Expression of recombinant Florida clade 2 hemagglutinin in baculovirus expression system: A step for subunit vaccine development against H3N8 equine influenza virus.

Background: Equine influenza (EI) is a transmissible viral respiratory sickness of the Equidae family. Two viruses, H7N7 and H3N8 caused EI; however, H7N7 has not been detected for decades. H3N8 has circulated and bifurcated into Eurasian and American lineages. The latter subsequently diversified into Kentucky, South America, and Florida sub-lineages. Florida clade 1 (FC1) and Florida clade 2 (FC2) strains are the only circulating EI viruses (EIVs) in the meantime. Immunization is considered the major means for the prevention and control of EI infection. Using disparate technologies and platforms, several vaccines have been developed and commercialized. According to the recommendations of the World Organization for Animal Health (WOAH), all commercial vaccines shall comprise representatives of both FC1 and FC2 strains. Unfortunately, most of the commercially available vaccines were not updated to incorporate a representative of FC2 strains. Aim: The purpose of this research was to develop a new EI vaccine candidate that incorporates the hemagglutinin (HA) antigen from the currently circulating FC2. Methods: In this study, we report the expression of the full-length recombinant HA gene of FC2 in the baculovirus expression system. Results: The HA recombinant protein has been proven to maintain its biological characteristics by hemadsorption (HAD) and hemagglutination tests. Moreover, using a reference-specific serum, the specificity of the HA has been confirmed through the implementation of immunoperoxidase and western immunoblotting assays. Conclusion: In conclusion, we report the expression of specific biologically active recombinant HA of FC2, which would act as a foundation for the generation of an updated EI subunit or virus vector vaccine candidates.

Genomic characterization of equine influenza A subtype H3N8 viruses by long read sequencing and functional analyses of the PB1-F2 virulence factor of A/equine/Paris/1/2018.

Equine influenza virus (EIV) remains a threat to horses, despite the availability of vaccines. Strategies to monitor the virus and prevent potential vaccine failure revolve around serological assays, RT-qPCR amplification, and sequencing the viral hemagglutinin (HA) and neuraminidase (NA) genes. These approaches overlook the contribution of other viral proteins in driving virulence. This study assesses the potential of long-read nanopore sequencing for fast and precise sequencing of circulating equine influenza viruses. Therefore, two French Florida Clade 1 strains, including the one circulating in winter 2018-2019 exhibiting more pronounced pathogenicity than usual, as well as the two currently OIE-recommended vaccine strains, were sequenced. Our results demonstrated the reliability of this sequencing method in generating accurate sequences. Sequence analysis of HA revealed a subtle antigenic drift in the French EIV strains, with specific substitutions, such as T163I in A/equine/Paris/1/2018 and the N188T mutation in post-2015 strains; both substitutions were in antigenic site B. Antigenic site E exhibited modifications in post-2018 strains, with the N63D substitution. Segment 2 sequencing also revealed that the A/equine/Paris/1/2018 strain encodes a longer variant of the PB1-F2 protein when compared to other Florida clade 1 strains (90 amino acids long versus 81 amino acids long). Further biological and biochemistry assays demonstrated that this PB1-F2 variant has enhanced abilities to abolish the mitochondrial membrane potential ΔΨm and permeabilize synthetic membranes. Altogether, our results highlight the interest in rapidly characterizing the complete genome of circulating strains with next-generation sequencing technologies to adapt vaccines and identify specific virulence markers of EIV.

First Known Human Death After Infection With the Avian Influenza A/H3N8 Virus: Guangdong Province, China, March 2023.

Here, we report on a case of human infection with the H3N8 avian influenza virus. The patient had multiple myeloma and died of severe infection. Genome analysis showed multiple gene mutations and reassortments without mammalian-adaptive mutations. This suggests that avian influenza (A/H3N8) virus infection could be lethal for immunocompromised persons.

Equine influenza outbreak in Eastern of Algeria in 2021: The first introduction of Florida Clade 1 to Maghreb area.

We have performed an equine influenza (EI) serological study of the equine population in Algeria by testing 298 serum samples collected between February and August 2021 from 5 provinces. The results were obtained performing an NP-ELISA. Our results revealed that 49.3% (147/298) samples positive for antibodies to EI (H3N8). During this study and after a gap of one decade an outbreak of EI was reported in Algeria in the first week of March 2021. The disease was confirmed by virus detection from the nasal swabs (n = 39) by qRT-PCR and by identifying 5 EI seroconversion. The virus sequences were identified as H3N8 by sequencing the haemagglutinin (HA) and neuraminidase (NA) genes. Alignment of HA1 amino acid sequence confirmed that viruses belong to Clade 1 of the Florida sublineage in the American lineage. This study indicate the first detection of FC1 strain of EIV in Maghreb area.

Molecular characterization and phylogenetic analysis of avian influenza H3N8 virus isolated from imported waterfowl in Malaysia.

Wild aquatic birds are natural reservoirs of influenza A viruses and H3 subtype is one of the most prevalent subtypes in waterfowl. Two H3N8 viruses of low pathogenic avian influenza (LPAI) were isolated via egg inoculation technique from the fecal swab specimens from imported barnacle goose and paradise shelduck in Veterinary Research Institute Ipoh, Malaysia. The full length of eight gene segments of the two viruses were amplified and sequenced with specific primers. The sequences were molecularly characterized, and the sequence identity were assessed with other published sequences. The two viruses are identical and they possess the same amino acid sequences for all the eight gene segments. The viruses were highly similar to the H3 virus from Netherlands and N8 virus from Belgium respectively. Phylogenetic analysis revealed that all the eight gene segments were grouped in the Eurasian lineage, and genetic reassortment may occur between the internal genes of the H3 viruses and other AI subtypes. Though four amino acid substitutions were identified in the hemagglutinin gene, the viruses retained most of the avian-type receptor binding preference. Few amino acid substitutions were observed in all internal genes. Most of the neuraminidase inhibitors and adamantine resistance related mutation were not seen in the viruses. The replicative capacity, cross species transmissibility, and potential zoonotic risk of the viruses are worth further investigation. As H3 virus poses potential threats to both human and animals, and with the increase in the international trade of birds; strict quarantine practice at the entry point and good laboratory diagnostic capabilities is crucial to prevent the introduction of new AI virus into our country.

Evolution of Avian Influenza Virus (H3) with Spillover into Humans, China.

The continuous evolution of avian influenza viruses (AIVs) of subtype H3 in China and the emergence of human infection with AIV subtype H3N8 highlight their threat to public health. Through surveillance in poultry-associated environments during 2009-2022, we isolated and sequenced 188 H3 AIVs across China. Performing large-scale sequence analysis with publicly available data, we identified 4 sublineages of H3 AIVs established in domestic ducks in China via multiple introductions from wild birds from Eurasia. Using full-genome analysis, we identified 126 distinct genotypes, of which the H3N2 G23 genotype predominated recently. H3N8 G25 viruses, which spilled over from birds to humans, might have been generated by reassortment between H3N2 G23, wild bird H3N8, and poultry H9N2 before February 2021. Mammal-adapted and drug-resistance substitutions occasionally occurred in H3 AIVs. Ongoing surveillance for H3 AIVs and risk assessment are imperative for potential pandemic preparedness.

Prevalence, evolution, replication and transmission of H3N8 avian influenza viruses isolated from migratory birds in eastern China from 2017 to 2021.

The continued evolution and emergence of novel influenza viruses in wild and domestic animals poses an increasing public health risk. Two human cases of H3N8 avian influenza virus infection in China in 2022 have caused public concern regarding the risk of transmission between birds and humans. However, the prevalence of H3N8 avian influenza viruses in their natural reservoirs and their biological characteristics are largely unknown. To elucidate the potential threat of H3N8 viruses, we analyzed five years of surveillance data obtained from an important wetland region in eastern China and evaluated the evolutionary and biological characteristics of 21 H3N8 viruses isolated from 15,899 migratory bird samples between 2017 and 2021. Genetic and phylogenetic analyses showed that the H3N8 viruses circulating in migratory birds and ducks have evolved into different branches and have undergone complicated reassortment with viruses in waterfowl. The 21 viruses belonged to 12 genotypes, and some strains induced body weight loss and pneumonia in mice. All the tested H3N8 viruses preferentially bind to avian-type receptors, although they have acquired the ability to bind human-type receptors. Infection studies in ducks, chickens and pigeons demonstrated that the currently circulating H3N8 viruses in migratory birds have a high possibility of infecting domestic waterfowl and a low possibility of infecting chickens and pigeons. Our findings imply that circulating H3N8 viruses in migratory birds continue to evolve and pose a high infection risk in domestic ducks. These results further emphasize the importance of avian influenza surveillance at the wild bird and poultry interface.

Genetic Characterization and Pathogenesis of Avian Influenza Virus H3N8 Isolated from Chinese pond heron in China in 2021.

In October 2021, a wild bird-origin H3N8 influenza virus-A/Chinese pond heron/Jiangxi 5-1/2021 (H3N8)-was isolated from Chinese pond heron in China. Phylogenetic and molecular analyses were performed to characterize the genetic origin of the H3N8 strain. Phylogenetic analysis revealed that eight gene segments of this avian influenza virus H3N8 belong to Eurasian lineages. HA gene clustered with avian influenza viruses is circulating in poultry in southern China. The NA gene possibly originated from wild ducks in South Korea and has the highest homology (99.3%) with A/Wild duck/South Korea/KNU2020-104/2020 (H3N8), while other internal genes have a complex and wide range of origins. The HA cleavage site is PEKQTR↓GLF with one basic amino acid, Q226 and T228 at HA preferentially bind to the alpha-2,3-linked sialic acid receptor, non-deletion of the stalk region in the NA gene and no mutations at E627K and D701N of the PB2 protein, indicating that isolate A/Chinese pond heron/Jiangxi 5-1/2021 (H3N8) was a typical avian influenza with low pathogenicity. However, there are some mutations that may increase pathogenicity and transmission in mammals, such as N30D, T215A of M1 protein, and P42S of NS1 protein. In animal studies, A/Chinese pond heron/Jiangxi 5-1/2021 (H3N8) replicates inefficiently in the mouse lung and does not adapt well to the mammalian host. Overall, A/Chinese pond heron/Jiangxi 5-1/2021 (H3N8) is a novel wild bird-origin H3N8 influenza virus reassortant from influenza viruses of poultry and wild birds. This wild bird-origin avian influenza virus is associated with wild birds along the East Asian-Australasian flyway. Therefore, surveillance of avian influenza viruses in wild birds should be strengthened to assess their mutation and pandemic risk in advance.

Evolution and mammalian adaptation of H3 and H10 subtype avian influenza viruses in wild birds in Yancheng Wetland of China.

Wild birds play a critical role in avian influenza virus (AIV) ecology and some outbreaks of avian influenza in human originate from wild birds, suggesting that epidemiological surveillance and interspecies-transmission analysis of AIVs in wild birds are critical. Since 2019, we have performed sampling in Yancheng Wetland along the East Asian-Australasian Flyway. Totally, 2054 fecal swabs were collected and one H3N8, two H3N1, one H10N8, and three H10N1 were isolated. Three H3 gene of AIVs we isolated belonged to Eurasian lineage, but the four H10 gene clustered into North American lineage. What's more, the H3 and the foreign H10 gene had generated novel reassortants in Yancheng wetland. Receptor binding assay indicated that nearly all strains, except D369/H10N1, presented a dual receptor-binding profile and bound to avian-type receptor preferentially. In animal experiment, all isolates could infect mice without prior adaptation and induce histopathological changes in mice lungs, moreover, all H3 subtype AIVs obviously triggered weight loss of mice. In addition to lung and turbinate, D322/H3N1, D338/H3N8, D211/H10N8 and D266/H10N1 could spread to brain and kidney or liver or spleen, showing a wider range of tissue tropism. Multiple mutants associated with mammalian adaptation were also detected in all isolates according to molecular analysis. These findings revealed that H3 and H10 AIVs circulating in wild birds in Yancheng Wetland underwent complex reassortment and increased mammalian adaptation, which highlighted the necessity to monitor the diverse reassortment of AIVs in wild birds and evaluate the risks of H3 and H10 viruses to human health.

Human infection with a reassortment avian influenza A H3N8 virus: an epidemiological investigation study.

A four-year-old boy developed recurrent fever and severe pneumonia in April, 2022. High-throughput sequencing revealed a reassortant avian influenza A-H3N8 virus (A/Henan/ZMD-22-2/2022(H3N8) with avian-origin HA and NA genes. The six internal genes were acquired from Eurasian lineage H9N2 viruses. Molecular substitutions analysis revealed the haemagglutin retained avian-like receptor binding specificity but that PB2 genes possessed sequence changes (E627K) associated with increased virulence and transmissibility in mammalian animal models. The patient developed respiratory failure, liver, renal, coagulation dysfunction and sepsis. Endotracheal intubation and extracorporeal membrane oxygenation were administered. H3N8 RNA was detected from nasopharyngeal swab of a dog, anal swab of a cat, and environmental samples collected in the patient's house. The full-length HA sequences from the dog and cat were identical to the sequence from the patient. No influenza-like illness was developed and no H3N8 RNA was identified in family members. Serological testing revealed neutralizing antibody response against ZMD-22-2 virus in the patient and three family members. Our results suggest that a triple reassortant H3N8 caused severe human disease. There is some evidence of mammalian adaptation, possible via an intermediary mammalian species, but no evidence of person-to-person transmission. The potential threat from avian influenza viruses warrants continuous evaluation and mitigation.

Emergence of equine influenza virus H3Nx Florida clade 2 in Arabian racehorses in Egypt.

BACKGROUND: Equine influenza is an important cause of respiratory disease in equids. The causative virus; EIV, is highly variable and can evolve by accumulation of mutations, particularly in the haemagglutinin (HA) gene. Currently, H3N8 is the sole subtype circulating worldwide with Florida clade 1 (FC1) is most prevalent in the Americas and FC2 in Asia and Europe. In Egypt, EIV was detected in two occasions: subtype H7N7 in 1989 and subtype H3N8 (FC1) in 2008. No data is available on the circulation pattern of EIV during the last decade despite frequent observation of suspected cases. METHODS: Twenty-two nasal swabs were collected from vaccinated and non-vaccinated horses showing respiratory signs suggestive of EIV infection in 2017-18. Three additional swabs were retrieved during a national race event in January 2018 from Arabian mares with high fever, gait stiffness and dry cough. Samples were screened by RT-qPCR and HA1 domain of the hemagglutinin gene was amplified and sequenced for sequence and phylogenetic analysis. RESULTS: RT-qPCR screening revealed that only the 3 samples from the race were positive with cycle thresholds ranging from 16 to 21 indicating high viral load. Isolation attempts in hen's eggs were unsuccessful. Sequence analysis of the HA1 domain gene has revealed two identical nucleotide sequences, while the third contained 3 synonymous mutations. Phylogenetic analysis clustered study sequences with recent FC2 sequences from Europe. Amino acid alignments revealed 14 and 13 amino acid differences in the study sequences compared to A/equine/Egypt/6066NANRU-VSVRI/08 (H3N8) and A/equine/Kentucky/1997 (H3N8), respectively, available as EIV vaccines in Egypt. Nine amino acids were different from A/equine/Richmond/1/2007 (H3N8), the recommended FC2 vaccine strain by the world organization of animal health expert surveillance panel (OIE-ESP), two of which were unique to the Egyptian sequences while the remaining 7 changes were shared with the FC2-144V subgroup detected in the United Kingdom from late 2015 to 2016. CONCLUSIONS: The study represents the first reported detection of FC2-144V related EIV from Arabian mares in Egypt, and probably from the entire middle east region. The presented information about EIV epidemiology and spread may require reconsideration of the vaccine strains used in the national vaccination programs.

Disease progression, pathologic, and virologic findings of an equine influenza outbreak in rescue donkeys.

BACKGROUND: Equine influenza virus is a common cause of respiratory disease in equids. Few reports describe clinical presentation and disease progression in donkeys. HYPOTHESIS/OBJECTIVES: Describe the clinical and diagnostic findings, outcome, and pathologic lesions associated with influenza pneumonia in donkeys. ANIMALS: Thirteen unvaccinated donkeys ranging from 1 week to 12 years of age and sharing clinical signs and exposure history. METHODS: Retrospective case series. Medical records from June to July 2020 at the Colorado State Veterinary Teaching Hospital and collaborating referring veterinary practices were reviewed. The diagnosis was confirmed by molecular testing, virus isolation, and partial genetic and phylogenetic analysis of the virus. RESULTS: Survival in donkeys <1 year old was 16.6% (1/6) whereas survival in animals >1 year of age was 85.7% (6/7). Hemagglutinin gene sequencing and phylogenetic analysis confirmed a contemporary clade 1 Florida sublineage H3 virus as the causative agent. CONCLUSIONS AND CLINICAL IMPORTANCE: Clinical signs of equine influenza virus infection in donkeys are similar to those observed in horses. Prognosis for survival generally is good, but deaths have been observed especially in foals born to seronegative dams. This finding emphasizes the importance of prenatal vaccination protocols in all equids, including donkeys.

Human infection of avian influenza A H3N8 virus and the viral origins: a descriptive study.

BACKGROUND: The H3N8 avian influenza virus (AIV) has been circulating in wild birds, with occasional interspecies transmission to mammals. The first human infection of H3N8 subtype occurred in Henan Province, China, in April, 2022. We aimed to investigate clinical, epidemiological, and virological data related to a second case identified soon afterwards in Hunan Province, China. METHODS: We analysed clinical, epidemiological, and virological data for a 5-year-old boy diagnosed with H3N8 AIV infection in May, 2022, during influenza-like illness surveillance in Changsha City, Hunan Province, China. H3N8 virus strains from chicken flocks from January, 2021, to April, 2022, were retrospectively investigated in China. The genomes of the viruses were sequenced for phylogenetic analysis of all the eight gene segments. We evaluated the receptor-binding properties of the H3N8 viruses by using a solid-phase binding assay. We used sequence alignment and homology-modelling methods to study the effect of specific mutations on the human receptor-binding properties. We also conducted serological surveillance to detect the H3N8 infections among poultry workers in the two provinces with H3N8 cases. FINDINGS: The clinical symptoms of the patient were mild, including fever, sore throat, chills, and a runny nose. The patient's fever subsided on the same day of hospitalisation, and these symptoms disappeared 7 days later, presenting mild influenza symptoms, with no pneumonia. An H3N8 virus was isolated from the patient's throat swab specimen. The novel H3N8 virus causing human infection was first detected in a chicken farm in Guangdong Province in December, 2021, and subsequently emerged in several provinces. Sequence analyses revealed the novel H3N8 AIVs originated from multiple reassortment events. The haemagglutinin gene could have originated from H3Ny AIVs of duck origin. The neuraminidase gene belongs to North American lineage, and might have originated in Alaska (USA) and been transferred by migratory birds along the east Asian flyway. The six internal genes had originated from G57 genotype H9N2 AIVs that were endemic in chicken flocks. Reassortment events might have occurred in domestic ducks or chickens in the Pearl River Delta area in southern China. The novel H3N8 viruses possess the ability to bind to both avian-type and human-type sialic acid receptors, which pose a threat to human health. No poultry worker in our study was positive for antibodies against the H3N8 virus. INTERPRETATION: The novel H3N8 virus that caused human infection had originated from chickens, a typical spillover. The virus is a triple reassortment strain with the Eurasian avian H3 gene, North American avian N8 gene, and dynamic internal genes of the H9N2 viruses. The virus already possesses binding ability to human-type receptors, though the risk of the H3N8 virus infection in humans was low, and the cases are rare and sporadic at present. Considering the pandemic potential, comprehensive surveillance of the H3N8 virus in poultry flocks and the environment is imperative, and poultry-to-human transmission should be closely monitored. FUNDING: National Natural Science Foundation of China, National Key Research and Development Program of China, Strategic Priority Research Program of the Chinese Academy of Sciences, Hunan Provincial Innovative Construction Special Fund: Emergency response to COVID-19 outbreak, Scientific Research Fund of Hunan Provincial Health Department, and the Hunan Provincial Health Commission Foundation.

Protective efficacy of a reverse genetics-derived inactivated vaccine against equine influenza virus in horses.

Updating vaccine strains is essential to control equine influenza. We evaluated the protective efficacy of an inactivated equine influenza vaccine derived from viruses generated by reverse genetics (RG) in horses in an experimental viral challenge study. Wild-type (WT) virus (A/equine/Tipperary/1/2019) and virus generated by RG (consisting of hemagglutinin and neuraminidase genes from A/equine/Tipperary/1/2019 and six other genes from high-growth A/Puerto Rico/8/34) were inactivated by formalin for vaccine use. Twelve 1-year-old naïve horses with no antibodies against equine influenza virus were assigned to three groups (each n = 4): control, WT, and RG. They were vaccinated twice, 4 weeks apart, and were challenged with A/equine/Tipperary/1/2019 2 weeks after the second vaccination. All four horses in the control group and one horse in the WT group had pyrexia for multiple days and respiratory illness, and one horse in the RG group had pyrexia for 2 days without respiratory illness. The mean rectal temperatures and the mean concentrations of serum amyloid A in the WT and RG groups were significantly lower than those in the control group, with no significant differences between them. The WT and RG vaccines significantly reduced viral shedding relative to the control. The protective efficacy of the RG-derived inactivated vaccine against equine influenza virus is comparable to that of the vaccine derived from WT viruses in horses. The RG technique can make it easy to update equine influenza vaccine strains.

Evidence for flock transmission of individual subtypes and strains of avian influenza viruses: A monitoring study of wild birds in Kazakhstan.

An active surveillance study of avian influenza viruses (AIVs) in wild birds was carried out in Kazakhstan in 2018-2019. In total, 866 samples were collected from wild birds and analyzed for influenza viruses using molecular and virological tests. Genome segments of Asian, European, and Australian lineages were detected in 25 (4.6%) out of 541 waterfowl samples positive for subtype H3N8, and in two (0.6%) out of 325 H3N8 positive samples from terrestrial birds. No highly pathogenic avian influenza virus (AIV) was detected. The results indicated transmission of closely related strains or identical subtypes of AIVs by a flock-unit of migratory birds or annual cyclical pattern of subtype dominance. The simultaneous circulation of genome segments of the Asian, European and Australian genetic lineages of H3N8 AIVs in wild birds in Kazakhstan indicated the important role of Central Asia as a transmission hub of AI viruses linking the East Asian migratory flyways with European flyways and vice versa.

Investigating Influenza Virus Polymerase Activity in Feline Cells Based on the Influenza Virus Minigenome Replication System Driven by the Feline RNA Polymerase I Promoter.

Emerging influenza virus poses a health threat to humans and animals. Domestic cats have recently been identified as a potential source of zoonotic influenza virus. The influenza virus minigenome replication system based on the ribonucleic acid (RNA) polymerase I (PolI) promoter is the most widely used tool for investigating polymerase activity. It could help determine host factors or viral proteins influencing influenza virus polymerase activity in vitro. However, influenza virus polymerase activity has never been studied in feline cells thus far. In the present study, the feline RNA PolI promoter was identified in the intergenic spacer regions between adjacent upstream 28S and downstream 18S rRNA genes in the cat (Felis catus) genome using bioinformatics strategies. The transcription initiation site of the feline RNA PolI promoter was predicted. The feline RNA PolI promoter was cloned from CRFK cells, and a promoter size of 250 bp contained a sequence with sufficient PolI promoter activity by a dual-luciferase reporter assay. The influenza virus minigenome replication system based on the feline RNA PolI promoter was then established. Using this system, the feline RNA PolI promoter was determined to have significantly higher transcriptional activity than the human and chicken RNA PolI promoters in feline cells, and equine (H3N8) influenza virus presented higher polymerase activity than human (H1N1) and canine (H3N2) influenza viruses. In addition, feline myxovirus resistance protein 1 (Mx1) and baloxavir were observed to inhibit influenza virus polymerase activity in vitro in a dose-dependent manner. Our study will help further investigations on the molecular mechanism of host adaptation and cross-species transmission of influenza virus in cats.

Adaptation of Two Wild Bird-Origin H3N8 Avian Influenza Viruses to Mammalian Hosts.

Wild birds play an important role in the emergence, evolution, and spread of zoonotic avian influenza viruses (AIVs). However, there are few studies on the cross-species transmission of the H3N8 AIV originating from wild birds. In this study, we investigated the transmissibility and pathogenicity of two H3N8 low pathogenic avian influenza viruses (LPAIVs) isolated from wild birds, GZA1 and XJ47, to mammals. The HA genes of both strains belonged to Eurasian isolates, while the other genes were derived from a variety of other subtypes of AIVs. Both strains can infect specific-pathogen-free (SPF) chickens, BALB/c mice, and guinea pigs. The XJ47 strain spread horizontally in SPF chickens and guinea pigs. The GZA1 strain did not spread horizontally but caused higher weight loss and mild lung inflammation in mice. P12-GZA1- and P12-XJ47-adapted strains obtained after 12 passages in the lung of mice showed enhanced pathogenicity in mice, which led to obvious clinical symptoms, lung inflammation, and 100% death. Both adapted strains have the reported mutation T97I in the PA, and the reported mutation D701N in PB2 has been found in the P12-GZA1-adapted strain. This study provides an important scientific basis for the continuous monitoring of wild AIVs and the mechanism underlying AIV cross-species transmission.

Human genes with codon usage bias similar to that of the nonstructural protein 1 gene of influenza A viruses are conjointly involved in the infectious pathogenesis of influenza A viruses.

Molecular mechanisms of the non-structural protein 1 (NS1) in influenza A-induced pathological changes remain ambiguous. This study explored the pathogenesis of human infection by influenza A viruses (IAVs) through identifying human genes with codon usage bias (CUB) similar to NS1 gene of these viruses based on the relative synonymous codon usage (RSCU). CUB of the IAV subtypes H1N1, H3N2, H3N8, H5N1, H5N2, H5N8, H7N9 and H9N2 was analyzed and the correlation of RSCU values of NS1 sequences with those of the human genes was calculated. The CUB of NS1 was uneven and codons ending with A/U were preferred. The ENC-GC3 and neutrality plots suggested natural selection as the main determinant for CUB. The RCDI, CAI and SiD values showed that the viruses had a high degree of adaptability to human. A total of 2155 human genes showed significant RSCU-based correlation (p < 0.05 and r > 0.5) with NS1 coding sequences and was considered as human genes with CUB similar to NS1 gene of IAV subtypes. Differences and similarities in the subtype-specific human protein-protein interaction (PPI) networks and their functions were recorded among IAVs subtypes, indicating that NS1 of each IAV subtype has a specific pathogenic mechanism. Processes and pathways involved in influenza, transcription, immune response and cell cycle were enriched in human gene sets retrieved based on the CUB of NS1 gene of IAV subtypes. The present work may advance our understanding on the mechanism of NS1 in human infections of IAV subtypes and shed light on the therapeutic options.

Investigation of cross-regional spread and evolution of equine influenza H3N8 at US and global scales using Bayesian phylogeography based on balanced subsampling.

Equine influenza virus (EIV) is a highly contagious pathogen of equids, and a well-known burden in global equine health. EIV H3N8 variants seasonally emerged and resulted in EIV outbreaks in the United States and worldwide. The present study evaluated the pattern of cross-regional EIV H3N8 spread and evolutionary characteristics at US and global scales using Bayesian phylogeography with balanced subsampling based on regional horse population size. A total of 297 haemagglutinin (HA) sequences of global EIV H3N8 were collected from 1963 to 2019 and subsampled to global subset (n = 67), raw US sequences (n = 100) and US subset (n = 44) datasets. Discrete trait phylogeography analysis was used to estimate the transmission history of EIV using four global and US genome datasets. The North American lineage was the major source of globally dominant EIV variants and spread to other global regions. The US EIV strains generally spread from the southern and midwestern regions to other regions. The EIV H3N8 accumulated approximately three nucleotide substitutions per year in the HA gene under heterogeneous local positive selection. Our findings will guide better decision making of target intervention strategies of EIV H3N8 infection and provide the better scheme of genomic surveillance in the United States and global equine health.

Generation and Characterization of Single-Cycle Infectious Canine Influenza A Virus (sciCIV) and Its Use as Vaccine Platform.

Influenza A viruses (IAVs) infect a broad range of hosts, including multiple avian and mammalian species. The frequent emergence of novel IAV strains in different hosts, including in humans, results in the need for vigilance and ongoing development of new approaches to fighting or prevent those infections. Canine influenza is a contagious respiratory disease in dogs caused by two subtypes of IAV, the equine-origin H3N8 canine influenza virus (CIV), and the avian-origin H3N2 CIV. A novel approach to influenza vaccination involves single-cycle infectious influenza A viruses (sciIAVs), which are defective for an essential viral gene. They are propagated in complementing cell lines which provide the missing gene in trans. As sciIAV cannot complete their replication cycle in regular cells they are limited to a single round of viral replication. Because of their safety profile and ability to express foreign antigens inside infected cells, sciIAVs have served both as live-attenuated vaccines and as vaccine vectors for the expression of heterologous antigens. Here, we describe experimental procedures for the generation of a single-cycle infectious CIV (sciCIV), where the viral hemagglutinin (HA) gene was exchanged for the gene for green fluorescent protein (GFP). Complementation of the viral HA protein is provided in trans by stable HA-expressing cell lines. Methods for the in vitro characterization of HA deficient but GFP-expressing sciCIV (sciCIV ΔHA/GFP) are described, as well as its use as a potential vaccine.