HylS', a fragment of truncated hyaluronidase of Streptococcus suis, contributes to immune evasion by interaction with host complement factor C3b.

Pathogenic bacteria have evolved many strategies to evade surveillance and attack by complements. Streptococcus suis is an important zoonotic pathogen that infects humans and pigs. Hyaluronidase (HylA) has been reported to be a potential virulence factor of S. suis. However, in this study, it was discovered that the genomic region encoding HylA of the virulent S. suis strain SC19 and other ST1 strains was truncated into four fragments when aligned with a strain containing intact HylA and possessing hyaluronidase activity. As a result, SC19 had no hyaluronidase activity, but one truncated HylA fragment, designated as HylS,' directly interacted with complement C3b, as confirmed by western ligand blotting, pull-down, and ELISA assays. The deposition of C3b and membrane attack complex (MAC) formation on the surface of a HylS'-deleted mutant (ΔhylS') was significantly increased compared to wild-type SC19. In human sera and whole blood, ΔhylS' survival was significantly reduced compared to that in SC19. The resistance of ΔhylS' to macrophages and human polymorphonuclear neutrophil PMNs also decreased. In a mouse infection model, ΔhylS' showed reduced lethality and lower bacterial load in the organs compared to that of SC19. We conclude that the truncated hyaluronidase HylS' fragment contributes to complement evasion and the pathogenesis of S. suis.

Native ApxIIA secreted by Actinobacillus pleuropneumoniae induces apoptosis in porcine alveolar macrophages dependent on concentration and acylation.

Actinobacillus pleuropneumoniae is an important swine respiratory pathogen causing substantial economic losses to the global pig industry. The Apx toxins of A. pleuropneumoniae belong to the RTX toxin family and are major virulence factors. In addition to hemolysis and/or cytotoxicity via pore-forming activity, RTX toxins, such as ApxIA of A. pleuropneumoniae, have been reported to cause other effects on target cells, e.g., apoptosis. A. pleuropneumoniae ApxIIA is expressed by most serotypes and has moderate hemolytic and cytotoxic activities. In this study, porcine alveolar macrophages (3D4/21) were stimulated with different concentrations of purified native ApxIIA from the serotype 7 strain AP76 which only secretes ApxIIA. By observation of nuclear condensation via fluorescent staining and detection of apoptosis and necrosis by flow cytometry, it was found that high and low concentrations of native ApxIIA mainly caused necrosis or apoptosis of 3D4/21 cells, respectively. ApxIIA purified from an AP76 mutant with a deleted acetyltransferase gene (apxIIC) did not induce necrosis nor apoptosis. Western blot analysis using specific antibodies showed that a cleaved caspase 3 and activated capase 9 was detected after treatment of cells with a low concentration of native ApxIIA, while general or specific inhibitors of caspase 3, 8, 9 blocked these effects. ApxIIA-induced apoptosis of macrophages may be a mechanism of A. pleuropneumoniae to escape host immune clearance.

The morphology and metabolic changes of Actinobacillus pleuropneumoniae during its growth as a biofilm.

Actinobacillus pleuropneumoniae is an important swine respiratory pathogen. Previous studies have suggested that growth as a biofilm is a natural state of A. pleuropneumoniae infection. To understand the survival features involved in the biofilm state, the growth features, morphology and gene expression profiles of planktonic and biofilm A. pleuropneumoniae were compared. A. pleuropneumoniae in biofilms showed reduced viability but maintained the presence of extracellular polymeric substances (EPS) after late log-phase. Under the microscope, bacteria in biofilms formed dense aggregated structures that were connected by abundant EPS, with reduced condensed chromatin. By construction of Δpga and ΔdspB mutants, polymeric ß-1,6-linked N-acetylglucosamine and dispersin B were confirmed to be critical for normal biofilm formation. RNA-seq analysis indicated that, compared to their planktonic counterparts, A. pleuropneumoniae in biofilms had an extensively altered transcriptome. Carbohydrate metabolism, energy metabolism and translation were significantly repressed, while fermentation and genes contributing to EPS synthesis and translocation were up-regulated. The regulators Fnr (HlyX) and Fis were found to be up-regulated and their binding motifs were identified in the majority of the differentially expressed genes, suggesting their coordinated global role in regulating biofilm metabolism. By comparing the transcriptome of wild-type biofilm and Δpga, the utilization of oligosaccharides, iron and sulfur and fermentation were found to be important in adhesion and aggregation during biofilm formation. Additionally, when used as inocula, biofilm bacteria showed reduced virulence in mouse, compared with planktonic grown cells. Thus, these results have identified new facets of A. pleuropneumoniae biofilm maintenance and regulation.

Genome-wide identification of genes critical for in vivo fitness of multi-drug resistant porcine extraintestinal pathogenic Escherichia coli by transposon-directed insertion site sequencing using a mouse infection model.

Extraintestinal pathogenic Escherichia coli (ExPEC) is an important zoonotic pathogen. Recently, ExPEC has been reported to be an emerging problem in pig farming. However, the mechanism of pathogenicity of porcine ExPEC remains to be revealed. In this study, we constructed a transposon (Tn) mutagenesis library covering Tn insertion in over 72% of the chromosome-encoded genes of a virulent and multi-drug resistant porcine ExPEC strain PCN033. By using a mouse infection model, a transposon-directed insertion site sequencing (TraDIS) assay was performed to identify in vivo fitness factors. By comparing the Tn insertion frequencies between the input Tn library and the recovered library from different organs, 64 genes were identified to be involved in fitness during systemic infection. 15 genes were selected and individual gene deletion mutants were constructed. The in vivo fitness was evaluated by using a competitive infection assay. Among them, ΔfimG was significantly outcompeted by the WT strain in vivo and showed defective adhesion to host cells. rfa which was involved in lipopolysaccharide biosynthesis was shown to be critical for in vivo fitness which may have resulted from its role in the resistance to serum killing. In addition, several metabolic genes including fepB, sdhC, fepG, gltS, dcuA, ccmH, ddpD, narU, glpD, malM, and yabL and two regulatory genes metJ and baeS were shown as important determinants of in vivo fitness of porcine ExPEC. Collectively, this study performed a genome-wide screening for in vivo fitness factors which will be important for understanding the pathogenicity of porcine ExPEC.

The Feed Additive Potassium Diformate Prevents Salmonella enterica Serovar Pullorum Infection and Affects Intestinal Flora in Chickens.

Extensive studies have shown that potassium diformate (KDF), an antibiotic substitute used as a feed additive, improves animal growth performance, although there is less direct evidence of its preventive effect on bacterial infections and its influence on the intestinal flora of animals. In this study, the inhibition effect of KDF on Salmonella enterica serovar Pullorum, an important enteric pathogen causing pullorum disease, was investigated in vitro and on a chicken infection model. The effect of KDF on the diversities and structures of chicken duodenal and cecum flora were also investigated using 16S rRNA gene sequencing. The results showed that addition of 0.5% KDF in feed or 0.1% KDF in drinking water significantly reduced the bacterial loads and the degree of pathological changes in the cecum, improved digestion and reduced the pH of the gastrointestinal tract of chickens infected with S. pullorum. KDF also significantly modified the diversity and abundance of intestinal microflorae in chickens. In particular, it promoted the colonization of several probiotics, such as Bacteroides, Blautia, Ruminococcus_torques_group and Faecalibacteriumm, which are involved in maintenance of the intestinal barrier, modulation of inflammation, energy supply for intestinal cells and pathogen resistance. These results enrich the theoretical basis for the clinical application of KDF in chickens.

The Metabolic Adaptation in Response to Nitrate Is Critical for Actinobacillus pleuropneumoniae Growth and Pathogenicity under the Regulation of NarQ/P.

Nitrate metabolism is an adaptation mechanism used by many bacteria for survival in anaerobic environments. As a by-product of inflammation, nitrate is used by the intestinal bacterial pathogens to enable gut infection. However, the responses of bacterial respiratory pathogens to nitrate are less well understood. Actinobacillus pleuropneumoniae is an important bacterial respiratory pathogen of swine. Previous studies have suggested that adaptation of A. pleuropneumoniae to anaerobiosis is important for infection. In this work, A. pleuropneumoniae growth and pathogenesis in response to the nitrate were investigated. Nitrate significantly promoted A. pleuropneumoniae growth under anaerobic conditions in vitro and lethality in mice. By using narQ and narP deletion mutants and single-residue-mutated complementary strains of ΔnarQ, the two-component system NarQ/P was confirmed to be critical for nitrate-induced growth, with Arg50 in NarQ as an essential functional residue. Transcriptome analysis showed that nitrate upregulated multiple energy-generating pathways, including nitrate metabolism, mannose and pentose metabolism, and glycerolipid metabolism via the regulation of NarQ/P. Furthermore, narQ, narP, and its target gene encoding the nitrate reductase Nap contributed to the pathogenicity of A. pleuropneumoniae. The Nap inhibitor tungstate significantly reduced the survival of A. pleuropneumoniae in vivo, suggesting that Nap is a potential drug target. These results give new insights into how the respiratory pathogen A. pleuropneumoniae utilizes the alternative electron acceptor nitrate to overcome the hypoxia microenvironment, which can occur in the inflammatory or necrotic infected tissues.

Involvement of the Actinobacillus pleuropneumoniae ompW Gene in Confrontation of Environmental Pressure.

Actinobacillus pleuropneumoniae causes porcine pleuropneumonia. The function of the outer membrane protein W gene (ompW) of A. pleuropneumoniae has not been evaluated. Thus a deletion mutant of ompW, ΔompW, was constructed to explore the effect of ompW gene deletion on bacterial growth, biofilm formation, bacterial morphology, oxidative tolerance, susceptibility to antibiotics, and the expression of ribosome synthesis and ABC transporter related genes. Results showed that the ompW gene deletion did not affect biofilm formation and the growth of A. pleuropneumoniae but did affect bacterial morphology during steady growth, oxidative tolerance, and bacterial susceptibility to polymyxin B, kanamycin, and penicillin. The ompW gene deletion also affected the expression of ribosome synthesis and ABC transporter related genes. These results suggested that ompW may regulate the biological phenotype of A. pleuropneumoniae.

The Rcs System Contributes to the Motility Defects of the Twin-Arginine Translocation System Mutant of Extraintestinal Pathogenic Escherichia coli.

Flagellum-mediated bacterial motility is important for bacteria to take up nutrients, adapt to environmental changes, and establish infection. The twin-arginine translocation system (Tat) is an important protein export system, playing a critical role in bacterial physiology and pathogenesis. It has been observed for a long time that the Tat system is critical for bacterial motility. However, the underlying mechanism remains unrevealed. In this study, a comparative transcriptomics analysis was performed with extraintestinal pathogenic Escherichia coli (ExPEC), which identified a considerable number of genes differentially expressed when the Tat system was disrupted. Among them, a large proportion of flagellar biosynthesis genes showed downregulation, indicating that transcription regulation plays an important role in mediating the motility defects. We further identified three Tat substrate proteins, MdoD, AmiA, and AmiC, that were responsible for the nonmotile phenotype. The Rcs system was deleted in the Δtat, the ΔmdoD, and the ΔamiAΔamiC strains, which restored the motility of ΔmdoD and partially restored the motility of Δtat and ΔamiAΔamiC. The flagella were also observed in all of the ΔtatΔrcsDB, ΔmdoDΔrcsDB, and ΔamiAΔamiCΔrcsDB strains, but not in the Δtat, ΔmdoD, and ΔamiAΔamiC strains, by using transmission electron microscopy. Quantitative reverse transcription-PCR data revealed that the regulons of the Rcs system displayed differential expression in the tat mutant, indicating that the Rcs signaling was activated. Our results suggest that the Rcs system plays an important role in mediating the motility defects of the tat mutant of ExPEC. IMPORTANCE The Tat system is an important protein export system critical for bacterial physiology and pathogenesis. It has been observed for a long time that the Tat system is critical for bacterial motility. However, the underlying mechanism remains unrevealed. In this study, we combine transcriptomics analysis and bacterial genetics, which reveal that transcription regulation plays an important role in mediating the motility defects of the tat mutant of extraintestinal pathogenic Escherichia coli. The Tat substrate proteins responsible for the motility defects are identified. We further show that the Rcs system contributes to the motility suppression. We for the first time reveal the link between the Tat system and bacterial motility, which is important for understanding the physiological functions of the Tat system.

An Engineered Outer Membrane-Defective Escherichia coli Secreting Protective Antigens against Streptococcus suis via the Twin-Arginine Translocation Pathway as a Vaccine.

Live bacterial vector vaccines are one of the most promising vaccine types and have the advantages of low cost, flexibility, and good safety. Meanwhile, protein secretion systems have been reported as useful tools to facilitate the release of heterologous antigen proteins from bacterial vectors. The twin-arginine translocation (Tat) system is an important protein export system that transports fully folded proteins in a signal peptide-dependent manner. In this study, we constructed a live vector vaccine using an engineered commensal Escherichia coli strain in which amiA and amiC genes were deleted, resulting in a leaky outer membrane that allows the release of periplasmic proteins to the extracellular environment. The protective antigen proteins SLY, enolase, and Sbp against Streptococcus suis were targeted to the Tat pathway by fusing a Tat signal peptide. Our results showed that by exploiting the Tat pathway and the outer membrane-defective E. coli strain, the antigen proteins were successfully secreted. The strains secreting the antigen proteins were used to vaccinate mice. After S. suis challenge, the vaccinated group showed significantly higher survival and milder clinical symptoms compared with the vector group. Further analysis showed that the mice in the vaccinated group had lower burdens of bacteria load and slighter pathological changes. Our study reports a novel live bacterial vector vaccine that uses the Tat system and provides a new alternative for developing S. suis vaccine.

Anti-inflammatory activity of arctigenin against PCV2 infection in a mouse model.

Arctigenin (ACT) is a novel anti-inflammatory lignan extracted from Arctium lappa L, a herb commonly used in traditional Chinese herbal medicine. In this study, we investigated the molecular mechanism whereby ACT inhibits PCV2 infection-induced proinflammatory cytokine production in vitro and in vivo. We observed that in PCV2 infection+ACT treated PK-15 cells, proinflammatory cytokine production was significantly reduced, compared to the PCV2-infected cells. The transfection and luciferase reporter assay confirmed that ACT suppressed NF-κB signalling pathway activation following PCV2 infection in PK-15 cells. Furthermore, western blotting demonstrated that ACT suppressed the NF-κB signal pathway in PCV2 infection-stimulated PK-15 cells by inhibiting the translocation of p65 from the cytoplasm to the nucleus and IκBα phosphorylation. BALB/c mice were used as a model to evaluate the anti-inflammatory effect of ACT in vivo. We found that the BALB/c mice inoculated with PCV2 infection + ACT treated showed a significant reduction of proinflammatory cytokine production in serum, lung and spleen tissue, compared to the PCV2-infected mice. Western blotting confirmed that ACT suppressed the NF-κB signal pathway in PCV2-infected mice by inhibiting the translocation of p65 from the cytoplasm to the nucleus and IκBα phosphorylation in lung tissue. Our studies first demonstrate that ACT inhibits PCV2 infection-induced proinflammatory cytokine production by suppressing the phosphorylation and nuclear translocation of NF-κB in vitro and in vivo. These results will help further develop ACT as a Traditional Chinese herbal medicine remedy in the treatment of porcine circovirus-associated diseases.

Genome analysis provides insight into hyper-virulence of Streptococcus suis LSM178, a human strain with a novel sequence type 1005.

Streptococcus suis has been well-recognized as a zoonotic pathogen worldwide, and the diversity and unpredictable adaptive potential of sporadic human strains represent a great risk to the public health. In this study, S. suis LSM178, isolated from a patient in contact with pigs and raw pork, was assessed as a hyper-virulent strain and interpreted for the virulence based on its genetic information. The strain was more invasive for Caco-2 cells than two other S. suis strains, SC19 and P1/7. Sequence analysis designated LSM178 with serotype 2 and a novel sequence type 1005. Phylogenetic analysis showed that LSM178 clustered with highly virulent strains including all human strains and epidemic strains. Compared with other strains, these S. suis have the most and the same virulent factors and a type I-89 K pathogenicity island. Further, groups of genes were identified to distinguish these highly virulent strains from other generally virulent strains, emphasizing the key roles of genes modeling transcription, cell barrier, replication, recombination and repair on virulence regulation. Additionally, LSM178 contains a novel prophage conducive potentially to pathogenicity.

A survey of Prototheca bovis infection in dairy farms of the Hubei province, China.

Bovine mastitis, caused by Prototheca bovis, has received much attention worldwide. To investigate the status of P. bovis infection in dairy farms of Hubei, we collected 1,158 milk samples and 90 environmental samples from 14 dairy farms of Hubei, China. The isolates were identified with traditional biological methods and molecular biological techniques, and their pathogenicity was tested through mice infection experiments. Isolates from 57 milk and 20 environmental samples were identified as P. bovis. The mice infection tests proved that the isolated P. bovis could cause mastitis in mice, manifesting as severe red swelling of the mammary glands. Histopathological analysis of tissue sections showed necrosis and nodules lesions formed in the infected mice mammary tissue, accompanied by macrophage and neutrophil infiltration. These results suggested the existence of pathogenic P. bovis in dairy farms of the Hubei province, China, with brewer's grains and fresh feces possibly playing important roles in the spread of this disease.

Optimisation of the Conversion and Extraction of Arctigenin From Fructus arctii Into Arctiin Using Fungi.

Fructus arctii is commonly used in Chinese medicine, and arctiin and arctigenin are its main active ingredients. Arctiin has low bioavailability in the human body and needs to be converted into arctigenin by intestinal microbes before it can be absorbed into the blood. Arctigenin has antiviral, anti-inflammatory, and anti-tumour effects and its development has important value. In this study, we used external microbial fermentation with Aspergillus awamori and Trichoderma reesei to process and convert arctiin from F. arctii powder into arctigenin, hence increasing its bioavailability. We developed a fermentation process by optimising the carbon and nitrogen source/ratio, fermentation time, pH, liquid volume, inoculation volume, and substrate solid-liquid ratio. This allowed for an arctiin conversion rate of 99.84%, and the dissolution rate of the final product was 95.74%, with a loss rate as low as 4.26%. After the fermentation of F. arctii powder, the average yield of arctigenin is 19.51 mg/g. Crude fermented F. arctii extract was purified by silica gel column chromatography, and we observed an arctigenin purity of 99.33%. Our technique effectively converts arctiin and extracts arctigenin from F. arctii and provides a solid basis for further development and industrialisation.

Pharmacokinetics of Arctigenin and Fructus Arctii Powder in Piglets.

Fructus arctii, also known as great power seed, is the dried fruit of Arctium lappa of the family Compositae. It is a commonly used veterinary herbal medicine, and arctigenin is the main active ingredient. The aim of this study was to characterize the absorption, distribution, metabolism, and excretion of arctigenin and Fructus arctii powder in piglets. These data were used to provide a theoretical reference for the development and clinical use of new veterinary drugs. Sixteen healthy piglets (mean weight 30.0 ± 5.0 kg) were divided into two groups. One group was administered 2.0 mg/kg body weight (bw) arctigenin intravenously, and the other was administered 1.0 g/kg.bw Fructus arctii powder by gavage. Blood samples were collected from the anterior vena cava at different time points, and the concentration of arctigenin in the plasma of the piglets was determined using high-performance liquid chromatography (HPLC). Arctigenin conformed to a two-compartment model with no absorption, and the main pharmacokinetic parameters were as follows: distribution half-life (t 1/2α)-0.166 ± 0.022 h; elimination half-life (t 1/2ß)-3.161 ± 0.296 h; apparent volume of distribution (V d)-0.231 ± 0.033 L/kg; clearance rate (CLb)-0.057 ± 0.003 L/(h.kg); and area under the curve (AUC)-1.189 ± 0.057 g.h/mL. The pharmacokinetic parameters of arctigenin following oral administration of the Fructus arctii powder were as follows: absorption half-life (t 1/2ka)-0.274 ± 0.102 h, t 1/2α-1.435 ± 0.725 h, t 1/2ß-63.467 ± 29.115 h, V d-1.680 ± 0.402 L/kg, CLb-0.076 ± 0.028 L/(h kg), peak time (t max)-0.853 ± 0.211 h, peak concentration (C max)-0.430 ± 0.035 g/mL, and AUC-14.672 ± 4.813 g/mL. These results indicated that intravenous arctigenin was sparingly distributed in tissues. In contrast, orally administered Fructus arctii powder was rapidly absorbed, more widely distributed, and more slowly eliminated than the intravenous arctigenin, which may indicate its sustained pharmacological effects.

Tea polyphenols suppress growth and virulence-related factors of Haemophilus parasuis.

The bacterium Haemophilus parasuis (H. parasuis) is the primary cause of Glässer's disease. Currently, there are no effective vaccines that can confer protection against all H. parasuis serovars. Therefore, the present study aimed to investigate the effect of tea polyphenols on growth, expression of virulence-related factors, and biofilm formation of H. parasuis, as well as to evaluate their protective effects against H. parasuis challenge. Our findings demonstrated that tea polyphenols can inhibit H. parasuis growth in a dose-dependent manner and attenuate the biofilm formation of H. parasuis. In addition, tea polyphenols exerted inhibitory effects on the expression of H. parasuis virulence-related factors. Moreover, tea polyphenols could confer protection against a lethal dose of H. parasuis and can reduce pathological tissue damage induced by H. parasuis. In summary, our findings demonstrated the promising use of tea polyphenols as a novel treatment for H. parasuis infection in pigs.

Characterization of a reassortant H11N9 subtype avian influenza virus isolated from bean goose along the East Asian-Australian flyway.

During the surveillance of avian influenza viruses in the Dongxi Lake wetland of Hubei in 2015-2016, an H11N9 avian influenza virus was isolated from a bean goose (Anser fabalis). Phylogenetic analysis showed that the HA gene of this isolate belongs to the North American lineage; however, the NA and the internal genes of the isolate were generated from the Eurasian lineage. This strain had reduced pathogenicity in mice and was capable of replication in the mouse lung without prior adaptation. This is the first report detecting H11N9 subtype influenza virus from migratory birds in central China. These findings highlight the transmission of avian influenza virus along the East Asian-Australian flyway and the need for continuing surveillance in central China.

The antiviral activity of arctigenin in traditional Chinese medicine on porcine circovirus type 2.

Arctigenin (ACT) is a phenylpropanoid dibenzylbutyrolactone lignan extracted from the traditional herb Arctium lappa L. (Compositae) with anti-viral and anti-inflammatory effects. Here, we investigated the antiviral activity of ACT found in traditional Chinese medicine on porcine circovirus type 2 (PCV2) in vitro and in vivo. Results showed that dosing of 15.6-62.5µg/mL ACT could significantly inhibit the PCV2 proliferation in PK-15 cells (P<0.01). Dosing of 62.5µg/mL ACT 0, 4 or 8h after challenge inoculation significantly inhibited the proliferation of 1MOI and 10MOI in PK-15 cells (P<0.01), and the inhibitory effect of ACT dosing 4h or 8h post-inoculation was greater than 0h after dosing (P<0.01). In vivo test with mice challenge against PCV2 infection demonstrated that intraperitoneal injection of 200µg/kg ACT significantly inhibited PCV2 proliferation in the lungs, spleens and inguinal lymph nodes, with an effect similar to ribavirin, demonstrating the effectiveness of ACT as an antiviral agent against PCV2 in vitro and in vivo. This compound, therefore, may have the potential to serve as a drug for protection of pigs against the infection of PCV2.

[Molecular Phylogenetic Analysis of a Highly Pathogenic H5N2 Avian Influenza Virus Isolated from Duck].

In 2016,routine influenza virus surveillance was conducted in the live poultry markets of Wuhan, Hubei Province. An H5N2 subtype avian influenza virus(AIV)was isolated from ducks in Wuhan. The entire genome of this virus isolate was sequenced,and molecular phylogenetic analysis performed. The results indicated that the HA gene belonged to clade 2.3.4.4and contained multiple basic amino acids at the cleavage site, which is characteristic of highly pathogenic AIV. Sequence alignment revealed that the isolate shared a high degree of homology with different H5 subtype AIVs isolated from waterfowl in southern China in recent years. This isolate was likely a natural reassortant from different subtype AIVs. This study provides epidemiological evidence of influenza evolution. Continuation of molecular epidemiology studies of H5 subtype influenza viruses in live poultry markets is important for understanding their role in the variation and evolution of highly pathogenic AIVs and their potential hazardous effects on human health. Furthermore, this information is important for strengthening comprehensive AIV surveillance and control measures.

Complete Genome Sequences of an H5N1 Highly Pathogenic Avian Influenza Virus Isolated from Pigeon in China in 2012.

An avian influenza virus strain, A/pigeon/Hubei/RP25/2012 (H5N1), was isolated from pigeons in Hubei province, China. Phylogenetic analysis indicates that the HA gene belongs to clade 2.3.4 and the other internal genes present different recombination events. Information about the strain provides basic research data for epidemiological evidences for revealing influenza evolution.