Identification of dual receptor-binding specific strains of human H5N1 viruses in China.

OBJECTIVE: Both the 2, 6 linkage and its topology on target cells are critical for the recognition by human influenza virus. The binding preference of avian flu virus H5N1 HA to the 2, 3-linked sialylated glycans is considered the major factor limiting its efficient infection and transmission in humans. To monitor potential adaptation of H5N1 virus in human population, the surveillance of receptor-binding specificity was undertaken in China. METHODS: The binding specificity of 32 human H5N1 virus strains isolated from 2003 to 2009 was tested by 2, 3-specific sialidase-treated chicken red blood cell (CRBC) agglutination assay and a solid-phase direct binding assay with synthetic sialylglycopolymers. RESULTS: Dual binding preference to 2, 3 and 2, 6-glycans were found in two strains: A/Guangdong/1/06 (A/GD/1/06) and A/Guangxi/1/08 (A/GX/1/08). Though minor effect of short-2, 6-binding was detected in A/GX/1/08 at a low virus titer, both showed high affinity to the oligosaccharide at a high load. Notably both are of the long-2, 6-recognition, with the same topology as that of human H1N1 and H3N2 viruses. CONCLUSION: The findings suggest that human H5N1 virus in China likely acquired the potential human-adaptation ability. Further research and surveillance on receptor-binding specificity of H5N1 viruses are required.

[The proliferation of H1N1 subtype influenza viruses in A549 and BEAS-2B cells].

OBJECTIVE: Analyze the proliferation of different host H1N1 subtype influenza viruses in A549 and BEAS-2B cells. METHODS: Human, avain and swine three hosts of the H1N1 influenza viruses infected A549 and BEAS-2B cells and analyze the characteristics of different periods after inocubation. Determine the receptor binding specificity of influenza virus by hemagglutination (HA) test with RBCs with two types of receptor. And the receptors on surfaces of A549 and BEAS-2B cells were tested by flow cytometry. RESULTS: The Cell Pathologic Effect (CPE) is obvious after 24 h inoculation in A549 cells by all the H1N1 influenza viruses, moreover, the peak hemagglutinin (HA) and 50% tissue culture cell infected dose (TCID50) titers was observed after 36 h of culturing in A549 cells. Otherwise, the CPE is not typical from 24 h-120 h inoculated by the same viruses and the HA, TCID50 titers were keep low all the periods in the BEAS-2B cell after inoculation. The receptor-binding preference of H1N1 viruses used in the study was screened by HA assay and some were found with 2-6-receptor binding affinity. Both SA a-2, 3Gal and SA a-2, 6Gal receptors were detected on A549 and BEAS-2B, furthermore, receptor density on A549 cells was significantly higher than that of BEAS-2B cells. CONCLUSION: A549 cells were susceptible to human, avian and swine H1N1 influenza viruses infection and permissively for viral replication. However, BEASE-2B cells with similar receptor pattern and epithelium-derived propriety as A549 cells were unsusceptible to their infection and replication. Possible host factors involved in effective viral infection and replication were needed further study.

[Expression and purification of avian influenza virus H5N1 NP protein, and screening interaction proteins of host cells in vitro].

OBJECTIVE: To express and purify H5N1 influenza virus (A/Anhui/1/2005) NP in prokaryotic system and to explore the NP-interacting proteins of human bronchial epithelial cells BEAS-2B in vitro. METHODS: The full length H5N1 NP gene fragment was amplified by PCR, inserted into prokaryotic expression vector (pET30a) to generate NP expression plasmid pET30a-NP. After transforming pET30a-NP into E. coli (BL21), the expression of soluble NP protein was induced by IPTG. The expressed NP protein was purified by two steps with metal chelation chromatography and ion exchange chromatography. Then the total proteins of BEAS-2B cells was extracted for screening the components which have protein-protein interaction with purified NP by pull-down and LC-MS/MS methods. RESULTS: The expression of H5N1 NP protein could be induced by IPTG in bacterial system using expression plasmid pET30a-NP. The soluble NP was purified. Twenty proteins were found by pull-down and LC-MS/MS, the further experiments may be needed to prove protein-protein interaction between them. CONCLUSION: The soluble H5N1 NP fusion protein with high purity was obtained and twenty proteins were found which could interact with it by pull-down and LC-MS/MS.

Visual detection of pandemic influenza A H1N1 Virus 2009 by reverse-transcription loop-mediated isothermal amplification with hydroxynaphthol blue dye.

A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of pandemic influenza A H1N1 virus infection. The reaction was performed in one step in a single tube at 65 degrees C for 60 min with the addition of hydroxynaphthol blue (HNB) dye prior to amplification. The detection limit of the RT-LAMP assay was approximately 60 copies, and no cross-detection was observed. The assay was evaluated further with 50 clinical specimens diagnosed clinically with seasonal influenza or pandemic influenza A H1N1 virus infection. RT-LAMP with HNB dye was demonstrated to be a sensitive and easy assay for rapid detection of pandemic influenza A H1N1 virus.

[The study of multiple RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses].

OBJECTIVE: To establish a multiplex RT-PCR-based reverse dot blot hybridization technique to detect influenza viruses. METHODS: Obtain the HA nucleotide sequences of seasonal influenza H1N1, seasonal influenza H3N2, influenza H1N1 and human avian influenza H5N1 from GenBank. Design primers in conservative district and probes t in high variable region respectively, after analyzing the HA nucleotide sequences of influenza virus through the Vector NTI 9.0. Establish and optimize multiple RT-PCR system by comparing amplification efficiency and specificity at different primer concentrations. Establish the reverse dot hybridization system after optimizing the concentration of probes. To compare the sensitivity and specificity of this technique and the general RT-PCR Method through extracting the viral RNA of the mentioned influenza virus which are to be the reference substance. RESULTS: Successfully establish a multiplex RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses. This technique is 100-1000 times more sensitive than gel electrophoresis method, and it has a good specificity. CONCLUSION: Successfully established multiplex RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses.

[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.

[Characterization of pseudotyped viruses coated with hemagglutinin of H5N1 avian influenza].

OBJECTIVE: To construct pseudovirus bearing H5N1 HA based on a lentivirus vector system. Then we study the biological feature of the pseudovirus. With the newly established viral particles, we performed the serological tests. METHODS: H5N1 avian influenza virus that isolated from human case was cloned to construct pLP-HA, then pLP-HA co-transfected with lentivirus vector plasmids pLP1, pLP2 and pEmGFP into 293T cells. The supernatant was harvested 48h post-transfection. Concentrated by super centrifuge, the pseudotyped viruses were analyzed by infection test, HA test and micro-neutralization test. At the same time, optimized HA gene and a Vietnam H5N1 HA gene were used to construct pseudotyped virus for comparison. RESULTS: Pseudotyped virus particles can be observed with electronic microscope. Western-blot revealed that HA glycoprotein can be expressed in the virions. Our neutralization assay by using the pseudoviruses was comparable with the conventional microneutralization assay with wild-type viruses. A high degree of correlation was detected. CONCLUSION: Pseudotyped Viruses coated with HA of H5N1 High Pathogenic Avian Influenza were successfully constructed; it can be used to for the microneutralization assay. The HA gene from different sources affect the efficiency of the packaging of the pseudovirus. But the optimized HA gene can not obviously improve packaging efficiency of the pseudovirus.

[The virus isolation analysis of the H5N1 subtype human avain influenza cases in mainland China from 2005 to 2009].

OBJECTIVE: To analyse the correlation between the virus isolation and the specimen collection of the H5N1 human high pathogenic avain influenza cases in Mainland China. METHODS: The specimens were collected in Mainland China from 2005.10 to 2009.3 and the H5N1 viruses were isolated by passage in embryonated chicken eggs. RESULTS: Most specimens were obtained within 14 days after disease onset. For the specimens collected within 7 days, the isolation rate was relatively high and the difference of the positive rate between different years was lower than those specimens collected after 7 days. Most of the samples in our study were collected from the upper or lower respiratory tract with few from blood, feces, et al. The isolation rate of lower respiratory specimens was higher and the difference of the positive rate between different years was relatively lower than those from upper respiratory specimens. CONCLUSION: We suggest that the samples should be collected from lower respiratory tract during the acute phase to get the higher isolation rate.

[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.

[The firstly confirmed pregnant woman case of avian influenza A (H5N1) by etiological research in China].

To investigate the cause of death of a pregnant woman with undefined pneumonia reported from the People's Hospital of Tongling City in Anhui Province on November 8, 2005, the patient's tracheal aspirates and serum samples were collected and tested by RT-PCR and Real-time PCR to detect viral nucleic acids of HA of A/H5N1, A/H7N7, A/H9N1 and A/M. Tracheal aspirates were inoculated into special pathogen free (SPF) embryonated eggs for cultivation and identification of virus. The HA gene of the virus was sequenced and analyzed. Serum samples were tested by HI assay to detect antibody of H5N1. The results showed that HA gene of A/H5N1 virus and A/M were positive in tracheal aspirates by both PCR tests. The serum sample collected on Nov. 9 was A/M gene positive by Real-time PCR. The analysis of HA gene of A/AnHui/1/2005 sequence showed that the receptor specificity and the connecting peptide between HA1 and HA2 were still avian influenza origin. The HI antibody of H5N1 was negative at 7th, 8th, 9th d of disease onset. This undefined pneumonia case was confirmed as the first pregnant woman case of avian influenza (H5N1) virus infection by etiology in the mainland of China.

[Laboratory diagnosis and molecular characterization of highly pathogenic avian influenza virus (H5N1) in human in Hunan Province in 2005-2006].

To determine the etiologic agents of two atypical pneumonia human cases in Hunan Province in 2005-2006 and to study their pathogenic potential, the patients' respiratory tract samples and sera were collected. The respiratory tract samples were tested by real-time RT-PCR and RT-PCR methods, and the sera by hemagglutination-inhibition assay. Virus was isolated from case 2 and its genome was sequenced and analyzed. Results showed the H5 nucleic acid tests of two cases were positive. The H5-specific antibody titer of the convalescence serum of case 1 showed a 4-fold greater rise than that of the acute phase. And case 2's antibody titer of acute phase was negative. The two atypical pneumonia cases were confirmed as the avian influenza A (H5N1) infection cases. Viral strain A/Hunan/1/2006 was isolated from case 2. Phylogenetic and molecular analysis suggested that 8 gene segments of A/Hunan/1/2006 originated from avian viruses. And A/Hunan/1/2006 was similar with viruses isolated from avian in Hunan Province. The isolated virus did not recombine with human influenza viruses and no obvious variation was observed.

[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.

[Study on a fatal pregnant woman died from by avian influenza (H5N1)].

OBJECTIVE: To ascertain the causation of a pregnant woman with undefined pneumonia reported from the People's Hospital of Tongling city in Anhui province on November 2005. METHODS: Epidemiological and clinical information of the case was collected from the keypersons close to the case and referring to the medical record. A medical observation was carried out on the close contacts of the case and sick or dead poultry. Tracheal aspirates being collected were tested by both RT-PCR and real-time PCR to detect viral nucleic acids of A/H5N1, and were inoculated into special pathogen free (SPF) embryonated hens' eggs. RESULTS: The pregnant woman was found to have been contacted with the sick/dead poultry directly on the 4th day before onset of illness. All the 122 close contacts were healthy after a 10-day medical observation. The major clinical features of the case were viral pneumonia with rapidly developed leukopenia and lymphopenia. The progress to acute respiratory distress syndrome and multiple organ dysfunction syndromes was found at clinical presentation. HA and NA gene of A/H5N1 virus were positive. The 8 gene fragments of A/Anhui/1/2005 (H5N1) isolated from the tracheal aspirates had not carried genes from a human virus through reassortment, and the receptor-binding site of the hemagglutinin was polybasic cleavage site. CONCLUSION: This was the first documented case of H5N1 infection in pregnant woman. The immunotolerant state of pregnancy might have predisposed to the fatal outcome of the patient.

[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.