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Reassortment is an evolutionary mechanism by which influenza A viruses (IAV) generate genetic novelty. Reassortment is an important driver of host jumps and is widespread according to retrospective surveillance studies. However, predicting the epidemiological risk of reassortant emergence in novel hosts from surveillance data remains challenging. IAV strains persist and co-occur in the environment, promoting co-infection during environmental transmission. These conditions offer opportunity to understand reassortant emergence in reservoir and spillover hosts. Specifically, environmental RNA could provide rich information for understanding the evolutionary ecology of segmented viruses, and transform our ability to quantify epidemiological risk to spillover hosts. However, significant challenges with recovering and interpreting genomic RNA from the environment have impeded progress towards predicting reassortant emergence from environmental surveillance data. We discuss how the fields of genomics, experimental ecology and epidemiological modelling are well positioned to address these challenges. Coupling quantitative disease models and natural transmission studies with new molecular technologies, such as deep-mutational scanning and single-virus sequencing of environmental samples, should dramatically improve our understanding of viral co-occurrence and reassortment. We define observable risk metrics for emerging molecular technologies and propose a conceptual research framework for improving accuracy and efficiency of risk prediction. This article is part of the theme issue Dynamic and integrative approaches to understanding pathogen spillover.

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. [ABSTRACT FROM AUTHOR]

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-PCRbased 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. IMPORTANCE Highly 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Coronaviruses of animals periodically transmit to humans (1), as recently occurred with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 was recognized in December 2019 in cases of atypical pneumonia [...]

• Four vaccines were tested for efficacy against clade 2.3.4.4b in chickens. • All four vaccines protected against morbidity and mortality. • All four vaccines reduced virus shed compared to sham vaccinates. • Antibodies to the challenge virus could be detected at 7 days post challenge. • These licensed vaccines are efficacious for current epornitic H5 avian influenza. 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. [ABSTRACT FROM AUTHOR]

Sequence data was obtained from the capsid (ORF-2) and the polymerase (ORF-lb) genes of 23 turkey astrovirus (TAstV) isolates collected from commercial turkey flocks around the United States between 2003 and 2004. A high level of genetic variation was observed among the isolates, particularly in the capsid gene, where nucleotide sequence identity among them was as low as 69%. Isolates collected on the same farm, on the same day, but from different houses could have as little as 72% identity between their capsid gene sequences when compared. Phylogenetic analysis of the capsid gene revealed no clear assortment by geographic region or isolation date. The polymerase gene was more conserved with between 86 and 99% nucleotide identity and did assort in a geographic manner. Based on differing topologies of the capsid and polymerase gene phylogenetic trees, TAstV appears to undergo recombination.