Recombinant parainfluenza virus 5 expressing clade 2.3.4.4b H5 hemagglutinin protein confers broad protection against H5Ny influenza viruses.

The global circulation of clade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) in poultry and wild birds, increasing mammal infections, continues to pose a public health threat and may even form a pandemic. An efficacious vaccine against H5Ny HPAIVs is crucial for emergency use and pandemic preparedness. In this study, we developed a parainfluenza virus 5 (PIV5)-based vaccine candidate expressing hemagglutinin (HA) protein of clade 2.3.4.4b H5 HPAIV, termed rPIV5-H5, and evaluated its safety and efficacy in mice and ferrets. Our results demonstrated that intranasal immunization with a single dose of rPIV5-H5 could stimulate H5-specific antibody responses, moreover, a prime-boost regimen using rPIV5-H5 stimulated robust humoral, cellular, and mucosal immune responses in mice. Challenge study showed that rPIV5-H5 prime-boost regimen provided sterile immunity against lethal clade 2.3.4.4b H5N1 virus infection in mice and ferrets. Notably, rPIV5-H5 prime-boost regimen provided protection in mice against challenge with lethal doses of heterologous clades 2.2, 2.3.2, and 2.3.4 H5N1, and clade 2.3.4.4h H5N6 viruses. These results revealed that rPIV5-H5 can elicit protective immunity against a diverse clade of highly pathogenic H5Ny virus infection in mammals, highlighting the potential of rPIV5-H5 as a pan-H5 influenza vaccine candidate for emergency use.IMPORTANCEClade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) have been widely circulating in wild birds and domestic poultry all over the world, leading to infections in mammals, including humans. Here, we developed a recombinant PIV5-vectored vaccine candidate expressing the HA protein of clade 2.3.4.4b H5 virus. Intranasal immunization with rPIV5-H5 in mice induced airway mucosal IgA responses, high levels of antibodies, and robust T-cell responses. Importantly, rPIV5-H5 conferred complete protection in mice and ferrets against clade 2.3.4.4b H5N1 virus challenge, the protective immunity was extended against heterologous H5Ny viruses. Taken together, our data demonstrate that rPIV5-H5 is a promising vaccine candidate against diverse H5Ny influenza viruses in mammals.

An Update on Highly Pathogenic Avian Influenza A(H5N1) Virus, Clade 2.3.4.4b.

Since the resurgence of highly pathogenic avian influenza (HPAI) A(H5N1) virus, clade 2.3.4.4b, during 2021, these viruses have spread widely among birds worldwide, causing poultry outbreaks and infections of a wide range of terrestrial and marine mammal species. During 2024, HPAI A(H5N1) virus, clade 2.3.4.4b, was detected in dairy cattle for the first time and caused an ongoing multistate outbreak, with high levels of virus documented in raw cow milk. Human infections with clade 2.3.4.4b viruses from exposures to infected poultry or dairy cattle have resulted in a wide spectrum of illness severity, from conjunctivitis or mild respiratory illness to severe and fatal pneumonia in different countries. Vigilance, and stronger global virologic surveillance among birds, poultry, terrestrial and marine mammals, and humans, with virus characterization and rapid data sharing, is needed to inform the threat of clade 2.3.4.4b viruses, as they continue to evolve, to public health.

Highly Pathogenic Avian Influenza A Virus in Wild Migratory Birds, Qinghai Lake, China, 2022.

In July 2022, an outbreak of highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b occurred among migratory birds at Qinghai Lake in China. The virus circulated in June, and reassortants emerged after its introduction into the area. Surveillance in 2023 showed that the virus did not establish a stable presence in wild waterfowl.

Highly Pathogenic Avian Influenza Virus A(H5N1) Clade 2.3.4.4b Infection in Free-Ranging Polar Bear, Alaska, USA.

We report a natural infection with a Eurasian highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus in a free-ranging juvenile polar bear (Ursus maritimus) found dead in North Slope Borough, Alaska, USA. Continued community and hunter-based participation in wildlife health surveillance is key to detecting emerging pathogens in the Arctic.

Evolution and Spread of Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus in Wild Birds, South Korea, 2022-2023.

During October 2022-March 2023, highly pathogenic avian influenza (HPAI) A(H5N1) clade 2.3.4.4b virus caused outbreaks in South Korea, including 174 cases in wild birds. To understand the origin and role of wild birds in the evolution and spread of HPAI viruses, we sequenced 113 HPAI isolates from wild birds and performed phylogenetic analysis. We identified 16 different genotypes, indicating extensive genetic reassortment with viruses in wild birds. Phylodynamic analysis showed that the viruses were most likely introduced to the southern Gyeonggi-do/northern Chungcheongnam-do area through whooper swans (Cygnus cygnus) and spread southward. Cross-species transmission occurred between various wild bird species, including waterfowl and raptors, resulting in the persistence of HPAI in wild bird populations and further geographic spread as these birds migrated throughout South Korea. Enhanced genomic surveillance was an integral part of the HPAI outbreak response, aiding in timely understanding of the origin, evolution, and spread of the virus.

Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus Infection in Domestic Dairy Cattle and Cats, United States, 2024.

We report highly pathogenic avian influenza A(H5N1) virus in dairy cattle and cats in Kansas and Texas, United States, which reflects the continued spread of clade 2.3.4.4b viruses that entered the country in late 2021. Infected cattle experienced nonspecific illness, reduced feed intake and rumination, and an abrupt drop in milk production, but fatal systemic influenza infection developed in domestic cats fed raw (unpasteurized) colostrum and milk from affected cows. Cow-to-cow transmission appears to have occurred because infections were observed in cattle on Michigan, Idaho, and Ohio farms where avian influenza virus-infected cows were transported. Although the US Food and Drug Administration has indicated the commercial milk supply remains safe, the detection of influenza virus in unpasteurized bovine milk is a concern because of potential cross-species transmission. Continued surveillance of highly pathogenic avian influenza viruses in domestic production animals is needed to prevent cross-species and mammal-to-mammal transmission.

Highly Pathogenic Avian Influenza A(H5N1) Virus Clade 2.3.4.4b in Domestic Ducks, Indonesia, 2022.

Highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b viruses were isolated from domestic ducks in South Kalimantan, Indonesia, during April 2022. The viruses were genetically similar to those detected in East Asia during 2021-2022. Molecular surveillance of wild birds is needed to detect potential pandemic threats from avian influenza virus.

Highly Pathogenic Avian Influenza A(H5N1) Virus Clade 2.3.4.4b Infections in Seals, Russia, 2023.

Highly pathogenic avian influenza A(H5N1) virus was detected in dead seals on Tyuleniy Island in eastern Russia, in the Sea of Okhotsk. Viruses isolated from dead northern fur seals belong to clade 2.3.4.4b and are closely related to viruses detected predominantly in the Russian Far East and Japan in 2022-2023.

Avian Influenza A(H5N1) Neuraminidase Inhibition Antibodies in Healthy Adults after Exposure to Influenza A(H1N1)pdm09.

We detected high titers of cross-reactive neuraminidase inhibition antibodies to influenza A(H5N1) virus clade 2.3.4.4b in 96.8% (61/63) of serum samples from healthy adults in Hong Kong in 2020. In contrast, antibodies at low titers were detected in 42% (21/50) of serum samples collected in 2009. Influenza A(H1N1)pdm09 and A(H5N1) titers were correlated.

Transmission dynamics and pathogenesis differ between pheasants and partridges infected with clade 2.3.4.4b H5N8 and H5N1 high-pathogenicity avian influenza viruses.

During the UK 2020-2021 epizootic of H5Nx clade 2.3.4.4b high-pathogenicity avian influenza viruses (HPAIVs), high mortality occurred during incursions in commercially farmed common pheasants (Phasianus colchicus). Two pheasant farms, affected separately by H5N8 and H5N1 subtypes, included adjacently housed red-legged partridges (Alectoris rufa), which appeared to be unaffected. Despite extensive ongoing epizootics, H5Nx HPAIV partridge outbreaks were not reported during 2020-2021 and 2021-2022 in the UK, so it is postulated that partridges are more resistant to HPAIV infection than other gamebirds. To assess this, pathogenesis and both intra- and inter-species transmission of UK pheasant-origin H5N8-2021 and H5N1-2021 HPAIVs were investigated. Onward transmission to chickens was also assessed to better understand the risk of spread from gamebirds to other commercial poultry sectors. A lower infectious dose was required to infect pheasants with H5N8-2021 compared to H5N1-2021. However, HPAIV systemic dissemination to multiple organs within pheasants was more rapid following infection with H5N1-2021 than H5N8-2021, with the former attaining generally higher viral RNA levels in tissues. Intraspecies transmission to contact pheasants was successful for both viruses and associated with viral environmental contamination, while interspecies transmission to a first chicken-contact group was also efficient. However, further onward transmission to additional chicken contacts was only achieved with H5N1-2021. Intra-partridge transmission was only successful when high-dose H5N1-2021 was administered, while partridges inoculated with H5N8-2021 failed to shed and transmit, although extensive tissue tropism was observed for both viruses. Mortalities among infected partridges featured a longer incubation period compared to that in pheasants, for both viruses. Therefore, the susceptibility of different gamebird species and pathogenicity outcomes to the ongoing H5Nx clade 2.3.4.4b HPAIVs varies, but pheasants represent a greater likelihood of H5Nx HPAIV introduction into galliforme poultry settings. Consequently, viral maintenance within gamebird populations and risks to poultry species warrant enhanced investigation.

A real-time colourimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of highly pathogenic H5 clade 2.3.4.4b avian influenza viruses.

Highly pathogenic avian influenza viruses (HPAIV) are a major threat to the global poultry industry and public health due to their zoonotic potential. Since 2016, Europe and France have faced major epizootics caused by clade 2.3.4.4b H5 HPAIV. To reduce sample-to-result times, point-of-care testing is urgently needed to help prevent further outbreaks and the propagation of the virus. This study presents the design of a novel real-time colourimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of clade 2.3.4.4b H5 HPAIV. A clinical validation of this RT-LAMP assay was performed on 198 pools of clinical swabs sampled in 52 poultry flocks during the H5 HPAI 2020-2022 epizootics in France. This RT-LAMP assay allowed the specific detection of HPAIV H5Nx clade 2.3.4.4b within 30 min with a sensitivity of 86.11%. This rapid, easy-to-perform, inexpensive, molecular detection assay could be included in the HPAIV surveillance toolbox.

Strain-dependent variations in replication of European clade 2.3.4.4b influenza A(H5N1) viruses in bovine cells and thermal inactivation in semi-skimmed or whole milk.

We investigated the thermostability of four European avian influenza A(H5N1) viruses in whole and semi-skimmed milk and their replication in bovine kidney and lung cells amid the current influenza A(H5N1) dairy cattle outbreak in the United States. Results showed strain-dependent differences in thermal inactivation, particularly in whole milk, and variable replication efficacy in lung cells. These findings support assessing the inactivation of European H5N1 viruses in milk and their replication in bovine cells, aiding biosafety protocols and public health measures.

Avian Influenza Virus A(H5Nx) and Prepandemic Candidate Vaccines: State of the Art.

Avian influenza virus has been long considered the main threat for a future pandemic. Among the possible avian influenza virus subtypes, A(H5N1) clade 2.3.4.4b is becoming enzootic in mammals, representing an alarming step towards a pandemic. In particular, genotype B3.13 has recently caused an outbreak in US dairy cattle. Since pandemic preparedness is largely based on the availability of prepandemic candidate vaccine viruses, in this review we will summarize the current status of the enzootics, and challenges for H5 vaccine manufacturing and delivery.

Antiviral Susceptibility of Highly Pathogenic Avian Influenza A(H5N1) Viruses Circulating Globally in 2022-2023.

The antiviral susceptibility of currently circulating (2022-2023) highly pathogenic avian influenza (HPAI) A(H5N1) viruses was assessed by genotypic and phenotypic approaches. The frequency of neuraminidase (NA) and polymerase acidic (PA) substitutions associated with reduced inhibition by NA inhibitors (NAIs) (21/2698, 0.78%) or by the PA inhibitor baloxavir (14/2600, 0.54%) was low. Phenotypic testing of 22 clade 2.3.2.1a and 2.3.4.4b viruses revealed broad susceptibility to NAIs and baloxavir concluding that most contemporary HPAI A(H5N1) viruses retain susceptibility to antiviral drugs. Novel NA-K432E and NA-T438I substitutions (N2 numbering) were identified at elevated frequencies (104/2698, 3.85%) and caused reduced zanamivir and peramivir inhibition.

Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus in Domestic Cat, France, 2022.

We detected highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus in a domestic cat that lived near a duck farm infected by a closely related virus in France during December 2022. Enhanced surveillance of symptomatic domestic carnivores in contact with infected birds is recommended to prevent further spread to mammals and humans.

Highly Pathogenic Avian Influenza Virus (H5N1) Clade 2.3.4.4b Introduced by Wild Birds, China, 2021.

Highly pathogenic avian influenza (HPAI) subtype H5N1 clade 2.3.4.4b virus has spread globally, causing unprecedented large-scale avian influenza outbreaks since 2020. In 2021, we isolated 17 highly pathogenic avian influenza H5N1 viruses from wild birds in China. To determine virus origin, we genetically analyzed 1,529 clade 2.3.4.4b H5N1 viruses reported globally since October 2020 and found that they formed 35 genotypes. The 17 viruses belonged to genotypes G07, which originated from eastern Asia, and G10, which originated from Russia. The viruses were moderately pathogenic in mice but were highly lethal in ducks. The viruses were in the same antigenic cluster as the current vaccine strain (H5-Re14) used in China. In chickens, the H5/H7 trivalent vaccine provided complete protection against clade 2.3.4.4b H5N1 virus challenge. Our data indicate that vaccination is an effective strategy for preventing and controlling the globally prevalent clade 2.3.4.4b H5N1 virus.

Novel Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus in Wild Birds, South Korea.

We isolated 5 highly pathogenic avian influenza A(H5N1) clade 2.3.4.4.b viruses from wild waterfowl feces in South Korea during November 2022. Whole-genome sequencing and phylogenetic analysis revealed novel genotypes produced by reassortment with Eurasian low pathogenicity avian influenza viruses. Enhanced surveillance will be required to improve prevention and control strategies.

Novel Avian Influenza Virus (H5N1) Clade 2.3.4.4b Reassortants in Migratory Birds, China.

Two novel reassortant highly pathogenic avian influenza viruses (H5N1) clade 2.3.4.4b.2 were identified in dead migratory birds in China in November 2021. The viruses probably evolved among wild birds through different flyways connecting Europe and Asia. Their low antigenic reaction to vaccine antiserum indicates high risks to poultry and to public health.

Highly Pathogenic Avian Influenza A(H5N1) Virus-Induced Mass Death of Wild Birds, Caspian Sea, Russia, 2022.

In May 2022, we observed a substantial die-off of wild migratory waterbirds on Maliy Zhemchuzhniy Island in the Caspian Sea, Russia. The deaths were caused by highly pathogenic avian influenza A(H5N1) clade 2.3.4.4.b virus. Continued surveillance of influenza viruses in wild bird populations is needed to predict virus spread over long distances.

Highly Pathogenic Avian Influenza A(H5N1) Virus Clade 2.3.4.4b Infections in Wild Terrestrial Mammals, United States, 2022.

We describe the pathology of natural infection with highly pathogenic avian influenza A(H5N1) virus of Eurasian lineage Goose/Guangdong clade 2.3.4.4b in 67 wild terrestrial mammals throughout the United States during April 1‒July 21, 2022. Affected mammals include 50 red foxes (Vulpes vulpes), 6 striped skunks (Mephitis mephitis), 4 raccoons (Procyon lotor), 2 bobcats (Lynx rufus), 2 Virginia opossums (Didelphis virginiana), 1 coyote (Canis latrans), 1 fisher (Pekania pennanti), and 1 gray fox (Urocyon cinereoargenteus). Infected mammals showed primarily neurologic signs. Necrotizing meningoencephalitis, interstitial pneumonia, and myocardial necrosis were the most common lesions; however, species variations in lesion distribution were observed. Genotype analysis of sequences from 48 animals indicates that these cases represent spillover infections from wild birds.