply before control measures for HPAIV were implemented in dairy cattle.IMPORTANCEHighly pathogenic avian influenza virus (HPAIV) infections in US dairy cattle were first confirmed in March 2024. Because the virus could be detected in raw milk, a study was conducted to determine whether it had entered the retail food supply. Pasteurized dairy products were collected from 17 states in April 2024. Viral RNA was detected in one in five samples, but infectious virus was not detected. This provides a snapshot of HPAIV in milk products early in the event and reinforces that with current safety measures, infectious viruses in milk are unlikely to enter the food supply.

Inactivation of Avian Influenza Virus Inoculated into Ground Beef Patties Cooked on a Commercial Open-Flame Gas Grill.

With the emergence of clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus (AIV) infection of dairy cattle and its subsequent detection in raw milk, coupled with recent AIV infections affecting dairy farm workers, experiments were conducted to affirm the safety of cooked ground beef related to AIV because such meat is often derived from cull dairy cows. Specifically, retail ground beef (percent lean:fat = ca. 80:20) was inoculated with a low pathogenic AIV (LPAIV) isolate to an initial level of 5.6 log10 50% egg infectious doses (EID50)  per 300 g patty. The inoculated meat was pressed into patties (ca. 2.54 cm thick, ca. 300 g each) and then held at 4 °C for up to 60 min. In each of the two trials, two patties for each of the following three treatments were cooked on a commercial open-flame gas grill to internal instantaneous temperatures of 48.9 °C (120°F), 62.8 °C (145°F), or 71.1 °C (160°F), but without any dwell time. Cooking inoculated ground beef patties to 48.9 °C (ave. cooking time of ca. 15 min) resulted in a mean reduction of ≥2.5 ± 0.9 log10 EID50 per 300 g of ground beef as assessed via quantification of virus in embryonating chicken eggs (ECEs). Likewise, cooking patties on a gas grill to 62.8 °C (ave. cooking time of ca. 21 min) or to the USDA FSIS recommended minimum internal temperature for ground beef of 71.1 °C (ave. cooking time of ca. 24 min) resulted in a reduction to nondetectable levels from initial levels of ≥5.6 log10 EID50 per 300 g. These data establish that levels of infectious AIV are substantially reduced within inoculated ground beef patties (20% fat) using recommended cooking procedures.

Epitopes in the HA and NA of H5 and H7 avian influenza viruses that are important for antigenic drift.

Avian influenza viruses evolve antigenically to evade host immunity. Two influenza A virus surface glycoproteins, the haemagglutinin and neuraminidase, are the major targets of host immunity and undergo antigenic drift in response to host pre-existing humoral and cellular immune responses. Specific sites have been identified as important epitopes in prominent subtypes such as H5 and H7, which are of animal and public health significance due to their panzootic and pandemic potential. The haemagglutinin is the immunodominant immunogen, it has been extensively studied, and the antigenic reactivity is closely monitored to ensure candidate vaccine viruses are protective. More recently, the neuraminidase has received increasing attention for its role as a protective immunogen. The neuraminidase is expressed at a lower abundance than the haemagglutinin on the virus surface but does elicit a robust antibody response. This review aims to compile the current information on haemagglutinin and neuraminidase epitopes and immune escape mutants of H5 and H7 highly pathogenic avian influenza viruses. Understanding the evolution of immune escape mutants and the location of epitopes is critical for identification of vaccine strains and development of broadly reactive vaccines that can be utilized in humans and animals.

Duration of Highly Pathogenic Avian Influenza Virus and Newcastle Disease Virus Infectivity in Dried Ornithologic Study Skins.

Ornithologic study skins are specimens of avian skins that have been preserved by drying after removing the viscera and muscle. Because of the high value of study skins for scientific studies, specimens are shared among researchers. There is concern that study skins might be contaminated with high-consequence diseases such as highly pathogenic avian influenza virus (HPAIV) or Newcastle disease virus (NDV). To mitigate risk, thermal or chemical treatment of study skins may be required before transfer; however, such treatments might damage the specimens. Therefore, a study was conducted to evaluate the duration of infectivity of HPAIV and NDV in study skins prepared from infected chickens (Gallus gallus). Study skins were prepared from 10 chickens infected with each virus. Skin and feather pulp samples were taken at the time of study skin preparation to establish starting titers. Mean starting titers in the skin was 4.2 log10 and 5.1 log10 50% egg infectious doses (EID50) for HPAIV and NDV groups respectively, and were 6.7 log10 EID50 for HPAIV, and 6.4 log10 EID50 for NDV in feather pulp. Samples were collected at 2 and 4 wk of drying to quantify viable virus. At 2 wk, fewer samples had detectable virus and mean titers were 1.8 log10 (skin) and 2.1 log10 (feathers) EID50 for HPAIV, and 1.7 log10 (skin) and 3.5 log10 (feathers) EID50 for NDV. At 4 wk viable virus could not be detected in either tissue type.

The 28S rRNA RT-qPCR assay for host depletion evaluation to enhance avian virus detection in Illumina and Nanopore sequencing.

Abundant host and bacterial sequences can obscure the detection of less prevalent viruses in untargeted next-generation sequencing (NGS). Efficient removal of these non-targeted sequences is vital for accurate viral detection. This study presents a novel 28S ribosomal RNA (rRNA) RT-qPCR assay designed to assess the efficiency of avian rRNA depletion before conducting costly NGS for the detection of avian RNA viruses. The comprehensive evaluation of this 28S-test focuses on substituting DNase I with alternative DNases in our established depletion protocols and finetuning essential parameters for reliable host rRNA depletion. To validate the effectiveness of the 28S-test, we compared its performance with NGS results obtained from both Illumina and Nanopore sequencing platforms. This evaluation utilized swab samples from chickens infected with highly pathogenic avian influenza virus, subjected to established and modified depletion protocols. Both methods significantly reduced host rRNA levels, but using the alternative DNase had superior performance. Additionally, utilizing the 28S-test, we explored cost- and time-effective strategies, such as reduced probe concentrations and other alternative DNase usage, assessed the impact of filtration pre-treatment, and evaluated various experimental parameters to further optimize the depletion protocol. Our findings underscore the value of the 28S-test in optimizing depletion methods for advancing improvements in avian disease research through NGS.

Efficacy of inactivated and RNA particle vaccines against a North American Clade 2.3.4.4b H5 highly pathogenic avian influenza virus in chickens.

Highly pathogenic avian influenza virus (HPAIV) has caused widespread outbreaks in poultry in the Americas. Because of the duration and extent of these outbreaks, vaccine use may be an additional tool to limit virus spread. Three vaccines were evaluated for efficacy in chickens against a current North American clade 2.3.4.4b H5 HPAIV isolate, A/turkey/Indiana/3703-003/2022 H5N1. The vaccines included: 1) a commercial inactivated reverse genetics (rg) generated H5N1 product with a clade 2.3.4.4c H5 hemagglutinin (HA) (rgH5N1); 2) a commercial alphavirus RNA particle (RP) vaccine with the TK/IN/22 HA; and 3) an in-house inactivated rg produced vaccine with the TK/IN/22 HA and a North American lineage N9 neuraminidase (NA) (SEP-22-N9). Both inactivated vaccines were produced with HA genes that were modified to be low pathogenic and with the remaining genes from the PR8 influenza strain. All vaccines provided 100% protection against mortality and morbidity and all vaccines reduced virus shed by the oropharyngeal and cloacal routes significantly compared to sham vaccinates. However, differences were observed among the vaccines in quantities of virus shed at two- and four-days post challenge (DPC). To determine if infected birds could be identified after vaccination to aid surveillance programs, serum was collected from the RP and SEP-22-N9 vaccine groups at 7, 10, and 14 DPC to detect antibody to the NA and nucleoprotein (NP) of the challenge virus by enzyme linked lectin assay (ELLA) and ELISA. As early as 7DPC ELLA detected antibody in sera from 100% of the chickens in the RP vaccinated group and 70% of the chickens in the SEP-22-N9 vaccinated group. Antibody to the NP was detected by commercial ELISA in more than 50% of the birds in the RP vaccinated group at each time point.

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.

SARS-CoV-2 utilization of ACE2 from different bat species allows for virus entry and replication in vitro.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is believed to have a zoonotic origin with bats suspected as a natural host. In this work, we individually express the ACE2 of seven bat species including, little brown, great roundleaf, Pearson's horseshoe, greater horseshoe, Brazilian free-tailed, Egyptian rousette, and Chinese rufous horseshoe in DF1 cells and determine their ability to support attachment and replication of SARS-CoV-2 viruses. We demonstrate that the ACE2 receptor of all seven species made DF1 cells permissible to SARS-CoV-2. The level of virus replication differed between bat species and variants tested. The Wuhan lineage SARS-CoV-2 virus replicated to higher titers than either variant virus tested. All viruses tested grew to higher titers in cells expressing the human ACE2 gene compared to a bat ACE2. This study provides a practical in vitromethod for further testing of animal species for potential susceptibility to current and emerging SARS-CoV-2 viruses.

Prediction of highly pathogenic avian influenza vaccine efficacy in chickens by comparison of in vitro and in vivo data: A meta-analysis and systematic review.

Vaccines for avian influenza (AI) can protect poultry against disease, mortality, and virus transmission. Numerous factors, including: vaccine platform, immunogenicity, and relatedness to the field strain, are known to be important to achieving optimal AI vaccine efficacy. To better understand how these factors contribute to vaccine protection, a systematic meta-analysis was conducted to evaluate efficacy data for vaccines in chickens challenged with highly pathogenic (HP) AI. Data from a total of 120 individual trials from 25 publications were selected and evaluated. Two vaccine criteria were evaluated for their effects on two metrics of protection. The vaccine criteria were: 1) the relatedness of the vaccine antigen and challenge strain in the hemagglutinin 1 domain (HA1) protein sequence; 2) vaccine-induced antibody titers to the challenge virus (VIAC). The metrics of protection were: A) survival of vaccinated chickens vs unvaccinated controls; and B) reduction in oral virus-shedding by vaccinated vs unvaccinated controls 2-4 days post challenge. Three vaccine platforms were evaluated: oil-adjuvanted inactivated whole AI virus, recombinant herpes virus of turkeys (rHVT) vectored, and a non-replicating alpha-virus vectored RNA particle (RP) vaccine. Higher VIAC correlated with greater reduction of virus-shed and vaccine efficacy by all vaccine platforms. Both higher HA1 relatedness and higher VIAC using challenge virus as antigen correlated with better survival by inactivated vaccines and rHVT-vectored vaccines. However, rHVT-vectored and RP based vaccines were more tolerant of variation in the HA1; the relatedness of the HA1 of the vaccine and challenge virus did not significantly correlate with survival with rHVT-vectored vaccines. Protection was achieved with the lowest aa similarity for which there was data, 90-93 % for rHVT vaccines and 88 % for the RP vaccine.

The pathogenesis of a 2022 North American highly pathogenic clade 2.3.4.4b H5N1 avian influenza virus in mallards (Anas platyrhynchos).

Highly pathogenic (HP) avian influenza viruses (AIVs) of the clade 2.3.4.4 goose/Guangdong/1996 H5 lineage continue to be a problem in poultry and wild birds in much of the world. The recent incursion of a H5N1 clade 2.3.4.4b HP AIV from this lineage into North America has resulted in widespread outbreaks in poultry and consistent detections of the virus across diverse families of birds and occasionally mammals. To characterize the pathobiology of this virus in mallards (Anas platyrhynchos), which are a primary reservoir of AIV, a challenge study was conducted with 2-week-old birds. The 50% bird infectious dose was determined to be < 2 log10 50% egg infectious doses (EID50) and all exposed ducks, including ducks co-housed with inoculated ducks, were infected. Infection appeared to be subclinical for 58.8% (20/34) of the ducks, one duck was lethargic, about 20% developed neurological signs and were euthanized, and 18% developed corneal opacity. The mallards shed virus by both the oral and cloacal routes within 24-48 h post-infection. Oral shedding substantially decreased by 6-7 days post-infection, but 65% of the ducks continued to shed virus cloacally through 14 days post-exposure (DPE) for the direct inoculates and 13 DPE for contact-exposed ducks. Based on the high transmissibility, high virus shed titres, and mild-to-moderate disease, mallards could serve as efficient reservoirs to amplify and disseminate recent North American clade 2.3.4.4b viruses.

A Review of the Stability of Avian Influenza Virus in Materials from Poultry Farms.

Avian influenza virus (AIV) is widespread among poultry and wild waterfowl. The severity of the disease is variable and the highly pathogenic form can rapidly kill numerous avian species. Understanding the stability of AIV infectivity in different substrates in the environment of poultry facilities is critical to developing processes to effectively decontaminate or safely dispose of potentially contaminated material. This review aims to compile the current information on the stability of AIV in materials from poultry farms that cannot be disinfected with chemicals or fumigants: water, litter/bedding, soil, feed, feathers, carcasses/meat, manure/feces, and eggs. There are still important gaps in the data, but available data will inform risk assessments, biosecurity, and procedures to dispose of potentially contaminated material. Among the parameters and conditions reported, temperature is a nearly universal factor where, regardless of substrate, the virus will inactivate faster under a given set of conditions as the temperature increases, and freeze-thaw cycles can facilitate virus inactivation.

Estudio recapitulativo- Una revisión de la estabilidad del virus de la influenza aviar en materiales de granjas avícolas. El virus de la influenza aviar (AIV) está muy extendido en la avicultura comercial y en las aves acuáticas silvestres. La severidad de la enfermedad es variable y la forma altamente patógena puede matar rápidamente a numerosas especies de aves. Comprender la estabilidad de la infectividad del virus de la influenza en diferentes sustratos en el ambiente de las instalaciones avícolas es fundamental para desarrollar procesos para descontaminar de manera efectiva o para eliminar de manera segura el material potencialmente contaminado. Esta revisión tiene como objetivo recopilar la información actual sobre la estabilidad del virus de la influenza aviar en materiales de granjas avícolas que no se pueden desinfectar con productos químicos o fumigantes: agua, heces/ material de cama, suelo, alimento, plumas, canales/carne, estiércol/heces y huevos. Todavía existen vacíos importantes en la información, pero los datos disponibles pueden proporcionar información durante las evaluaciones de riesgos, la bioseguridad y los procedimientos para eliminar material potencialmente contaminado. Entre los parámetros y condiciones que se han reportado, la temperatura es un factor casi universal donde, independientemente del sustrato, el virus se inactivará más rápido bajo un conjunto determinado de condiciones a medida que aumenta la temperatura, y los ciclos de congelación y descongelación pueden facilitar la inactivación del virus.

The Thermal Stability of Newcastle Disease Virus in Poultry Litter.

Sanitary disposal of contaminated organic material during recovery from an animal disease outbreak is costly and laborious. Characterizing the thermal stability of avian paramyxovirus type 1 (APMV-1; virulent APMV-1 strains cause Newcastle disease in poultry) will help inform risk assessments on the presence of viable virus on infected premises or in organic waste from infected premises. In some environments and housing types, heat may also be used as a decontamination method. Therefore, the objective of this study was to characterize the thermal stability (i.e., decimal reduction values [D values]) of APMV-1 in poultry litter. Virus inactivation was evaluated at seven temperatures from 10.0 C through 43.3 C, at 5.5 C intervals (50-110 F in 10 F intervals), using the I2 isolate of APMV-1, a vaccine strain known to be thermally stable. A high titer of virus (approximately 108 50% egg infectious doses) was added to wood shavings based, soiled chicken litter (poultry litter). Litter with both low and high moisture levels were evaluated. Samples were collected at different time intervals, and infectious virus was titrated in embryonated chicken eggs. At high temperatures (37.8 C-43.3 C), infectious virus could not be detected after 2-7 days, whereas at lower temperatures (10 C-21.1 C), it took up to 112 days for virus to decrease to undetectable levels. Furthermore, the D values were almost always shorter in the high moisture litter.

Estabilidad térmica del virus de la enfermedad de Newcastle en la cama avícola. La eliminación sanitaria de material orgánico contaminado durante la recuperación de un brote de enfermedad en los animales es costosa y laboriosa. La caracterización de la estabilidad térmica del paramixovirus aviar tipo 1 (APMV-1; las cepas virulentas de APMV-1 que causan la enfermedad de Newcastle en avicultura) contribuirá con información para las evaluaciones de riesgo sobre la presencia de virus viables en las instalaciones infectadas o en los desechos orgánicos de las instalaciones infectadas. En algunos entornos y tipos de casetas, el calor también se puede utilizar como método de descontaminación. Por lo tanto, el objetivo de este estudio fue caracterizar la estabilidad térmica (es decir, valores de reducción decimal [valores D]) del APMV-1 en la cama de aves. La inactivación del virus se evaluó a siete temperaturas, desde 10.0 C hasta 43.3 C, en intervalos de 5.5 C (50­110 F en intervalos de 10 F), utilizando el aislamiento I2 de APMV-1, que es una cepa vacunal conocida por ser térmicamente estable. Se añadió el virus con un título alto (aproximadamente 108 dosis infecciosas de embrión de pollo al 50%) a la cama de pollo con base de virutas de madera (cama avícola). Se evaluaron camas con niveles de humedad altos y bajos. Se recolectaron muestras en diferentes intervalos de tiempo y se tituló el virus infeccioso en huevos embrionados de pollo. A altas temperaturas (37.8 C­43.3 C), el virus infeccioso no se pudo detectar después de dos a siete días, mientras que a temperaturas más bajas (10 C­21.1 C), el virus tardó hasta 112 días en disminuir a niveles no detectables. Además, los valores D fueron casi siempre más cortos en la cama con alta humedad.

Optimizing sample collection methods for detection of respiratory viruses in poultry housing environments.

Viral respiratory diseases, such as avian influenza, Newcastle disease, infectious bronchitis and infectious laryngotracheitis, have considerable negative economic implications for poultry. Ensuring the virus-free status of premises by environmental sampling after cleaning and disinfection is essential for lifting a quarantine and/or safely restocking the premises following an outbreak. The objectives of this study were to identify optimal sample collection devices and to determine the locations in poultry housing which are best for poultry respiratory virus sample collection. Chickens exposed to infectious bronchitis virus, which was used as a representative virus for enveloped poultry respiratory viruses, were housed in floor-pens in either a curtain-sided wood framed house or a cement block house. Foam swabs, cellulose sponges, polyester swabs, dry cotton gauze and pre-moistened cotton gauze were evaluated for comparative efficiency in recovering viral RNA. Cotton gauze pre-moistened with the viral transport media had the highest sensitivity among the devices (wood-framed house: 78% positive, geometric mean titre [GMT] of 2.6 log10 50% egg infectious doses [EID50 ] equivalents/ml; cement block houses: 55% positive, GMT of 1.7 log10 EID50 equivalents/ml). Targeting virus deposition sites is also crucial for efficient virus elimination procedures and subsequent testing; therefore, 10 locations within the houses were compared for virus detection. In both housing types, the highest viral RNA loads were recovered from the tops of drinker lines within the pen. Places the chickens could contact directly (e.g., feeder rim) or were contacted by caretaker feet (hallway floor) also yielded higher levels of viral RNA more consistently. These results will facilitate the establishment of efficient environmental sampling procedures for respiratory viruses of poultry.

Development of an in vitro model for animal species susceptibility to SARS-CoV-2 replication based on expression of ACE2 and TMPRSS2 in avian cells.

The SARS-CoV-2 (SARS-CoV-2) virus has caused a worldwide pandemic because of the virus's ability to transmit efficiently human-to-human. A key determinant of infection is the attachment of the viral spike protein to the host receptor angiotensin-converting enzyme 2 (ACE2). Because of the presumed zoonotic origin of SARS-CoV-2, there is no practical way to assess the susceptibility of every species to SARS-CoV-2 by direct challenge studies. In an effort to have a better predictive model of animal host susceptibility to SARS-CoV-2, we expressed the ACE2 and/or transmembrane serine protease 2 (TMPRSS2) genes from humans and other animal species in the avian fibroblast cell line, DF1, that is not permissive to infection. We demonstrated that expression of both human ACE2 and TMPRSS2 genes is necessary to support SARS-CoV-2 infection and replication in DF1 and a non-permissive sub-lineage of MDCK cells. Titers of SARS-CoV-2 in these cell lines were comparable to those observed in control Vero cells. To further test the model, we developed seven additional transgenic cell lines expressing the ACE2 and TMPRSS2 derived from Felis catus (cat), Equus caballus (horse), Sus domesticus (pig), Capra hircus (goat), Mesocricetus auratus (Golden hamster), Myotis lucifugus (Little Brown bat) and Hipposideros armiger (Great Roundleaf bat) in DF1 cells. Results demonstrate permissive replication of SARS-CoV-2 in cat, Golden hamster, and goat species, but not pig or horse, which correlated with the results of reported challenge studies. Cells expressing genes from either bat species tested demonstrated temporal replication of SARS-CoV-2 that peaked early and was not sustained. The development of this cell culture model allows for more efficient testing of the potential susceptibility of many different animal species for SARS-CoV-2 and emerging variant viruses.

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.

Identification of Efficacious Vaccines Against Contemporary North American H7 Avian Influenza Viruses.

Five vaccines, including four inactivated, whole-virus water-in-oil adjuvanted vaccines and a commercial nonreplicating alphavirus-vectored RNA particle (RP) vaccine were evaluated in chickens for their ability to provide protection against challenge with a recent H7 highly pathogenic avian influenza virus (AIV) from the United States (A/turkey/IN/1403-1/2016 H7N8). One of the inactivated vaccines and the RP vaccine were prepared with A/turkey/IN/16-01571-6/2016 H7N8 low pathogenic AIV (LPAIV; TK/IN/16), which is identical to the challenge virus, except for the proteolytic cleavage site of the hemagglutinin protein. The remaining three inactivated vaccines were prepared with other North American H7 LPAIVs. The hemagglutination inhibition assay was used to evaluate the antigenic relationships among the vaccines and selected recent H7 AIV isolates. All five vaccines provided protection against mortality. The inactivated vaccines reduced virus shedding significantly at 2 and 4 days post challenge compared with sham-vaccinated chickens. In contrast, the RP vaccine did not significantly reduce virus shedding. The inactivated vaccine prepared with TK/IN/16 elicited the highest antibody responses, which suggests it is a strong candidate for use as an antigen for North American H7 AIVs. Antigenic distance calculations showed that the four inactivated vaccine strains and other recent North American H7 isolates are antigenically similar, which suggests that the vaccines evaluated here would be similar enough to provide protection to other North American H7 AIVs. If future H7 outbreaks in poultry warrant vaccination, the field strain can be rapidly evaluated with these antigens and, if adequately related, one of these characterized strains may be used.

Artículo reguarl­Identificación de vacunas eficaces contra los virus contemporáneos de la influenza aviar H7 de América del Norte. Se evaluaron cinco vacunas, incluidas cuatro vacunas inactivadas con virus completo y con adyuvante de agua en aceite y una vacuna comercial de partículas de ARN en un vector de alfavirus no replicante (RP) en pollos para determinar su capacidad para brindar protección contra el desafío con un virus de influenza aviar altamente patógena H7 (AIV) de los Estados Unidos (A/pavo/IN/1403-1/2016 H7N8). Una de las vacunas inactivadas y la vacuna de partículas de ARN se prepararon con un virus de influenza aviar de baja patogenicidad A/pavo/IN/16-01571-6/2016 AIV de baja patogenicidad H7N8 (LPAIV; TK/IN/16), que es idéntico al virus de desafío, excepto por el sitio de disociación proteolítica de la proteína hemaglutinina. Las tres vacunas inactivadas restantes se prepararon con otros virus de baja patogenicidad H7 de América del Norte. El ensayo de inhibición de la hemaglutinación se utilizó para evaluar las relaciones antigénicas entre las vacunas y los aislados del virus de influenza aviar H7 recientes seleccionados. Las cinco vacunas proporcionaron protección contra la mortalidad. Las vacunas inactivadas redujeron significativamente la diseminación del virus a los 2 y 4 días posteriores al desafío en comparación con los pollos que recibieron la vacunación falsa. Por el contrario, la vacuna de partículas de ARN no redujo significativamente la diseminación del virus. La vacuna inactivada preparada con el virus TK/IN/16 provocó las respuestas de anticuerpos más altas, lo que indica que es un fuerte candidato para su uso como antígeno contra los virus de influenza aviar H7 de América del Norte. Los cálculos de la distancia antigénica mostraron que las cuatro cepas de vacunas inactivadas y otros aislados recientes del subtipo H7 de América del Norte son antigénicamente similares, lo que sugiere que las vacunas evaluadas en este estudio serían lo suficientemente similares para brindar protección a otros virus de influenza aviar de H7 de América del Norte. Si futuros brotes de H7 en la avicultura justifican la vacunación, la cepa de campo se puede evaluar rápidamente con estos antígenos y si están adecuadamente relacionados, se puede utilizar una de estas cepas caracterizadas.

The pathogenicity and transmission of live bird market H2N2 avian influenza viruses in chickens, Pekin ducks, and guinea fowl.

H2N2 subtype low pathogenic avian influenza viruses (LPAIVs) have persisted in live bird markets (LBMs) in the Northeastern United States since 2014. Although unrelated to the 1957 pandemic H2N2 lineage, there is concern that the virus could have animal and public health consequences because of high contact with humans and numerous species in the LBM system. The pathogenicity, infectivity, and transmissibility of six LBM H2N2 viruses isolated from three avian species in LBMs were examined in chickens. Two of these isolates were also tested in Pekin ducks and guinea fowl. Full genome sequence was obtained from all 6 isolates and evaluated for genetic markers for host adaptation and pathogenicity in poultry. Clinical signs were not observed in any host with any of the isolates, however one recent isolate was shed at higher titers than the other isolates and had the lowest bird infectious dose of all the isolates tested in all three species. This isolate, A/chicken/NY/19-012787-1/2019, was also the only isolate with a deletion in the stalk region of the neuraminidase protein (NA). This supports the theory that the NA stalk deletion is evidence of adaptation to gallinaceous poultry.

Efficacy of two vaccines against recent emergent antigenic variants of clade 2.3.2.1a highly pathogenic avian influenza viruses in Bangladesh.

H5N1 highly pathogenic avian influenza viruses (HPAIVs) have caused outbreaks in poultry in Bangladesh since 2007. While clade 2.2.2 and 2.3.4.2 HPAIVs have not been detected since 2012, clade 2.3.2.1a viruses have caused continuous outbreaks since 2012 despite the use of vaccines. In this study, we evaluated the efficacy of two H5 vaccines licensed in Bangladesh, RE-6 inactivated vaccine, and a recombinant herpesvirus of turkeys vaccine with an H5 insert (rHVT-H5), for protection against recent field viruses in chickens. We selected three viruses for efficacy tests (A/chicken/Bangladesh/NRL-AI-3237/2017, A/crow/Bangladesh/NRL-AI-8471/2017 and A/chicken/Bangladesh/NRL-AI-8323/2017) from 36 H5 viruses isolated from Bangladesh between 2016 and 2018 by comparing the amino acid sequences at five antigenic sites (A-E) and analyzing hemagglutination inhibition (HI) titers with reference antisera. The RE-6 and rHVT-H5 vaccines both conferred 80-100% clinical protection (i.e. reduced morbidity and mortality) against the three challenge viruses with no significant differences in protection. In addition, both vaccines significantly decreased viral shedding from infected chickens as compared to challenge control chickens. Based on these metrics, the current licensed H5 vaccines protected chickens against the recent field viruses. However, the A/crow/Bangladesh/NRL-AI-8471/2017 virus exhibited antigenic divergence including: several unique amino acid changes in antigenic epitope sites A and B and was a serological outlier in cross HI tests as visualized on the antigenic map. The continuing emergence of such antigenic variants which could alter the dominant antigenicity of field viruses should be continuously monitored and vaccines should be updated if field efficacy declines.

Estimating epidemiological parameters using diagnostic testing data from low pathogenicity avian influenza infected turkey houses.

Limiting spread of low pathogenicity avian influenza (LPAI) during an outbreak is critical to reduce the negative impact on poultry producers and local economies. Mathematical models of disease transmission can support outbreak control efforts by estimating relevant epidemiological parameters. In this article, diagnostic testing data from each house on a premises infected during a LPAI H5N2 outbreak in the state of Minnesota in the United States in 2018 was used to estimate the time of virus introduction and adequate contact rate, which determines the rate of disease spread. A well-defined most likely time of virus introduction, and upper and lower 95% credibility intervals were estimated for each house. The length of the 95% credibility intervals ranged from 11 to 22 with a mean of 17 days. In some houses the contact rate estimates were also well-defined; however, the estimated upper 95% credibility interval bound for the contact rate was occasionally dependent on the upper bound of the prior distribution. The estimated modes ranged from 0.5 to 6.0 with a mean of 2.8 contacts per day. These estimates can be improved with early detection, increased testing of monitored premises, and combining the results of multiple barns that possess similar production systems.