Divergent Pathogenesis and Transmission of Highly Pathogenic Avian Influenza A(H5N1) in Swine.

Highly pathogenic avian influenza (HPAI) viruses have potential to cross species barriers and cause pandemics. Since 2022, HPAI A(H5N1) belonging to the goose/Guangdong 2.3.4.4b hemagglutinin phylogenetic clade have infected poultry, wild birds, and mammals across North America. Continued circulation in birds and infection of multiple mammalian species with strains possessing adaptation mutations increase the risk for infection and subsequent reassortment with influenza A viruses endemic in swine. We assessed the susceptibility of swine to avian and mammalian HPAI H5N1 clade 2.3.4.4b strains using a pathogenesis and transmission model. All strains replicated in the lung of pigs and caused lesions consistent with influenza A infection. However, viral replication in the nasal cavity and transmission was only observed with mammalian isolates. Mammalian adaptation and reassortment may increase the risk for incursion and transmission of HPAI viruses in feral, backyard, or commercial swine.

Dual Gene Detection of H5N1 Avian Influenza Virus Based on Dual RT-RPA.

The H5N1 avian influenza virus seriously affects the health of poultry and humans. Once infected, the mortality rate is very high. Therefore, accurate and timely detection of the H5N1 avian influenza virus is beneficial for controlling its spread. This article establishes a dual gene detection method based on dual RPA for simultaneously detecting the HA and M2 genes of H5N1 avian influenza virus, for the detection of H5N1 avian influenza virus. Design specific primers for the conserved regions of the HA and M2 genes. The sensitivity of the dual RT-RPA detection method for HA and M2 genes is 1 × 10-7 ng/µL. The optimal primer ratio is 1:1, the optimal reaction temperature is 40 °C, and the optimal reaction time is 20 min. Dual RT-RPA was used to detect 72 samples, and compared with RT-qPCR detection, the Kappa value was 1 (p value < 0.05), and the clinical sample detection sensitivity and specificity were both 100%. The dual RT-RPA method is used for the first time to simultaneously detect two genes of the H5N1 avian influenza virus. As an accurate and convenient diagnostic tool, it can be used to diagnose the H5N1 avian influenza virus.

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

In December 2022, highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus emerged in Chile. We detected H5N1 virus in 93 samples and obtained 9 whole-genome sequences of strains from wild birds. Phylogenetic analysis suggests multiple viral introductions into South America. Continued surveillance is needed to assess risks to humans and domestic poultry.

Highly Pathogenic Avian Influenza A(H5N1) Virus in a Harbor Porpoise, Sweden.

We found highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b associated with meningoencephalitis in a stranded harbor porpoise (Phocoena phocoena). The virus was closely related to strains responsible for a concurrent avian influenza outbreak in wild birds. This case highlights the potential risk for virus spillover to mammalian hosts.

Advances in Detection Techniques for the H5N1 Avian Influenza Virus.

Avian influenza is caused by avian influenza virus infection; the H5N1 avian influenza virus is a highly pathogenic subtype, affecting poultry and human health. Since the discovery of the highly pathogenic subtype of the H5N1 avian influenza virus, it has caused enormous losses to the poultry farming industry. It was recently found that the H5N1 avian influenza virus tends to spread among mammals. Therefore, early rapid detection methods are highly significant for effectively preventing the spread of H5N1. This paper discusses the detection technologies used in the detection of the H5N1 avian influenza virus, including serological detection technology, immunological detection technology, molecular biology detection technology, genetic detection technology, and biosensors. Comparisons of these detection technologies were analyzed, aiming to provide some recommendations for the detection of the H5N1 avian influenza virus.

Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus in Poultry, Benin, 2021.

In August 2021, we detected highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b viruses in poultry in southern Benin. The isolates were genetically similar to H5N1 viruses of clade 2.3.4.4b isolated during the same period in Africa and Europe. We also found evidence for 2 separate introductions of these viruses into Benin.

Mass Mortality Caused by Highly Pathogenic Influenza A(H5N1) Virus in Sandwich Terns, the Netherlands, 2022.

We collected data on mass mortality in Sandwich terns (Thalasseus sandvicensis) during the 2022 breeding season in the Netherlands. Mortality was associated with at least 2 variants of highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b. We report on carcass removal efforts relative to survival in colonies. Mitigation strategies urgently require structured research.

Highly Pathogenic Avian Influenza A(H5N1) Virus in Wild Red Foxes, the Netherlands, 2021.

We detected infection with highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b in 2 red fox (Vulpes vulpes) cubs found in the wild with neurologic signs in the Netherlands. The virus is related to avian influenza viruses found in wild birds in the same area.

Diversity of Influenza A(H5N1) Viruses in Infected Humans, Northern Vietnam, 2004-2010.

Influenza viruses exist in each host as a collection of genetically diverse variants, which might enhance their adaptive potential. To assess the genetic and functional diversity of highly pathogenic avian influenza A(H5N1) viruses within infected humans, we used deep-sequencing methods to characterize samples obtained from infected patients in northern Vietnam during 2004-2010 on different days after infection, from different anatomic sites, or both. We detected changes in virus genes that affected receptor binding, polymerase activity, or interferon antagonism, suggesting that these factors could play roles in influenza virus adaptation to humans. However, the frequency of most of these mutations remained low in the samples tested, implying that they were not efficiently selected within these hosts. Our data suggest that adaptation of influenza A(H5N1) viruses is probably stepwise and depends on accumulating combinations of mutations that alter function while maintaining fitness.

Containment of Highly Pathogenic Avian Influenza A(H5N1) Virus, Lebanon, 20161.

A preparedness plan for avian influenza A(H5N1) virus infection was activated in Lebanon in 2016 after reported cases in poultry. Exposed persons were given prophylaxis and monitored daily. A total of 185 exposed persons were identified: 180 received prophylaxis, 181 were monitored, and 41 suspected cases were reported. All collected specimens were negative for virus by PCR.

Predicting risk of avian influenza a(H5N1) in Egypt: the creation of a community level metric.

BACKGROUND: Efficient A(H5N1) control is unlikely to be based on epidemiological data alone. Such control depends on a thorough understanding and appreciation of the interconnectedness of epidemiological, social, and economic factors that contribute to A(H5N1) vulnerability. To date, the control of A(H5N1) in Egypt has been challenging. The disease has been endemic for more than 10 years with a dramatic increase in human cases between December 2014 and March 2015. Part of the problem has been a lack of understanding of the inter-play of drivers, conditions and motives that influence preventive behaviours at the household level. METHODS: To address this issue, the authors developed a Composite Risk Index (CRI) to inform decision-makers of critical epidemiological, livelihood, food security and risk perception factors that were found to contribute to A(H5N1) vulnerability at the community level. The CRI consists of seven constructs that were individually scored for each community. The seven constructs included poultry sales, previous flock exposure to A(H5N1), human risk probability, sense of control over the disease, preventative actions taken, level of household food insecurity and community norms toward certain handling and disposal practices. One hundred forty female poultry keepers across four governorates were interviewed in 2010 using a mix of random and purposive sampling techniques. A mixed method approach underpinned the analysis. The study used wealth ranking in order to help decision-makers in understanding the specific constraints of different wealth groups and aid better targeting of A(H5N1) control and prevention strategies. RESULTS: Poverty, widowhood and lack of education were among the factors associated with high risk scores. CRI scores in those villages where awareness raising had taken place were not significantly different compared to those villages where awareness raising had not taken place. CONCLUSIONS: The aim of the tool is to enable targeting those communities that are likely to be highly vulnerable to A(H5N1) outbreaks and where control and awareness-raising efforts are expected to be most effective. In this manner, policy makers and practitioners will be able to better allocate limited resources to those communities most vulnerable to the negative impact of A(H5N1).

[Comparison of epidemiological characteristics of human infection with avian influenza A (H5N1) virus in five countries of Asia and Africa].

Objective: To understand characteristics of demographic, seasonal and spatial distribution of H5N1 cases in major countries of Asia (Indonesia, Cambodia, Vietnam, China) and Africa (Egypt). Methods: Through searching public data resource and published papers, we collected cases information in five countries from May 1st, 1997 to November 6th, 2017, including general characteristics, diagnosis, onset and exposure history, etc. Different characteristics of survived and death cases in different countries were described and χ(2) test was used to compare the differences among death cases and odds ratio (OR) and 95%CI value was used to compare death risk in different countries. Results: A total of 856 cases were reported in five countries with Egypt had the most cases (44.3%). The highest number of cases were reported in 2015 (18.3%). 53% cases were reported from January to March, and 96.1% of cases had the history of poultry exposure. 64.2% (43 cases) cases in China had live poultry market exposure, but the sick/dead poultry exposure was the major exposure for cases in other four countries. 452 death cases were reported in five countries, and the fatality rate was 52.8%. With Egypt as the reference group, the highest death risk was seen in Indonesia (OR (95%CI): 11.52 (7.46-17.77)), followed by Cambodia (OR (95%CI): 4.27(2.37-7.69)) and China (OR (95%CI): 2.87 (1.73-4.74)). The age distribution of death cases among 5 countries was statistically significant, and the highest fatality rate was in 15-54 years group in Egypt (83.6%, 102 cases), while in Cambodia the highest fatality rate was in 0-14 years group (76.9%, 30 cases). The highest number of deaths were reported in 2006, and 48.3% were reported from January to March. There was difference in exposure routes among 5 countries (χ(2)=43.85, P=0.001), 63.2% (24 cases) of the death cases in China had live poultry market exposure. 92.9% (79 cases), 83.3% (40 cases) and 100.0% (38 cases) death cases in Indonesia, Vietnam and Camodia had sick/dead poultry exposure, respectively;and 81.6% (31 cases) of the death cases in Egypt had backyard poultry exposure. Conclusion: The geographical distribution, seasonal age, gender, exposure matter and outcome of H5N1 cases in five countries were different.

Mild Respiratory Illness Among Young Children Caused by Highly Pathogenic Avian Influenza A (H5N1) Virus Infection in Dhaka, Bangladesh, 2011.

Background: In March 2011, a multidisciplinary team investigated 2 human cases of highly pathogenic avian influenza A(H5N1) virus infection, detected through population-based active surveillance for influenza in Bangladesh, to assess transmission and contain further spread. Methods: We collected clinical and exposure history of the case patients and monitored persons coming within 1 m of a case patient during their infectious period. Nasopharyngeal wash specimens from case patients and contacts were tested with real-time reverse-transcription polymerase chain reaction, and virus culture and isolates were characterized. Serum samples were tested with microneutralization and hemagglutination inhibition assays. We tested poultry, wild bird, and environmental samples from case patient households and surrounding areas for influenza viruses. Results: Two previously healthy case patients, aged 13 and 31 months, had influenzalike illness and fully recovered. They had contact with poultry 7 and 10 days before illness onset, respectively. None of their 57 contacts were subsequently ill. Clade 2.2.2.1 highly pathogenic avian influenza H5N1 viruses were isolated from the case patients and from chicken fecal samples collected at the live bird markets near the patients' dwellings. Conclusion: Identification of H5N1 cases through population-based surveillance suggests possible additional undetected cases throughout Bangladesh and highlights the importance of surveillance for mild respiratory illness among populations frequently exposed to infected poultry.

Survival of Highly Pathogenic Avian Influenza H5N1 Virus in Tissues Derived from Experimentally Infected Chickens.

Eurasian lineage highly pathogenic avian influenza (HPAI) H5N1 virus has been a severe threat to the poultry industry since its emergence in 1996. The carcass or tissues derived from infected birds may present the risk of the virus spreading to humans, animals, and the surrounding environment. In this study, we investigated the survival of the virus in feather, muscle, and liver tissues collected from six chickens (Gallus gallus) experimentally infected with HPAI H5N1 virus. The tissues were stored at +4°C or +20°C, and viral isolation was performed at different times for 360 days. The maximum periods for viral survival were observed in samples stored at +4°C in all tissue types and were 240 days in feather tissues, 160 days in muscle, and 20 days in liver. The viral infectivity at +20°C was maintained for a maximum of 30 days in the feather tissues, 20 days in muscle, and 3 days in liver. The viral inactivation rates partly overlapped in the feather and muscle tissues at the two temperatures. The virus was inactivated rapidly in the liver. Our experimental results indicate that the tissue type and temperature can greatly influence the survival of HPAI H5N1 virus in the tissues of infected chickens.IMPORTANCE Highly pathogenic avian influenza virus of the H5N1 subtype can cause massive losses of poultry, and people need to handle a large number of chicken carcasses contaminated with the virus at outbreak sites. This study evaluated how long the virus can keep its infectivity in the three types of tissues derived from chickens infected with the virus. Our experimental results indicate that the virus can survive in tissues for a specific period of time depending on the tissue type and temperature. Our results are valuable for better understanding of viral ecology in the environment and for reducing the risk of the virus spreading via bird tissues contaminated with the virus.

Annexin A2 (ANXA2) interacts with nonstructural protein 1 and promotes the replication of highly pathogenic H5N1 avian influenza virus.

BACKGROUND: Non-structural protein 1 (NS1) is a multifunctional protein and a crucial regulatory factor in the replication and pathogenesis of avian influenza virus (AIV). Studies have shown that NS1 can interact with a variety of host proteins to modulate the viral life cycle. We previously generated a monoclonal antibody against NS1 protein; In the current research study, using this antibody, we immunoprecipitated host proteins that interact with NS1 to better understand the roles played by NS1 in communications between virus and host. RESULTS: Co-immunoprecipitation experiments identified annexin A2 (ANXA2) as a target molecule interacting with NS1. Results from confocal laser scanning microscopy indicated that NS1 co-localized with ANXA2 in the cell cytoplasm. Overexpression of ANXA2 significantly increased the titer of H5N1 subtype HPAIV, whereas siRNA-mediated knockdown of ANXA2 markedly inhibited the expression of viral proteins and reduced the progeny virus titer. CONCLUSIONS: Our results indicate that ANXA2 interacts with NS1 and ANXA2 expression increases HPAIV replication.

Genetically Different Highly Pathogenic Avian Influenza A(H5N1) Viruses in West Africa, 2015.

To trace the evolution of highly pathogenic influenza A(H5N1) virus in West Africa, we sequenced genomes of 43 viruses collected during 2015 from poultry and wild birds in 5 countries. We found 2 co-circulating genetic groups within clade 2.3.2.1c. Mutations that may increase adaptation to mammals raise concern over possible risk for humans.

Corneal Opacity in Domestic Ducks Experimentally Infected With H5N1 Highly Pathogenic Avian Influenza Virus.

Domestic ducks can be a key factor in the regional spread of H5N1 highly pathogenic avian influenza (HPAI) virus in Asia. The authors performed experimental infections to examine the relationship between corneal opacity and H5N1 HPAI virus infection in domestic ducks (Anas platyrhyncha var domestica). A total of 99 domestic ducks, including 3 control birds, were used in the study. In experiment 1, when domestic ducks were inoculated intranasally with 2 H5N1 HPAI viruses, corneal opacity appeared more frequently than neurologic signs and mortality. Corneal ulceration and exophthalmos were rare findings. Histopathologic examinations of the eyes of domestic ducks in experiment 2 revealed that corneal opacity was due to the loss of corneal endothelial cells and subsequent keratitis with edema. Influenza viral antigen was detected in corneal endothelial cells and some other ocular cells by immunohistochemistry. Results suggest that corneal opacity is a characteristic and frequent finding in domestic ducks infected with the H5N1 HPAI virus. Confirming this ocular change may improve the detection rate of infected domestic ducks in the field.

High Antibody-Dependent Cellular Cytotoxicity Antibody Titers to H5N1 and H7N9 Avian Influenza A Viruses in Healthy US Adults and Older Children.

Human influenza is a highly contagious acute respiratory illness that is responsible for significant morbidity and excess mortality worldwide. In addition to neutralizing antibodies, there are antibodies that bind to influenza virus-infected cells and mediate lysis of the infected cells by natural killer (NK) cells (antibody-dependent cellular cytotoxicity [ADCC]) or complement (complement-dependent lysis [CDL]). We analyzed sera obtained from 16 healthy adults (18-63 years of age), 52 children (2-17 years of age), and 10 infants (0.75-1 year of age) in the United States, who were unlikely to have been exposed to the avian H7N9 subtype of influenza A virus, by ADCC and CDL assays. As expected, none of these sera had detectable levels of hemagglutination-inhibiting antibodies against the H7N9 virus, but we unexpectedly found high titers of ADCC antibodies to the H7N9 subtype virus in all sera from adults and children aged ≥8 years.

Full-genome analysis of avian influenza A(H5N1) virus from a human, North America, 2013.

Full-genome analysis was conducted on the first isolate of a highly pathogenic avian influenza A(H5N1) virus from a human in North America. The virus has a hemagglutinin gene of clade 2.3.2.1c and is a reassortant with an H9N2 subtype lineage polymerase basic 2 gene. No mutations conferring resistance to adamantanes or neuraminidase inhibitors were found.

Susceptibility of Synanthropic Rodents (Mus musculus, Rattus norvegicus and Rattus rattus) to H5N1 Subtype High Pathogenicity Avian Influenza Viruses.

Synanthropic wild rodents associated with agricultural operations may represent a risk path for transmission of high pathogenicity avian influenza viruses (HPAIVs) from wild birds to poultry birds. However, their susceptibility to HPAIVs remains unclear. In the present study, house mice (Mus musculus), brown rats (Rattus norvegicus), and black rats (Rattus rattus) were experimentally exposed to H5N1 subtype HPAIVs to evaluate their vulnerability to infection. After intranasal inoculation with HA clade 2.2 and 2.3.2.1 H5N1 subtype HPAIVs, wild rodents did not show any clinical signs and survived for 10- and 12-day observation periods. Viruses were isolated from oral swabs for several days after inoculation, while little or no virus was detected in their feces or rectal swabs. In euthanized animals at 3 days post-inoculation, HPAIVs were primarily detected in respiratory tract tissues such as the nasal turbinates, trachea, and lungs. Serum HI antibodies were detected in HA clade 2.2 HPAIV-inoculated rodents. These results strongly suggest that synanthropic wild rodents are susceptible to infection of avian-origin H5N1 subtype HPAIVs and contribute to the virus ecosystem as replication-competent hosts. Detection of infectious viruses in oral swabs indicates that wild rodents exposed to HPAIVs could contaminate food, water, and the environment in poultry houses and play roles in the introduction and spread of HPAIVs in farms.