Biological Characteristics of H9N2 Avian Influenza Viruses from Healthy Chickens in Shanghai, China.

BACKGROUND H9N2 avian influenza viruses that circulate in domestic poultry in eastern China pose challenges to human health. However, few studies have compared the biological characteristics of H9N2 viruses isolated from healthy chickens in Shanghai. MATERIAL AND METHODS Three H9N2 viruses - CK/SH/Y1/07, CK/SH/Y1/02, and CK/SH/23/13 - isolated from healthy chickens in Shanghai between 2002 and 2013, were selected and their biological characteristics were determined. RESULTS All 3 H9N2 viruses showed a preference for both the avian- and human-like receptors, and they replicated well in MDCK and A549 cells. All H9N2 viruses were non-pathogenic to mini-pigs and were detected in the trachea and lung tissues. The CK/SH/Y1/07 and CK/SH/Y1/02 viruses were transmitted to mini-pigs through direct-contact or respiratory droplet exposure, but CK/SH/23/13 virus was not. CONCLUSIONS These results suggest that H9N2 viruses isolated from healthy chickens in Shanghai efficiently replicate and transmit among pigs and other mammals.

Detection and identification of viral pathogens in patients with hand, foot, and mouth disease by multilocus PCR, reverse-transcription PCR and electrospray ionization mass spectrometry.

BACKGROUND: Rapid detection and identification of viruses are important for early diagnosis and effective surveillance of hand, foot, and mouth disease (HFMD). We described a novel assay using multilocus PCR and reverse transcription-PCR coupled with electrospray ionization mass spectrometry (RT-PCR/ESI-MS) to simultaneously detect and identify human enterovirus A-D, adenovirus A-F, human herpesvirus 1-8, parvovirus B19 and polyomavirus. OBJECTIVES: To evaluate the accuracy and efficacy of the RT-PCR/ESI-MS method, to detect and type enterovirus from specimens of clinical diagnosed HFMD patients. STUDY DESIGN: In this study, 152 specimens of clinically diagnosed HFMD patients were studied by the assay using RT-PCR/ESI-MS method. The detection and typing of enterovirus by RT-PCR/ESI-MS were compared with results from reference molecular methods. RESULTS: The assay detected enteroviruses in 97 (63.8%) specimens, resulting in a sensitivity of 93.8% (95% CI: 91.8-95.7%) and a specificity of 87.5% (95% CI: 84.8-90.2%) compared with a reference clinical diagnostic test. Most enterovirus genotypes (65/84; 77%) determined by the platform were consistent with 5' UTR sequence analysis, and most misidentifications resulted from the virus library, which could be further improved by updating the enterovirus database. In addition to enteroviruses, herpesviruses, polyomaviruses, adenoviruses and human rhinoviruses were detected and identified in 55 (36%) HFMD specimens by RT-PCR/ESI-MS. CONCLUSION: With the capability of high throughput and detection and typing of multiple clinically relevant viruses simultaneously, RT-PCR/ESI-MS can be a rapid and robust laboratory tool for identifying viral pathogens.

[Correlation between acute schistosomiasis japonica and serum interleukin-33 levels].

OBJECTIVE: To study the function of interleukin-33 (IL-33) in the development of human acute schistosomiasis japonica by determining the serum IL-33 levels in acute schistosomiasis japonica patients. METHODS: Four patients with acute schistosomiasis japonica were recruited from schistosomiasis endemic lake areas, and 15 controls were recruited outside the schistosomiasis endemic areas. The demographic data and venous blood were collected from all the subjects. The serum IL-33 levels of all the subjects were tested by using enzyme-linked immunosorbent assay. All the results were statistically analyzed with Stata 10.0 software. RESULTS: The serum IL-33 levels of the patients with acute schistosomiasis japonica [517.33 (334.65, 1 056.88) pg/ml] were significantly higher than those of the controls [1.66(1.66, 6.35) pg/ml] (Z = -3.207, P = 0.001). The correlation coefficients between serum IL-33 levels and numbers of eosinophils, serum IL-33 levels and duration of infection were both 0.8 (P = 0.2). CONCLUSIONS: The serum IL-33 level is significantly elevated in the patients with acute schistosomiasis japonica, indicating that IL-33 may play a pro-inflammatory role in the acute stage of schistosomiasis japonica and participate in initiating the Th2 type immune responses between 7 and 9 weeks after the infection.

Expression and immunogenicity of novel subunit enterovirus 71 VP1 antigens.

Hand, foot, and mouth disease (HFMD) is a common viral illness in young children. HFMD is caused by viruses belonging to the enterovirus genus of the picornavirus family. Recently, enterovirus 71 (EV71) has emerged as a virulent agent for HFMD with severe clinical outcomes. In the current report, we conducted a pilot antigen engineering study to optimize the expression and immunogenicity of subunit VP1 antigen for the design of EV71 vaccines. DNA immunization was adopted as a simple technical approach to test different designs of VP1 antigens without the need to express VP1 protein in vitro first. Our studies indicated that the expression and immunogenicity of VP1 protein can be improved with alternated VP1 antigen designs. Data presented in the current report revealed novel pathways to optimize the design of VP1 antigen-based EV71 vaccines.

[Heat impact upon the infectivity of hepatitis B virus in serum].

OBJECTIVE: This article was to explore the impact of temperature on hepatitis B virus infectivity. METHODS: HBV positive serum with a HBV DNA titer of 1.33 × 10(8) copies/ml was aliquots into 23 Ep tubes with 1.5 ml, 100 µl in one tube.15 tubes were incubated at 37, 56 and 65°C for 0, 30, 60, 120 and 600 minutes, respectively. The other 8 tubes were incubated at 98°C for 0, 5, 10 and 30 minutes, respectively. Post-treated serum at all time points were selected to infect HepG-2 cell. When 18 hours after infection, these cells were extensively washed with phosphate buffered saline. Cells were harvested after the addition of fresh culture medium to culture cells for 48 hours. HBV DNA was detected by FQ-PCR. RESULTS: HBV DNA was detected in cells that were infected by serum at 37°C and 56°C for 30, 60, 120 and 600 minutes, respectively. The titers for the cells incubated at 37°C were (4.85 ± 1.71) × 10(5), (3.85 ± 1.76) × 10(5), (1.67 ± 0.67) × 10(5), (7.86 ± 1.03) × 10(4) copies/ml, and those for the cells incubated at 56°C were (4.01 ± 0.16) × 10(5), (9.77 ± 0.97) × 10(4), (6.36 ± 0.65) × 10(4), (5.05 ± 0.24) × 10(3) copies/ml at different incubation time points. For the cells incubated at 65°C for 60 and 120 minutes, HBV DNAs were (5.15 ± 7.28) × 10(3) and (7.56 ± 10.60) × 10(2) copies/ml, respectively, which were much lower than those in the controls cells ((6.79 ± 1.48) × 10(5) copies/ml). The results of HBV DNA were different (F = 104.4, P < 0.001) in groups treated with different temperature, and results of HBV DNA were also different (F = 144.0, P < 0.001) in groups processed for different period of time. Temperature and processing time had interaction (F = 23.6, P < 0.001). After heating at 98°C for 10 minutes and boiling for 5 minutes, the HBV DNA copy number ((3.02 ± 4.26) × 10(2), (4.31 ± 6.09) × 10(2) copies/ml) in infected cells decreased by about 10 folds than that in the control group ((6.79 ± 1.48) × 10(5) copies/ml). HBV DNAs were not detected in cells that were infected by serum which was heated at 98°C for 30 minutes and boiled for 10 minutes. CONCLUSION: The infectivity of HBV serum in vitro was relatively stable at low temperature, and it would lose its infectivity in short period of time at high temperature.

Antigenic and genetic variation in the hemagglutinins of H1N1 and H3N2 human influenza a viruses in the Shanghai area from 2005 to 2008.

Continued rapid evolution of the influenza A virus is responsible for annual epidemics and occasional pandemics in the Shanghai area. In the present study, the representative strains of A/H1N1 and A/H3N2 influenza viruses isolated in the Shanghai area from 2005 to 2008 were antigenically and genetically characterized. The antigenic cartography method was carried out to visualize the hemagglutination-inhibition data. Antigenic differences were detected between circulating A/H1N1 strains isolated from 2005 to 2006 and the epidemic A/H1N1 strains isolated in 2008, which were found to be associated with the amino acid substitution K140E in HA1. The present vaccine strain A/Brisbane/59/2007 is considered to be capable of providing sufficient immunity against most of the circulating A/H1N1 viruses isolated in 2008 from the Shanghai population. The study showed that there were significant antigenic differences between the epidemic A/H3N2 strains isolated in 2007 and 2008, suggesting that antigenic drift had occurred in the A/H3N2 strains isolated in 2008. The P194L mutation was thought to be responsible for the antigenic evolution of influenza A/H3N2 viruses isolated from Shanghai in 2008. Evidence of antigenic drift suggests that the influenza A/H3N2 vaccine component needs to be updated.

The epidemiology of hospitalized influenza in children, a two year population-based study in the People's Republic of China.

BACKGROUND: The epidemiology and disease burden of annual influenza in children in mainland People's Republic of China have not been reported in detail. To understand the incidence and epidemiology of laboratory-proven influenza hospitalization in children in China, a review of available laboratory and hospital admission data was undertaken. METHODS: We conducted a retrospective population-based study in Suzhou and the surrounding area of Jiangsu province, China for hospitalized cases of respiratory illness at Suzhou Children's Hospital. Cases of pneumonia or respiratory illness were identified from hospital computer data bases. Routine virological testing by fluorescent monoclonal antibody assay of all hospitalized children identified influenza and other viruses. We calculated incidence rates using census population denominators. RESULTS: Of 7,789 specimens obtained during 2007 and 2008, 85 were positive for influenza A and 25 for influenza B. There were 282 specimens with parainfluenza virus and 1392 with RSV. Influenza occurred throughout the year, with peaks in the winter, and in August/September. Overall estimated annual incidence of laboratory-proven influenza hospitalization was 23-27/100,000 children 0-4 years old, and 60/100,000 in infants 0-6 months, with an average hospitalization of 9 days. CONCLUSIONS: Influenza disease in young children in this part of China is a relatively common cause of hospitalization, and occurs throughout the year. The use of influenza vaccine in Chinese children has the potential to reduce the effect of influenza in the children, as well as in their communities. Studies are needed to further assess the burden of influenza, and to develop and refine effective strategies of immunization of young children in China.

Co-infection with influenza A/H1N1 and A/H3N2 viruses in a patient with influenza-like illness during the winter/spring of 2008 in Shanghai, China.

Co-infection with different influenza viruses occurs naturally and plays an important role in epidemiology and pathogenicity. To monitor the prevalence of influenza viruses in humans during seasonal influenza epidemics in Shanghai, China, and to analyze the genetic characteristics of the viruses, 365 nasopharyngeal swabs collected from patients with influenza-like illness between January and April 2008, were tested by a colloidal gold assay, viral isolation in Madin-Darby canine kidney (MDCK) cells, direct immunofluorescence assay and multiplex RT-PCR. The genetic characteristics of the viruses were analyzed by full-length PCR amplification of the HA segment. One case of co-infection with influenza A/H1N1 and A/H3N2 viruses was detected among the 7 cases of A/H1N1, 84 cases of A/H3N2 and 48 cases of influenza B virus during the winter/spring of 2008. All influenza A/H1N1 and A/H3N2 isolates were similar, including the co-infecting isolates. The present study demonstrates the possibility of natural co-infection with different types of influenza viruses in humans, which could provide the opportunity for the occurrence of viral genetic reassortment within the human respiratory tract.

[Study on evolutionary origin of influenza A virus (H1N1) based on HA gene].

OBJECTIVE: To determine the evolutionary rate and divergence time of influenza A virus HA gene isolated recently worldwide pandemic and explore the origin and its transmission. METHODS: A total of 344 H1 sequences available in the GenBank (including 248 isolated from human, 84 from swine, 11 from avian, and 1 from ferret) and 7 isolated in Shanghai were collected. The nucleotide substitution rate and time to most recent common ancestor (tMRCA) was calculated using molecular clock theory and Bayesian Skyline Plot (BSP) based on Markov chain Monte Carlo. Then genetic phylogeny was constructed referring to posterior distribution. RESULTS: It was found that H1 sequences in the US from human, swine and avian were clustered significantly with swine H1 ones from Asia phylogenetically (Cluster US). The second cluster (Cluster Eurasian Human) nearly consisted of human H1 sequences isolated in other regions. The third cluster (Cluster Eurasian Animal) consisted of swine and avian H1 sequences from China and Italy respectively. As for all the H1 sequences, the evolutionary rate was of 2.57 x 10(-3) substitutions/site per year averagely (95% Highest Posterior Density: 1.96 x 10(-3) - 3.03 x 10(-3)/site per year). The estimated dates for tMRCA of human H1 in Europe and swine H1 in the mainland of China were the earliest, with the corresponding rates of 6.46 x 10(-3)/site per year and 0.97 x 10(-3)/site per year respectively. The tMRCAs of human and swine H1 sequences from the US were similar, with the rates of 5.86 x 10(-3)/site per year and 5.02 x 10(-3)/site per year. CONCLUSION: The present flu outbreak was possibly induced by long-term circulation of influenza A virus (H1N1) in human population and swine herds in America. There was no evidence proving that influenza virus in China involved in the present outbreak.

[Type and subtype distribution of influenza virus and genetic evolution of hemagglutinin in Shanghai area in duration of 2004 - 2008].

OBJECTIVE: To analyze the type and subtype distribution of influenza virus and the genetic evolution of hemagglutinin (HA) in Shanghai area during 2004 to 2008. METHODS: All 962 throat swabs were collected from influenza-like patients in 5 influenza sentry hospitals and influenza outbreaks. Influenza viruses were isolated in MDCK cell lines, and then viral types and subtypes were identified. The HA of influenza A isolates selected by outbreak or sporadic patients in different areas and epidemic seasons were sequenced and analyzed by phylogenetic trees. RESULTS: A/H3N2, accounting for 54.9% (162/295), was the dominate subtype in recent years, but less popular in the end of 2005 to the middle of 2006 with 0% (0/16)and 23.5% (8/34) of positive specimen, respectively. There were more A/H1N1 isolates in 2005 - 2006 with 21.4% (12/56), 43.8% (7/16) and 76.5% (26/34) of positive specimen, respectively, but declined obviously in 2007 - 2008 accounting for only 0% (0/44) and 5.0% (7/139). Influenza B virus was more popular in 2004 to 2005 with 42.9% (24/56) and 56.2% (9/16), respectively, and not isolated from 2006 to 2007, then increased in 2008 accounting for 34.5% (48/139). Phylogenetic tree of HA showed that A/H1N1 isolates in the same year clustered from 2005 to 2008, and most A/H3N2 isolated were homologous in the same year during 2004 - 2008 while some were inserted to the clusters of near years and more distinguished sequences appeared. A/H1N1 and A/H3N2 isolates were all similar to the vaccine strains recommended by WHO. CONCLUSION: The distribution of influenza type and subtype kept on changing each year, but A/H3N2 dominated in most years. A/H1N1 and A/H3N2 in the same year clustered, but some A/H3N2 of near years were and evolved faster with more distinguished strains appeared in same interval. Generally, HA of influenza A isolates in Shanghai during 2004 to 2008 were similar to the WHO reference strains.

[Etiology monitoring of influenza virus and variation in hemagglutinin genes of H13 subtype in Wuxi area].

OBJECTIVE: To monitor the seasonal distribution of influenza types and subtypes in Wuxi area during 2005-2008, and to investigate the variation in hemagglutinin (HA) genes of A/H3N2 strains in 2008. METHODS: Nose-throat swab specimens were collected in Wuxi area from flu-like patients from outpatient departments of hospitals as well as from clustering flu-like outbreak patients from workspace, followed by MDCK cell inoculation. Types and subtypes of positive influenza isolates were identified using standard antiserum. We then sequenced the HA genes for H3 subtype influenza viruses isolated from 2008 specimens to investigate the variation in HA genes. RESULTS: During 2005 and September 2008, 435 strains of influenza viruses were isolated from flu-like patients in Wuxi Area, among which 164 isolates are of A/H1N1 subtype, 80 isolates are of A/H3N2 subtype, and 191 isolates are of B type. These types/subtypes have significant seasonal distributions. Sequences of HA genes for H3 subtype show that the 9 strains isolated in Wuxi area are similar to those of strains isolated in Shanghai within the same period. Many of the sequences belong to the same branch of the phylogenetic tree, and are similar to sequences of vaccine strains in WHO 2008-2009 repositories. CONCLUSION: A/H1N1, A/H3N2 and B still attribute to most of the sporadic and local outbreaks of influenza infection in Wuxi area in recent years. HA genes of A/H3N2 strains isolated in Wuxi area are similar to those of strains isolated in Shanghai in the same period, and also similar to those of vaccine strains recommended by WHO for 2008-2009.

[Sequence analysis on measles viruses isolated in Shanghai in 2005].

OBJECTIVE: To ascertain the genetic characterization and genotype of measles viruses isolated in Shanghai region, in 2005. METHODS: Measles virus was isolated from throat swab specimens collected from suspected measles cases and 450 bp fragment of C terminus of nucleprotein (N) gene was amplified by RT-PCR. Sequence analysis was conducted to ascertain the genotype and to compare the difference of nucleotide with other measles virus strain published in GenBank. RESULTS: 4 measles viruses were isolated from 10 throat swab specimens, and the sequence analysis indicated that they belonged to H1 genotype. The homogeneity of 450 nucleotides in the C terminal of the N gene was at 98%-98.2% as compared to H1 genotype (China93-7). They differed from genotype H2 (China94-1) at 6.4%-6.9% and from genotype A (Edmonston) at 6.7%-6.9%, from measles vaccine (Shanghail91) at 7.6%-8.0%. They differed from the other measles viral strain isolated in China in 1993 - 2005 at 0.2%-3.7%. The variation within 4 isolated measles viruses was at 0.7%-1.3%. CONCLUSION: It was H1 genotype measles viruses,which are the native viruses in China that led to the outbreak of measles in Shanghai, in 2005.

Serologic study on the outbreak of acute upper respiratory tract infections caused by adenovirus 3.

From April to June, 2004, an outbreak of acute upper respiratory tract infections (AURTI) occurred in the north area of Jiangsu Province, China. Twenty throat swabs were collected with 13 of them presenting an adenovirus (Ad)-like cytopathogenic effect on HEp-2. These were verified as Ad by the electron microscope, direct immunofluorescence assay and Ad primer-mediated PCR. Moreover, they were identified as adenovirus type 3 (Ad3) by type-specific PCR and sequencing of the amplification products. Subsequent serologic studies were carried out to finally diagnose and document the outbreak. The neutralization test of paired serum of six in nine cases show obviously increased antibodies titers. The positive rate of IgM, IgG and recovery phase neutralization antibodies of the cases were 3.7%, 44.4% and 59.5% respectively while those of the controls were 0%, 8.3% and 33.3% respectively. The P values of Chi-Square were 0.510, 0.018 and 0.226 respectively. The concordance between IgG detected by ELISA and neutralization anti bodies detected by the neutralization test was 61.4% and the P value of Kappa was 0.070. By the serologic study, we can definitively diagnose that this outbreak of acute respiratory infections was caused by Adenovirus 3.

[Application of gene sequence cluster in research for H3 antigenic evolution of influenza A virus].

OBJECTIVE: Gene sequence data were clustered to explore evolution lineages of H3 antigen of influenza A virus. METHODS: All data of H3 RNA sequence in NCBI Genbank and Influenza sequence database were downloaded and aligned in ClustalX while two step cluster method were applied to explore the data. RESULTS: All sequences were aggregated into ten clusters, while seven of them mainly were human virus. Human virus and avian/other mammal virus were separated into different clusters distinctively, but coexisted into same clusters with swine virus. Time and host distribution were very distinctive in these clusters, but no geographic distribution features were found. CONCLUSION: With the interaction of human immunity system, H3 antigen mutated significantly every 5 - 7 years, and the speed of mutation had accelerated with the application of influenza vaccines in recent years. Mean while, human and swine influenza virus were not separated distinctly between clusters indicating that they had short inheritance distance. Result showed again that swine served as the mixer for antigenic recombination of different influenza virus.