Genome sequences of haemagglutinin cleavage site predict the pathogenicity phenotype of avian influenza virus: statistically validated data for facilitating rapid declarations and reducing reliance on in vivo testing.

Based on the pathogenicity in chickens, most H1-H16 avian influenza viruses (AIV) cause mild diseases, whereas some of the H5 and H7 AI viruses cause severe, systemic disease. The number of basic amino acids in the haemagglutinin (HA) cleavage site of AIV plays a critical role in pathogenicity. As we gain a greater understanding of the molecular mechanisms of pathogenicity, genome sequencing of the HA0 cleavage site has assumed a greater role in assessment of the potential pathogenicity of H5 and H7 viruses. We validated the use of HA cleavage site motif analysis by comparing molecular pathotyping data against experimental in vivo (intravenous pathogenicity index [IVPI] and lethality) data for determination of both low pathogenicity and high pathogenicity AI virus declaration with the goal of expediting pathotype confirmation and further reducing the reliance on in vivo testing. Our data provide statistical support to the continued use of molecular determination of pathotype for AI viruses based on the HA cleavage site sequence in the absence of an in vivo study determination. This approach not only expedites the declaration process of highly pathogenic AIV (HPAIV) but also reduces the need for experimental in vivo testing of H5 and H7 viruses.

Opposite Outcomes of the Within-Host Competition between High- and Low-Pathogenic H5N8 Avian Influenza Viruses in Chickens Compared to Ducks.

Highly pathogenic avian influenza viruses (HPAIV) emerge from low-pathogenic avian influenza viruses (LPAIV) through the introduction of basic amino acids at the hemagglutinin (HA) cleavage site. Following viral evolution, the newly formed HPAIV likely represents a minority variant within the index host, predominantly infected with the LPAIV precursor. Using reverse genetics-engineered H5N8 viruses differing solely at the HA cleavage, we tested the hypothesis that the interaction between the minority HPAIV and the majority LPAIV could modulate the risk of HPAIV emergence and that the nature of the interaction could depend on the host species. In chickens, we observed that the H5N8LP increased H5N8HP replication and pathogenesis. In contrast, the H5N8LP antagonized H5N8HP replication and pathogenesis in ducks. Ducks mounted a more potent antiviral innate immune response than chickens against the H5N8LP, which correlated with H5N8HP inhibition. These data provide experimental evidence that HPAIV may be more likely to emerge in chickens than in ducks and underscore the importance of within-host viral variant interactions in viral evolution. IMPORTANCE Highly pathogenic avian influenza viruses represent a threat to poultry production systems and to human health because of their impact on food security and because of their zoonotic potential. It is therefore crucial to better understand how these viruses emerge. Using a within-host competition model between high- and low-pathogenic avian influenza viruses, we provide evidence that highly pathogenic avian influenza viruses could be more likely to emerge in chickens than in ducks. These results have important implications for highly pathogenic avian influenza virus emergence prevention, and they underscore the importance of within-host viral variant interactions in virus evolution.

Emergence of a Novel Reassortant H5N3 Avian Influenza Virus in Korean Mallard Ducks in 2018.

INTRODUCTION: The avian influenza (AI) virus causes a highly contagious disease which is common in wild and domestic birds and sporadic in humans. Mutations and genetic reassortments among the 8 negative-sense RNA segments of the viral genome alter its pathogenic potential, demanding well-targeted, active surveillance for infection control. METHODS: Wild duck fecal samples were collected during the 2018 bird health annual surveillance in South Korea for tracking variations of the AI virus. One low-pathogenic avian influenza H5N3 reassortment virus (A/mallard duck/South Korea/KNU18-91/2018 [H5N3]) was isolated and genomically characterized by phylogenetic and molecular analyses in this study. RESULTS: It was devoid of polybasic amino acids at the hemagglutinin (HA) cleavage site and exhibited a stalk region without deletion in the neuraminidase (NA) gene and NA inhibitor resistance-linked E/D627K/N and D701N marker mutations in the PB2 gene, suggesting its low-pathogenic AI. It showed a potential of a reassortment where only HA originated from the H5N3 poultry virus of China and other genes were derived from Mongolia. In phylogenetic analysis, HA was different from that of the isolate of H5N3 in Korea, 2015. In addition, this novel virus showed adaptation in Madin-Darby canine kidney cells, with 8.05 ± 0.14 log10 50% tissue culture infectious dose (TCID50) /mL at 36 h postinfection. However, it could not replicate in mice well, showing positive growth at 3 days postinfection (dpi) (2.1 ± 0.13 log10 TCID50/mL) but not at 6 dpi. CONCLUSIONS: The HA antigenic relationship of A/mallard duck/South Korea/KNU18-91/2018 (H5N3) showed differences toward one of the old low-pathogenic H5N3 viruses in Korea. These results indicated that a novel reassortment low-pathogenic avian influenza H5N3 subtype virus emerged in South Korea in 2018 via novel multiple reassortments with Eurasian viruses, rather than one of old Korean H5N3 strains.

Enterotropism of highly pathogenic avian influenza virus H5N8 from the 2016/2017 epidemic in some wild bird species.

In 2016/2017, H5N8 highly pathogenic avian influenza (HPAI) virus of the Goose/Guangdong lineage spread from Asia to Europe, causing the biggest and most widespread HPAI epidemic on record in wild and domestic birds in Europe. We hypothesized that the wide dissemination of the 2016 H5N8 virus resulted at least partly from a change in tissue tropism from the respiratory tract, as in older HPAIV viruses, to the intestinal tract, as in low pathogenic avian influenza (LPAI) viruses, allowing more efficient faecal-oral transmission. Therefore, we determined the tissue tropism and associated lesions in wild birds found dead during the 2016 H5N8 epidemic, as well as the pattern of attachment of 2016 H5N8 virus to respiratory and intestinal tissues of four key wild duck species. We found that, out of 39 H5N8-infected wild birds of 12 species, four species expressed virus antigen in both respiratory and intestinal epithelium, one species only in respiratory epithelium, and one species only in intestinal epithelium. Virus antigen expression was association with inflammation and necrosis in multiple tissues. The level of attachment to wild duck intestinal epithelia of 2016 H5N8 virus was comparable to that of LPAI H4N5 virus, and higher than that of 2005 H5N1 virus for two of the four duck species and chicken tested. Overall, these results indicate that 2016 H5N8 may have acquired a similar enterotropism to LPAI viruses, without having lost the respirotropism of older HPAI viruses of the Goose/Guangdong lineage. The increased enterotropism of 2016 H5N8 implies that this virus had an increased chance to persist long term in the wild waterbird reservoir.

Major difference in clinical outcome and replication of a H3N1 avian influenza strain in young pullets and adult layers.

In this study, we investigated the pathogenicity, replication and tropism of the low pathogenic avian influenza (LPAI) strain A/chicken/Belgium/460/2019(H3N1) in adult SPF layers and young SPF males. The inoculated hens showed 58% mortality and a 100% drop in egg production in the second week post inoculation. The high viral loads in the cloacal samples coincided with the period of the positive immunohistochemistry of the oviduct, acute peritonitis and time of mortality, suggesting that the replication of H3N1 in the oviduct was a major component of the onset of clinical disease and increased level of excretion of the virus. In the inoculated young birds, the clinical signs were very mild with the exception of one bird. The results suggest that the time of replication of the virus was much shorter than in the adult layers; some of the young males did not show any proof of being infected at all. To conclude, the results of the study in young birds confirmed the intravenous pathogenicity test results but also showed that the clinical signs in adult layers were very severe. Based on the mortality without a bacterial component, complete drop of egg production and post mortem findings, this H3N1 strain is a moderately virulent strain, the highest category for LPAI strains. It is important to realize that if HPAI did not exist, this moderately virulent H3N1 virus would most likely to be considered as a very virulent virus.

Insertion of Basic Amino Acids in the Hemagglutinin Cleavage Site of H4N2 Avian Influenza Virus (AIV)-Reduced Virus Fitness in Chickens is Restored by Reassortment with Highly Pathogenic H5N1 AIV.

Highly pathogenic (HP) avian influenza viruses (AIVs) are naturally restricted to H5 and H7 subtypes with a polybasic cleavage site (CS) in hemagglutinin (HA) and any AIV with an intravenous pathogenicity index (IVPI) ≥ 1.2. Although only a few non-H5/H7 viruses fulfill the criteria of HPAIV; it remains unclear why these viruses did not spread in domestic birds. In 2012, a unique H4N2 virus with a polybasic CS 322PEKRRTR/G329 was isolated from quails in California which, however, was avirulent in chickens. This is the only known non-H5/H7 virus with four basic amino acids in the HACS. Here, we investigated the virulence of this virus in chickens after expansion of the polybasic CS by substitution of T327R (322PEKRRRR/G329) or T327K (322PEKRRKR/G329) with or without reassortment with HPAIV H5N1 and H7N7. The impact of single mutations or reassortment on virus fitness in vitro and in vivo was studied. Efficient cell culture replication of T327R/K carrying H4N2 viruses increased by treatment with trypsin, particularly in MDCK cells, and reassortment with HPAIV H5N1. Replication, virus excretion and bird-to-bird transmission of H4N2 was remarkably compromised by the CS mutations, but restored after reassortment with HPAIV H5N1, although not with HPAIV H7N7. Viruses carrying the H4-HA with or without R327 or K327 mutations and the other seven gene segments from HPAIV H5N1 exhibited high virulence and efficient transmission in chickens. Together, increasing the number of basic amino acids in the H4N2 HACS was detrimental for viral fitness particularly in vivo but compensated by reassortment with HPAIV H5N1. This may explain the absence of non-H5/H7 HPAIV in poultry.

Genetic Evidence Supports Sporadic and Independent Introductions of Subtype H5 Low-Pathogenic Avian Influenza A Viruses from Wild Birds to Domestic Poultry in North America.

Wild-bird origin influenza A viruses (IAVs or avian influenza) have led to sporadic outbreaks among domestic poultry in the United States and Canada, resulting in economic losses through the implementation of costly containment practices and destruction of birds. We used evolutionary analyses of virus sequence data to determine that 78 H5 low-pathogenic avian influenza viruses (LPAIVs) isolated from domestic poultry in the United States and Canada during 2001 to 2017 resulted from 18 independent virus introductions from wild birds. Within the wild-bird reservoir, the hemagglutinin gene segments of H5 LPAIVs exist primarily as two cocirculating genetic sublineages, and our findings suggest that the H5 gene segments flow within each migratory bird flyway and among adjacent flyways, with limited exchange between the nonadjacent Atlantic and Pacific Flyways. Phylogeographic analyses provided evidence that IAVs from dabbling ducks and swans/geese contributed to the emergence of viruses among domestic poultry. H5 LPAIVs isolated from commercial farm poultry (i.e., turkey) that were descended from a single introduction typically remained a single genotype, whereas those from live-bird markets sometimes led to multiple genotypes, reflecting the potential for reassortment with other IAVs circulating within live-bird markets. H5 LPAIVs introduced from wild birds to domestic poultry represent economic threats to the U.S. poultry industry, and our data suggest that such introductions have been sporadic, controlled effectively through production monitoring and a stamping-out policy, and are, therefore, unlikely to result in sustained detections in commercial poultry operations.IMPORTANCE Integration of viral genome sequencing into influenza surveillance for wild birds and domestic poultry can elucidate evolutionary pathways of economically costly poultry pathogens. Evolutionary analyses of H5 LPAIVs detected in domestic poultry in the United States and Canada during 2001 to 2017 suggest that these viruses originated from repeated introductions of IAVs from wild birds, followed by various degrees of reassortment. Reassortment was observed where biosecurity was low and where opportunities for more than one virus to circulate existed (e.g., congregations of birds from different premises, such as live-bird markets). None of the H5 lineages identified were maintained for the long term in domestic poultry, suggesting that management strategies have been effective in minimizing the impacts of virus introductions on U.S. poultry production.

[Comparative analysis of methods efficiency of virification of the tick-borne encephalite virus.]

Based on experimental studies with 10-fold dilutions of two the strains TBEV of the Far Eastern subtype, different in moleculargenetic characteristic complex data of simultaneously taking into account three indicators of their verification (virus titer, ELISA and PCR-RT) were obtained. The efficiency of detecting a genetic marker in PCR compared with ELISA for a weakly pathogenic strain with defects in the genetic structure was higher by a factor of 10, and for a highly pathogenic strain by a factor of 5,000. At the same time, positive results in both reactions with respect to two strains were detected with a virus titer of not less than 1-1.5 log TCID50, i.e. this level of virus in the sample is defined as epidemically significant. An algorithm for conducting research on the verification of TBEV is proposed: 1) Ticks collected from vegetation can be examined by ELISA or by PCR. All positive results can be summarized and considered viral ticks; 2) All samples with positive results only in PCR or ELISA must be investigated in two reactions in order to obtain confirmation of the possible infectivity of the pathogen; 3) To obtain a fast complex result of infection of the removed ticks from patients or blood after a tick bite, studies should be carried out simultaneously in two reactions simultaneously in ELISA and PCR; 4) Isolation virus should be carried out in biological samples (ticks collected from vegetation, ticks removed from patients, the blood of patients with suspected TBE, mammals) only with the same results in PCR and ELISA. Thus, such an approach to verifying TBEV in a tick or in the blood of patients will improve the reliability of laboratory diagnostics, identifying not only markers of TBEV, but also determining the infectivity of the pathogen, which may be the basis for the appointment of early intensive antiviral therapy.

A Novel A(H7N2) Influenza Virus Isolated from a Veterinarian Caring for Cats in a New York City Animal Shelter Causes Mild Disease and Transmits Poorly in the Ferret Model.

In December 2016, a low-pathogenic avian influenza (LPAI) A(H7N2) virus was identified to be the causative source of an outbreak in a cat shelter in New York City, which subsequently spread to multiple shelters in the states of New York and Pennsylvania. One person with occupational exposure to infected cats became infected with the virus, representing the first LPAI H7N2 virus infection in a human in North America since 2003. Considering the close contact that frequently occurs between companion animals and humans, it was critical to assess the relative risk of this novel virus to public health. The virus isolated from the human case, A/New York/108/2016 (NY/108), caused mild and transient illness in ferrets and mice but did not transmit to naive cohoused ferrets following traditional or aerosol-based inoculation methods. The environmental persistence of NY/108 virus was generally comparable to that of other LPAI H7N2 viruses. However, NY/108 virus replicated in human bronchial epithelial cells with an increased efficiency compared with that of previously isolated H7N2 viruses. Furthermore, the novel H7N2 virus was found to utilize a relatively lower pH for hemagglutinin activation, similar to human influenza viruses. Our data suggest that the LPAI H7N2 virus requires further adaptation before representing a substantial threat to public health. However, the reemergence of an LPAI H7N2 virus in the northeastern United States underscores the need for continuous surveillance of emerging zoonotic influenza viruses inclusive of mammalian species, such as domestic felines, that are not commonly considered intermediate hosts for avian influenza viruses.IMPORTANCE Avian influenza viruses are capable of crossing the species barrier to infect mammals, an event of public health concern due to the potential acquisition of a pandemic phenotype. In December 2016, an H7N2 virus caused an outbreak in cats in multiple animal shelters in New York State. This was the first detection of this virus in the northeastern United States in over a decade and the first documented infection of a felid with an H7N2 virus. A veterinarian became infected following occupational exposure to H7N2 virus-infected cats, necessitating the evaluation of this virus for its capacity to cause disease in mammals. While the H7N2 virus was associated with mild illness in mice and ferrets and did not spread well between ferrets, it nonetheless possessed several markers of virulence for mammals. These data highlight the promiscuity of influenza viruses and the need for diligent surveillance across multiple species to quickly identify an emerging strain with pandemic potential.

Low-Pathogenic Influenza A Viruses in North American Diving Ducks Contribute to the Emergence of a Novel Highly Pathogenic Influenza A(H7N8) Virus.

Introductions of low-pathogenic avian influenza (LPAI) viruses of subtypes H5 and H7 into poultry from wild birds have the potential to mutate to highly pathogenic avian influenza (HPAI) viruses, but such viruses' origins are often unclear. In January 2016, a novel H7N8 HPAI virus caused an outbreak in turkeys in Indiana, USA. To determine the virus's origin, we sequenced the genomes of 441 wild-bird origin influenza A viruses (IAVs) from North America and subjected them to evolutionary analyses. The results showed that the H7N8 LPAI virus most likely circulated among diving ducks in the Mississippi flyway during autumn 2015 and was subsequently introduced to Indiana turkeys, in which it evolved high pathogenicity. Preceding the outbreak, an isolate with six gene segments (PB2, PB1, PA, HA, NA, and NS) sharing >99% sequence identity with those of H7N8 turkey isolates was recovered from a diving duck sampled in Kentucky, USA. H4N8 IAVs from other diving ducks possessed five H7N8-like gene segments (PB2, PB1, NA, MP, and NS; >98% sequence identity). Our findings suggest that viral gene constellations circulating among diving ducks can contribute to the emergence of IAVs that affect poultry. Therefore, diving ducks may serve an important and understudied role in the maintenance, diversification, and transmission of IAVs in the wild-bird reservoir.IMPORTANCE In January 2016, a novel H7N8 HPAI virus caused a disease outbreak in turkeys in Indiana, USA. To determine the origin of this virus, we sequenced and analyzed 441 wild-bird origin influenza virus strains isolated from wild birds inhabiting North America. We found that the H7N8 LPAI virus most likely circulated among diving ducks in the Mississippi flyway during autumn 2015 and was subsequently introduced to Indiana turkeys, in which it evolved high pathogenicity. Our results suggest that viral gene constellations circulating among diving ducks can contribute to the emergence of IAVs that affect poultry. Therefore, diving ducks may play an important and understudied role in the maintenance, diversification, and transmission of IAVs in the wild-bird reservoir. Our study also highlights the importance of a coordinated, systematic, and collaborative surveillance for IAVs in both poultry and wild-bird populations.

Potential mediators of in ovo delivered double stranded (ds) RNA-induced innate response against low pathogenic avian influenza virus infection.

BACKGROUND: Toll like receptor (TLR) 3 is a critically important innate pattern recognizing receptor that senses many viral infections. Although, it has been shown that double stranded (ds) RNA can be used for the stimulation of TLR3 signaling pathway in a number of host-viral infection models, it's effectiveness as an antiviral agent against low pathogenic avian influenza virus (LPAIV) needs further investigation. METHODS: In this study, first, we delivered TLR3 ligand, dsRNA, in ovo at embryo day (ED)18 since in ovo route is routinely used for vaccination against poultry viral and parasitic infections and infected with H4N6 LPAIV 24-h post-treatment. A subset of in ovo dsRNA treated and control groups were observed for the expressions of TLR3 and type I interferon (IFN)s, mRNA expression of interleukin (IL)-1ß and macrophage recruitment coinciding with the time of H4N6 LPAIV infection (24 h post-treatment). Additionally, Day 1 chickens were given dsRNA intra-tracheally along with a control group and a subset of chickens were infected with H4N6 LPAIV 24-h post-treatment whereas the rest of the animals were observed for macrophage and type 1 IFN responses coinciding with the time of viral infection. RESULTS: Our results demonstrate that the pre-hatch treatment of eggs with dsRNA reduces H4N6 replication in lungs. Further studies revealed that in ovo delivery of dsRNA increases TLR3 expression, type I IFN production and number of macrophages in addition to mRNA expression of IL-1ß in lung 24-h post-treatment. The same level of induction of innate response was not evident in the spleen. Moreover, we discovered that dsRNA elicits antiviral response against LPAIV correlating with type I IFN activity in macrophages in vitro. Post-hatch, we found no difference in H4N6 LPAIV genome loads between dsRNA treated and control chickens although we observed higher macrophage recruitment and IFN-ß response coinciding with the time of viral infection. CONCLUSIONS: Our findings imply that the TLR3 ligand, dsRNA has antiviral activity in ovo and in vitro but not in chickens post-hatch and dsRNA-mediated innate host response is characterized by macrophage recruitment and expressions of TLR3 and type 1 IFNs.

Development of a quadruple qRT-PCR assay for simultaneous identification of highly and low pathogenic H7N9 avian influenza viruses and characterization against oseltamivir resistance.

BACKGROUND: During the fifth wave of human H7N9 infections, a novel highly pathogenic (HP) H7N9 variant emerged with an insertion of multiple basic amino acids in the HA cleavage site. Moreover, a neuraminidase inhibitor (NAI) resistance (R292K in NA) mutation was found in H7N9 isolates from humans, poultry and the environment. In this study, we set out to develop and validate a multiplex quantitative reverse transcript polymerase chain reaction (qRT-PCR) to simultaneously detect the presence of H7N9 and further identify the HP and NAI-resistance mutations. METHODS: A quadruple qRT-PCR to simultaneously detect the presence of H7N9 and further identify the HP and NAI-resistance mutations was designed based on the analyses of the HA and NA genes of H7N9. This assay was further tested for specificity and sensitivity, and validated using clinical samples. RESULTS: The assay was highly specific and able to detect low pathogenic (LP)- or HP-H7N9 with/without the NAI-resistance mutation. The detection limit of the assay was determined to be 50 genome-equivalent copies and 2.8 × 10- 3 50% tissue culture infectious doses (TCID50) of live H7N9 per reaction. Clinical validation was confirmed by commercial kits and Sanger sequencing with ten clinical samples. CONCLUSIONS: We developed and validated a rapid, single-reaction, one-step, quadruple real-time qRT-PCR to simultaneously detect the presence of H7N9 and further identify the HP- and NAI-resistance strains with excellent performance in specificity and sensitivity. This assay could be used to monitor the evolution of H7N9 viruses in the laboratory, field and the clinic for early-warning and the prevention of H7N9 infections.

Domestic canaries (Serinus canaria forma domestica) are susceptible to low pathogenic avian influenza virus infections.

Avian influenza viruses have been isolated from many bird species; however, little is known about the susceptibility of pet birds to low pathogenic avian influenza (LPAI) viruses. To address this research gap, domestic canaries (Serinus canaria forma domestica) were experimentally infected with H5 and H7 LPAI viruses to determine susceptibility and to evaluate samples for diagnostic purposes. Clinical evidence of infection (e.g. ruffled plumage and apathy) and mortality were noted for the canaries inoculated with chicken-adapted LPAI viruses. Real-time reverse transcription-polymerase chain reaction (RRT-PCR) demonstrated higher viral RNA levels in buccal compared to faecal samples. No clinical signs or mortality were observed in canaries inoculated with LPAI virus originating from wild birds; however, the canaries in this group did have evidence of viral RNA in buccal and faecal samples. Overall, this study showed that domestic canaries are susceptible to LPAI virus infections and that they can shed large amounts of viral RNA, primarily through the respiratory route. Thus, buccal swabs might be better samples than faeces for efficient detection of some LPAI virus infections in these birds. Although canaries have not been identified as a significant reservoir for LPAI viruses, they may be infected by LPAI viruses. Thus, the importance of the control of domestic canaries for detection of LPAI viruses should not be underestimated, especially in the contexts of international commercial exchange and outbreaks. RESEARCH HIGHLIGHTS Canaries are susceptible to infection with H5/H7 LPAI viruses. Canaries inoculated with LPAI viruses excrete large amounts of viral RNA. Buccal swabs may be appropriate specimens for AI virus detection in canaries. The control of canaries for LPAI virus detection should not be overlooked.

Risk of Human Infections With Highly Pathogenic H5N2 and Low Pathogenic H7N1 Avian Influenza Strains During Outbreaks in Ostriches in South Africa.

Background: Risk factors for human infection with highly pathogenic (HP) and low-pathogenic (LP) avian influenza (AI) H5N2 and H7N1 were investigated during outbreaks in ostriches in the Western Cape province, South Africa. Methods: Serum surveys were conducted for veterinarians, farmworkers, and laboratory and abattoir workers involved in 2 AI outbreaks in the Western Cape province: (1) controlling and culling of 42000 ostriches during (HPAI)H5N2 outbreaks in ostriches (2011) (n = 207); (2) movement control during (LPAI)H7N1 outbreaks in 2012 (n = 66). A third serosurvey was conducted on state veterinarians from across the country in 2012 tasked with disease control in general (n = 37). Antibodies to H5 and H7 were measured by means of hemagglutination inhibition and microneutralization assays, with microneutralization assay titers >40 considered positive. Results: Two of 207 (1%) participants were seropositive for H5 and 4 of 207 (2%) for H7 in 2011, compared with 1 of 66 (1.5%) and 8 of 66 (13%) in 2012. Although individuals in all professions tested seropositive, abattoir workers (10 of 97; 10.3%) were significantly more at risk of influenza A(H7N1) infection (P = .001) than those in other professions (2 of 171;1.2%). Among state veterinarians, 4 of 37(11%) were seropositive for H7 and 1 of 37 (2.7%) for H5. Investigations of (LP)H7N1-associated fatalities in wild birds and quarantined exotic birds in Gauteng, AI outbreaks in poultry in KwaZulu-Natal, and ostriches in Western Cape province provide possible exposure events. Conclusion: (LPAI)H7N1 strains pose a greater infection-risk than (HPAI)H5N2 strains to persons involved in control of outbreaks in infected birds, with ostrich abattoir workers at highest risk.

Characterization of Avian Influenza Viruses Detected in Kenyan Live Bird Markets and Wild Bird Habitats Reveal Genetically Diverse Subtypes and High Proportion of A(H9N2), 2018-2020.

Following the detection of highly pathogenic avian influenza (HPAI) virus in countries bordering Kenya to the west, we conducted surveillance among domestic and wild birds along the shores of Lake Victoria. In addition, between 2018 and 2020, we conducted surveillance among poultry and poultry workers in live bird markets and among wild migratory birds in various lakes that are resting sites during migration to assess introduction and circulation of avian influenza viruses in these populations. We tested 7464 specimens (oropharyngeal (OP) and cloacal specimens) from poultry and 6531 fresh fecal specimens from wild birds for influenza A viruses by real-time RT-PCR. Influenza was detected in 3.9% (n = 292) of specimens collected from poultry and 0.2% (n = 10) of fecal specimens from wild birds. On hemagglutinin subtyping, most of the influenza A positives from poultry (274/292, 93.8%) were H9. Of 34 H9 specimens randomly selected for further subtyping, all were H9N2. On phylogenetic analysis, these viruses were genetically similar to other H9 viruses detected in East Africa. Only two of the ten influenza A-positive specimens from the wild bird fecal specimens were successfully subtyped; sequencing analysis of one specimen collected in 2018 was identified as a low-pathogenicity avian influenza H5N2 virus of the Eurasian lineage, and the second specimen, collected in 2020, was subtyped as H11. A total of 18 OP and nasal specimens from poultry workers with acute respiratory illness (12%) were collected; none were positive for influenza A virus. We observed significant circulation of H9N2 influenza viruses in poultry in live bird markets in Kenya. During the same period, low-pathogenic H5N2 virus was detected in a fecal specimen collected in a site hosting a variety of migratory and resident birds. Although HPAI H5N8 was not detected in this survey, these results highlight the potential for the introduction and establishment of highly pathogenic avian influenza viruses in poultry populations and the associated risk of spillover to human populations.

Inactivation of Avian Influenza Virus Inoculated into Ground Beef Patties Cooked on a Commercial Open-Flame Gas Grill.

With the emergence of clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus (AIV) infection of dairy cattle and its subsequent detection in raw milk, coupled with recent AIV infections affecting dairy farm workers, experiments were conducted to affirm the safety of cooked ground beef related to AIV because such meat is often derived from cull dairy cows. Specifically, retail ground beef (percent lean:fat = ca. 80:20) was inoculated with a low pathogenic AIV (LPAIV) isolate to an initial level of 5.6 log10 50% egg infectious doses (EID50)  per 300 g patty. The inoculated meat was pressed into patties (ca. 2.54 cm thick, ca. 300 g each) and then held at 4 °C for up to 60 min. In each of the two trials, two patties for each of the following three treatments were cooked on a commercial open-flame gas grill to internal instantaneous temperatures of 48.9 °C (120°F), 62.8 °C (145°F), or 71.1 °C (160°F), but without any dwell time. Cooking inoculated ground beef patties to 48.9 °C (ave. cooking time of ca. 15 min) resulted in a mean reduction of ≥2.5 ± 0.9 log10 EID50 per 300 g of ground beef as assessed via quantification of virus in embryonating chicken eggs (ECEs). Likewise, cooking patties on a gas grill to 62.8 °C (ave. cooking time of ca. 21 min) or to the USDA FSIS recommended minimum internal temperature for ground beef of 71.1 °C (ave. cooking time of ca. 24 min) resulted in a reduction to nondetectable levels from initial levels of ≥5.6 log10 EID50 per 300 g. These data establish that levels of infectious AIV are substantially reduced within inoculated ground beef patties (20% fat) using recommended cooking procedures.

Diagnostic Detection of H7N3 Low Pathogenicity Avian Influenza in a Commercial Game Bird Flock.

Low pathogenicity avian influenza (LPAI) H7N3 was diagnosed in a flock of commercially raised Japanese quail (Coturnix japonica) breeder hens. The birds were submitted with an initial complaint of a drop in egg production and watery droppings. Initial PCR testing of the flock for avian influenza (AI) was negative; however, mortality started increasing drastically, prompting further examination and the submission of more birds to the diagnostic laboratory. On antemortem examination, the birds appeared lethargic, with ruffled feathers, and had labored breathing. Gross examination revealed poor body condition, moderate dehydration, splenomegaly, ovarian regression, and airsacculitis. Several hens produced thin-shelled and shell-less eggs. Microscopically, lymphoplasmacytic encephalitis with neuronal cell necrosis, fibrinoheterophilic pneumonia, and splenic lymphoid depletion with amyloid deposition were seen. AI virus was detected in a follow-up submission and characterized as LPAI H7N3. Quail are a species of interest in the pathobiology of avian influenza viruses, underlined by the ability to serve as an amplification host.

Reporte de caso- Detección diagnóstica de influenza aviar de baja patogenicidad H7N3 en una parvada comercial de aves de caza. Se diagnosticó influenza aviar de baja patogenicidad (LPAI) H7N3 en una parvada de gallinas reproductoras de codornices japonesas criadas comercialmente. Las aves se recibieron con una historia inicial de caída en la producción de huevo y excrementos acuosos. La prueba inicial de PCR para influenza aviar de la parvada fue negativa; sin embargo, la mortalidad comenzó a aumentar drásticamente, lo que generó posteriores evaluaciones y el envío de más aves al laboratorio de diagnóstico. En el examen ante mortem, las aves estaban letárgicas, con las plumas erizadas y tenían dificultad para respirar. El examen macroscópico reveló mala condición corporal, deshidratación moderada, esplenomegalia, regresión ovárica y aerosaculitis. Varias gallinas produjeron huevos con cascarón delgado o sin cascarón. Microscópicamente, se observaron encefalitis linfoplasmocítica con necrosis de células neuronales, neumonía fibrinoheterófila y depleción linfoide esplénica con depósito de amiloide. El virus de influenza aviar se detectó en un caso de seguimiento y se caracterizó como de baja patogenicidad subtipo H7N3. Las codornices son una especie de interés en la patobiología de los virus de la influenza aviar, subrayada por la capacidad de servir como huésped de amplificación.

Homo- and Heterosubtypic Immunity to Low Pathogenic Avian Influenza Virus Mitigates the Clinical Outcome of Infection with Highly Pathogenic Avian Influenza H5N8 Clade 2.3.4.4.b in Captive Mallards (Anas platyrhynchos).

In this study, we investigated the clinical response, viral shedding, transmissibility, pathologic lesions, and tropism of HPAIV Gs/Gd H5N8 subtype (clade 2.3.4.4b), following experimental infection of three groups of captive mallards (Anas platyrhynchos): (i) fully susceptible, (ii) pre-exposed to low pathogenic avian influenza virus (LPAIV) H5N1 subtype, and (iii) pre-exposed to LPAIV H3N8 subtype. Infection of naïve mallards with HPAIV H5N8 resulted in ~60% mortality, neurological signs, abundant shedding, and transmission to contact ducks, who also became sick and died. High amounts of viral RNA were found in all collected organs, with the highest RNA load recorded in the brain. The IHC examinations performed on tissues collected at 4 and 14 days post-infection (dpi) revealed tropism to nervous tissue, myocardium, respiratory epithelium, and hepatic and pancreatic cells. The mallards pre-exposed to LPAIV H5N1 and challenged with HPAIV H5N8 were asymptomatic and showed a significant reduction of viral RNA shedding, yet still sufficient to cause infection (but no disease) in the contact ducks. The AIV antigen was not detected in organs at 4 and 14 dpi, and microscopic lesions were mild and scarce. Similarly, mallards previously inoculated with LPAIV H3N8 remained healthy after challenge with HPAIV H5N8, but viral RNA was detected in large quantities in swabs and organs, particularly in the early phase of infection. However, in contrast to mallards from group I, the IHC staining yielded negative results at the selected timepoints. The virus was transmitted to contact birds, which remained symptomless but demonstrated low levels of viral RNA shedding and mild- to moderate tissue damage despite negative IHC staining. The results indicate that naïve mallards are highly susceptible to HPAIV H5N8 clade 2.3.4.4b and that homo- and heterosubtypic immunity to LPAIV can mitigate the clinical outcomes of infection.

A Review of the Stability of Avian Influenza Virus in Materials from Poultry Farms.

Avian influenza virus (AIV) is widespread among poultry and wild waterfowl. The severity of the disease is variable and the highly pathogenic form can rapidly kill numerous avian species. Understanding the stability of AIV infectivity in different substrates in the environment of poultry facilities is critical to developing processes to effectively decontaminate or safely dispose of potentially contaminated material. This review aims to compile the current information on the stability of AIV in materials from poultry farms that cannot be disinfected with chemicals or fumigants: water, litter/bedding, soil, feed, feathers, carcasses/meat, manure/feces, and eggs. There are still important gaps in the data, but available data will inform risk assessments, biosecurity, and procedures to dispose of potentially contaminated material. Among the parameters and conditions reported, temperature is a nearly universal factor where, regardless of substrate, the virus will inactivate faster under a given set of conditions as the temperature increases, and freeze-thaw cycles can facilitate virus inactivation.

Estudio recapitulativo- Una revisión de la estabilidad del virus de la influenza aviar en materiales de granjas avícolas. El virus de la influenza aviar (AIV) está muy extendido en la avicultura comercial y en las aves acuáticas silvestres. La severidad de la enfermedad es variable y la forma altamente patógena puede matar rápidamente a numerosas especies de aves. Comprender la estabilidad de la infectividad del virus de la influenza en diferentes sustratos en el ambiente de las instalaciones avícolas es fundamental para desarrollar procesos para descontaminar de manera efectiva o para eliminar de manera segura el material potencialmente contaminado. Esta revisión tiene como objetivo recopilar la información actual sobre la estabilidad del virus de la influenza aviar en materiales de granjas avícolas que no se pueden desinfectar con productos químicos o fumigantes: agua, heces/ material de cama, suelo, alimento, plumas, canales/carne, estiércol/heces y huevos. Todavía existen vacíos importantes en la información, pero los datos disponibles pueden proporcionar información durante las evaluaciones de riesgos, la bioseguridad y los procedimientos para eliminar material potencialmente contaminado. Entre los parámetros y condiciones que se han reportado, la temperatura es un factor casi universal donde, independientemente del sustrato, el virus se inactivará más rápido bajo un conjunto determinado de condiciones a medida que aumenta la temperatura, y los ciclos de congelación y descongelación pueden facilitar la inactivación del virus.

Prevalence of major infectious diseases in backyard chickens from rural markets in Morocco.

Background and Aim: Raising backyard chickens is a common practice in Morocco, mainly in rural or periurban areas. Constraints due to devastating avian diseases have been recognized as a major limiting factor in backyard poultry production. Consequently, these flocks could potentially be implicated as reservoirs for poultry diseases. However, there is a considerable lack of information on disease prevalence in this production system, and the risk represented by these small flocks remains under debate. This study aimed to estimate the seroprevalence and identify related risk factors of a range of bacterial and viral pathogens of outstanding importance for the economy and public health in backyard poultry in Morocco. Materials and Methods: A total of 712 sera samples and 258 cloacal swabs were collected from 712 backyard chickens from 15 rural markets in the Khemisset and Skhirat-Temara provinces. None of the sampled chickens received any vaccination. Sera samples were screened for antibodies against Newcastle disease virus (NDV) and low pathogenic avian influenza H9N2 subtype (LPAI H9N2) using a hemagglutination-inhibition test, against bursal infectious disease virus (IBDV) and infectious bronchitis virus (IBV) using enzyme-linked immunosorbent assay, and against Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) using a rapid serum agglutination test. Swab samples were compiled into 86 pools and submitted for molecular detection using real-time reverse-transcription-polymerase chain reaction (RT-PCR). Results: The seroprevalences in backyard chickens for NDV, LPAI H9N2, IBDV, IBV, MG, and MS were 52.1% (371/712), 63.5% (452/712), 84.7% (603/712), 82.2% (585/712), 58% (413/712), and 74.8% (533/712), respectively. Based on the RT-PCR results, 2.3% (2/86), 62.8% (54/86), 2.3% (2/86), 63.9% (55/86), 40.7% (35/86), and 29.1% (25/86) of the pools were positive for NDV, H9N2 LPAI, IBDV, IBV, MG, and MS, respectively. Multiple coinfections (H9N2-IBV-MG), (H9N2-IBV-MS), or (IBV-MG-MS) were observed in 15.1%, 8.5%, and 8.5% of the tested samples, respectively. Conclusion: The results show that backyard chicken flocks and rural markets have the potential to serve as reservoirs or amplifiers for poultry pathogens and could pose a risk to the commercial poultry sector. This highlights the need for a comprehensive and adapted vaccination plan for backyard chickens, and extension of efforts to increase flock owners' awareness of avian diseases and incite the implementation of biosecurity measures at the farm level.