Red knots in Europe: a dead end host species or a new niche for highly pathogenic avian influenza?

The 2020/2021 epidemic in Europe of highly pathogenic avian influenza virus (HPAIV) of subtype H5 surpassed all previously recorded European outbreaks in size, genotype constellations and reassortment frequency and continued into 2022 and 2023. The causative 2.3.4.4b viral lineage proved to be highly proficient with respect to reassortment with cocirculating low pathogenic avian influenza viruses and seems to establish an endemic status in northern Europe. A specific HPAIV reassortant of the subtype H5N3 was detected almost exclusively in red knots (Calidris canutus islandica) in December 2020. It caused systemic and rapidly fatal disease leading to a singular and self-limiting mass mortality affecting about 3500 birds in the German Wadden Sea, roughly 1 % of the entire flyway population of islandica red knots. Phylogenetic analyses revealed that the H5N3 reassortant very likely had formed in red knots and remained confined to this species. While mechanisms of virus circulation in potential reservoir species, dynamics of spill-over and reassortment events and the roles of environmental virus sources remain to be identified, the year-round infection pressure poses severe threats to endangered avian species and prompts adaptation of habitat and species conservation practices.

Genomic diversity and evolutionary dynamics of Influenza A viruses in Colombian swine: implications for one health surveillance and control.

Influenza A viruses (IAV) impose significant respiratory disease burdens in both swine and humans worldwide, with frequent human-to-swine transmission driving viral evolution in pigs and highlighting the risk at the animal-human interface. Therefore, a comprehensive One Health approach (interconnection among human, animal, and environmental health) is needed for IAV prevention, control, and response. Animal influenza genomic surveillance remains limited in many Latin American countries, including Colombia. To address this gap, we genetically characterized 170 swine specimens from Colombia (2011-2017). Whole genome sequencing revealed a predominance of pandemic-like H1N1 lineage, with a minority belonging to H3N2 and H1N2 human seasonal-like lineage and H1N1 early classical swine lineages. Significantly, we have identified reassortant and recombinant viruses (H3N2, H1N1) not previously reported in Colombia. This suggests a broad genotypic viral diversity, likely resulting from reassortment between classical endemic viruses and new introductions established in Colombia's swine population (e.g. the 2009 H1N1 pandemic). Our study highlights the importance of a One Health approach in disease control, particularly in an ecosystem where humans are a main source of IAV to swine populations, and emphasizes the need for continued surveillance and enhanced biosecurity measures. The co-circulation of multiple subtypes in regions with high swine density facilitates viral exchange, underscoring the importance of monitoring viral evolution to inform vaccine selection and public health policies locally and globally.

Phylogenetic and mutational analysis of H10N3 avian influenza A virus in China: potential threats to human health.

In recent years, the avian influenza virus has emerged as a significant threat to both human and public health. This study focuses on a patient infected with the H10N3 subtype of avian influenza virus, admitted to the Third People's Hospital of Kunming City on March 6, 2024. Metagenomic RNA sequencing and polymerase chain reaction (PCR) analysis were conducted on the patient's sputum, confirming the H10N3 infection. The patient presented severe pneumonia symptoms such as fever, expectoration, chest tightness, shortness of breath, and cough. Phylogenetic analysis of the Haemagglutinin (HA) and neuraminidase (NA) genes of the virus showed that the virus was most closely related to a case of human infection with the H10N3 subtype of avian influenza virus found in Zhejiang Province, China. Analysis of amino acid mutation sites identified four mutations potentially hazardous to human health. Consequently, this underscores the importance of continuous and vigilant monitoring of the dynamics surrounding the H10N3 subtype of avian influenza virus, utilizing advanced genomic surveillance techniques.

CRISPR-Based Assays for Point-of-Need Detection and Subtyping of Influenza.

The high disease burden of influenza virus poses a significant threat to human health. Optimized diagnostic technologies that combine speed, sensitivity, and specificity with minimal equipment requirements are urgently needed to detect the many circulating species, subtypes, and variants of influenza at the point of need. Here, we introduce such a method using Streamlined Highlighting of Infections to Navigate Epidemics (SHINE), a clustered regularly interspaced short palindromic repeats (CRISPR)-based RNA detection platform. Four SHINE assays were designed and validated for the detection and differentiation of clinically relevant influenza species (A and B) and subtypes (H1N1 and H3N2). When tested on clinical samples, these optimized assays achieved 100% concordance with quantitative RT-PCR. Duplex Cas12a/Cas13a SHINE assays were also developed to detect two targets simultaneously. This study demonstrates the utility of this duplex assay in discriminating two alleles of an oseltamivir resistance (H275Y) mutation as well as in simultaneously detecting influenza A and human RNAse P in patient samples. These assays have the potential to expand influenza detection outside of clinical laboratories for enhanced influenza diagnosis and surveillance.

Epidemic patterns of the different influenza virus types and subtypes/lineages for 10 years in Chongqing, China, 2010-2019.

To optimize seasonal influenza control and prevention programs in regions with potentially complicated seasonal patterns. Descriptive epidemiology was used to analyze the etiology of influenza, and chi-square tests were used to compare the epidemic patterns among different influenza virus types and subtypes/lineages. From January 2010 to December 2019, a total of 63,626 ILI cases were reported in Chongqing and 14,136 (22.22%) were laboratory-confirmed influenza cases. The proportions of specimens positive for influenza A and influenza B were 13.32% (8,478/63,626) and 8.86% (5,639/63,626), respectively. The proportion of positive specimens for influenza A reached the highest in winter (23.33%), while the proportion of positive specimens for influenza B reached the highest in spring (11.88%). Children aged 5-14 years old had the highest proportion of positive specimens for influenza. The influenza virus types/subtypes positive was significantly different by seasons and age groups (P<.001), but not by gender (p = .436). The vaccine strains were matched to the circulating influenza virus strains in all other years except for 2018 (vaccine strain was B/Colorado/06/2017; circulating strain was B/Yamagata). The study showed significant variations in epidemic patterns, including seasonal epidemic period and age distributions, among different influenza types, subtypes/lineages in Chongqing. Influenza vaccines matched to the circulating influenza virus strain in nine of the ten years. To prevent and mitigate the influenza outbreaks in this area, high risk population, especially children aged 5-14 years, are encouraged to get vaccinated against influenza before the epidemic seasons.

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

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

Epidemiological and Genetic Characteristics of Respiratory Viral Coinfections with Different Variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

This study aimed to determine the incidence and etiological, seasonal, and genetic characteristics of respiratory viral coinfections involving severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Between October 2020 and January 2024, nasopharyngeal samples were collected from 2277 SARS-CoV-2-positive patients. Two multiplex approaches were used to detect and sequence SARS-CoV-2, influenza A/B viruses, and other seasonal respiratory viruses: multiplex real-time polymerase chain reaction (PCR) and multiplex next-generation sequencing. Coinfections of SARS-CoV-2 with other respiratory viruses were detected in 164 (7.2%) patients. The most common co-infecting virus was respiratory syncytial virus (RSV) (38 cases, 1.7%), followed by bocavirus (BoV) (1.2%) and rhinovirus (RV) (1.1%). Patients ≤ 16 years of age had the highest rate (15%) of mixed infections. Whole-genome sequencing produced 19 complete genomes of seasonal respiratory viral co-pathogens, which were subjected to phylogenetic and amino acid analyses. The detected influenza viruses were classified into the genetic groups 6B.1A.5a.2a and 6B.1A.5a.2a.1 for A(H1N1)pdm09, 3C.2a1b.2a.2a.1 and 3C.2a.2b for A(H3N2), and V1A.3a.2 for the B/Victoria lineage. The RSV-B sequences belonged to the genetic group GB5.0.5a, with HAdV-C belonging to type 1, BoV to genotype VP1, and PIV3 to lineage 1a(i). Multiple amino acid substitutions were identified, including at the antibody-binding sites. This study provides insights into respiratory viral coinfections involving SARS-CoV-2 and reinforces the importance of genetic characterization of co-pathogens in the development of therapeutic and preventive strategies.

Avian influenza viruses in New Zealand wild birds, with an emphasis on subtypes H5 and H7: Their distinctive epidemiology and genomic properties.

The rapid spread of highly pathogenic avian influenza (HPAI) A (H5N1) viruses in Southeast Asia in 2004 prompted the New Zealand Ministry for Primary Industries to expand its avian influenza surveillance in wild birds. A total of 18,693 birds were sampled between 2004 and 2020, including migratory shorebirds (in 2004-2009), other coastal species (in 2009-2010), and resident waterfowl (in 2004-2020). No avian influenza viruses (AIVs) were isolated from cloacal or oropharyngeal samples from migratory shorebirds or resident coastal species. Two samples from red knots (Calidris canutus) tested positive by influenza A RT-qPCR, but virus could not be isolated and no further characterization could be undertaken. In contrast, 6179 samples from 15,740 mallards (Anas platyrhynchos) tested positive by influenza A RT-qPCR. Of these, 344 were positive for H5 and 51 for H7. All H5 and H7 viruses detected were of low pathogenicity confirmed by a lack of multiple basic amino acids at the hemagglutinin (HA) cleavage site. Twenty H5 viruses (six different neuraminidase [NA] subtypes) and 10 H7 viruses (two different NA subtypes) were propagated and characterized genetically. From H5- or H7-negative samples that tested positive by influenza A RT-qPCR, 326 AIVs were isolated, representing 41 HA/NA combinations. The most frequently isolated subtypes were H4N6, H3N8, H3N2, and H10N3. Multivariable logistic regression analysis of the relations between the location and year of sampling, and presence of AIV in individual waterfowl showed that the AIV risk at a given location varied from year to year. The H5 and H7 isolates both formed monophyletic HA groups. The H5 viruses were most closely related to North American lineages, whereas the H7 viruses formed a sister cluster relationship with wild bird viruses of the Eurasian and Australian lineages. Bayesian analysis indicates that the H5 and H7 viruses have circulated in resident mallards in New Zealand for some time. Correspondingly, we found limited evidence of influenza viruses in the major migratory bird populations visiting New Zealand. Findings suggest a low probability of introduction of HPAI viruses via long-distance bird migration and a unique epidemiology of AIV in New Zealand.

IAVCP (Influenza A Virus Consensus and Phylogeny): Automatic Identification of the Genomic Sequence of the Influenza A Virus from High-Throughput Sequencing Data.

Recently, high-throughput sequencing of influenza A viruses has become a routine test. It should be noted that the extremely high diversity of the influenza A virus complicates the task of determining the sequences of all eight genome segments. For a fast and accurate analysis, it is necessary to select the most suitable reference for each segment. At the same time, there is no standardized method in the field of decoding sequencing results that allows the user to update the sequence databases to which the reads obtained by virus sequencing are compared. The IAVCP (influenza A virus consensus and phylogeny) was developed with the goal of automatically analyzing high-throughput sequencing data of influenza A viruses. Its goals include the extraction of a consensus genome directly from paired raw reads. In addition, the pipeline enables the identification of potential reassortment events in the evolutionary history of the virus of interest by analyzing the topological structure of phylogenetic trees that are automatically reconstructed.

Simultaneous and ultrafast detection of pan-SARS-coronaviruses and influenza A/B viruses by a novel multiplex real-time RT-PCR assay.

Here we report an ultrafast quadruplex RT-qPCR assay with robust diagnostic ability to detect and distinguish pan-SARS-CoVs and influenza A/B viruses within 35 min. This quadruplex RT-qPCR assay comprised of one novel RNA-based internal control targeting human ß2-microglobulin (B2M) for process accuracy and three newly-designed primers-probe sets targeting the envelope protein (E) of pan-SARS-CoV, matrix protein (MP) of influenza A virus and non-structural (NS) region of influenza B virus. This quadruplex assay exhibited a sensitivity comparable to its singleplex counterparts and a slightly higher to that of the Centers for Disease Control and Prevention-recommended SARS-CoV-2 and influenza A/B assays. The novel assay showed no false-positive amplifications with other common respiratory viruses, and its 95 % limits of detection for pan-SARS-CoV and influenza A/B virus was 4.26-4.52 copies/reaction. Moreover, the assay was reproducible with less than 1 % coefficient of variation and adaptable testing different clinical and environmental samples. Our ultrafast quadruplex RT-qPCR assay can serve as an attractive tool for effective differentiation of influenza A/B virus and SARS-CoV-2, but more importantly prognose the reemergence/emergence of SARS and novel coronaviruses or influenza viruses from animal spillover.

H6N2 reassortant avian influenza virus isolate in wild birds in Jiangxi Province, China.

H6 avian influenza virus is widely prevalent in wild birds and poultry and has caused human infection in 2013 in Taiwan, China. During our active influenza surveillance program in wild waterfowl at Poyang Lake, Jiangxi Province, an H6N2 AIV was isolated and named A/bean goose/JiangXi/452-4/2013(H6N2). The isolate was characterized as a typical low pathogenic avian influenza virus (LPAIV) due to the presence of the amino acid sequence PQIETR↓GLFGAI at the cleavage site of the hemagglutinin (HA) protein. The genetic evolution analysis revealed that the NA gene of the isolate originated from North America and exhibited the highest nucleotide identity (99.29%) with a virus recovered from wild bird samples in North America, specifically A/bufflehead/California/4935/2012(H11N2). Additionally, while the HA and PB1 genes belonged to the Eurasian lineage, they displayed frequent genetic interactions with the North American lineage. The remaining genes showed close genetic relationships with Eurasian viruses. The H6N2 isolate possessed a complex genome, indicating it is a multi-gene recombinant virus with genetic material from both Eurasian and North American lineages.

Iceland: an underestimated hub for the spread of high-pathogenicity avian influenza viruses in the North Atlantic.

High-pathogenicity avian influenza viruses (HPAIVs) of the goose/Guangdong lineage are enzootically circulating in wild bird populations worldwide. This increases the risk of entry into poultry production and spill-over to mammalian species, including humans. Better understanding of the ecological and epizootiological networks of these viruses is essential to optimize mitigation measures. Based on full genome sequences of 26 HPAIV samples from Iceland, which were collected between spring and autumn 2022, as well as 1 sample from the 2023 summer period, we show that 3 different genotypes of HPAIV H5N1 clade 2.3.4.4b were circulating within the wild bird population in Iceland in 2022. Furthermore, in 2023 we observed a novel introduction of HPAIV H5N5 of the same clade to Iceland. The data support the role of Iceland as an utmost northwestern distribution area in Europe that might act also as a potential bridging point for intercontinental spread of HPAIV across the North Atlantic.

Pathological and phylogenetic characteristics of fowl AOAV-1 and H5 isolated from naturally infected Meleagris Gallopavo.

BACKGROUND: In this study, we investigated the prevalence of respiratory viruses in four Hybrid Converter Turkey (Meleagris gallopavo) farms in Egypt. The infected birds displayed severe respiratory signs, accompanied by high mortality rates, suggesting viral infections. Five representative samples from each farm were pooled and tested for H5 & H9 subtypes of avian influenza viruses (AIVs), Avian Orthoavulavirus-1 (AOAV-1), and turkey rhinotracheitis (TRT) using real-time RT-PCR and conventional RT-PCR. Representative tissue samples from positive cases were subjected to histopathology and immunohistochemistry (IHC). RESULTS: The PCR techniques confirmed the presence of AOAV-1 and H5 AIV genes, while none of the tested samples were positive for H9 or TRT. Microscopic examination of tissue samples revealed congestion and hemorrhage in the lungs, liver, and intestines with leukocytic infiltration. IHC revealed viral antigens in the lungs, liver, and intestines. Phylogenetic analysis revealed that H5 HA belonged to 2.3.4.4b H5 sublineage and AOAV-1 belonged to VII 1.1 genotype. CONCLUSIONS: The study highlights the need for proper monitoring of hybrid converter breeds for viral diseases, and the importance of vaccination programs to prevent unnecessary losses. To our knowledge, this is the first study that reports the isolation of AOAV-1 and H5Nx viruses from Hybrid Converter Turkeys in Egypt.

Influenza Virus Genomic Surveillance, Arizona, USA, 2023-2024.

Influenza viruses are constantly evolving and are therefore monitored worldwide in the hope to reduce the burden of disease by annual updates to vaccine recommendations. We conducted genomic sequencing of 110 influenza A and 30 influenza B viruses from specimens collected between October 2023 and February 2024 in Arizona, USA. We identified mutations in the hemagglutinin (HA) antigenic sites as well as the neuraminidase (NA) gene in our samples. We also found no unique HA and NA mutations in vaccinated yet influenza-infected individuals. Real-time genomic sequencing surveillance is important to ensure influenza vaccine effectiveness.

Genetic Diversity and Detection of Respiratory Viruses Excluding SARS-CoV-2 during the COVID-19 Pandemic in Gabon, 2020-2021.

Acute respiratory infections are a major global burden in resource-limited countries, including countries in Africa. Although COVID-19 has been well studied since the pandemic emerged in Gabon, Central Africa, less attention has been paid to other respiratory viral diseases, and very little data are available. Herein, we provide the first data on the genetic diversity and detection of 18 major respiratory viruses in Gabon during the COVID-19 pandemic. Of 582 nasopharyngeal swab specimens collected from March 2020 to July 2021, which were SARS-CoV-2 negative, 156 were positive (26%) for the following viruses: enterovirus (20.3%), human rhinovirus (HRV) (4.6%), human coronavirus OC43 (1.2%), human adenovirus (0.9%), human metapneumovirus (hMPV) (0.5%), influenza A virus (IAV) (0.3%), and human parainfluenza viruses (0.5%). To determine the genetic diversity and transmission route of the viruses, phylogenetic analyses were performed using genome sequences of the detected viruses. The IAV strain detected in this study was genetically similar to strains isolated in the USA, whereas the hMPV strain belonging to the A2b subtype formed a cluster with Kenyan strains. This study provides the first complete genomic sequences of HRV, IAV, and hMPV detected in Gabon, and provides insight into the circulation of respiratory viruses in the country.

On-Site and Visual Detection of the H5 Subtype Avian Influenza Virus Based on RT-RPA and CRISPR/Cas12a.

Avian influenza viruses (AIVs) of the H5 subtype rank among the most serious pathogens, leading to significant economic losses in the global poultry industry and posing risks to human health. Therefore, rapid and accurate virus detection is crucial for the prevention and control of H5 AIVs. In this study, we established a novel detection method for H5 viruses by utilizing the precision of CRISPR/Cas12a and the efficiency of RT-RPA technologies. This assay facilitates the direct visualization of detection results through blue light and lateral flow strips, accurately identifying H5 viruses with high specificity and without cross-reactivity against other AIV subtypes, NDV, IBV, and IBDV. With detection thresholds of 1.9 copies/µL (blue light) and 1.9 × 103 copies/µL (lateral flow strips), our method not only competes with but also slightly surpasses RT-qPCR, demonstrating an 80.70% positive detection rate across 81 clinical samples. The RT-RPA/CRISPR-based detection method is characterized by high sensitivity, specificity, and independence from specialized equipment. The immediate field applicability of the RT-RPA/CRISPR approach underscores its importance as an effective tool for the early detection and management of outbreaks caused by the H5 subtype of AIVs.

Nanopore sequencing of influenza A and B in Oxfordshire and the United Kingdom, 2022-23.

OBJECTIVES: We evaluated Nanopore sequencing for influenza surveillance. METHODS: Influenza A and B PCR-positive samples from hospital patients in Oxfordshire, UK, and a UK-wide population survey from winter 2022-23 underwent Nanopore sequencing following targeted rt-PCR amplification. RESULTS: From 941 infections, successful sequencing was achieved in 292/388 (75 %) available Oxfordshire samples: 231 (79 %) A/H3N2, 53 (18 %) A/H1N1, and 8 (3 %) B/Victoria and in 53/113 (47 %) UK-wide samples. Sequencing was more successful at lower Ct values. Most same-sample replicate sequences had identical haemagglutinin segments (124/141, 88 %); 36/39 (92 %) Illumina vs. Nanopore comparisons were identical, and 3 (8 %) differed by 1 variant. Comparison of Oxfordshire and UK-wide sequences showed frequent inter-regional transmission. Infections were closely-related to 2022-23 vaccine strains. Only one sample had a neuraminidase inhibitor resistance mutation. 849/941 (90 %) Oxfordshire infections were community-acquired. 63/88 (72 %) potentially healthcare-associated cases shared a hospital ward with ≥ 1 known infectious case. 33 epidemiologically-plausible transmission links had sequencing data for both source and recipient: 8 were within ≤ 5 SNPs, of these, 5 (63 %) involved potential sources that were also hospital-acquired. CONCLUSIONS: Nanopore influenza sequencing was reproducible and antiviral resistance rare. Inter-regional transmission was common; most infections were genomically similar. Hospital-acquired infections are likely an important source of nosocomial transmission and should be prioritised for infection prevention and control.

Concurrent Infection with Clade 2.3.4.4b Highly Pathogenic Avian Influenza H5N6 and H5N1 Viruses, South Korea, 2023.

Highly pathogenic avian influenza H5N6 and H5N1 viruses of clade 2.3.4.4b were simultaneously introduced into South Korea at the end of 2023. An outbreak at a broiler duck farm consisted of concurrent infection by both viruses. Sharing genetic information and international surveillance of such viruses in wild birds and poultry is critical.

Novel Avian Influenza A(H5N6) Virus in Wild Birds, South Korea, 2023.

We isolated novel reassortant avian influenza A(H5N6) viruses containing genes from clade 2.3.4.4b H5N1 virus and low pathogenicity avian influenza viruses in carcasses of whooper swans and bean geese in South Korea during December 2023. Neuraminidase gene was from a clade 2.3.4.4b H5N6 virus infecting poultry and humans in China.