Assessment of Survival Kinetics for Emergent Highly Pathogenic Clade 2.3.4.4 H5Nx Avian Influenza Viruses.

High pathogenicity avian influenza viruses (HPAIVs) cause high morbidity and mortality in poultry species. HPAIV prevalence means high numbers of infected wild birds could lead to spill over events for farmed poultry. How these pathogens survive in the environment is important for disease maintenance and potential dissemination. We evaluated the temperature-associated survival kinetics for five clade 2.3.4.4 H5Nx HPAIVs (UK field strains between 2014 and 2021) incubated at up to three temperatures for up to ten weeks. The selected temperatures represented northern European winter (4 °C) and summer (20 °C); and a southern European summer temperature (30 °C). For each clade 2.3.4.4 HPAIV, the time in days to reduce the viral infectivity by 90% at temperature T was established (DT), showing that a lower incubation temperature prolonged virus survival (stability), where DT ranged from days to weeks. The fastest loss of viral infectivity was observed at 30 °C. Extrapolation of the graphical DT plots to the x-axis intercept provided the corresponding time to extinction for viral decay. Statistical tests of the difference between the DT values and extinction times of each clade 2.3.4.4 strain at each temperature indicated that the majority displayed different survival kinetics from the other strains at 4 °C and 20 °C.

Efficient and Informative Laboratory Testing for Rapid Confirmation of H5N1 (Clade 2.3.4.4) High-Pathogenicity Avian Influenza Outbreaks in the United Kingdom.

During the early stages of the UK 2021-2022 H5N1 high-pathogenicity avian influenza virus (HPAIV) epizootic in commercial poultry, 12 infected premises (IPs) were confirmed by four real-time reverse-transcription-polymerase chain reaction (RRT)-PCRs, which identified the viral subtype and pathotype. An assessment was undertaken to evaluate whether a large sample throughput would challenge laboratory capacity during an exceptionally large epizootic; hence, assay performance across our test portfolio was investigated. Statistical analysis of RRT-PCR swab testing supported it to be focused on a three-test approach, featuring the matrix (M)-gene, H5 HPAIV-specific (H5-HP) and N1 RRT-PCRs, which was successfully assessed at 29 subsequent commercial IPs. The absence of nucleotide mismatches in the primer/probe binding regions for the M-gene and limited mismatches for the H5-HP RRT-PCR underlined their high sensitivity. Although less sensitive, the N1 RRT-PCR remained effective at flock level. The analyses also guided successful surveillance testing of apparently healthy commercial ducks from at-risk premises, with pools of five oropharyngeal swabs tested by the H5-HP RRT-PCR to exclude evidence of infection. Serological testing at anseriform H5N1 HPAIV outbreaks, together with quantitative comparisons of oropharyngeal and cloacal shedding, provided epidemiological information concerning the chronology of initial H5N1 HPAIV incursion and onward spread within an IP.

Investigating the Genetic Diversity of H5 Avian Influenza Viruses in the United Kingdom from 2020-2022.

Since 2020, the United Kingdom and Europe have experienced annual epizootics of high-pathogenicity avian influenza virus (HPAIV). The first epizootic, during the autumn/winter of 2020-2021, involved six H5Nx subtypes, although H5N8 HPAIV dominated in the United Kingdom. While genetic assessments of the H5N8 HPAIVs within the United Kingdom demonstrated relative homogeneity, there was a background of other genotypes circulating at a lower degree with different neuraminidase and internal genes.  Following a small number of detections of H5N1 in wild birds over the summer of 2021, the autumn/winter of 2021-2022 saw another European H5 HPAIV epizootic that dwarfed the prior epizootic. This second epizootic was dominated almost exclusively by H5N1 HPAIV, although six distinct genotypes were defined. We have used genetic analysis to evaluate the emergence of different genotypes and proposed reassortment events that have been observed. The existing data suggest that the H5N1 viruses circulating in Europe during late 2020 continued to circulate in wild birds throughout 2021, with minimal adaptation, but then went on to reassort with AIVs in the wild bird population. We have undertaken an in-depth genetic assessment of H5 HPAIVs detected in the United Kingdom over two winter seasons and demonstrate the utility of in-depth genetic analyses in defining the diversity of H5 HPAIVs circulating in avian species, the potential for zoonotic risk, and whether incidents of lateral spread can be defined over independent incursions of infections from wild birds. This provides key supporting data for mitigation activities. IMPORTANCE High-pathogenicity avian influenza virus (HPAIV) outbreaks devastate avian species across all sectors, having both economic and ecological impacts through mortalities in poultry and wild birds, respectively. These viruses can also represent a significant zoonotic risk. Since 2020, the United Kingdom has experienced two successive outbreaks of H5 HPAIV. While H5N8 HPAIV was predominant during the 2020-2021 outbreak, other H5 subtypes were also detected. The following year, there was a shift in the subtype dominance to H5N1 HPAIV, but multiple H5N1 genotypes were detected. Through the thorough utilization of whole-genome sequencing, it was possible to track and characterize the genetic evolution of these H5 HPAIVs in United Kingdom poultry and wild birds. This enabled us to assess the risk posed by these viruses at the poultry-wild bird and the avian-human interfaces and to investigate the potential lateral spread between infected premises, a key factor in understanding the threat to the commercial sector.

JMM Profile: Swine influenza A virus: a neglected virus with pandemic potential.

Swine influenza is an acute respiratory disease of swine caused by swine influenza A virus (SwIAV). The ability of SwIAV to spread bidirectionally from animals to humans (zoonotic), and from humans to animals (reverse zoonotic), drives coinfection that can result in gene segment exchange and elevates the risk of generating viruses with pandemic potential. Compared to human-origin influenza A viruses, current data indicate a greater diversity amongst circulating SwIAVs, with three major subtypes (classified by haemagglutinin and neuraminidase) circulating globally in swine (H1N1, H1N2 and H3N2). The lack of protection afforded by human seasonal influenza vaccines against SwIAVs exacerbates the risk associated with reassortment of human, swine and potentially avian viruses. As such, global monitoring of SwIAVs is important for both human and animal health as they represent a true 'One Health' challenge with pandemic potential.

High pathogenicity avian influenza: targeted active surveillance of wild birds to enable early detection of emerging disease threats.

Avian influenza (AI) is an important disease that has significant implications for animal and human health. High pathogenicity AI (HPAI) has emerged in consecutive seasons within the UK to cause the largest outbreaks recorded. Statutory measures to control outbreaks of AI virus (AIV) at poultry farms involve disposal of all birds on infected premises. Understanding of the timing of incursions into the UK could facilitate decisions on improved responses. During the autumnal migration and wintering period (autumn 2019- spring 2020), three active sampling approaches were trialled for wild bird species considered likely to be involved in captive AI outbreaks with retrospective laboratory testing undertaken to define the presence of AIV.Faecal sampling of birds (n = 594) caught during routine and responsive mist net sampling failed to detect AIV. Cloacal sampling of hunter-harvested waterfowl (n = 146) detected seven positive samples from three species with the earliest detection on the 17 October 2020. Statutory sampling first detected AIV in wild and captive birds on 3 November 2020. We conclude that hunter sourced sampling of waterfowl presents an opportunity to detect AI within the UK in advance of outbreaks on poultry farms and allow for early intervention measures to protect the national poultry flock.

JMM Profile: Avian paramyxovirus type-1 and Newcastle disease: a highly infectious vaccine-preventable viral disease of poultry with low zoonotic potential.

Newcastle disease (ND) is a highly contagious disease of poultry caused by virulent avian paramyxovirus-1 (APMV-1) (previously termed avian avulavirus-1 and avian orthoavulavirus-1). APMV-1 is endemic in poultry in many developing countries, whilst outbreaks still occur in developed countries, affecting both commercial and backyard flocks. ND outbreaks can have substantial economic consequences due to high mortality rates and the imposition of trade restrictions. APMV-1 nucleic acid can be detected from swabs or tissues of suspected cases by PCR. Evidence of infection or vaccination may be demonstrated by the presence of specific antibodies against HN in serum samples. No anti-viral treatments exist, but vaccines are available, although there are currently concerns over their efficacy.

Gross pathology of high pathogenicity avian influenza virus H5N1 2021-2022 epizootic in naturally infected birds in the United Kingdom.

High pathogenicity avian influenza virus (HPAIV) clade 2.3.4.4b has re-emerged in the United Kingdom in 2021-2022 winter season, with over 90 cases of HPAIV detected among poultry and captive birds in England, Scotland, Wales, and Northern Ireland. Globally, HPAIV H5N1 has also had a wide geographical dispersion, causing outbreaks in Europe, North America, Asia, and Africa, impacting on socioeconomic and wildlife conservation. It is important to raise awareness of the gross pathological features of HPAIV and subsequently aid disease investigation through definition of pathological indicators following natural infection. In this study, we report on the gross pathology of HPAI H5N1 in poultry species (chicken, turkey, pheasant, guineafowl, duck, goose), and captive or wild birds (mute swan, tufted duck, jackdaw, peahen, white-tailed eagle) that tested positive between October 2021 and February 2022. Pancreatic and splenic necrosis were the common pathological findings in both Galliformes and Anseriformes. In addition to the more severe lesions documented in Galliformes, we also noted increased detection of pathological changes in a broader range of Anseriformes particularly in domestic ducks, in contrast to those reported in previous seasons with other H5Nx HPAIV subtypes. A continual effort to characterise the pathological impact of the disease is necessary to update on the presentation of HPAIV for both domestic/captive and wild birds whilst guiding early presumptive diagnosis.

Has Epizootic Become Enzootic? Evidence for a Fundamental Change in the Infection Dynamics of Highly Pathogenic Avian Influenza in Europe, 2021.

Phylogenetic evidence from the recent resurgence of high-pathogenicity avian influenza (HPAI) virus subtype H5N1, clade 2.3.4.4b, observed in European wild birds and poultry since October 2021, suggests at least two different and distinct reservoirs. We propose contrasting hypotheses for this emergence: (i) resident viruses have been maintained, presumably in wild birds, in northern Europe throughout the summer of 2021 to cause some of the outbreaks that are part of the most recent autumn/winter 2021 epizootic, or (ii) further virus variants were reintroduced by migratory birds, and these two sources of reintroduction have driven the HPAI resurgence. Viruses from these two principal sources can be distinguished by their hemagglutinin genes, which segregate into two distinct sublineages (termed B1 and B2) within clade 2.3.4.4b, as well as their different internal gene compositions. The evidence of enzootic HPAI virus circulation during the summer of 2021 indicates a possible paradigm shift in the epidemiology of HPAI in Europe.

JMM Profile: Avian influenza: a veterinary pathogen with zoonotic potential.

Avian influenza viruses (AIVs) are classified as either low pathogenicity (LP; generally causing sub-clinical to mild infections) or high pathogenicity (HP; capable of causing significant mortality events in birds). To date, HPAIVs appear o be restricted to the haemagglutinin (HA) glycoprotein H5 and H7 AIV subtypes. Both LPAIV and HPAIV H5 and H7 AIV subtypes are classified as the causative agents of notifiable disease in poultry. A broad range of non-H5/non-H7 LPAIVs also exist that have been associated with more severe disease outcomes in avian species. As a result, the constant threat from AIVs causes significant economic damage in poultry production systems worldwide. The close proximity between mammalian and susceptible avian species in some environments provides the opportunity for both inter-host transmission and mammalian adaptation, potentially resulting in novel AIV strains capable of infecting humans.

Detection of Highly Pathogenic Avian Influenza Virus H5N1 Clade 2.3.4.4b in Great Skuas: A Species of Conservation Concern in Great Britain.

The UK and Europe have seen successive outbreaks of highly pathogenic avian influenza across the 2020/21 and 2021/22 autumn/winter seasons. Understanding both the epidemiology and transmission of these viruses in different species is critical to aid mitigating measures where outbreaks cause extensive mortalities in both land- and waterfowl. Infection of different species can result in mild or asymptomatic outcomes, or acute infections that result in high morbidity and mortality levels. Definition of disease outcome in different species is of great importance to understanding the role different species play in the maintenance and transmission of these pathogens. Further, the infection of species that have conservation value is also important to recognise and characterise to understand the impact on what might be limited wild populations. Highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b has been detected in great skuas (Stercorarius skua) across different colonies on islands off the shore of Scotland, Great Britain during summer 2021. A large number of great skuas were observed as developing severe clinical disease and dying during the epizootic and mortalities were estimated to be high where monitored. Of eight skuas submitted for post-mortem examination, seven were confirmed as being infected with this virus using a range of diagnostic assays. Here we overview the outbreak event that occurred in this species, listed as species of conservation concern in Great Britain and outline the importance of this finding with respect to virus transmission and maintenance.

Rapid and sensitive detection of high pathogenicity Eurasian clade 2.3.4.4b avian influenza viruses in wild birds and poultry.

Avian influenza virus (AIV) is classified as high or low pathogenicity AIV (HPAIV/LPAIV) based on intravenous pathogenicity in chickens and/or the presence or absence of multiple basic residues at the heamagglutinin (HA) cleavage site (CS). Since 2014, Europe has experienced waves of incursions of H5Nx HPAIV. Between November 2020 and March 2021, these included HPAIV H5N8, with sporadic of H5N1 and H5N5 (all clade 2.3.4.4b), detected in more than 300 "found dead" wild birds submitted through a passive surveillance programme in the United Kingdom. Currently, H5Nx HPAIV detection relies on identification of AIV RNA and H5 subtyping using real-time reverse transcription PCR (rRT-PCR) assays. The pathotype is subsequently determined by Sanger sequencing of the HA CS. Here, we report the validation and application of a rapid, more cost-effective HP H5-detection rRT-PCR assay. The HP H5 rRT-PCR assay specifically, sensitively and reproducibly detected RNA from contemporary clade 2.3.4.4b H5 HPAIVs with comparable sensitivity to the diagnostic H5-specific rRT-PCR; LPAIV H5 RNA and non-AIV RNA were not detected. On material from "found-dead" wild birds, and for statutory disease diagnosis on poultry, the HP H5 rRT-PCR results provided 100% discrimination when compared to conventional CS sequencing, significantly reducing time-to-pathotype determination and cost, enhancing the diagnostic workflow.

Gross pathology associated with highly pathogenic avian influenza H5N8 and H5N1 in naturally infected birds in the UK (2020-2021).

BACKGROUND: Multiple outbreaks with highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4b viruses, including H5N8 and H5N1, have occurred in the United Kingdom, as well as in other European countries, since late 2020. METHODS: This report describes the pathology among poultry species (chickens, turkeys, ducks, and pheasants) and captive birds (Black Swans, a whistling duck and peregrine falcons) naturally infected with HPAIV from 22 cases of HPAIV H5N8 and two cases of HPAIV H5N1 outbreaks investigated between October 2020 and April 2021. RESULTS: On gross examination, pancreatic necrosis was easily identified and most commonly observed in galliformes infected with both subtypes of HPAIV but rarely in anseriformes. In addition, splenic necrosis was also frequently observed in chickens and turkeys infected with HPAIV H5N8. Other less common lesions included cardiac petechiae, serosal haemorrhages and ascites in a variety of species. CONCLUSION: Given the widespread dissemination of HPAIV infection in susceptible avian species during autumn/winter 2020-2021, these data, when evaluated along with clinical information, is a valuable first step for both veterinarians and field services to evaluate gross pathology at post-mortem to support the diagnosis of HPAIV infection.

A worm's best friend: recruitment of neutrophils by Wolbachia confounds eosinophil degranulation against the filarial nematode Onchocerca ochengi.

Onchocerca ochengi, a filarial parasite of cattle, represents the closest relative of the human pathogen, Onchocerca volvulus. Both species harbour Wolbachia endosymbionts and are remarkable in that adult female worms remain viable but sessile for many years while surrounded by host cells and antibodies. The basis of the symbiosis between filariae and Wolbachia is thought to be metabolic, although a role for Wolbachia in immune evasion has received little attention. Neutrophils are attracted to Wolbachia, but following antibiotic chemotherapy they are replaced by eosinophils that degranulate on the worm cuticle. However, it is unclear whether the eosinophils are involved in parasite killing or if they are attracted secondarily to dying worms. In this study, cattle infected with Onchocerca ochengi received adulticidal regimens of oxytetracycline or melarsomine. In contrast to oxytetracycline, melarsomine did not directly affect Wolbachia viability. Eosinophil degranulation increased significantly only in the oxytetracycline group; whereas nodular gene expression of bovine neutrophilic chemokines was lowest in this group. Moreover, intense eosinophil degranulation was initially associated with worm vitality, not degeneration. Taken together, these data offer strong support for the hypothesis that Wolbachia confers longevity on O. ochengi through a defensive mutualism, which diverts a potentially lethal effector cell response.