Role of serotonin on the intestinal mucosal immune response to stress-induced diarrhea in weaning mice.

BACKGROUND: During weaning, babies and young animal often experience diarrhea from food intolerance and/or decreasing levels of maternal antibodies, and diarrhea tends to be particularly severe during the early-weaned period, which often exhibits an underdeveloped immune system, a disturbed gut environment and results in nutrient malabsorption and dehydration. It was deduced that neuroendocrine might have close relation with diarrhea, especially 5-HT. METHODS: To explore the role of serotonin (5-HT) in weaning mice subjected to stress-induced diarrhea, 21-day-old weaned mice were divided into the following groups: control group, stress-induced diarrhea group (restrained by binding the hind limbs and intragastric administration of folium sennae with 0.4 g/mL, 15 mL/kg body weight) and para-chlorophenylalanine (PCPA) + stress-induced diarrhea group (30 mg/mL, 300 mg/kg body weight PCPA intraperitoneal injection before stress-induced diarrhea treatment). RESULTS: Based on results from enzyme-linked immunosorbent assays, histological staining, lymphocyte proliferation assays and flow cytometry analysis, we found that the mice experienced increases in several stress markers, which coincided with severe diarrhea and an increase in 5-HT levels. However, pre-treatment with PCPA resulted in a decrease in the stress indicators and the severity of diarrhea, which correlated with decreased 5-HT levels. Interestingly, stress-induced diarrhea caused changes in various aspects of the immune system, including the amount of intraepithelium lymphocytes, CD4+/CD8+ T lymphocyte populations, B and T lymphocyte proliferation, and the secretion of sIgA and cytokines in the small intestine and ileum. However, these immune system changes could be reversed upon treatment with PCPA. CONCLUSIONS: We observed a distinct correlation between 5-HT levels and the occurrence of stress-induced diarrhea in weaning mice, which may result in the deregulation of the mucosal immune system.

[Genetic evolution and substitution frequency of avian influenza virus HA gene in chicken H9N2 subtype in China in the last 20 years].

OBJECTIVE: Low pathogenic avian influenza (LPAI) H9N2 subtype virus has been prevalent in domestic poultry in China over two decades. This study was to determine the genetic evolution trend of H9N2 avian influenza virus (AIV) under immune pressure of vaccine. METHODS: H9 HA sequences of 40 isolates from the present study and 136 pandemic strains and 7 classical strains from China downloaded from GenBank, were genetically analyzed to determine evolution, molecular characteristic, and mutation frequency. RESULTS: Phylogenetic trees analysis suggested that H9N2 subtypes AIV could be clustered into 5 distinct lineages: G1-like, BJ94-like, Y280-like, S2-like and Americans lineages. Most H9N2 isolates in 2005-2014 belonged to S2-like sub-genotype, suggesting that this genotype was the dominate isolates in China. Further more, comparison based on the amino acid sequence showed that different lineages have their distinct characteristics, and significant accumulations of amino acid variation were also found. In addition, in comparison with reference Ck/BJ/1/1994 HA gene, average annual substitution rates of H9N2 pandemic strain nucleotide and amino acid were 5.73 x 10⁻³ and 4.25 x 10⁻³ from 1994 to 2014, respectively. Substitution rate during 2011-2014 were 6.35 x 10⁻³ and 5.32 x 10⁻³, higher than that during the period of 2006-2010 (5.22 x 10⁻³ and 3.70 x 10⁻³) and even much higher than that during the 1999-2005 (0.74 x 10⁻³ and 0.50 x 10⁻³), when the vaccines were initially applied in the field. CONCLUSION: Overall, these data indicate that the mismatch between H9N2 vaccine strains and pandemic strains drives the virus to quickly mutate.

Characterization of two pigeon paramyxovirus type 1 isolates in China.

For over three decades, there has been a continuing panzootic caused by a virulent variant avian paramyxovirus type 1 strain, the so-called pigeon paramyxovirus type 1. It is found primarily in racing pigeons, but it has also spread to wild birds and poultry. In this study, two pigeon paramyxovirus type 1 strains, SD12 and BJ13, obtained from diseased pigeons in China, were characterized. Phylogenetic analysis based on complete sequences allowed characterization of both strains as genotype VI, class II. Further phylogenetic analysis of a 374-nucleotide section of the fusion gene showed that SD12 fell into lineage VIbii-d and BJ13 into VIbii-f. The deduced amino acid sequence of the cleavage site of the fusion protein confirmed that both isolates contained the virulent motif (112)K/RRQKR↓F(117) at the cleavage site. Nevertheless, the values of intracerebral pathogenicity indices showed the SD12 isolate to be a velogenic strain and BJ13 isolate to be a mesogenic strain. The SD12 isolate was further investigated via clinical observation, RNA detection, histopathology and viral serology in experimentally infected 3-week-old chickens. It showed a mild pathological phenotype in chickens, with viral replication restricted to a few tissues. The molecular mechanism for the SD12 isolate to have a virulent motif but low levels of virulence for chickens requires further study.

Generation by reverse genetics of an effective attenuated Newcastle disease virus vaccine based on a prevalent highly virulent Chinese strain.

OBJECTIVES: To investigate whether the differences between the circulating Newcastle disease virus (NDV) isolates and the used vaccine might account for the current ND outbreaks in vaccinated poultry flocks. RESULTS: A reverse genetics system using prevalent genotype VIId isolate SG10 was constructed and a mutant virus, named aSG10, was developed by changing the virulent F protein cleavage site motif "(112)RRQKR↓F(117)" into an avirulent motif "(112)GRQGR↓L(117)". The attenuated pathogenicity of aSG10 was confirmed from the mean death time and intracerebral pathogenicity index. aSG10 and LaSota both protected vaccinated birds from death after challenge with highly virulent genotype VII NDV, strain SG10. However, aSG10 significantly reduced the challenge virus shedding from the vaccinated birds compared to LaSota vaccine. We also generated a recombinant virus, aSG10-enhanced green fluorescent protein (EGFP), which expresses EGFP. aSG10-EGFP stably expressed EGFP for at least 10 passages. CONCLUSIONS: The mutant, aSG10, can be safely used as a vaccine vector and is a potential vaccine candidate in increasing the protective efficacy for the control of current ND epidemic in China.

[Evolution of non-coding nucleotide sequences in Newcastle disease virus genomes ].

[OBJECTIVE] Although much is done in the coding genes of Newcastle disease virus (NDV) , limited papers can be found with non-coding sequences. In this paper, the evolution tendency of non-coding sequences was studied. [METHODS] NDV strain LC12 isolated from duck with egg drop syndrome in 2012, and others 35 strains genome cDNA of different NDV genotype were sought and obtained from GenBank. Analytical approaches including nucleotide homology, nucleotide alignment and phylogenetic tree were associated with the leading sequences, trailer sequences, intergenic sequences (IGS), and coding gene between 5 'and 3' UTR nucleotide, respectively. [RESULTS] The location and the length of the non-coding sequences highly conserve, and the variation trend of non-coding sequences is synchronous with the entire genomes and coding genes. [ CONCLUSION] The molecular variation of the coding gene was indistinguishable with the non-coding gene in view of the NDV genome.

Genetic and biological characterizations of a Newcastle disease virus from swine in China.

BACKGROUND: Newcastle Disease Virus (NDV) has been considered to only infect avian species. However, one paramyxovirus named as Xiny10 was isolated from swine. The differences of Xiny10, another previous swine NDV (JL01) and vaccine strain La Sota were compared on the basis of sequences of the whole-lengthen Fusion (F) gene and biological characteristics. FINDINGS: Through serologic tests and sequence alignment, Xiny10 was proved as NDV. It has great differences with JL01 in virulence, biological characteristics, genotype and amino acid homology of F gene. The sequence alignment showed Xiny10 and La Sota both belonged to genotype II. It shared 97.3% to 98.7% identities with genotype II NDVs, which was higher than these strains from the other genotypes. CONCLUSIONS: These above data suggested that the swine virus was NDV and it might be generated from La Sota.

Swine influenza H1N1 virus induces acute inflammatory immune responses in pig lungs: a potential animal model for human H1N1 influenza virus.

Pigs are capable of generating reassortant influenza viruses of pandemic potential, as both the avian and mammalian influenza viruses can infect pig epithelial cells in the respiratory tract. The source of the current influenza pandemic is H1N1 influenza A virus, possibly of swine origin. This study was conducted to understand better the pathogenesis of H1N1 influenza virus and associated host mucosal immune responses during acute infection in humans. Therefore, we chose a H1N1 swine influenza virus, Sw/OH/24366/07 (SwIV), which has a history of transmission to humans. Clinically, inoculated pigs had nasal discharge and fever and shed virus through nasal secretions. Like pandemic H1N1, SwIV also replicated extensively in both the upper and lower respiratory tracts, and lung lesions were typical of H1N1 infection. We detected innate, proinflammatory, Th1, Th2, and Th3 cytokines, as well as SwIV-specific IgA antibody in lungs of the virus-inoculated pigs. Production of IFN-γ by lymphocytes of the tracheobronchial lymph nodes was also detected. Higher frequencies of cytotoxic T lymphocytes, γδ T cells, dendritic cells, activated T cells, and CD4+ and CD8+ T cells were detected in SwIV-infected pig lungs. Concomitantly, higher frequencies of the immunosuppressive T regulatory cells were also detected in the virus-infected pig lungs. The findings of this study have relevance to pathogenesis of the pandemic H1N1 influenza virus in humans; thus, pigs may serve as a useful animal model to design and test effective mucosal vaccines and therapeutics against influenza virus.

Novel genetic reassortants in H9N2 influenza A viruses and their diverse pathogenicity to mice.

BACKGROUND: H9N2 influenza A viruses have undergone extensive reassortments in different host species, and could lead to the epidemics or pandemics with the potential emergence of novel viruses. METHODS: To understand the genetic and pathogenic features of early and current circulating H9N2 viruses, 15 representative H9N2 viruses isolated from diseased chickens in northern China between 1998 and 2010 were characterized and compared with all Chinese H9N2 viruses available in the NCBI database. Then, the representative viruses of different genotypes were selected to study the pathogenicity in mice with the aim to investigate the adaptation and the potential pathogenicity of the novel H9N2 reassortants to mammals. RESULTS: Our results demonstrated that most of the 15 isolates were reassortants and generated four novel genotypes (B62-B65), which incorporated the gene segments from Eurasian H9N2 lineage, North American H9N2 branch, and H5N1 viruses. It was noteworthy that the newly identified genotype B65 has been prevalent in China since 2007, and more importantly, different H9N2 influenza viruses displayed a diverse pathogenicity to mice. The isolates of the 2008-2010 epidemic (genotypes B55 and B65) were lowly infectious, while two representative viruses of genotypes B0 and G2 isolated from the late 1990s were highly pathogenic to mice. In addition, Ck/SD/LY-1/08 (genotype 63, containing H5N1-like NP and PA genes) was able to replicate well in mouse lungs with high virus titers but caused mild clinical signs. CONCLUSION: Several lines of evidence indicated that the H9N2 influenza viruses constantly change their genetics and pathogenicity. Thus, the genetic evolution of H9N2 viruses and their pathogenicity to mammals should be closely monitored to prevent the emergence of novel pandemic viruses.

Rapid detection of newly isolated Tembusu-related Flavivirus by reverse-transcription loop-mediated isothermal amplification assay.

BACKGROUND: From April 2010 to January 2011, a severe new viral disease had devastated most duck-farming regions in China. This disease affected not only laying ducks but also meat ducks, causing huge economic losses for the poultry industry. The objective of this study is to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of the new virus related to Tembusu-related Flavivirus. RESULTS: The RT-LAMP assay is very simple and rapid, and the amplification can be completed within 50 min under isothermal conditions at 63°C by a set of 6 primers targeting the E gene based on the sequences analysis of the newly isolated viruses and other closely related Flavivirus.The monitoring of gene amplification can also be visualized by using SYBR green I fluorescent dye. In addition, the RT-LAMP assay for newly isolated Tembusu-related Flavivirus showed higher sensitivity with an RNA detection-limit of 2 copies/µL compared with 190 copies/µL of the conventional RT-PCR method. The specificity was identified without cross reaction to other common avian pathogens. By screening a panel of clinical samples this method was more feasible in clinical settings and there was higher positive coincidence rate than conventional RT-PCR and virus isolation. CONCLUSION: The RT-LAMP assay for newly isolated Tembusu-related Flavivirus is a valuable tool for the rapid and real-time detection not only in well-equipped laboratories but also in general conditions.

[Prokaryotic expression and transmembrane transfer of fusion protein TAT-RIG-I-GFP].

We studied the construction of fusion protein TAT-RIG-I-GFP prokaryotic expression vector and verified the function of TAT in transmembrane delivery. First, four pairs of specific primers were designed, and the RIG-I gene of Mallard Duck (Anas platyrhynchos) was cloned. Then, the pET-TAT-RIG-I-GFP and pET-RIG-I-GFP prokaryotic expression vectors were constructed. Meanwhile, they were converted to E. coli BL21 (DE3), which were induced to be expressed after culture. After the purification of His-60 nickel affinity chromatography column and the identification of SDS-PAGE, the purified TAT-RIG-I-GFP and RIG-I-GFP proteins were incubated to DF-1 cells. Finally, fluorescence microscopy was used to observe whether the corresponding fluorescence was produced in DF-1 cells. The results showed that pET-TAT-RIG-I-GFP fusion with TAT showed obvious green fluorescence in DF-1 cells. However, the pET-RIG-I-GFP without TAT cannot display green fluorescence. This shows that TAT-fused protein have successfully delivered DF-1 cells and play a key role in transmembrane delivery. In conclusion, these results provide a solid material basis for further study of antiviral drugs in poultry.

Pathotypical characterization and molecular epidemiology of Newcastle disease virus isolates from different hosts in China from 1996 to 2005.

Thirty Newcastle disease virus (NDV) strains isolated from outbreaks in China during 1996 to 2005 were characterized pathotypically and genotypically. All strains except one were velogenic. An analysis of the variable region (nucleotides 47 to 420) of the F gene indicated that 6 isolates belonged to genotype II, 3 to genotype III, 1 (isolated from a pigeon) to genotype VI, and 20 to genotype VII. Isolates belonging to genotype VII were further divided into five subtypes, VIIa, VIIb, VIIc, VIId, and VIIe, and subtype VIId was made up of VIId1 to VIId5. These results showed that genotype VII isolates might have been the most prevalent in China during the past two decades. Genotype VII isolates shared high homology, but the homology was less than that between genotype VII viruses and the vaccine virus LaSota. Among these NDV isolates, 25 isolates had the velogenic motif (112)R/K-R-Q-K/R-R-F(117) that is consistent with results of the biological tests. However, four of five LaSota-type isolates that contained the lentogenic motif (112)G-R-Q-G-R-L(117) were velogenic, except SY/03, in the view of the biological test. The majority of genotype VII isolates had lost one or two N-glycosylation sites. Finally, a cross-protection experiment in which specific-pathogen-free chickens vaccinated with LaSota were challenged by six NDV isolates showed that more than three isolates were antigenic variants that could be responsible for recent outbreaks of Newcastle disease.

Molecular characterization of three new virulent Newcastle disease virus variants isolated in China.

Three cases of Newcastle disease virus (NDV) found in nature had the lentogenic motif (112)G-R-Q-G-R-L(117) in their fusion protein cleavage sites. However, both intracerebral pathogenicity and intravenous pathogenicity indexes showed that these NDV isolates were virulent. In comparison with the LaSota live virus vaccine, these viruses had significant genetic variations in the hemagglutinin-neuraminidase gene.

F gene recombination between genotype II and VII Newcastle disease virus.

A velogenic Newcastle disease virus (NDV) strain, designated as SRZ03, was isolated from an egg layer flock with NDV vaccine immunization failure in China in 2003. Recombination was found in the F gene of SRZ03. Complete genome sequences analysis indicated that the N-terminal of SRZ03 F gene originated from a genotype II NDV strain, whereas the C-terminal of F gene and the rest of the genes originated from a prevalent velogenic genotype VII NDV strain. It provides us valuable information for understanding the recombination of nonsegmented negative-sense RNA viruses.

[Correlation of the neutralization index in chicken embryo with the homologies of F and HN gene of different Newcastle-disease isolates].

Thirteen prevailed Newcastle-disease viruses (NDV) isolated in China during 2001-2004 were purified by chick embryo fibroblast (CEF) plaque assay and characterized pathotypically and genotypically. The biological tests showed that these viruses were highly virulent. Sequence analysis based on the variable region (nucleotide 47-420) of the F gene indicated that of the 13 NDV isolates 2 belonged to genotype II, 2 to genotype IX and 9 to genotype VII. Isolates with genotype VII shared 94.6%-99.3% nucleotide (nt) homology with the F gene, whereas for genotype VII and La Sota was only 82.7%-84.1%. In addition, these NDV isolates all shared 95.2%-100% nt homology with the hemagglutinin-neuraminidase (HN) gene, whereas only 79.1%-84.3% compared these viruses with La Sota. The cross neutralization assays were done using positive serums in specific pathogen free (SPF) chicken embryos respectively. Correlation of the neutralization index in chicken embryo with the homologies of F and HN gene of different NDV isolates were analyzed by SPSS8.0 software. The result showed that the neutralization index was closely correlated with nt sequence (P < 0.01, r = 0.35) or deduced amino acid sequence (P < 0.01, r = 0.34) of the HN gene, whereas weekly correlated (P < 0.05, r = 0.20 or 0.19) with the F gene, and non-correlated with 374 nt segment. This implied that the genetic mutations of HN resulted in antigenic variations of these viruses and the search for new vaccines would be necessary.

Role of serotonin in the intestinal mucosal epithelium barrier in weaning mice undergoing stress-induced diarrhea.

Stress-induced diarrhea is a frequent and challenging threat to humans and domestic animals. Serotonin (5-HT) has been shown to be involved in the pathological process of stress-induced diarrhea. However, the role of 5-HT in stress-induced diarrhea remains unclear. A stress-induced diarrhea model was established in 21-day-old ICR weaning mice through an intragastric administration of 0.25 mL of 0.4 g/mL folium sennae and restraint of the hind legs with adhesive tape for 4 h to determine whether 5-HT regulates the mucosal barrier to cause diarrhea. Mice with decreased levels of 5-HT were pretreated with an intraperitoneal injection of 300 mg/kg p-chlorophenylalanine (PCPA), a 5-HT synthesis inhibitor. After 5 days of treatment, the stress level, body weight and intestinal mucosal morphology indexes were measured. Compared to the controls, the mice with stress-induced diarrhea displayed a stress reaction, with increased corticosterone levels, as well as increased 5-HT-positive cells. However, the mice with stress-induced diarrhea exhibited decreased body weights, villus height to crypt depth ratios (V/C), and Occludin and Claudin1 expression. The PCPA injection reversed these effects in mice with different degrees of stress-induced diarrhea. Based on these findings, inhibition of 5-HT synthesis relieved the stress response and improved the health of the intestinal tract, including both the intestinal absorption capacity, as determined by the villus height and crypt depth, and the mucosal barrier function, as determined by the tight junction proteins of epithelial cell.

[Study on immune efficacy of Newcastle disease chitosan microsphere vaccine].

Newcastle disease is an acute and highly contagious disease caused by Newcastle disease virus (NDV), one of which does great harms to the poultry industry. The most basic measure of controlling New Castle disease is to alid vaccine, now we usually use La Sota live vaccine and inactivated NDV vaccine, but these two vaccines both have more or less limitation. It can produce higher mucosal immunity titers by taking vaccine orally, meanwhile it can induce humoral and cell-mediated immune response and mucosal immunity strongly. Therefore, it becomes the focus of the research, which prepare new pattern vaccines taking orally. NDV chitosan microsphere vaccine was prepared using chitosan as capsule wall material, NDV as core material, glutaraldehyde as cross-linking material, and its even particle diameter was 5.83um, and its surface was smooth and glossy, no obviously pore space, yellow brown pykno-ball, and its safety and potency were evaluated. The SPF chickens were immunized with NDV chitosan microsphere vaccine, La Sota live vaccine and inactivated NDV vaccine respectively. To evaluate vaccine's immune efficacy, using MTT to measure lymphocytes proliferation in vitro, using HI to measure serum special IgG and using ELISA tests to detect mucosal sIgA titers. The results show that NDV chitosan microsphere vaccine was safe, could induce humoral and cell-mediated immune response and mucosal immunity strongly. The results of the potency tests conformed that the vaccine could produce good protective effect.

[Newcastle disease virus heredity mutation and correlation of HN and F gene].

Prevailed Newcastle disease virus isolates were collected during 1999-2005 in China. These isolates were purified by CEF plaque assay and replicated in SPF embryos. The fusion protein (F) gene and hemagglutinin-neuraminidase (HN) gene of these isolated viruses were cloned and sequenced. Some of the F gene and HN gene sequences from GenBank were also used in this study. The homologies of nucleotide and amino acids were compared and correlations were analyzed by SPSS8.0 software among different length sequences of the F gene or HN gene. The nucleotide homologies and correlation among the F gene and HN gene were also analyzed. The results indicated there are good correlation among different length sequences of the F gene or HN gene and the F genome or HN genome (r > or = 0.973). There was also good correlations among different length amino acids of NDV F protein or HN protein (0.911< or = r < or = 0.968). But, there was only a less correlation between the whole F gene and HN gene (r = 0.312). The heredity mutation of HN genes had the character of geographical areas. The sequences of HN gene in Chinese isolates had an identity of more than 97%. But there was only 79.2% - 80.7% in HN nucleotide homology among the Chinese isolates and La Sota (vaccine).

[Molecular characterization of a genotype II virulent Newcastle disease virus isolate from chicken].

Newcastle disease virus (NDV) field strain SQZ04 was isolated from a broiler flock with typical symptoms and lesions, and cloned by plaque-purification three times. NDV SQZ04 was determined as a virulent strain with MDT of 50.5h and 51.2h, ICPI of 2.0 and 1.92, IVPI of 2.8 and 2.68 respectively before and after plaque-purification. Analysis of F gene indicated that SQZO4 was determined as a virulent gene type II , and its protein amino acid sequence has homologies of 99.3% , 98.7% and 96.9% with published gene type II vaccine strains LaSota, B1, virulent strain Taxas48,much higher than homologies of 88.3% - 88.6% or 91.3% - 92.1% with published gene types VI and IX. This is the first virulent field strain of gene type UI reported in China. Further more, the amino acid sequence 111 GGRQGRL117 in the F protein cleavage site in SQZ04 strain is identical to lentogenic strains of NDV, such as vaccine strains LaSota, B1. This is the first report that virulent NDV could have lentogenic amino acid sequence in the cleavage site of F protein, where HN genes was compared SQZ04 has a higher homologies of 95.3%- 97.3% with known velogenic strains, but lower homologies of 87.8% - 89.5% with published lentogenic strains.

Molecular Evolution and Genetic Analysis of the Major Capsid Protein VP1 of Duck Hepatitis A Viruses: Implications for Antigenic Stability.

The duck hepatitis A virus (DHAV), a member of the family Picornaviridae, is the major cause of outbreaks with high mortality rates in young ducklings. It has three distinctive serotypes and among them, serotypes 1 (DHAV-1) and 3 (DHAV-3) were recognized in China. To investigate evolutionary and antigenic properties of the major capsid protein VP1 of these two serotypes, a primary target of neutralizing antibodies, we determined the VP1 coding sequences of 19 DHAV-1 (spanning 2000-2012) and 11 DHAV-3 isolates (spanning 2008-2014) associated with disease outbreaks. By bioinformatics analysis of VP1 sequences of these isolates and other DHAV strains reported previously, we demonstrated that DHAV-1 viruses evolved into two genetic lineages, while DHAV-3 viruses exhibited three distinct lineages. The rate of nucleotide substitution for DHAV-1 VP1 genes was estimated to be 5.57 x 10(-4) per site per year, which was about one-third times slower than that for DHAV-3 VP1 genes. The population dynamics analysis showed an upward trend for infection of DHAV-1 viruses over time with little change observed for DHAV-3 viruses. Antigenic study of representative DHAV-1 and DHAV-3 strains covering all observed major lineages revealed no detectable changes in viral neutralization properties within the serotype, despite the lack of cross-neutralization between serotypes 1 and 3 strains. Structural analysis identified VP1 mutations in DHAV-1 and DHAV-3 viruses that underpin the observed antigenic phenotypes. Results of our experiments described here shall give novel insights into evolution and antigenicity of duck picornaviruses.

Genomic and phylogenetic characterization of novel, recombinant H5N2 avian influenza virus strains isolated from vaccinated chickens with clinical symptoms in China.

Infection of poultry with diverse lineages of H5N2 avian influenza viruses has been documented for over three decades in different parts of the world, with limited outbreaks caused by this highly pathogenic avian influenza virus. In the present study, three avian H5N2 influenza viruses, A/chicken/Shijiazhuang/1209/2013, A/chicken/Chiping/0321/2014, and A/chicken/Laiwu/0313/2014, were isolated from chickens with clinical symptoms of avian influenza. Complete genomic and phylogenetic analyses demonstrated that all three isolates are novel recombinant viruses with hemagglutinin (HA) and matrix (M) genes derived from H5N1, and remaining genes derived from H9N2-like viruses. The HA cleavage motif in all three strains (PQIEGRRRKR/GL) is characteristic of a highly pathogenic avian influenza virus strain. These results indicate the occurrence of H5N2 recombination and highlight the importance of continued surveillance of the H5N2 subtype virus and reformulation of vaccine strains.