Preventive, safety and control measures against Avian Influenza A(H5N1) in occupationally exposed groups: A scoping review.

Introduction: During the outbreak of avian influenza, A (H5N1) (IA) in wild and domestic birds recorded in January 2023, the epidemiological alert has been extended due to its potential contagion to humans, particularly in those exposed occupational groups. Objective: to identify the primary occupational risk groups, as well as the preventive, safety, and control measures against IA intended or implemented in these positions. Material and methods: A systematic search was conducted in Pubmed, Scopus, Web of science, Scielo and literature databases. Scientific articles, normative documents, and technical reports identifying vulnerable occupational groups and preventive measures against IA were included. Two authors conducted a full-text review, extracting information independently, and findings were summarized narratively. Results: A total of 5518 documents were identified, and 30 reports were included. 20% of the reports were published in 2023, 13/30 were affiliated to a university institution. Occupationally exposed groups were identified both directly and indirectly. 63.3% of reports identified breeders, poultry farmers and sellers as the most concerning occupational group, while 60% identified biosecurity practices (use of PPE, handwashing) as the primary measure against IA, followed by strategies such as education (training and capacity-building). Conclusion: Occupational groups of interest were identified, primarily those involved in sales, commerce, and the handling of bird waste with potential exposure to IA. Furthermore, the maintenance of biosecurity measures, cleaning-disinfection practices, and educational strategies in workplace settings are recommended.

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.

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.

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.

Molecular characterization and phylogenetic analysis of highly pathogenic H5N1 clade 2.3.4.4b virus in Bosnia and Herzegovina.

Influenza A virus continues to represent a growing problem affecting mainly birds but with an increasing number of mammal transmission events reported each year. Nevertheless, molecular characterization and phylogenetic analysis of influenza A viruses originating from all confirmed cases have not been systematically performed in all parts of the world. In this study, we investigated a sample originating from a mute swan that died in November 2021 in the northern part of Bosnia and Herzegovina with RT-qPCR and whole genome sequencing using ONT MinION. It was diagnosed as a highly pathogenic Influenza A virus, subtype H5N1 of 2.3.4.4b clade, and phylogenetic analysis revealed high sequence homology with other European HPAI H5N1 sequences at the time. The notable detected mutations in HA (N110S and T139P) and NA genes (H155Y), that facilitate the host specificity shift and enable the resistance to some antiviral drugs respectively, underscore the necessity of virus evolution surveillance. Therefore, the rapid dissemination of information, including virological and molecular data, is essential for the introduction of tailored prevention measures for infected animals, providing clearer insight and better awareness of a potential public health threat.

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.

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.

Two major epidemics of highly pathogenic avian influenza virus H5N8 and H5N1 in domestic poultry in France, 2020-2022.

The spread of highly pathogenic avian influenza (HPAI) viruses worldwide has serious consequences for animal health and a major economic impact on the poultry production sector. Since 2014, Europe has been severely hit by several HPAI epidemics, with France being the most affected country. Most recently, France was again affected by two devastating HPAI epidemics in 2020-21 and 2021-22. We conducted a descriptive analysis of the 2020-21 and 2021-22 epidemics, as a first step towards identifying the poultry sector's remaining vulnerabilities regarding HPAI viruses in France. We examined the spatio-temporal distribution of outbreaks that occurred in France in 2020-21 and 2021-22, and we assessed the outbreaks' spatial distribution in relation to the 2016-17 epidemic and to the two 'high-risk zones' recently incorporated into French legislation to strengthen HPAI prevention and control. There were 468 reported outbreaks during the 2020-21 epidemic and 1375 outbreaks during the 2021-22 epidemic. In both epidemics, the outbreaks' distribution matched extremely well that of 2016-17, and most outbreaks (80.6% and 68.4%) were located in the two high-risk zones. The southwestern high-risk zone was affected in both epidemics, while the western high-risk zone was affected for the first time in 2021-22, explaining the extremely high number of outbreaks reported. As soon as the virus reached the high-risk zones, it started to spread between farms at very high rates, with each infected farm infecting between two and three other farms at the peaks of transmission. We showed that the spatial distribution model used to create the two high-risk zones was able to predict the location of outbreaks for the 2020-21 and 2021-22 epidemics. These zones were characterized by high poultry farm densities; future efforts should, therefore, focus on reducing the density of susceptible poultry in highly dense areas.

Improving siRNA design targeting nucleoprotein gene as antiviral against the Indonesian H5N1 virus.

BACKGROUND: Small interfering RNA technology has been considered a prospective alternative antiviral treatment using gene silencing against influenza viruses with high mutations rates. On the other hand, there are no reports on its effectiveness against the highly pathogenic avian influenza H5N1 virus isolated from Indonesia. OBJECTIVES: The main objective of this study was to improve the siRNA design based on the nucleoprotein gene (siRNA-NP) for the Indonesian H5N1 virus. METHODS: The effectiveness of these siRNA-NPs (NP672, NP1433, and NP1469) was analyzed in vitro in Marbin-Darby canine kidney cells. RESULTS: The siRNA-NP672 caused the largest decrease in viral production and gene expression at 24, 48, and 72 h post-infection compared to the other siRNA-NPs. Moreover, three serial passages of the H5N1 virus in the presence of siRNA-NP672 did not induce any mutations within the nucleoprotein gene. CONCLUSIONS: These findings suggest that siRNA-NP672 can provide better protection against the Indonesian strain of the H5N1 virus.

Single Dose of Bivalent H5 and H7 Influenza Virus-Like Particle Protects Chickens Against Highly Pathogenic H5N1 and H7N9 Avian Influenza Viruses.

Both H5N1 and H7N9 subtype avian influenza viruses cause enormous economic losses and pose considerable threats to public health. Bivalent vaccines against both two subtypes are more effective in control of H5N1 and H7N9 viruses in poultry and novel egg-independent vaccines are needed. Herein, H5 and H7 virus like particle (VLP) were generated in a baculovirus expression system and a bivalent H5+H7 VLP vaccine candidate was prepared by combining these two antigens. Single immunization of the bivalent VLP or commercial inactivated vaccines elicited effective antibody immune responses, including hemagglutination inhibition, virus neutralizing and HA-specific IgG antibodies. All vaccinated birds survived lethal challenge with highly pathogenic H5N1 and H7N9 viruses. Furthermore, the bivalent VLP significantly reduced viral shedding and virus replication in chickens, which was comparable to that observed for the commercial inactivated vaccine. However, the bivalent VLP was better than the commercial vaccine in terms of alleviating pulmonary lesions caused by H7N9 virus infection in chickens. Therefore, our study suggests that the bivalent H5+H7 VLP vaccine candidate can serve as a critical alternative for the traditional egg-based inactivated vaccines against H5N1 and H7N9 avian influenza virus infection in poultry.

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.

Spatio-temporal distribution of outbreaks of highly pathogenic avian influenza virus subtype H5N1 in Vietnam, 2015- 2018.

Despite strong commitments of the central and provincial veterinary authorities in Vietnam to control highly pathogenic avian influenza H5N1 (HPAI-H5N1) in poultry and to diminish the risk of human infection, outbreaks continue to occur in poultry. This study describes the spatio-temporal distribution of HPAI-H5N1 outbreaks in Vietnam for the period December 2014-April 2018 using the space-time K-function and the space-time scan statistic. The space-time K-function analyses showed statistically significant spatio-temporal clustering of HPAI-H5N1 outbreaks in poultry during the study period of up to 50 days and 60 kilometres. The space-time scan statistic test identified three statistically significant space-time clusters of HPAI-H5N1 in the south of the country where the incidence of HPAI-H5N1 outbreaks was greater than that expected if outbreaks were randomly distributed in space and time. The analyses indicated shortfalls in the effectiveness of control measures used to control HPAI-H5N1 throughout the study period. Development of a better understanding of the relative impact of HPAI-H5N1 control measures (depopulation of infected flocks, vaccination, movement restrictions) on space-time interaction would allow animal health authorities to focus their efforts on control measures shown to have the greatest relative effect.

H5N1 avian influenza virus without 80-84 amino acid deletion at the NS1 protein hijacks the innate immune system of dendritic cells for an enhanced mammalian pathogenicity.

NS gene is generally considered to be related to the virulence of highly pathogenic avian influenza virus (AIV). In recent years, the strains with five amino acids added to the 80-84 positions of the NS1 protein have become prevalent in H5N1 subtype AIVs isolated from mammals. However, the pathogenicity and mechanism of this pattern in mammals remain unclear. In this study, H5N1 subtype AIVs without 80-84 amino acids of the NS1 protein (rNSΔ5aa ) and a mutant virus (rNS5aa-R ) with no deletion of 80-84 amino acids of the NS1 protein were used to determine the pathogenicity in mice. Our results showed that rNS5aa-R possessed an enhanced pathogenicity compared with rNSΔ5aa in vivo and in vitro, which was accompanied by high expression of IL-6, MX1 and CXCL10 in murine lungs. Furthermore, we found that rNS5aa-R increased the infection ability to dendritic cells (DCs). Besides, rNS5aa-R enhanced the expression of phenotypic markers (CD80, CD86, CD40 and MHCII), activation marker CD69, inflammatory cytokines (IL-6, TNF-α and IL-10) and antagonized interferon (IFN-α) of DCs, in comparison to rNSΔ5aa . Moreover, rNS5aa-R induced DCs to quickly migrate into nearby cervical lymph nodes by highly upregulating CCR7, and CD86 showed a high expression on the migrated DCs. We also found that rNS5aa-R -infected DCs significantly promoted the allogeneic CD4+ T-cell proliferation. These findings suggested that rNS5aa-R strongly induced the innate immune response compared with the rNSΔ5aa , which is conducive to activate a wide immune response, resulting in a strong cytokine storm and causing an enhanced pathogenicity of H5N1 subtype AIVs in mammals.

Truncation or Deglycosylation of the Neuraminidase Stalk Enhances the Pathogenicity of the H5N1 Subtype Avian Influenza Virus in Mallard Ducks.

H5N1 subtype avian influenza virus (AIV) with a deletion of 20 amino acids at residues 49-68 in the stalk region of neuraminidase (NA) became a major epidemic virus. To determine the effect of truncation or deglycosylation of the NA stalk on virulence, we used site-directed mutagenesis to insert 20 amino acids in the short-stalk virus A/mallard/Huadong/S/2005 (SY) to recover the long-stalk virus (rSNA+). A series of short-stalk or deglycosylated-stalk viruses were also constructed basing on the long-stalk virus, and then the characteristics and pathogenicity of the resulting viruses were evaluated. The results showed that most of the short-stalk or deglycosylated-stalk viruses had smaller plaques, and increased thermal and low-pH stability, and a decreased neuraminidase activity when compared with the virus rSNA+. In a mallard ducks challenge study, most of the short-stalk or deglycosylated-stalk viruses showed increased pathological lesions and virus titers in the organ tissues and increased virus shedding in the oropharynx and cloaca when compared with the rSNA+ virus, while most of the short-stalk viruses, especially rSNA-20, showed higher pathogenicity than the deglycosylated-stalk virus. In addition, the short-stalk viruses showed a significantly upregulated expression of the immune-related factors in the lungs of the infected mallard ducks, including IFN-α, Mx1, and IL-8. The results suggested that NA stalk truncation or deglycosylation increases the pathogenicity of H5N1 subtype AIV in mallard ducks, which will provide a pre-warning for prevention and control of H5N1 subtype avian influenza in the waterfowl.

Study on the Antiviral Activities and Hemagglutinin-Based Molecular Mechanism of Novel Chlorogenin 3-O-ß-Chacotrioside Derivatives Against H5N1 Subtype Viruses.

The objective of this study was to investigate the inhibitory effect of chlorogenin 3-O-ß-chacotrioside derivatives against H5N1 subtype of the highly pathogenic avian influenza (HPAI) viruses and its molecular mechanism. A series of novel small molecule pentacyclic triterpene derivatives were designed and synthesized and their antiviral activities on HPAI H5N1 viruses were detected. The results displayed that the derivatives UA-Nu-ph-5, XC-27-1 and XC-27-2 strongly inhibited wild-type A/Duck/Guangdong/212/2004 H5N1 viruses with the IC50 values of 15.59 ± 2.4 µM, 16.83 ± 1.45 µM, and 12.45 ± 2.27 µM, respectively, and had the selectivity index (SI) > 3, which was consistent with the efficacy against A/Thailand/kan353/2004 pseudo-typed viruses. Four dealt patterns were compared via PRNT. The prevention dealt pattern showed the strongest inhibitory effects than other patterns, suggesting that these derivatives act on the entry process at the early stages of H5N1 viral infection, providing protection for cells against infection. Further studies through hemagglutinin inhibition (HI) and neuraminidase inhibitory (NAI) assay confirmed that these derivatives inhibited H5N1 virus replication by interfering with the viral hemagglutinin function. The derivatives could recognize specifically HA protein with binding affinity constant KD values of 2.57 × 10-4 M and 3.67 × 10-4 M. In addition, through site-directed mutagenesis combined with a pseudovirion system, we identified that the high-affinity docking sites underlying interaction were closely associated with amino acid residues I391 and T395 of HA. However, the potential binding sites of the derivatives with HA did not locate at HA1 sialic acids receptor binding domain (RBD). Taken together, these study data manifested that chlorogenin 3-O-ß-chacotrioside derivatives generated antiviral effect against HPAI H5N1 viruses by targeting the hemagglutinin fusion machinery.

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.

[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.

Phylogeography of H5N1 avian influenza virus in Indonesia.

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype are a major concern to human and animal health in Indonesia. This study aimed to characterize transmission dynamics of H5N1 over time using novel Bayesian phylogeography methods to identify factors which have influenced the spread of H5N1 in Indonesia. We used publicly available hemagglutinin sequence data sampled between 2003 and 2016 to model ancestral state reconstruction of HPAI H5N1 evolution. We found strong support for H5N1 transmission routes between provinces in Java Island and inter-island transmissions, such as between Nusa Tenggara and Kalimantan Islands, not previously described. The spread is consistent with wild bird flyways and poultry trading routes. H5N1 migration was associated with the regions of high chicken densities and low human development indices. These results can be used to inform more targeted planning of H5N1 control and prevention activities in Indonesia.