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近日，军事医学科学院的祝庆余实验室从2003年的疑似SARS患者的呼吸道标本里面，利用Vero细胞，分离得到H5N1型流感病毒。

该患者入院4天后即以严重的呼吸道感染症状死亡。其时正值SARS流行之际，按照SARS诊断标准，虽临床症状相似，但是核酸扩增呈阴性。

祝等发现，以呼吸道标本接种Vero细胞，表现出流感病毒的CPE（A/Beijing/01/2003 ）。随后成功地从细胞培养物里利用PCR扩增得到流感病毒的全部8个基因片段。序列分析表明，该病毒的核酸序列和H5N1型禽流感病毒高度相似，但是其8个基因片段却分别与不同地区分离得到的H5N1的相应片段表现出序列相似性。分析HA基因，得知其和越南、泰国的H5N1属于同一簇（但是2005年的中国人禽流感病毒却属于另一簇）；NA基因则与Hongkong/156/97表现出高度同源性。

这个研究结果对疫苗株的选择有重要的启示作用。

全文：
Fatal Infection with Influenza A (H5N1) Virus in China

To the Editor: A 24-year-old man had pneumonia and respiratory distress in November 2003 and died four days after being hospitalized. Because the clinical manifestations were consistent with those of the severe acute respiratory syndrome (SARS) and occurred when sporadic cases of SARS were described in southern China,1 serum and lung tissue from the patient, as well as fluid aspirated from his chest, were examined for SARS coronavirus with the use of indirect fluorescence antibody tests and the reverse-transcriptase polymerase chain reaction (RT-PCR). All tests were negative for SARS.

A virus was isolated from the lung tissue in Vero-cell cultures and was characteristic of influenza A virus on electron microscopy. A serum sample collected on day 8 of the patient's illness was positive for IgM antibody against the isolated virus. Fragments of both the influenza A virus matrix gene (M) and the H5-subtype hemagglutinin gene (HA) were amplified from the infected Vero cells with the use of RT-PCR assay.2,3 The nucleotide sequences of the fragments were identical to those amplified from the stored specimens of the patient's serum, chest fluid, and lung tissue.

The genomic sequence of the virus (A/Beijing/01/2003) was determined, and its eight segments were genetically related most closely to corresponding sequences of influenza A (H5N1) viruses that had been isolated from chickens in various regions of China in 2004. The segments of the polymerase basic protein 1 gene (P and the nonstructural gene (NS) were most closely related to those from Guangdong Province (in southeastern China), with 99 percent identity. The segments of the polymerase basic protein 2 gene (PB2) and HA gene were closest to those from Jilin Province (in northeastern China), with 99 percent and 97 percent identity, respectively. The segments of the neuraminidase gene (NA), nucleoprotein gene (NP), and M gene were closest to those from Hubei Province (in mideastern China), with 98 percent, 98 percent, and 99 percent identity, respectively, and the polymerase acidic protein gene (PA) segment was closest to that from Japan, with 99 percent identity.

These findings suggest that influenza A/Beijing/01/2003 may be a mixed virus. Phylogenetic analyses of the HA and NA genes of the representative influenza A (H5N1) strains have revealed that the viruses isolated from patients in Thailand and Vietnam in 2004 and 2005 belong to the same clade, and those obtained from patients in Hong Kong in 1997 and 1998 are from another clade (Figure 1 in the Supplementary Appendix, available with the full text of this letter at www.nejm.org). A sample of virus obtained from a patient in Hong Kong in 2003 seems to represent a transitional genotype, of which the HA gene sequence was close to the cluster from southeastern Asia (Figure 1A in the Supplementary Appendix), whereas the NA gene sequence was close to that of the cluster from Hong Kong in 1997 and 1998 (Figure 1B in the Supplementary Appendix). Phylogenetic analyses of the HA or NA gene indicated that the influenza A/Beijing/01/2003 strain was genetically distant from viruses previously isolated from humans, although it appears to have originated from a lineage similar to the influenza A/goose/Guangdong/1/96 (Gs/GD) lineage.4

These findings have important implications for selecting viruses for the development of vaccines to prevent infection in humans. The genetic distance between the isolate reported and the strain currently proposed for vaccine development (A/Vietnam/1203/2004)5 implies that viruses from different regions may need to be considered in the development of an effective vaccine against influenza A virus.


http://content.nejm.org/cgi/content/full/354/25/2731


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