Correction: Establishment of an RAA-CRISPR/Cas12a-based diagnostic method for the detection of fowl adenovirus serotype 4 virus in chickens and wild birds

Fowl adenovirus (FAdV) serotype 4, recognized as the causative agent of hydropericardium syndrome (HPS) in chickens, causes substantial economic losses in poultry farming. To develop a simple, rapid, and reliable diagnostic method for the timely detection of FAdV-4 nucleic acid, we integrated the CRISPR/Cas12a system with recombinase-aided amplification (RAA). This approach enables visual detection of FAdV-4 with a sensitivity of one genome copy. The results can be obtained within 40 to 50 min without the need for complex instrumentation, making it ideal for remote field applications. Using this method, we investigated the prevalence of FAdV-4 in both common farm poultry and wild birds. Our results indicated that the FAdV-4-positive rate in wild birds was 51.19%, suggesting that wild birds may serve as specific reservoirs for this virus. In summary, we present a sensitive, swift, accurate, and inexpensive detection method for FAdV-4, along with an investigation of its epidemic situation in birds. Our study advances the detection and epidemiological understanding of FAdV-4 transmission among farm poultry and wild birds.

1. Modes of transmission and the origin of H5N1 virusesAvian Influenza (A.I.) is transmitted by infected birds and their excrements. Also, AI is mechanically transmitted by surface means via contaminated food, water, feeds, soil, vehicles, humans, animals, flies, feathers etc. AI viruses can be spread by national or international trades of infected birds and contaminated products. Wild birds, especially migratory waterfowl, are a recognized source and reservoir for all subtypes of AI viruses. Some mammals such as dogs and cats are susceptible to the virus, but they are usually considered as the dead ends.In 1996, H5N1 virus was first detected in Guangdong Province, China. In 1997 the virus became widespread in poultry markets in Hong Kong, and killed 6 of 18 infected persons. The virus was wiped out by culling all domestic poultry in Hong Kong. In 2002, a new H5N1 genotype appeared again in Hong Kong, and the variant strains spread across Southeast Asia and South Asia between 2002 and 2007. The viruses can be divided into several clades such as V1, V2, V3 and Indonesian clades. The strains of H5N1 virus appeared in Korea (2003) and Japan (2004) were closely related to Guangdong strain/174/04 which is distinct from the abov 4 clades.In April 2005, a new variant H5N1 virus, which caused high mortality in both wild birds and poultry, was observed in Quinghai Lake, China. The virus was spread westward through migratory birds into Siberia, Kazakhstan and Turkey. This unprecedented mortality of wild birds associated with H5N1 viruses opened a new window for its movement within wild and domestic birds across Eurasia, the Near East and Africa. Virus strains are divided into 3 clades (EMA 1, 2, 3). The virus isolated in 2007 in Japan is closely related to one of those viruses of Quinghai origin (EMA clades).2. AI situation in EuropeThe European Union decided to make risk assessments of H5N1 virus entering via migratory birds into Europe, and active and passive surveillance for AI virus in wild birds started in July 2005. The conclusion of this study indicated a high risk of introducing the virus via migratory birds, and also a risk of the infection to become enzootic in Europe.The EU encouraged each member country (a) to make an extensive survey of AI viruses in both wild and domestic birds, (b) to vaccinate zoo birds and poultry that cannot be kept in houses (c) to keep all domestic birds in closed housing in high risk areas or zones and (d) to vaccinate domestic birds that cannot be housed.Between 2005 and 2006, H5N1 viruses were detected in wild birds in 25 countries. AI outbreaks in poultry farms were reported from 13 countries in Eastern Europe, and 4 countries in Western Europe (Sweden, Denmark, France and Germany). It is considered that migratory birds played a major role in spreading H5N1 viruses in Europe.The results of the risk control measures in Western Europe can be summarized as follows : (i) It was successful to protect the zoo birds by vaccination, but several birds died due to trauma of vaccination.(ii) Surveillance of wild birds was useful in improving early warning systems for poultry producers, and was effective in reducing the exposure risks of poultry.(iii) Mass culling of poultry and ornamental birds could be avoided.In 2007, H5N1 virus surfaced again in Hungary, UK, Czech R., Germany and France. It seems that H5N1 viruses became enzootic in some countries in Eastern Europe including Russia.(View PDF for the rest of the abstract.)

The objective of the study was to detect the presence of avian influenza (AI) virus in wild aquatic birds found in Puerto Viejo wetlands, Lima-Peru. Fresh faecal samples (n=900) from 18 species of wild birds were collected from April 2008 to February 2009. Samples were analyzed by virus isolation in SPF embryonated chicken eggs. Seven strains of low pathogenicity AI viruses subtype H12N5 were isolated; six from the migratory species Arenaria interpres, and one from the resident species Fulica ardesiaca. The technique of risk assessment using Monte Carlo Simulation (program @ risk) indicated that the probability of finding the AI virus in wild birds from Puerto Viejo wetlands was 0.88% with a confidence interval of 0.15 to 2.53%. The results of the study showed that wild birds from Puerto Viejo wetlands constitute a reservoir for avian influenza virus in Peru.

Although extensive data exist on avian influenza in wild birds in North America, limited information is available from elsewhere, including Europe. Here, molecular diagnostic tools were employed for high-throughput surveillance of migratory birds, as an alternative to classical labor-intensive methods of virus isolation in eggs. This study included 36,809 samples from 323 bird species belonging to 18 orders, of which only 25 species of three orders were positive for influenza A virus. Information on species, locations, and timing is provided for all samples tested. Seven previously unknown host species for avian influenza virus were identified: barnacle goose, bean goose, brent goose, pink-footed goose, bewick's swan, common gull, and guillemot. Dabbling ducks were more frequently infected than other ducks and Anseriformes; this distinction was probably related to bird behavior rather than population sizes. Waders did not appear to play a role in the epidemiology of avian influenza in Europe, in contrast to the Americas. The high virus prevalence in ducks in Europe in spring as compared with North America could explain the differences in virus–host ecology between these continents. Most influenza A virus subtypes were detected in ducks, but H13 and H16 subtypes were detected primarily in gulls. Viruses of subtype H6 were more promiscuous in host range than other subtypes. Temporal and spatial variation in influenza virus prevalence in wild birds was observed, with influenza A virus prevalence varying by sampling location; this is probably related to migration patterns from northeast to southwest and a higher prevalence farther north along the flyways. We discuss the ecology and epidemiology of avian influenza A virus in wild birds in relation to host ecology and compare our results with published studies. These data are useful for designing new surveillance programs and are particularly relevant due to increased interest in avian influenza in wild birds.

There have been three waves of highly pathogenic avian influenza (HPAI) outbreaks in commercial, backyard poultry, and wild birds in Ukraine. The first (2005-2006) and second (2008) waves were caused by H5N1 HPAI virus, with 45 outbreaks among commercial poultry (chickens) and backyard fowl (chickens, ducks, and geese) in four regions of Ukraine (AR Crimea, Kherson, Odesa, and Sumy Oblast). H5N1 HPAI viruses were isolated from dead wild birds: cormorants (Phalacrocorax carbo) and great crested grebes (Podiceps cristatus) in 2006 and 2008. The third HPAI wave consisted of nine outbreaks of H5N8 HPAI in wild and domestic birds, beginning in November 2016 in the central and south regions (Kherson, Odesa, Chernivtsi, Ternopil, and Mykolaiv Oblast). H5N8 HPAI virus was detected in dead mute swans (Cygnus olor), peacocks (Pavo cristatus) (in zoo), ruddy shelducks (Tadorna ferruginea), white-fronted geese (Anser albifrons), and from environmental samples in 2016 and 2017. Wide wild bird surveillance for avian influenza (AI) virus was conducted from 2006 to 2016 in Ukraine regions suspected of being intercontinental (north-south and east-west) flyways. A total of 21 511 samples were collected from 105 species of wild birds representing 27 families and 11 orders. Ninety-five avian influenza (AI) viruses were isolated (including one H5N2 LPAI virus in 2010) from wild birds with a total of 26 antigenic hemagglutinin (HA) and neuraminidase (NA) combinations. Fifteen of 16 known avian HA subtypes were isolated. Two H5N8 HPAI viruses (2016-2017) and two H5N2 LPAI viruses (2016) were isolated from wild birds and environmental samples (fresh bird feces) during surveillance before the outbreak in poultry in 2016-2017. The Ukrainian H5N1, H5N8 HPAI, and H5N2 LPAI viruses belong to different H5 phylogenetic groups. Our results demonstrate the great diversity of AI viruses in wild birds in Ukraine, as well as the importance of this region for studying the ecology of avian influenza.

There have been three waves of highly pathogenic avian influenza (HPAI) outbreaks in commercial, backyard poultry, and wild birds in Ukraine. The first (2005-2006) and second (2008) waves were caused by H5N1 HPAI virus, with 45 outbreaks among commercial poultry (chickens) and backyard fowl (chickens, ducks, and geese) in four regions of Ukraine (AR Crimea, Kherson, Odesa, and Sumy Oblast). H5N1 HPAI viruses were isolated from dead wild birds: cormorants (Phalacrocorax carbo) and great crested grebes (Podiceps cristatus) in 2006 and 2008. The third HPAI wave consisted of nine outbreaks of H5N8 HPAI in wild and domestic birds, beginning in November 2016 in the central and south regions (Kherson, Odesa, Chernivtsi, Ternopil, and Mykolaiv Oblast). H5N8 HPAI virus was detected in dead mute swans (Cygnus olor), peacocks (Pavo cristatus) (in zoo), ruddy shelducks (Tadorna ferruginea), white-fronted geese (Anser albifrons), and from environmental samples in 2016 and 2017. Wide wild bird surveillance for avian influenza (AI) virus was conducted from 2006 to 2016 in Ukraine regions suspected of being intercontinental (north-south and east-west) flyways. A total of 21 511 samples were collected from 105 species of wild birds representing 27 families and 11 orders. Ninety-five avian influenza (AI) viruses were isolated (including one H5N2 LPAI virus in 2010) from wild birds with a total of 26 antigenic hemagglutinin (HA) and neuraminidase (NA) combinations. Fifteen of 16 known avian HA subtypes were isolated. Two H5N8 HPAI viruses (2016-2017) and two H5N2 LPAI viruses (2016) were isolated from wild birds and environmental samples (fresh bird feces) during surveillance before the outbreak in poultry in 2016-2017. The Ukrainian H5N1, H5N8 HPAI, and H5N2 LPAI viruses belong to different H5 phylogenetic groups. Our results demonstrate the great diversity of AI viruses in wild birds in Ukraine, as well as the importance of this region for studying the ecology of avian influenza.

Lessons learned from research and surveillance directed at highly pathogenic influenza A viruses in wild birds inhabiting North America

Despite the panzootic nature of emergent highly pathogenic avian influenza H5Nx viruses in wild migratory birds and domestic poultry, only a limited number of human infections with H5Nx viruses have been identified since its emergence in 1996. Few countries with endemic avian influenza viruses (AIVs) have implemented vaccination as a control strategy, while most of the countries have adopted a culling strategy for the infected flocks. To date, China and Egypt are the two major sites where vaccination has been adopted to control avian influenza H5Nx infections, especially with the widespread circulation of clade 2.3.4.4b H5N1 viruses. This virus is currently circulating among birds and poultry, with occasional spillovers to mammals, including humans. Herein, we will discuss the history of AIVs in Egypt as one of the hotspots for infections and the improper implementation of prophylactic and therapeutic control strategies, leading to continuous flock outbreaks with remarkable virus evolution scenarios. Along with current pre-pandemic preparedness efforts, comprehensive surveillance of H5Nx viruses in wild birds, domestic poultry, and mammals, including humans, in endemic areas is critical to explore the public health risk of the newly emerging immune-evasive or drug-resistant H5Nx variants.

The global spread of HPAI (H5N1) between 2005 and 2006 was blamed on movement of migratory wild birds and trade in live poultry across continents from infected regions. A survey was carried out to detect the presence of avian influenza (AI) antibodies in wild birds and AI viruses in poultry and wild birds from Kogi state, Nigeria. Haemagglutination inhibition (HI) test and enzyme link immunosorbent assay (ELISA) were used to detect AI antibodies in some species of apparently healthy wild birds during the survey. Using HI test, the wild birds were negative for AI (H5) antibodies but ELISA detected AI (NP) antibodies in Black Stork ( Ciconia nigra ) with an overall seroprevalence of 4.5% and mean titre of 24.50±2.400 EU. Cloacal swabs from the same species of wild birds that were tested for antibodies and 710 oropharyngeal swabs from poultry were tested for AI viruses using RT-PCR with primers targeting the AI matrix proteins but were negative for AI viruses. The detection of AI (NP) antibodies in wild birds but failure to detect the viruses showed that the exposure might not be recent. We recommend that poultry should be prevented from contact with wild water birds and a broad based surveillance for AI viruses in poultry and wild birds should be carried out in Kogi state, Nigeria. Keywords : Avian influenza, Black stork, ELISA, HI, RT-PCR

Summary The conceptual framework considering Anseriformes and Charadriiformes as the main maintenance hosts for influenza A viruses (IAV) in wild birds has shaped IAV research and surveillance over the last decades. We challenge this framework by reviewing the world‐wide surveillance data on non‐Anseriformes and non‐Charadriiformes (NANC) species, generally considered as playing little role in IAV maintenance, available in literature and online data bases (close to 200 sources). Globally, we found an IAV infection rate of 1·51% (95% CI, 1·44–1·59%) for c. 101 000 birds tested from NANC species. If Anseriformes have, as expected, a higher infection rate than any other bird orders, eight bird orders have an infection rate higher or close to the Charadriiformes infection rate, challenging the status of Charadriiformes. We interpret the attention paid in favour of Charadriiformes by an extrapolation bias from data collected in hotspots of IAV infection in Charadriiformes (e.g. Delaware Bay, USA). The growing data on IAV in wild birds world‐wide, summarised here, support two non‐exclusive hypotheses: (i) the quality of the diagnostic tools and techniques used explain the patterns observed; (ii) IAV maintenance is determined by complex multi‐host systems composed of multiple bird species, dependent on the ecosystem and its bird diversity and composition. Synthesis and applications. Our results have two main implications. First, new research and surveillance should be designed in order to understand influenza A viruses ecology in wild birds across the world, along with appropriate diagnostic tools and new hypotheses and dedicated protocols. This should be done in line with our new conceptual framework that conveys less a priori than its predecessor. Second, our results call for more bridging between biological and epidemiological sciences in order to tackle disease ecology in multi‐host systems.

Phylogeography of Highly Pathogenic H5 Avian Influenza Viruses in China.

A United States interagency avian influenza surveillance plan was initiated in 2006 for early detection of highly pathogenic avian influenza viruses (HPAIV) in wild birds. The plan included a variety of wild bird sampling strategies including the testing of fecal samples from aquatic areas throughout the United States from April 2006 through December 2007. Although HPAIV was not detected through this surveillance effort we were able to obtain 759 fecal samples that were positive for low pathogenic avian influenza virus (LPAIV). We used 136 DNA sequences obtained from these samples along with samples from a public influenza sequence database for a phylogenetic assessment of hemagglutinin (HA) diversity in the United States. We analyzed sequences from all HA subtypes except H5, H7, H14 and H15 to examine genetic variation, exchange between Eurasia and North America, and geographic distribution of LPAIV in wild birds in the United States. This study confirms intercontinental exchange of some HA subtypes (including a newly documented H9 exchange event), as well as identifies subtypes that do not regularly experience intercontinental gene flow but have been circulating and evolving in North America for at least the past 20 years. These HA subtypes have high levels of genetic diversity with many lineages co-circulating within the wild birds of North America. The surveillance effort that provided these samples demonstrates that such efforts, albeit labor-intensive, provide important information about the ecology of LPAIV circulating in North America.

Active surveillance and studying the virological features of avian-origin influenza viruses are essential for early warning and preparedness for the next potential pandemic. During our active surveillance of avian influenza viruses in wild birds in Egypt in the period 2014-2017, multiple reassortant low-pathogenic avian influenza H7N3 viruses were isolated. In this study, we investigated and compared the infectivity, pathogenicity, and transmission of four different constellation forms of Egyptian H7N3 viruses in chickens and mice and assessed the sensitivity of these viruses to different commercial antiviral drugs in vitro. Considerable variation in virus pathogenicity was observed in mice infected with different H7N3 viruses. The mortality rate ranged from 20 to 100% in infected mice. Infected chickens showed only ocular clinical signs at three days postinfection as well as systemic viral infection in different organs. Efficient virus replication and transmission in chickens was observed within each group, indicating that these subtypes can spread easily from wild birds to poultry without prior adaptation. Mutations in the viral proteins associated with antiviral drug resistance were not detected, and all strains were sensitive to the antiviral drugs tested. In conclusion, all of the viruses studied had the ability to infect mice and chickens. H7N3 viruses circulating among wild birds in Egypt could threaten poultry production and public health.

Avian influenza (AI) viruses have been routinely isolated from a wide diversity of free-living avian species, representing numerous taxonomic orders. Birds in orders Anseriformes and Charadriiformes are considered the natural reservoirs for all AI viruses; it is from these orders that AI viruses have been most frequently isolated. Since first recognized in the late 1800s, AI viruses have been an important cause of disease in poultry and, occasionally, in non-gallinaceous birds and mammals. While AI viruses tend to be of low pathogenicity (LP) in wild birds, the 2014-2015 incursion of highly pathogenic avian influenza (HPAI) clade 2.3.4.4 H5Nx viruses into North America and the recent circulation of HPAI H5 viruses in European wild birds highlight the need for targeted, thorough, and continuous surveillance programs in the wild bird reservoir. Such programs are crucial to understanding the potential risk for the incursion of AI into human and domestic animal populations. The aim of this chapter is to provide general concepts and guidelines for the planning and implementation of surveillance plans for AI viruses in wild birds.

BackgroundHighly pathogenic avian influenza virus (HPAIV) is a highly contagious disease which is a zoonotic pathogen of significant economic and public health concern. The outbreaks caused by HPAIV H5N1 of Asian origin have caused animal and human disease and mortality in several countries of Southeast Asia, such as Bangladesh, Cambodia, China, India, Indonesia, Laos, Myanmar, Thailand and Viet Nam. For the first time since 1961, this HPAIV has also caused extensive mortality in wild birds and has sparked debate of the role wild birds have played in the spread of this virus. Other than confirmed mortality events, little is known of this virus in wild birds. There is no report on the seroprevalence of avian influenza H5 infection in wild migratory birds in Yunnan Province. In this study we examined live wild birds in Yunnan Province for H5 specific antibody to better understand the occurrence of this disease in free living birds.MethodsSera from 440 wild birds were collected from in Kunming and Northern Ailaoshan of Yunnan Province, Southwestern China, and assayed for H5 antibodies using the hemagglutination inhibition (HI) assays.ResultsThe investigation revealed that the seroprevalence of avian influenza H5 was as following: Ciconiiformes 2.6%, Strigiformes 13.04%, Passeriformes 20%, Cuculiformes 21.74%, Gruiformes 0%, Columbiformes 0%, Charadriiformes 0% and Coraciiformes 0%. Statistical analyses showed that there was a significant difference of prevalence between the orders (P < 0.01). Specific avian influenza H5 antibodies were detected in 23 of 440 (5.23%) sera. Mean HI titer 23 positive sera against H5 were 5.4 log2.ConclusionsThe results of the present survey indicated that the proportion of wild birds had previously infected AIV H5 at other times of the year. To our knowledge, this is the first seroprevalence report of avian influenza H5 infection in wild migratory birds in China’ s southwestern Yunnan Province. The results of the present survey have significant public health concerns.