HPAI H5N1 Research Articles

Highly Pathogenic Avian Influenza Virus H5N1 in Double-crested Cormorants (Nannopterum auritum) of the Chesapeake Bay, USA.

Double-crested Cormorants (Nannopterum auritum) have historically exhibited low levels of infection and antibodies to avian influenza virus (AIV). The recent global expansion of clade 2.3.4.4b A/goose/Guangdong/1/1996 highly pathogenic (HP) avian influenza virus H5N1 (HPAI H5N1) has resulted in large-scale mortalities across diverse waterbird taxa including cormorants. We sampled 32 and 29 Double-crested Cormorants breeding in the Chesapeake Bay, US, during the summers of 2023 and 2024, respectively, to assess HPAI H5N1 infection and AIV antibodies. Although no mortality was observed in the area, one bird sampled in 2023 was infected with HPAI H5N1. Additionally, 21/31 individuals in 2023 and 10/25 individuals in 2024 for which sera were collected had AIV antibodies. Based on additional testing using hemagglutination inhibition, virus neutralization, and an enzyme-linked lectin assay, 94 and 100% (2023 and 2024, respectively) of the seropositive birds tested positive for antibodies to both H5 and N1, suggesting previous infection with HPAI H5N1. These results are consistent with survival and limited clinical effects related to HPAI H5N1 infections. Furthermore, these results suggest that population immunity to HPAI H5N1 within the Chesapeake Bay might reduce future infections and potential population impacts should HP H5N1 remain on the landscape, though immunity may be waning across time. Because results are based on a single population, additional testing for both infection and antibodies as well as continued monitoring could enhance understanding of antibody persistence.

Genetic characteristics and pathogenesis of clade 2.3.4.4b H5N1 high pathogenicity avian influenza virus isolated from poultry in South Korea, 2022-2023.

During the 2022-2023 winter season in South Korea, a novel clade 2.3.4.4b H5N1 HPAIV was first detected in wild birds, which then subsequently caused multiple outbreaks in poultry farms and wild birds. This study aimed to investigate the genetic characteristics of H5N1 HPAIVs isolated during the 2022-2023, along with their pathogenicity and transmissibility in chickens and ducks. The clade 2.3.4.4b H5N1 HPAIV viruses caused outbreaks in 75 poultry farms and detected in 174 wild bird cases. Phylogenetic analysis of hemagglutinin genes revealed that the South Korean H5N1 HPAIV isolates were closely related to Eurasian and American HPAIVs isolated between 2022 and 2023. In total, 21 diverse genotypes (22G0-22G20) were identified in virus isolates from poultry and wild birds, among which 22G7 was the dominant genotype. The 22G1 genotype (A/duck/Korea/H493/2022(H5N1)) caused high virulence and pathogenicity, with a 100% mortality rate in specific-pathogen-free chickens. Ducks inoculated with genotypes 22G1 or 22G7 (A/duck/Korea/H537/2022(H5N1)) showed neurological signs, with 60%-80% mortality rate. In the contact groups of ducks, 100% of transmissibility was observed. Notably, in the 22G7-inoculated group, viral shedding via the cloacal route was longer, and viral replication in the cecal tonsil was higher than that in the 22G1-inoculated group, which may have contributed to the dominancy of the 22G7 genotype. Therefore, better understanding of the genetic and pathogenic features of HPAI viruses is important for effective virus control in the field.

Pathogenesis of bovine H5N1 clade 2.3.4.4b infection in Macaques.

Since early 2022 highly pathogenic avian influenza (HPAI) H5N1 virus infections have been reported in wild aquatic birds and poultry throughout the United States (US) with spillover into several mammalian species1-6. In March 2024, HPAIV H5N1 clade 2.3.4.4b was first detected in dairy cows in Texas, US and continues to circulate on dairy farms in multiple states7,8. Milk production and quality are diminished in infected dairy cows, with high virus titers in milk raising concerns of exposure to mammals including humans through consumption9-12. Here we investigated routes of infection with bovine HPAIV H5N1 clade 2.3.4.4b in cynomolgus macaques, a surrogate model for human infection13. We show that intranasal or intratracheal inoculation of macaques could cause systemic infection resulting in mild and severe respiratory disease, respectively. In contrast, infection by the orogastric route resulted in limited infection and seroconversion of macaques which remained subclinical.

Natural infection of common cranes (Grus grus) with highly pathogenic avian influenza H5N1 in Serbia

IntroductionThe late autumn epizootic of the highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in Serbia in 2023 caused massive mortality in the migratory population of common cranes (Grus Grus). This is the first time HPAIV has been identified in the common crane in Serbia, leading to mass mortality of this bird species.MethodsTo understand the pathological impact of HPAIV in cranes, we evaluated the pathological changes in the tissues of common cranes. Additionally, we report genomic characterization of HPAI/H5N1. In total, 14 juvenile common crane carcasses were examined.ResultsInfected birds primarily exhibited neurologic signs, including ataxia and incoordination. Grossly, necrotizing pancreatitis was the most common finding, while microscopic lesions included necrosis, inflammation and hemorrhages in the lungs, spleen, brain, liver and kidneys. Based on RT-PCR, all birds were infected with the HPAI H5N1 virus, as viral RNA was detected in all 14 selected tissues. Genetic analysis revealed that our H5N1 isolate could be grouped with highly pathogenic avian influenza clade 2.3.4.4b, subgroup DA, and is very closely related to the H5N1 strains isolated from the common crane and turkey from Croatia, the common crane from Italy and the Ural owl from Slovakia.DiscussionOur findings showed that common cranes are highly susceptible to natural infection with the HPAI H5N1 virus of clade 2.3.4.4b and may serve as bio-sentinels for the presence of the HPAI virus in wildlife.

Analysis of the Monophyletic Lineage of Avian Influenza H5N1 Which Circulated in Venezuelan Birds During the 2022-2023 Outbreak.

Avian influenza subtype H5N1 has caused outbreaks worldwide since 1996, with the emergence of the Guandong lineage in China. The current clade 2.3.4.4b has evolved from this lineage, with increased virulence and mass mortality events in birds and mammals. The objective of this study was the analysis of 17 viral genomes of H5N1 avian influenza isolated in Venezuela during the 2022-2023 outbreak. The eight viral genomic segments were amplified using universal primers and sequenced via next-generation sequencing. The sequences were analyzed to confirm the H5 hemagglutinin clade, identify possible genetic reassortments, and perform a phylogenetic and docking analysis of the viral isolates. The viruses found in Venezuela belonged, as expected, to clade 2.3.4.4b and formed a monophyletic clade with North American influenza viruses, with no evidence of further reassortment. The introduction of the virus in South America is associated with bird migration through the Atlantic (Venezuela), Atlantic/Mississippi (Choco, Colombia), and Pacific migratory flyways, with the emergence of several viral lineages. Several mutations were found in all segments of the genome, although none of the key mutations was involved in mammalian adaptation. Moreover, in silico structural analysis suggests, as expected, that the viral hemagglutinin maintained a predilection for avian α2,3-linked sialic acid. The unprecedented pathogenic outbreak of avian influenza disease in South America was associated with the circulation of three different lineages, which maintain a lower affinity for the mammalian receptor.

PB2 627V and HA 217 sites synergistically affect the lethality of H9N2 in mice.

The H9N2 subtype avian influenza virus (AIV) continues to propagate and undergo evolution within China, thereby posing a significant threat to the poultry industry. This study encompassed the collection of 436 samples and swabs in East China over the period spanning 2018 to 2019, from which 31 strains of the H9N2 subtype viruses were isolated and purified. We revealed that the HA and NA genes of the 31 isolates categorized within the Y280 branch, while the PB2 and M genes were associated with the G1 branch, and the remaining genes aligned with the F/98 branch. Despite this alignment, antigenic mapping demonstrated differences between the 2018 and 2019 strains, with the early vaccine strains displaying low serological reactivity toward these isolates. Notably, the CK/SH/49/19 isolate exhibited lethality in mice, characterized by a PB2 E627V mutation and a HA deletion at amino acid position 217. Mechanistically, in vitro studies showed that the influenza virus CK/SH/49/19 carrying PB2 627V and HA 217M mutations displayed enhanced replication capacity, attributed to the heightened activity of the polymerase with PB2 627V. Moreover, the absence of the amino acid at the HA 217 site obstructed viral adsorption and internalization, resulted in lower activation pH, and impeded the virus budding process. Critically, in vivo experiments revealed that CK/SH/49/19 (PB2 627V, HA 217Δ) triggered a robust activation of interferon response and interferon-stimulated genes. This study furnished a theoretical foundation for the scientific prevention and control strategies against H9N2 subtype avian influenza.

Genotypic Clustering of H5N1 Avian Influenza Viruses in North America Evaluated by Ordination Analysis.

The introduction of HPAI H5N1 clade 2.3.4.4b viruses to North America in late 2021 resulted in avian influenza outbreaks in poultry, mortality events in many wild bird species, and spillovers into many mammalian species. Reassortment events with North American low-pathogenic virus were identified as early as February 2022 and over 100 genotypes have been characterized. Such diversity increases the complexity and time required for monitoring virus evolution. Here, we performed ordination and clustering analyses on sequence data from H5N1 viruses identified in North America between January 2020 and December 2023 to visualize the genotypic diversity of viruses in poultry and wildlife populations. Our results reveal that ordination- and cluster-based approaches can complement traditional phylogenetic analyses specifically for the preliminary assignment of H5N1 viruses to genotypic groups or to identify novel genotypes. Our study expands current knowledge on the genotypic diversity of H5N1 viruses in North America and describes a rapid approach for early virus genotype assignment.

Susceptibility of calf lung slice cultures to H5N1 influenza virus

ABSTRACT The current outbreak of HPAI H5N1 virus infections in dairy cattle in the USA underscores the need for easily accessible methods to rapidly assess host susceptibility for infection with known and emerging influenza viruses. Here we show that ex vivo lung slice cultures from calves provide a useful method to rapidly screen host susceptibility to a range of influenza A viruses.

Specific detection of duck adeno-associated virus using a TaqMan-based real-time PCR assay.

Duck adeno-associated Virus (DAAV) is a novel pathogen that was recently discovered in ducks. To establish a molecular detection assay for DAAV for further epidemiological investigation and pathogenic mechanism. Here, we designed specific primers and probes according to the sequence characteristics of the newly discovered DAAV and then established a TaqMan real-time PCR method (TaqMan-qPCR) for the detection of DAAV. Our data showed that the established TaqMan-qPCR for detecting DAAV had high sensitivity, with the lowest detection limit of 29.1 copies/μL. No cross reaction was found with duck circovirus (DuCV), H9N2 subtype avian influenza virus (AIV), avian Tembusu virus (ATmV). duck hepatitis A virus 1 and 3 (DHAV-1 and DHAV-3), duck adenovirus A (DAdV-A), duck adenovirus 3 (DAdV-3), or duck enteritis virus (DEV). The repeatability was excellent, with the coefficients of variation of repeated intragroup and intergroup tests ranging from 0.12-0.21% and 0.62-1.42%, respectively. Seventy-eight clinical samples collected from diseased or deceased ducklings were tested. The results showed that the DAAV positive rate was 21.79%, and a triple infection (DAAV+MDPV+GPV) was found. These data provide technical support for further molecular epidemiological surveillance and pathogenic mechanism studies of DAAV infection.

H9 Consensus Hemagglutinin Subunit Vaccine with Adjuvants Induces Robust Mucosal and Systemic Immune Responses in Mice by Intranasal Administration.

The H9N2 subtype avian influenza viruses mainly cause respiratory symptoms, reduce the egg production and fertility of poultry, and result in secondary infections, posing a great threat to the poultry industry and human health. Currently, all H9N2 avian influenza commercial vaccines are inactivated vaccines, which provide protection for immunized animals but cannot inhibit the spread of the virus and make it difficult to distinguish between the infected animals and vaccinated animals. In this study, a trimeric consensus H9 hemagglutinin (HA) subunit vaccine for the H9N2 subtype avian influenza virus based on a baculovirus expression system was first generated, and then the effects of three molecular adjuvants on the H9 HA subunit vaccine, Cholera toxin subunit B (CTB), flagellin, and granulocyte-macrophage colony-stimulating factor (GM-CSF) fused with H9 HA, and one synthetic compound, a polyinosinic-polycytidylic acid (PolyI:C) adjuvant, were evaluated in mice by intranasal administration. The results showed that these four adjuvants enhanced the immunogenicity of the H9 HA subunit vaccine for avian influenza viruses, and that GM-CSF and PolyI:C present better mucosal adjuvant activity for the H9 HA subunit vaccine. These results demonstrate that we have developed a potential universal H9 HA mucosal subunit vaccine with adjuvants in a baculovirus system that would be helpful for the prevention and control of H9N2 subtype avian influenza viruses.

Establishment and application of a duplex fluorescent quantitative PCR assay for H9N2 subtype avian influenza virus and infectious bronchitis virus.

The H9N2 subtype of avian influenza virus (AIV) and infectious bronchitis virus (IBV) are important avian viruses that cause respiratory symptoms in poultry, and can form mixed infections. In this study, primers and probes were designed based on the HA gene of H9N2 and the 5' noncoding region of IBV, respectively, and a fluorescent quantitative RT-PCR assay was established for simultaneous detection of these two pathogens. The reaction system and conditions were optimized. The method only detected AIV subtype H9N2 and IBV and no other viruses, confirming its high specificity. The assay detected 13.5 copies/μL and 1.66 copies/μL of H9N2 and IBV in clinical samples, respectively. The coefficients of variation for intra- and interassay repeatability were < 3%. The established method was used to analyze 254 clinical samples (oropharyngeal and cloacal swabs) from Hubei Province, China; 98.82% were positive for both pathogens. In summary, a duplex fluorescent quantitative RT-PCR method capable of simultaneously detecting AIV subtype H9N2 and IBV was established. It is specific, sensitive, and reproducible, and can be used for diagnosis of a variety of clinical samples. It provides a technological means for the rapid and simultaneous detection of both pathogens, and thus can facilitate clinical diagnosis and epidemiological investigations.

Epidemiological data of an influenza A/H5N1 outbreak in elephant seals in Argentina indicates mammal-to-mammal transmission

H5N1 high pathogenicity avian influenza virus has killed thousands of marine mammals in South America since 2022. Here we report epidemiological data and full genome characterization of clade 2.3.4.4b H5N1 HPAI viruses associated with a massive outbreak in southern elephant seals (Mirounga leonina) at Península Valdés, Argentina, in October 2023. We also report on H5N1 viruses in concurrently dead terns. Our genomic analysis shows that viruses from pinnipeds and terns in Argentina form a distinct clade with marine mammal viruses from Peru, Chile, Brazil and Uruguay. Additionally, these marine mammal clade viruses share an identical set of mammalian adaptation mutations which were also present in tern viruses. Our combined ecological and phylogenetic data support mammal-to-mammal transmission and occasional mammal-to-bird spillover and suggest multinational transmission of H5N1 viruses in mammals. We reflect that H5N1 viruses becoming more evolutionary flexible and adapting to mammals in new ways could have global consequences for wildlife, humans, and/or livestock.

A human isolate of bovine H5N1 is transmissible and lethal in animal models.

The outbreak of clade 2.3.4.4b highly pathogenic avian influenza viruses of the H5N1 subtype (HPAI H5N1) in dairy cattle in the USA has so far resulted in spillover infections of at least 14 farm workers1-3, who presented with mild respiratory symptoms or conjunctivitis, and one individual with no known animal exposure who was hospitalized but recovered3,4. Here we characterized A/Texas/37/2024 (huTX37-H5N1), a virus isolated from the eyes of an infected farm worker who developed conjunctivitis5. huTX37-H5N1 replicated efficiently in primary human alveolar epithelial cells, but less efficiently in corneal epithelial cells. Despite causing mild disease in the infected worker, huTX37-H5N1 proved lethal in mice and ferrets and spread systemically, with high titres in both respiratory and non-respiratory organs. Importantly, in four independent experiments in ferrets, huTX37-H5N1 transmitted by respiratory droplets in 17-33% of transmission pairs, and five of six exposed ferrets that became infected died. PB2-631L (encoded by bovine isolates) promoted influenza polymerase activity in human cells, suggesting a role in mammalian adaptation similar to that of PB2-627K (encoded by huTX37-H5N1). In addition, bovine HPAI H5N1 virus was found to be susceptible to polymerase inhibitors both in vitro and in mice. Thus, HPAI H5N1 virus derived from dairy cattle transmits by respiratory droplets in mammals without previous adaptation and causes lethal disease in animal models.

Recombinant avian-derived antiviral proteins cIFITM1, cIFITM3, and cViperin as effective adjuvants in inactivated H9N2 subtype avian influenza vaccines

Vaccine adjuvants, serving as non-specific immune enhancers, play a pivotal role in the immunoprevention and control of animal diseases. This study utilized prokaryotic expression systems to express and purify chicken-derived cIFITM1, cIFITM3, and cViperin, which were then formulated as adjuvants with H9N2 avian influenza virus antigens to create inactivated vaccines. These vaccines were administered to SPF chickens to investigate their immunopotentiating functions. Additionally, the proteins were assessed for their ability to act as standalone immune enhancers. The results demonstrated that cIFITM1, cIFITM3, and cViperin significantly elevated serum hemagglutination inhibition (HI) antibody titers. Notably, when used individually, these proteins markedly enhanced the antiviral capabilities of challenged chickens, leading to alleviated clinical symptoms, reduced tracheal virus replication, diminished virus shedding, and lessened histopathological damage, with cIFITM1 exhibiting the most pronounced effect. Furthermore, the protective efficacy of two H9N2 recombinant virus inactivated vaccines supplemented with cIFITM1 adjuvant was validated, achieving a 100 % vaccine protection efficiency. In conclusion, cIFITM1, cIFITM3, and cViperin as adjuvants for influenza vaccines effectively inhibit virus replication and shedding, highlighting their significant potential in influenza prevention and control.

Genetic characterization and receptor binding analysis of a novel H5N1 HPAI virus with a H6Nx-derived PA gene in Guangdong, China

Genetic characterization and receptor binding analysis of a novel H5N1 HPAI virus with a H6Nx-derived PA gene in Guangdong, China

Field production efficiency investigation of broilers immunized with a turkey herpesvirus vector vaccine expressing hemagglutinin from H9N2 subtype avian influenza virus

Turkey herpesvirus (HVT) vector vaccine expressing hemagglutinin from the H9N2 AIV, namely HVT-H9, were demonstrated to block H9N2 AIV infection and transmission in chickens. In this study, we evaluated the protection efficiency and production performance of broilers in HVT-H9 field trials in the presence or absence of the H9N2 AIV natural infection. HI titers against H9N2 AIV in broilers harboring maternal antibodies were successfully induced by HVT-H9. In the presence of H9N2 AIV natural infection, immunization with HVT-H9 blocked H9N2 AIV infection and reduced the mortality rate. Importantly, HVT-H9 vaccination slightly increased broiler weight and decreased the feed conversion rate in the absence of the H9N2 AIV natural infection but significantly reduced mortality rates and increased production efficiency during the H9N2 AIV natural infection. In summary, HVT-H9 immunization might block H9N2 AIV infection and improve production efficiency in the field, especially in the presence of H9N2 AIV natural infection.

Continuing evolution of H5N1 highly pathogenic avian influenza viruses of clade 2.3.2.1a G2 genotype in domestic poultry of Bangladesh during 2018–2021

ABSTRACT We characterized 15 H5N1 HPAI viruses from different small- and medium-scale poultry flocks across Bangladesh during 2018–2021 based on their complete genome sequences. The antigenic relatedness of H5N1 HPAI viruses from different timepoints was analysed. During 2020–2021, 42.11% of the flocks tested positive for at least one of the respiratory infections, with 15.79% showing influenza A virus, of which 8.77% tested positive for HPAIV H5N1. Co-infections with two to four pathogens were detected in 15.8% of flocks. Phylogeny and gene constellation analyses based on complete genome sequences of 15 HPAI viruses revealed the continuing circulation of H5 clade 2.3.2.1a genotype G2 viruses. In the HA protein of the study isolates, functionally meaningful mutations caused the loss of an N-linked glycosylation site (T156A), a modified antigenic site A (S141P), and a mutation in the receptor binding pocket (E193R/K). Consequently, antigenic analysis revealed a significant loss of cross-reactivity between viruses from different host species and periods. Most viruses displayed oseltamivir resistance markers at positions V96, I97, S227, and N275 (N1 numbering) of the NA protein. In addition, for the PB2, M1, and NS1 proteins, significant mutations were noticed that have been associated with polymerase activity and increased virulence for mammals in all study isolates. These results highlight the need for intensified genomic surveillance of HPAI circulating in poultry in Bangladesh and for establishing appropriate control measures to decrease the circulation of these viruses in poultry in the country.

Geographic, ecological, and temporal patterns of seabird mortality during the 2022 HPAI H5N1 outbreak on the island of Newfoundland

Highly pathogenic avian influenza (HPAI) H5N1 caused mass mortality of wildlife across the North Atlantic in 2022. Following European outbreaks, the first North American case was detected on the island of Newfoundland, Canada in November 2021, before spreading throughout North America. During the following summer, Newfoundland and surrounding islands (NFLD) experienced one of the most significant mortalities in Canadian provinces, with seabirds being the most affected taxa. From surveys and citizen reports, we estimate that 13 517 mortalities that can be attributed to HPAI occurred in NFLD from April to September 2022. Most estimated mortalities were among Northern Gannets ( Morus bassanus (Linnaeus, 1758)) (6596), Common Murres ( Uria aalge (Pontoppidan, 1763)) (5992), Atlantic Puffins ( Fratercula arctica (Linnaeus, 1758)) (282), and Black-legged Kittiwakes ( Rissa tridactyla (Linnaeus, 1758)) (217). Mortality reports moved from west to east along the southern, then eastern NFLD coast, and peaked in July and August. We formulated exploratory hypotheses regarding traits that could contribute to infection and mortality. Species differences in mortality most strongly associated with inter-nest distance, breeding phenology, and at-sea overlap with allospecifics from other colonies. Unprecedented seabird mortality and ongoing transmission within the circulating avian influenza viruses highlight the need for continued monitoring and development of conservation strategies.

Hot topic: Avian influenza subtype H5N1 in US dairy—A preliminary dairy foods perspective

In February and March of 2024, an unusual illness began affecting dairy herds primarily in Texas and neighboring states. The causative agent of this illness was ultimately confirmed in late March 2024 to be a strain of highly pathogenic avian influenza H5N1 belonging to clade 2.3.4.4b. In the months following the emergence of this viral disease in cattle, infections have spread to at least 191 herds in 13 states at the time of this writing in August 2024, primarily through cattle and human movement. Surprisingly, early examination of raw milk samples from clinically affected animals indicated that the virus had an affinity for the mammary tissue, and viral shedding into raw milk occurred at high levels, exceeding 108 log10 50% tissue culture infectious dose (TCID50) in some cases. These high viral loads coupled with evidence that farm cats who consumed raw milk from clinically ill animals were infected and exhibited high mortality rates, raised concerns about the safety of the US milk supply for human consumption. To date, 4 cow-associated human infections have been reported, all from farm employees with direct contact with infected animals. Several parameters ultimately affect the theoretical public health risk from consumption of dairy products manufactured from a milk supply containing H5N1, namely (1) initial viral load, (2) persistence of H5N1 in raw milk, (3) viral inactivation through processing practices including pasteurization, and (4) human susceptibility and infectious dose. In the short period since the emergence of this disease in dairy cattle in the United States, research has begun to answer these critical questions, although our knowledge is still quite limited at this time. Here we review the literature available from the current H5N1 outbreak in US dairy cattle, as well as selected relevant literature from previous research in other animal agriculture sectors, that affect our current understanding of the parameters associated with the food safety risk of this disease in the US dairy supply chain.

Evolution and mutational landscape of highly pathogenic avian influenza strain A(H5N1) in the current outbreak in the USA and global landscape

The emergence of highly pathogenic avian influenza strain A (H5N1) in the USA is a high concern. Here, we illustrated the evolution, divergence, transmission pattern, infection pattern, entropy diversity, nucleotide diversity, and mutational landscape of HPAI(H5N1). We depicted three phylogenetic trees, i.e., from three perspectives: considering the HPAI H5N1 genome of the current outbreak in the USA (n = 971), considering the HPAI H5N1 spared in different hosts (cattle, hunan, avian, and nonhuman primates) and using the global genome sequences (n = 3154). We found that the clade 2.3.4.4b was responsible for the present infection. We noted that the USA's divergence rate is 3.43e-3 subs per site per year, and the global divergence rate is 5.21e-3 subs per site per year. We reported significant nucleotide changes to illustrate the genome.Similarly, we observe several point mutations in some proteins, such as PB2, PA, HA, NA, and NS1. Among point mutations, some common mutations are noted in PB2 (E362G, M631L) and PA (L219I, K497R). However, elimination strategies should be a high priority for dairy farm workers, domestic cattle, and poultry birds to limit future outbreaks.