PA Genes Research Articles

Genetic characterization and receptor binding analysis of a novel H5N1 HPAI virus with a H6Nx-derived PA gene in Guangdong, China

Genetic characterization and receptor binding analysis of a novel H5N1 HPAI virus with a H6Nx-derived PA gene in Guangdong, China

Genetic Reassortment in a Child Coinfected with Two Influenza B Viruses, B/Yamagata Lineage and B/Victoria-Lineage Strains.

We identified a child coinfected with influenza B viruses of B/Yamagata and B/Victoria lineages, in whom we analyzed the occurrence of genetic reassortment. Plaque purification was performed using a throat swab specimen from a 9-year-old child, resulting in 34 well-isolated plaques. The genomic composition of eight gene segments (HA, NA, PB1, PB2, PA, NP, M, and NS genes) for each plaque was determined at the lineage level. Of the 34 plaques, 21 (61.8%) had B/Phuket/3073/2013 (B/Yamagata)-like sequences in all gene segments, while the other 13 (38.2%) were reassortants with B/Texas/02/2013 (B/Victoria)-like sequences in 1-5 of the 8 segments. The PB1 segment had the most B/Victoria lineage genes (23.5%; 8 of 34 plaques), while PB2 and PA had the least (2.9%; 1 of 34 plaques). Reassortants with B/Victoria lineage genes in 2-5 segments showed the same level of growth as viruses with B/Yamagata lineage genes in all segments. However, reassortants with B/Victoria lineage genes only in the NA, PB1, NP, or NS segments exhibited reduced or undetectable growth. We demonstrated that various gene reassortments occurred in a child. These results suggest that simultaneous outbreaks of two influenza B virus lineages increase genetic diversity and could promote the emergence of new epidemic strains.

Genetic Characterization and Phylogeographic Analysis of the First H13N6 Avian Influenza Virus Isolated from Vega Gull in South Korea.

Avian influenza virus (AIV) is a pathogen with zoonotic and pandemic potential. Migratory birds are natural reservoirs of all known subtypes of AIVs, except for H17N10 and H18N11, and they have been implicated in previous highly pathogenic avian influenza outbreaks worldwide. This study identified and characterized the first isolate of the H13N6 subtype from a Vega gull (Larus vegae mongolicus) in South Korea. The amino acid sequence of hemagglutinin gene showed a low pathogenic AIV subtype and various amino acid substitutions were found in the sequence compared to the reference sequence and known H13 isolates. High sequence homology with other H13N6 isolates was found in HA, NA, PB1, and PA genes, but not for PB2, NP, M, and NS genes. Interestingly, various point amino acid mutations were found on all gene segments, and some are linked to an increased binding to human-type receptors, resistance to antivirals, and virulence. Evolutionary and phylogenetic analyses showed that all gene segments are gull-adapted, with a phylogeographic origin of mostly Eurasian, except for PB2, PA, and M. Findings from this study support the evidence that reassortment of AIVs continuously occurs in nature, and migratory birds are vital in the intercontinental spread of avian influenza viruses.

Reassortant H9N2 canine influenza viruses containing the pandemic H1N1/2009 ribonucleoprotein complex circulating in pigs acquired enhanced virulence in mice

The reassortment between avian H9N2 and Eurasian avian-like (EA) H1N1 viruses may have potentially changed from avian-to-mammals adaptation. This study generated 20 reassortant viruses with the introduction of H1N1/2009 internal genes from EA H1N1 virus into H9N2 virus. 12 of these recovered the replication capability both in the lungs and turbinate samples. 10 of 12 obtained PA gene segments from the ribonucleoprotein (RNP) complexes of the EA H1N1 virus, and 3 exhibited extreme virulence. Specially, the combination of PB2, PA and NP genes could overcome the species-specific restriction in human cells. Analysis of the polymerase activities found that introduction of the PA gene resulted in increased polymerase activity. These findings indicated that RNP complexes from EA H1N1 virus could confer an adaptation advantage and high compatibility to avian H9N2 virus. This raises new concerns for public health due to the possible coexistence of H9N2 and EA H1N1 viruses in dogs.

Hemagglutinin affects replication, stability and airborne transmission of the H9N2 subtype avian influenza virus

H9N2 subtype avian influenza virus (AIV) can transmit by direct as well as airborne contacts. It has been widespread in poultry and continued to contribute to zoonotic spillover events by providing its six internal genes for the reassortment of novel influenza viruses (eg, H7N9) that infect poultry and humans. Compared to H7N9, H9N2 virus displays an efficient airborne transmissibility in poultry, but the mechanisms of transmission difference have been insufficiently studied. The Hemagglutinin (HA) and viral polymerase acidic protein (PA) have been implicated in the airborne transmission of influenza A viruses. Accordingly, we generated the reassortant viruses of circulating airborne transmissible H9N2 and non-airborne transmissible H7N9 viruses carrying HA and/or PA gene. The introduction of the PA gene from H7N9 into the genome of H9N2 virus resulted in a reduction in airborne transmission among chickens, while the isolated introduction of the HA gene segment completely eliminated airborne transmission among chickens. We further showed that introduction of HA gene of non-transmissible H7N9 did not influence the HA/NA balance of H9N2 virus, but increased the threshold for membrane fusion and decreased the acid stability. Thus, our results indicate that HA protein plays a key role in replication, stability, and airborne transmission of the H9N2 subtype AIV.

Newly emerged genotypes of highly pathogenic H5N8 avian influenza viruses in Kagoshima prefecture, Japan during winter 2020/21.

During the 2020/21 winter season, 29 and 10 H5N8 high pathogenicity avian influenza viruses (HPAIVs) were isolated from environmental water and wild birds, respectively, in Kagoshima prefecture, Japan. Furthermore, seven subtypes of low pathogenicity avian influenza viruses (LPAIVs) were also isolated; H1N1, H2N9, H3N2, H3N6, H3N8, H4N6, and H6N6 subtypes. While the H5 hemagglutinin (HA) genes of the G1 cluster were isolated throughout the winter season, those of the G2 cluster were also detected in late winter, suggesting that H5 HPAIVs possessing H5 HA genes from the two different clusters were individually introduced into Kagoshima prefecture. Intriguingly, genetic constellations revealed that the H5N8 HPAIVs could be classified into six genotypes, including four previously reported genotypes (E1, E2, E3, and E7), and two new genotypes (tentatively named E8 and E9). The PB1 and PA gene segments of genotypes E8 and E9 shared high similarity with those of LPAIVs, whereas the remaining gene segments were close to those of genotype E1. Furthermore, LPAIVs whose PA gene segment was close to that of genotype E9 were isolated from the environmental water. Overall, we revealed that various HPAIV genotypes circulated in Kagoshima prefecture during the 2020/21 winter season. This study highlights the importance of monitoring both HPAIV and LPAIV to better understand AIV ecology in migratory waterfowl populations.

Probiotic Properties and Antioxidant Activity In Vitro of Lactic Acid Bacteria.

The properties of probiotics such as lactic acid bacteria (LAB) have been widely studied over the last decades. In the present study, four different LAB species, namely Lactobacillus gasseri ATCC 33323, Lacticaseibacillus rhamnosus GG ATCC 53103, Levilactobacillus brevis ATCC 8287, and Lactiplantibacillus plantarum ATCC 14917, were investigated in order to determine their ability to survive in the human gut. They were evaluated based on their tolerance to acids, resistance to simulated gastrointestinal conditions, antibiotic resistance, and the identification of genes encoding bacteriocin production. All four tested strains demonstrated high resistance to simulated gastric juice after 3 h, and the viable counts revealed declines in cell concentrations of less than 1 log cycle. L. plantarum showed the highest level of survival in the human gut, with counts of 7.09 log CFU/mL. For the species L. rhamnosus and L. brevis, the values were 6.97 and 6.52, respectively. L. gasseri, after 12 h, showed a 3.96 log cycle drop in viable counts. None of the evaluated strains inhibited resistance to ampicillin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, or chloramphenicol. With regard to bacteriocin genes, the Pediocin PA gene was identified in Lactiplantibacillus plantarum ATCC 14917, Lacticaseibacillus rhamnosus GG ATCC 53103, and Lactobacillus gasseri ATCC 33323. The PlnEF gene was detected in Lactiplantibacillus plantarum ATCC 14917 and Lacticaseibacillus rhamnosus GG ATCC 53103. The Brevicin 174A and PlnA genes were not detected in any bacteria. Moreover, the potential antioxidant activity of LAB's metabolites was evaluated. At the same time, the possible antioxidant activity of metabolites of LAB was first tested using the free radical DDPH• (a, a-Diphenyl-β-Picrylhydrazyl) and then evaluated with regard to their radical scavenging activity and inhibition against peroxyl radical induced DNA scission. All strains showed antioxidant activity; however, the best antioxidant activity was achieved by L. brevis (94.47%) and L. gasseri (91.29%) at 210 min. This study provides a comprehensive approach to the action of these LAB and their use in the food industry.

Single nucleotide variants in Pseudomonas aeruginosa populations from sputum correlate with baseline lung function and predict disease progression in individuals with cystic fibrosis.

The severity and progression of lung disease are highly variable across individuals with cystic fibrosis (CF) and are imperfectly predicted by mutations in the human gene CFTR, lung microbiome variation or other clinical factors. The opportunistic pathogen Pseudomonas aeruginosa (Pa) dominates airway infections in most CF adults. Here we hypothesized that within-host genetic variation of Pa populations would be associated with lung disease severity. To quantify Pa genetic variation within CF sputum samples, we used deep amplicon sequencing (AmpliSeq) of 209 Pa genes previously associated with pathogenesis or adaptation to the CF lung. We trained machine learning models using Pa single nucleotide variants (SNVs), microbiome diversity data and clinical factors to classify lung disease severity at the time of sputum sampling, and to predict lung function decline after 5 years in a cohort of 54 adult CF patients with chronic Pa infection. Models using Pa SNVs alone classified lung disease severity with good sensitivity and specificity (area under the receiver operating characteristic curve: AUROC=0.87). Models were less predictive of lung function decline after 5 years (AUROC=0.74) but still significantly better than random. The addition of clinical data, but not sputum microbiome diversity data, yielded only modest improvements in classifying baseline lung function (AUROC=0.92) and predicting lung function decline (AUROC=0.79), suggesting that Pa AmpliSeq data account for most of the predictive value. Our work provides a proof of principle that Pa genetic variation in sputum tracks lung disease severity, moderately predicts lung function decline and could serve as a disease biomarker among CF patients with chronic Pa infections.

Phylogenetic and epidemiological characteristics of H9N2 avian influenza viruses in Shandong Province, China from 2019 to 2021

H9N2 avian influenza virus (AIV) has widely circulated in poultry worldwide and sporadic infections in humans and mammals. During our surveillance of chicken from 2019 to 2021 in Shandong Province, China, we isolated 11 H9N2 AIVs. Phylogenetic analyses showed that the 8 gene segments of the 11 isolates were closely related to several sublineages of Eurasian lineage: BJ/94-like clades (HA and NA genes), G1-like clades (PB2 and M genes), and SH/F/98-like clades (PB1, PA, NP and NS genes). The isolates showed mutation sites that preferentially bind to human-like receptors (HA) and mammalian fitness sites (PB2, PB1 and PA), as well as mutations in antigen and drug resistance sites. Moreover, studies with mice revealed 4 isolates with varying levels of pathogenicity. The average antibody titer of the H9N2 AIVs was 8.60 log2. Based on our results, the epidemiological surveillance of H9N2 AIVs should be strengthened.

Preparing for the Next Influenza Season: Monitoring the Emergence and Spread of Antiviral Resistance.

The relaxation of pandemic restrictions in 2022 has led to a reemergence of respiratory virus circulation worldwide and anticipation of substantial influenza waves for the 2022/2023 Northern Hemisphere winter. Therefore, the antiviral susceptibility profiles of human influenza viruses circulating in Germany were characterized. Between October 2019 (week 40/2019) and March 2022 (week 12/2022), nasal swabs from untreated patients with acute respiratory symptoms were collected in the national German influenza surveillance system. A total of 598 influenza viruses were isolated and analyzed for susceptibility to oseltamivir, zanamivir and peramivir, using a neuraminidase (NA) inhibition assay. In addition, next-generation sequencing was applied to assess molecular markers of resistance to NA, cap-dependent endonuclease (PA) and M2 ion channel inhibitors (NAI, PAI, M2I) in 367 primary clinical samples. Furthermore, a genotyping assay based on RT-PCR and pyrosequencing to rapidly assess the molecular resistance marker PA-I38X in PA genes was designed and established. While NAI resistance in the strict sense, defined by a ≥ 10-fold (influenza A) or ≥5-fold (influenza B) increase of NAI IC50, was not detected, a subtype A(H1N1)pdm09 isolate displayed 2.3- to 7.5-fold IC50 increase for all three NAI. This isolate carried the NA-S247N substitution, which is known to enhance NAI resistance induced by NA-H275Y. All sequenced influenza A viruses carried the M2-S31N substitution, which confers resistance to M2I. Of note, one A(H3N2) virus displayed the PA-I38M substitution, which is associated with reduced susceptibility to the PAI baloxavir marboxil. Pyrosequencing analysis confirmed these findings in the original clinical specimen and in cultured virus isolate, suggesting sufficient replicative fitness of this virus mutant. Over the last three influenza seasons, the vast majority of influenza viruses in this national-level sentinel were susceptible to NAIs and PAIs. These findings support the use of antivirals in the upcoming influenza season.

Evolutionary dynamics of the clade 2.3.4.4B H5N8 high-pathogenicity avian influenza outbreaks in coastal seabirds and other species in southern Africa from 2017 to 2019.

From late 2017 to early 2018, clade 2.3.4.4B H5N8 highly pathogenic avian influenza (HPAI) viruses caused mass die-offs of thousands of coastal seabirds along the southern coastline of South Africa. Terns (Laridae) especially were affected, but high mortalities in critically endangered and threatened species like African Penguins (Spheniscus demersus) caused international concern and, exactly a year later, the disease recurred at a key African Penguin breeding site on Halifax Island, Namibia. Twenty-five clade 2.3.4.4B H5N8 HPAI viruses from coastal seabirds and a Jackal Buzzard (Buteo rufofuscus) were isolated and/or sequenced in this study. Phylogenetic analyses of the full viral genomes and time to the most recent common ancestor (tMRCA) analyses of the HA, NA, PB1 and PA genes determined that the South African coastal seabird viruses formed a monophyletic group nested within the South African genotype 4 viruses. This sub-lineage likely originated from a single introduction by terrestrial birds around October 2017. Only the HA and NA sequences were available for the Namibian penguin viruses, but the phylogenetic data confirmed that the South African coastal seabird viruses from 2017 to 2018 were the source and the most closely related South African virus was found in a gull. tMRCA analyses furthermore determined that the progenitors of the five genotypes implicated in the earlier 2017 South African outbreaks in wild birds and poultry were dated at between 2 and 4months prior to the index cases. tMRCA and phylogenetic data also showed that the novel genotype 6 virus introduced to South Africa in 2018, and later also detected in Nigeria and Poland in 2019, most likely arose in late 2017 in West, Central or East Africa. We propose that it continued to circulate there, and that an unidentified reservoir was the source of both the South African outbreaks in early 2018 and in Nigeria in mid-2019.

Host adaptive mutations in the 2009 H1N1 pandemic influenza A virus PA gene regulate translation efficiency of viral mRNAs via GRSF1

Avian species are the major natural reservoir from which pandemic influenza A viruses can be introduced to humans. Avian influenza A virus genes, including the three viral polymerase genes, PA, PB1 and PB2, require host-adaptive mutations to allow for viral replication and transmission in humans. Previously, PA from the 2009 pH1N1 viral polymerase was found to harbor host-adaptive mutations leading to enhanced viral polymerase activity. By quantifying translation and mRNA transcription, we found that the 2009 pH1N1 PA, and the associated host-adaptive mutations, led to greater translation efficiency. This was due to enhanced cytosolic accumulation of viral mRNA, which was dependent on the host RNA binding protein GRSF1. Mutations to the GRSF1 binding site in viral mRNA, as well as GRSF1 knockdown, reduced cytosolic accumulation and translation efficiency of viral mRNAs. This study identifies a previously unrecognized mechanism by which host-adaptive mutations in PA regulate viral replication and host adaptation. Importantly, these results provide greater insight into the host adaptation process of IAVs and reveal the importance of GRSF1 in the lifecycle of IAV.

Phylogeography and Biological Characterizations of H12 Influenza A Viruses.

Influenza A virus (IAV) is widespread in wild bird reservoirs. Sixteen hemagglutinin subtypes are associated with wild waterfowl hosts; some subtypes are isolated infrequently, one of which is H12 IAV. In this study, we detected three H12 IAVs from Anas crecca and Anas formosa in Poyang Lake, China, in 2018, one of which was isolated. Phylogenetic analysis revealed that the genome sequences of the three H12 viruses belonged to the Eurasian lineage, except for PA genes and one NP gene, which belonged to the North American lineage. The growth kinetics showed that the H12 isolate grew better in A549 than MDCK cells. Moreover, although the H12 isolate cannot efficiently replicate in BALB/c mice, it can bind to both α-2,6 sialic acid (SA) and α-2,SA-linked receptors. In addition, we examined the phylodynamics of H12 viruses by Bayesian phylogeographic analysis. The results show that two major transmission routes of H12 IAVs were from Asia to Oceania and from Europe to South America, and Anas and Arenaria genera were the major hosts of the viral transmission. Our findings help us better understand the evolution of H12 IAV and highlight the need for the continued surveillance of IAVs circulating in wild birds.

Emergence of a Reassortant 2.3.4.4b Highly Pathogenic H5N1 Avian Influenza Virus Containing H9N2 PA Gene in Burkina Faso, West Africa, in 2021.

Since 2006, the poultry population in Burkina Faso has been seriously hit by different waves of Highly Pathogenic Avian Influenza (HPAI) H5N1 epizootics. In December 2021, three distinct regions of Burkina Faso, namely, Gomboussougou, Bonyollo, and Koubri, detected HPAI H5N1 viruses in poultry. Whole genome characterization and statistical phylogenetic approaches were applied to shed light on the potential origin of these viruses and estimate the time of virus emergence. Our results revealed that the HPAI H5N1 viruses reported in the three affected regions of Burkina Faso cluster together within clade 2.3.4.4b, and are closely related to HPAI H5N1 viruses identified in Nigeria and Niger in the period 2021–2022, except for the PA gene, which clusters with H9N2 viruses of the zoonotic G1 lineage collected in West Africa between 2017 and 2020. These reassortant viruses possess several mutations that may be associated with an increased zoonotic potential. Although it is difficult to ascertain where and when the reassortment event occurred, the emergence of a H5N1/H9N2 reassortant virus in a vulnerable region, such as West Africa, raises concerns about its possible impact on animal and human health. These findings also highlight the risk that West Africa may become a new hotspot for the emergence of new genotypes of HPAI viruses.

PA-X protein assists H9N2 subtype avian influenza virus in escaping immune response of mucosal dendritic cells.

H9N2 subtype low pathogenicity avian influenza virus (AIV) poses a potential zoonotic risk. PA-X, a novel protein generated by PA gene ribosomal frameshift, is considered to be the virulence factor of H9N2 subtype AIVs. Our study found that rTX possessing PA-X protein enhanced the mammalian pathogenicity of H9N2 subtype AIVs compared with PA-X-deficient virus (rTX-FS). Furthermore, PA-X protein inhibited H9N2 subtype AIVs to infect dendritic cells (DCs), but not nonimmune cells (MDCK cells). Meanwhile, PA-X protein suppressed the phenotypic expression (CD80, CD86, CD40 and MHCII), early activation marker (CD69) and pro-inflammatory cytokines (IL-6 and TNF-α), whereas increased anti-inflammatory cytokine (IL-10) in DCs. After intranasally viral infection in mice, we found that PA-X protein of H9N2 subtype AIVs reduced CD11b+ and CD103+ subtype mucosal DCs recruitment to the nasal submucosa by inhibiting CCL20 expression. Moreover, PA-X protein abolished the migratory ability of CD11b+ and CD103+ DCs into draining cervical lymph nodes by down-regulating CCR7 expression. The rTX-infected DCs significantly impaired the allogeneic CD4+ T cell proliferation, suggesting PA-X protein suppressed the immune functions of DCs for hindering the downstream immune activation. These findings indicated that PA-X protein assisted H9N2 subtype AIVs in escaping immune response of mucosal DCs for enhancing the pathogenicity.

Genetic characterization and evolution of H6N6 subtype avian influenza viruses.

H6-subtype avian influenza virus (AIV) was prevalent in the world and could sporadically infect humans. Here, a new chicken-derived H6N6-subtype AIV strain A/chicken/Zhejiang/49/2021 (ZJ49) was isolated in Zhejiang Province, China in 2021. Phylogenetic analysis by Maximum likelihood methods showed that H6-subtype AIVs were classed into 13 groups according to HA gene. The ZJ49 strain belonged to the G12 group, which mainly consisted of strains from Asian and dominated in recent years. Based on NA gene, H6-subtype AIVs were divided into N6.1 and N6.2 clades according to the NA gene. The ZJ49 isolate was located in the N6.2e clade, which mainly consisted of the H5N6-subtype AIVs. Phylogenetic analysis by Bayesian methods showed that the effective quantity size of H6-subtype AIVs increased around 1990, reached a peak around 2015, declined after 2015, then kept in a stable level after 2018. The reassortment analysis predicted that the PB2, PA, and NA genes of ZJ49 may recombine with H5-subtype AIVs. The amino acid at 222 position of HA gene of ZJ49 strain mutated from A to V, suggesting that ZJ49 has a potential ability to cross species barriers. The four glycosylation sites were highly conserved, implying less impact on the fold and conception of HA stem structure. Our results revealed the complicated evolution, reassortment, and mutations of receptor binding sites of H6-subtype AIVs, which emphasize the importance to continuously monitor the epidemiology and evolution of H6-subtype AIVs.

Genome characterization and mutation analysis of human influenza A virus in Thailand.

The influenza A viruses have high mutation rates and cause a serious health problem worldwide. Therefore, this study focused on genome characterization of the viruses isolated from Thai patients based on the next-generation sequencing technology. The nasal swabs were collected from patients with influenza-like illness in Thailand during 2017-2018. Then, the influenza A viruses were detected by reverse transcription-quantitative polymerase chain reaction and isolated by MDCK cells. The viral genomes were amplified and sequenced by Illumina MiSeq platform. Whole genome sequences were used for characterization, phylogenetic construction, mutation analysis and nucleotide diversity of the viruses. The result revealed that 90 samples were positive for the viruses including 44 of A/H1N1 and 46 of A/H3N2. Among these, 43 samples were successfully isolated and then the viral genomes of 25 samples were completely amplified. Finally, 17 whole genomes of the viruses (A/H1N1, n=12 and A/H3N2, n=5) were successfully sequenced with an average of 232,578 mapped reads and 1,720 genome coverage per sample. Phylogenetic analysis demonstrated that the A/H1N1 viruses were distinguishable from the recommended vaccine strains. However, the A/H3N2 viruses from this study were closely related to the recommended vaccine strains. The nonsynonymous mutations were found in all genes of both viruses, especially in HA and NA genes. The nucleotide diversity analysis revealed negative selection in the PB1, PA, hemagglutinin (HA) and neuraminidase (NA) genes of the A/H1N1 viruses. High-throughput data in this study allow for genetic characterization of circulating influenza viruses which would be crucial for preparation against pandemic and epidemic outbreaks in the future.

Genome-Wide Reassortment Analysis of Influenza A H7N9 Viruses Circulating in China during 2013-2019.

Reassortment with the H9N2 virus gave rise to the zoonotic H7N9 avian influenza virus (AIV), which caused more than five outbreak waves in humans, with high mortality. The frequent exchange of genomic segments between H7N9 and H9N2 has been well-documented. However, the reassortment patterns have not been described and are not yet fully understood. Here, we used phylogenetic analyses to investigate the patterns of intersubtype and intrasubtype/intralineage reassortment across the eight viral segments. The H7N9 virus and its progeny frequently exchanged internal genes with the H9N2 virus but rarely with the other AIV subtypes. Before beginning the intrasubtype/intralineage reassortment analyses, five Yangtze River Delta (YRD A-E) and two Pearl River Delta (PRD A-B) clusters were divided according to the HA gene phylogeny. The seven reset segment genes were also nomenclatured consistently. As revealed by the tanglegram results, high intralineage reassortment rates were determined in waves 2–3 and 5. Additionally, the clusters of PB2 c05 and M c02 were the most dominant in wave 5, which could have contributed to the onset of the largest H7N9 outbreak in 2016–2017. Meanwhile, a portion of the YRD-C cluster (HP H7N9) inherited their PB2, PA, and M segments from the co-circulating YRD-E (LP H7N9) cluster during wave 5. Untanglegram results revealed that the reassortment rate between HA and NA was lower than HA with any of the other six segments. A multidimensional scaling plot revealed a robust genetic linkage between the PB2 and PA genes, indicating that they may share a co-evolutionary history. Furthermore, we observed relatively more robust positive selection pressure on HA, NA, M2, and NS1 proteins. Our findings demonstrate that frequent reassortment, particular reassorted patterns, and adaptive mutations shaped the H7N9 viral genetic diversity and evolution. Increased surveillance is required immediately to better understand the current state of the HP H7N9 AIV.

Enhancing the yield of seasonal influenza viruses through manipulation of microRNAs in Madin-Darby canine kidney cells.

Annual influenza vaccine is recommended to reduce the occurrence of seasonal influenza and its complications. Thus far, Madin-Darby canine kidney (MDCK) cell line has been used to manufacture cell-based influenza vaccines. Even though host microRNAs may facilitate viral replication, the interaction between MDCK cells-derived microRNAs and seasonal influenza viruses has been less frequently investigated. Therefore, this study highlighted microRNA profiles of MDCK cells to increase the yield of seasonal influenza virus production by manipulating cellular microRNAs. MDCK cells were infected with influenza A or B virus at a multiplicity of infection (MOI) of 0.01, and microRNA collections were then subjected to MiSeq (Illumina) Sequencing. The validated profiles revealed that cfa-miR-340, cfa-miR-146b, cfa-miR-197, and cfa-miR-215 were the most frequently upregulated microRNAs. The effect of candidate microRNA inhibition and overexpression on viral replication was determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). The hybridization pattern between candidate miRNAs and viral genes was performed using miRBase and RNAhybrid web-based programs. Moreover, the predicted microRNA-binding sites were validated by a 3'-UTR reporter assay. The results indicated that cfa-miR-146b could directly target the PB1 gene of A/pH1N1 and the PA gene of B/Yamagata. Furthermore, cfa-miR-215 could silence the PB1 gene of A/pH1N1 and the PB1 gene of B/Victoria. However, the PB2 gene of the A/H3N2 virus was silenced by cfa-miR-197. In addition, the HA and NA sequences of influenza viruses harvested from the cell cultures treated with microRNA inhibitors were analyzed. The sequencing results revealed no difference in the antigenic HA and NA sequences between viruses isolated from the cells treated with microRNA inhibitors and the parental viruses. In conclusion, these findings suggested that MDCK cell-derived microRNAs target viral genes in a strain-specific manner for suppressing viral replication. Conversely, the use of such microRNA inhibitors may facilitate the production of influenza viruses.

Novel Low Pathogenic Avian Influenza H6N1 in Backyard Chicken in Easter Island (Rapa Nui), Chilean Polynesia.

Little is known about the prevalence of avian influenza viruses (AIV) in wildlife and domestic animals in Polynesia. Here, we present the results of active AIV surveillance performed during two sampling seasons in 2019 on Easter Island (Rapa Nui). Tracheal and cloacal swabs as well as sera samples were obtained from domestic backyard poultry, while fresh faeces were collected from wild birds. In addition to detecting antibodies against AIV in 46% of the domestic chickens in backyard production systems tested, we isolated a novel low pathogenic H6N1 virus from a chicken. Phylogenetic analysis of all genetic segments revealed that the virus was closely related to AIV’s circulating in South America. Our analysis showed different geographical origins of the genetic segments, with the PA, HA, NA, NP, and MP gene segments coming from central Chile and the PB2, PB1, and NS being closely related to viruses isolated in Argentina. While the route of introduction can only be speculated, our analysis shows the persistence and independent evolution of this strain in the island since its putative introduction between 2015 and 2016. The results of this research are the first evidence of AIV circulation in domestic birds on a Polynesian island and increase our understanding of AIV ecology in region, warranting further surveillance on Rapa Nui and beyond.