[Chlamydia-like and coronavirus-like agents found in dead cases of atypical pneumonia by electron microscopy].

OBJECTIVE: To explore the causative agents of the atypical pneumonia (also SARS) occurred recently in some regions of our country. METHOD: Organ samples of 7 dead cases of SARS were collected from Guangdong, Shanxi, Sichuan Provinces and Beijing for electron microscopic examination. 293 cell line was inoculated with the materials derived from the lungs to isolate causative agent(s). The agents in the organs and cell cultures were revealed by immunoassay. RESULTS: Both Chlamydia-like and coronavirus-like particles were found in EM. Inclusion bodies containing elementary bodies, reticulate antibodies and intermediate bodies of Chlamydia-like agent were visualized in multiple organs from the 7 dead cases, including lungs (7 cases), spleens (2 cases), livers (2 cases), kidneys (3 cases) and lymph nodes (1 cases), by ultrathin section electron microscopy (EM). In some few sections, coronavirus-like particles were concurrently seen. A coronavirus RNA- polymerase segment (440 bp) was amplified from the lung tissues of two cases of the SARS. After inoculated with materials from the lung samples, the similar Chlamydia-like particles were also found in the inoculated 293 cells. Since the Chlamydia-like agents visualized in both organs and cell cultures could not react with the genus specific antibodies against Chlamydia and monoclonal antibodies against C. pneumoniae and C. psittaci, the results might well be suggestive of a novel Chlamydia-like agent. CONCLUSION: Since the novel Chlamydia-like agent was found co-existing with a coronavirus-like agent in the dead cases of SARS, it looks most likely that both the agents play some roles in the disease. At the present time, however, one can hardly determining how did these agents interact each other synergetically, or one follows another, need further study.

[Study on the antigenicity and HA1 gene characteristics of influenza A viruses during 2004-2008 year in China].

OBJECTIVE: To under stand influenza A viruses epidemic, antigenicity and genetic characteristics variation between the vaccine and Circulation strains during 2004-2008 year in China. METHODS: The influenza A viruses (H1N1, H3N2) isolated from 2004-2008 year were under took antigenic and sequence analysis. Influenza A virus antigenicity and genetic characteristics were analyzed thought amino acid variation compassion of HA1 protein of influenza A virus isolates. RESULTS: The antigenicity of influenza H1N1 subtype viruses isolated from 2004 to 2007 is very similar with vaccine strain A/New Caledonia/20/1999 (HIN1)-like virus. The influenza H1N1 viruses circulated in 2008 year had similar antigenic characteristics with A/Brisben/59/2007 (H1N1) which is component of influenza vaccines for northern hemisphere 2008-2009 year. The influenza H3N2 subtype viruses of 2004-2005 year had antigenic variation comparatively with vaccine strain A/Fujian/411/12002 (H3N2), The antigenicity of 2006-2007 H3N2 viruses and 2008 year's is A/Wiscansin/67/2006(H3N2) and A/ Brisben/10/2006(H3N2) respectively. CONCLUSION: There is change of influenza A viruses (H1N1, H3N2) antigenic and genetic characteristics during 2004-2008 in China.

[Construction and characterization of reverse genetic system for H9N2 avian influenza].

OBJECTIVE: To provide a technology platform for vaccine development as well as the research on transmission and pathogenesis, the reverse genetic system for H9N2 avian influenza virus was established. METHODS: Eight full-length cDNAs of avian influenza virus A/Guangzhou/333/99 (H9N2) were amplified by RT-PCR and separately cloned into the transcription/expression vector, pCI-polI. The 8 plasmids DNA was cotransfected into 293T cell, the cell supernatant was collected and inoculated into embryonated eggs, the rescued virus from the allantoic fluid was identified by hemagglutinination assay. RESULTS: The avian influenza H9N2 virus was successfully rescued by 8 plasmids co-transfection in 293T cells. The hemagglutinination titer of the rescued virus is up to 2(9)/50 microl and its growth curve remained relatively as to the wild-type virus. CONCLUSION: The reverse genetic for avian influenza H9N2 subtype virus has been established successfully.

Development of a new candidate H5N1 avian influenza virus for pre-pandemic vaccine production.

BACKGROUND: Highly pathogenic H5N1 avian influenza viruses currently circulating in birds have caused hundreds of human infections, and pose a significant pandemic threat. Vaccines are a major component of the public health preparedness for this likely event. The rapid evolution of H5N1 viruses has resulted in the emergence of multiple clades with distinct antigenic characteristics that require clade-specific vaccines. A variant H5N1 virus termed clade 2.3.4 emerged in 2005 and has caused multiple fatal infections. Vaccine candidates that match the antigenic properties of variant viruses are necessary because inactivated influenza vaccines elicit strain-specific protection. OBJECTIVE: To address the need for a suitable seed for manufacturing a clade 2.3.4 vaccine, we developed a new H5N1 pre-pandemic candidate vaccine by reverse genetics and evaluated its safety and replication in vitro and in vivo. METHODS: A reassortant virus termed, Anhui/PR8, was produced by reverse genetics in compliance with WHO pandemic vaccine development guidelines and contains six genes from A/Puerto Rico/8/34 as well as the neuraminidase and hemagglutinin (HA) genomic segments from the A/Anhui/01/2005 virus. The multi-basic cleavage site of HA was removed to reduce virulence. RESULTS: The reassortant Anhui/PR8 grows well in eggs and is avirulent to chicken and ferrets but retains the antigenicity of the parental A/Anhui/01/2005 virus. CONCLUSION: These results indicate that the Anhui/PR8 reassortant lost a major virulent determinant and it is suitable for its use in vaccine manufacturing and as a reference vaccine virus against the H5N1 clade 2.3.4 viruses circulating in eastern China, Vietnam, Thailand, and Laos.

[Establishment of a method for rapid detection of the nucleic acid of the novel A (H1N1) influenza virus].

A new flu caused by a novel influenza A(H1N1) virus has spread over the United States, Mexico and more than 40 other countries. And because of the immediate global concern, WHO has announced that the current level of influenza pandemic alert is raised to phase 5, indicating approaching of an influenza pandemic. As patients suffering from the influenza A (H1N1) have the similar symptoms as patients with seasonal influenza, differential detection and identification of the influenza virus have to depend on specific laboratory tests. We have successfully developed a RT-PCR based method for detection of the influenza A (H1N1) virus, and had applied the method to detection of clinical samples.

[Laboratory confirmation of the first influenza A (H1N1) imported case in Mainland China].

The clinical throat swab specimen of an imported suspected case of influenza A (H1N1) was detec ted with real-time PCR, RT-PCR and subsequently confirmed by gene sequencing. The presence of influ enza A (H1N1) virus confirmed the first case with A (H1N1) infection in Mainland China.

[Detection of influenza viruses/avian influenza viruses and identification of virulence using a microarray].

OBJECTIVE: To establish the DNA microarray to detect influenza viruses and avian influenza viruses, and identify their virulence. METHODS: Hemagglutinin (HA), neuramidinase (NA) and nucleoprotein(NP) genes were chosen simultaneously as targets for designing a microarray used for detection of viruses and identification virulence. The nucleic acid were amplified by single primer amplication (SPA). And then its specificity,sensitivity and reproducibility were evaluated. RESULTS: The microarray was able to specially detect H1N1, H3N2, B influenza viruses and H5N1, H9N2 avian influenza viruses. Their limits were 8HAU, 16HAU, 32HAU, and 8HAU, 8HAU respectively. The limit for virulence was 32HAU. When samples were analyzed by both RT-PCR and microarray in parallel, the results agreed in 83.9% (47/56). CONCLUSION: The microarray can detect and distinguish five tested viruses, and especially identify virulence. It not only supplies an assistant tool for clinical diagnosis and control of infectious disease, but also is valuable for controlling and preventing outbreak of avian influenza epidemic.

[Antigenic and genetic study of influenza virus circulated in China in 2006].

OBJECTIVE: To analyse seasonal influenza epidemic situation in 2006, and to analyse the genetic and antigenic characteristics of viral hemagglutinin (HA) gene. METHODS: The single-way hemagglutination inhibition (HI) tests were used to test the antigenic characteristics of these viruses from influenza surveillance network, and the HA1 genes were sequenced based on the antigenic test results according to different isolation times and sites. RESULTS: The influenza virus types A and B co-circulated in 2006. influenza A H1N1 subtype and Victoria-like B influenza circulated preponderantly during this epidemic season. The HA1 gene sequence of H1N1 viruses showed that 192, 193, 196, 198 positions (located at antigenic site B) have an amino acid substitute, compared with the last circulating strain A/Hubeihongshan/53/2005(H1N1). Two amino acid changes at 142 and 144 positions compared with A/Yunnan/1145/2005 (H3N2). There was no change in influenza B viruses either Victoria-like B or Yamagata-like B virus, i.e . antigenic characteristics is analogous to B/shenzhen/155/2005 and B/tianjin/144/2005, respectively. CONCLUSION: The H1N1 and H3N2 influenza viruses had changing antigenic and genetic characteristics in 2006. Influenza virus types B did not change in 2006.

[Study on the correlation of human influenza A/H3N2 hemagglutinin gene variation and the epidemic from 1995 to 2005 in China].

To study the correlation of human influenza A/H3N2 hemagglutinin gene variation and the epidemic from 1995 to 2005 in China, 550 HA1 sequences of H3N2 viruses isolated in China were analyzed with phylogenetic tree. The results showed that the evolution of HA1 represented a long trunk with short side branches. The animo acid changes of HA1 mostly located at the antigenic sites or aside of them, but also may occur at the other sites simultaneously. The analysis also showed three possibilities of HA1 variation to cause H3N2 epidemic, the first is multiple site mutations happened simultaneously; the second is mutation sites happened gradually and then accumulated to multiple sites; the third is a single antigenic site mutation occurred simultaneously with the receptor binding site variation.

[Characteristic analysis of NA gene of human influenza viruses (H3N2) isolated from 1996 to 2005 in China].

The NA genes of 395 strains of human H3N2 influenza virus isolated from 1996 to 2005 in China were sequenced, analyzed with bioinformatics tools. The NA nucleotide sequence of phylogenetic tree showed a main evolution branch with multiple short side branches. The strains in the same year may be divided into several branches. There was an obvious lag between vaccine strains recommended by WHO and the Chinese circulating strains in phylogenetic tree of the NA nucleotide. The result also showed no amino acid deletion and insertion in the NA. In NA antigen sites, where including residues 197-199 aa, 431-434 aa and 339-347aa the mutation was higher, in contrast, the residues including 153 aa, 328-336 aa, 367-370aa and 400-403 aa, the mutation was lower. Besides the antigenic determinant sites, there also had the other amino acid mutated highly, such as 18, 23, 30, 93, 143, 208, 216, 221, 249, 265, 267, 307, 385 and 437 aa, among them 143 and 267 mutation were higher than that in antigenic determinant sites, their biological significance are not clear yet. The neuraminidase active-site residues in NA were highly conservative and the same were the disulphide bond and the glycosylation sites in NA. In conclusion, our analysis provides some information for influenza prevention and control and the NA inhibitor medicine application.

[Application of single radial hemolysis technique for diagnosis of influenza A (H5N1)].

BACKGROUND: To understand the optimal condition of single radial hemolysis (SRH) for diagnosis of avian influenza A (H5N1) virus in order that SRH could be performed in general laboratories. METHODS: The effect of different concentration of virus and species of red blood cells, as well as kind and concentration of agarose on testing sensitivity of SRH was determined. Meanwhile the sensitivity and specificity of this method were compared with those of micro-neutralization test. RESULTS: The optimal condition of SRH included the viral concentration of 1000 HA units per 0.1 ml packed chicken red blood cells, the agarose concentration of 1.0%, the compliment added into agarose-virus-rbc slides after diffusion of sera. The sensitivity and specificity of SRH were very similar to those of micro-neutralization test. Meanwhile, no cross reaction between antibodies, especially antibodies against N1 antigens, H5N1 and H1N1 viruses was detected. CONCLUSION: The sensitivity and specificity of SRH were very similar to those of micro-neutralization assay. SRH could be performed in normal laboratories and be used for testing large scale serum samples.

[Adamantane resistance among influenza A (H3N2) viruses isolated from the mainland of China].

BACKGROUND: To study the incidence of adamantane resistance among influenza A (H3N2) viruses isolated from the mainland of China since 1989 through our influenza surveillance system, and to provide more information for the clinical usage of adamantane drugs. METHODS: Totally 584 influenza A (H3N2) virus strains were randomly selected from our surveillance network since 1989, the adamantane drug resistance related gene M2 of all 584 strains was sequenced, and the drug sensitivity of viruses was also assayed by using biological methods in cells. RESULTS: No adamantane resistant strains were detected among the strains isolated from 1989 to 1999, but there was a surprisingly increased resistance rate of 56% in 2003 compared with 3.4% in 2002, and in 2005 the resistance rate increased to 77.6%. CONCLUSION: Over 50% of virus among the strains isolated showed adamantane resistance since 2003, and the incidence rate is increasing.

[Analysis of human H5N1 virus hemagglutinin gene isolated from the mainland of China].

BACKGROUND: To analyze the genetic and antigenic characteristics of human H5N1 virus isolated from the mainland of China. METHODS: The hemagglutinin (HA) gene of human H5N1 virus were sequenced and analyzed. RESULTS: The results of HA gene sequencing showed that all the virus isolates belong to the same group because of the high similarity, but they were different from the virus isolated from Thailand and Vietnam. The sequence data also showed that the receptor specificity and the connecting peptide between HA1 and HA2 are still avian influenza origin. CONCLUSION: The virus isolates from mainland of China until now belong to the same group and are different from the virus isolated from Thailand and Vietnam, and there is no evidence showing the human-avian influenza reassortant and recombination.

[Antigenic and genetic study of influenza virus B circulated in China in 2004-2005].

BACKGROUND: To analyze the genetic and antigenic characteristics of hemagglutinin (HA) gene of human influenza B virus isolated from the mainland of China since 2004-2005. METHODS: The single-way hemagglutinin inhibition (HI) tests were used to test the antigenic characteristics, and the HA1 gene was sequenced based on the antigenic results. RESULTS: The Yamagata-like and Victoria-like viruses co-circulated in 2004-2005. For the Yamagata-like virus, the single-way HI results showed that 3.7% and 4.5% of the viruses had 4-fold greater HI titer difference compared with B/Shanghai/361/02 in 2004 and 2005, respectively. The HA1 sequence data showed that the virus had amino acid mutation, and there was one more glycosylation site at 196th site. For the Victoria-like virus, the single-way HI results showed that 8.5% and 20.6% of the viruses had 4-fold greater HI titer difference compared with B/Hong kong/330/01 in 2004 and 2005, respectively. The HA1 sequence data showed that the virus had replacement of 9 amino acids, and there was one more glycosylation site at 197th site. CONCLUSION: The results showed that influenza B viruses had changed antigenic and genetic characteristics compared with B/Shanghai/361/02, B/Hong kong/330/01 in 2004-2005.

[Development of methods for detection of H5N1 from human clinical specimens].

BACKGROUND: To establish a method for H5N1 RNA detection and laboratory diagnosis of suspected human avian influenza (H5N1) virus infected cases. METHODS: The typing and sub-typing primers were designed according to M and H5 and N1 gene respectively, and the RT-PCR and real-time PCR were developed using these primers. RESULTS: The RT-PCR and real-time PCR could be used for H5N1 detection specifically, and there was no cross reaction with other influenza subtypes such as H1 and H3. The sensitivity for RT-PCR and real-time PCR was 1 TCID50 and 0.01 TCID50 respectively. Thirteen laboratory confirmed human H5N1 cases were detected from 42 suspected cases by using these methods. CONCLUSION: The established RT-PCR and real-time PCR methods can be used for laboratory detection of suspected human H5N1 cases.

[Antigenic and genetic study of influenza virus (H1N1) circulated in China in 2004-2005].

BACKGROUND: To analyze the genetic and antigenic characteristics of hemagglutinin (HA) gene of human influenza (H1N1) virus isolated from the mainland of China since 2004 to 2005. METHODS: The single-way hemagglutination inhibition (HI) tests were used to test the antigenic characteristics, and the HA1 gene was sequenced based on the antigenic results. RESULTS: The single-way HI results showed that no virus isolates had 4-folds greater HI titer compared with A/Shanghai/1/1999 (H1N1) in 2004, but there was 6.3% virus had 4-fold greater difference in 2005. The HA1 sequence data showed that the H1N1 virus had the following amino acid mutations such as 54 K > R, 90 T > K, 101 Y > H, 149 R > K, 169 V > A, 190 D > N, 212 R > K, 219 K > R, 245 W > R, 246 Y > F, 258 T > N, 318 V > A and the 54 and 190 amino acids located in antigenic group of HA1. CONCLUSION: The H1N1 virus was changing in antigenic and genetic characteristics.

[Laboratory diagnoses of the first confirmed human case of avian influenza A (H5N1) in the mainland of China].

BACKGROUND: To determine the etiologic agent of an atypical pneumonia human case admitted to Xiangtan City hospital, Hunan Province in Oct. 2005. METHODS: The patient's respiratory tract samples and serum were collected. Throat swabs were tested by microneutralization and hemagglutination-inhibition assays. RESULTS: The results of nucleic acid detection of all respiratory samples were negative and virus isolation was also negative. The H5-specific antibodies of convalescence showed a 4-fold greater rise than acute phase. CONCLUSION: The atypical pneumonias case was confirmed as the first human case of avian influenza A (H5N1) infection in the mainland of China.

[Origin of internal genes of two strains of swine influenza A (H1N1) virus].

BACKGROUND: Through the analysis of internal genes of viruses to understand whether the internal genes of two strains of swine influenza A(H1N1) virus contain the gene segment deriving from avian influenza A viruses, and whether the reassortment of the internal genes occurred between swine H1N1 and swine H9N2 viruses. METHODS: Viruses were passaged in embryonated hen eggs and virion RNA was extracted from allantoic fluids and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards, RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (version 1.03). RESULTS: All the six, but PB2 gene segment internal gene segments were the same between A/swine/Beijing/1/2002(H1N1) and A/Swine/Beijing/3/2002(H1N1) viruses. Whereas all the six internal gene segments in two trains of swine (H1N1) virus were similar to those of swine H1N1 viruses, but different from those of classical strain of swine (H1N1) virus. CONCLUSION: Two strains of Beijing swine H1N1 influenza A virus were not reassortant, All the six internal gene segments were closely related to swine influenza A (H1N1) viruses.

[Do pigs play a role in human infection with avian influenza A H9N2 viruses].

OBJECTIVE: To understand whether pigs play a role in human infection with avian influenza A H9N2 viruses. METHODS: The target gene was amplified by RT-PCR, and the PCR product was linked to PGEM-T Vector (Promega, USA) at 4 degrees C, the recombined plasmid was transferred into dH5a bacteria, and the positive colonies were selected and identified with restriction endonuclease. Afterwards, they were sent to Liu He Tong Company in Beijing for nucleotide sequencing. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (Version 1.03) and Editseq (Version 3.69) software. RESULTS: The genomic characterizations of A/Swine/Shandong /5/2002(H9N2) and A/Swine/Shandong/10/2002(H9N2) viruses were different from those of H9N2 viruses which were isolated either from men or from chickens. The genomic characteristics of H9N2 viruses isolated from humans in China mainland were similar to those of H9N2 viruses isolated from chickens. Whereas, the genomes of H9N2 viruses isolated from men in Hong Kong, China were closely related to those of H9N2 viruses isolated from quails. Avian influenza A H9N2 viruses not only have wide range of host, but their genomes are also diverse. CONCLUSION: Avian influenza A H9N2 viruses can directly infect human. Avian influenza A H9N2 viruses did not require to pass through the pigs as mixing vessels prior to infecting man.

[Antigenic and genetic study of hemagglutinin gene of influenza virus (H3N2) circulated in China in 2004].

BACKGROUND: To study the antigenic and genetic characteristics of influenza (H3N2) virus circulated in China in 2004. METHODS: Single-way and cross-way hemagglutination inhibition (HI) tests were firstly used to determine the reactivity with the reference serum of virus isolates. Based on the serological results, virus isolates were selected according to the different time and location in China in 2004. The HA1 domain of HA gene of those virus isolates were then sequenced in order to analyze the gene characterization. RESULTS: Single-way HI test results showed that 52.3% of isolates showed 4 folds or more HI titer difference compared to A/Fujian/411/2002 (H3N2) itself (international reference strain in 2004). Cross-way HI test results showed that the antigenic ratio was 4. The nucleic acid and amino acid sequence data of HA1 domain showed that the mutated virus appeared in early February of 2004, and became the dominant circulating strain gradually. There were four important mutant positions, they were 159 Y>F, 189 S>N, 145 K>N, 226 V>I, respectively. The results also indicated that the mutated viruses originated from southern China, then transmitted to northern China, according to the analysis of time and location distribution. CONCLUSION: The HA1 domain of HA gene of influenza virus (H3N2) isolated from 2004 in China showed mutation and antigenic drift, and the mutated viruses were becoming the dominant circulating strain in China, and showed amino acid sequence difference compared to A/Fujian/411/2002 (H3N2) A/Wellington/1/2004 (H3N2), the vaccine components pronounced by WHO for 2004-2005 northern hemisphere and 2005 southern hemisphere respectively, which suggested that further surveillance should be conducted to monitor the virus mutation in circulation.