To the Editor: A 24-year-old man had pneumonia and respiratory
distress in November 2003 and died 4 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 (ref 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 (ref 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 8 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 (PB1) 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 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,
98, 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 Viet Nam 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 ). 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 /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 (ref 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.

From: Qing-Yu Zhu, M.D., State Key Laboratory of Pathogens and
Biosecurity, Beijing 100071, China; E-De Qin, M.D., Beijing Institute
of Microbiology and Epidemiology, Beijing 100071, China; Wei Wang,
M.D, 309th Hospital of the People´s Liberation Army, Beijing 100091,
China; Jun Yu, Ph.D., Beijing Genomics Institute, Beijing 101300,
China; Bo-Hua Liu, Ph.D, State Key Laboratory of Pathogens and
Biosecurity, Beijing 100071, China; Yi Hu, Ph.D., Beijing Institute
of Microbiology and Epidemiology, Beijing 100071, China; Jian-Fei Hu,
Ph.D.,Beijing Genomics Institute, Beijing 101300, China; and Wu-Chun
Cao, M.D., Ph.D., State Key Laboratory of Pathogens and Biosecurity,
Beijing 100071, China

References
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sporadic cases of community-acquired SARS, Guangdong Province, China.
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2. Poddar SK. Influenza virus types and subtypes detection by single
step single tube multiplex reverse transcription-polymerase chain
reaction (RT-PCR) and agarose gel electrophoresis. J Virol Methods
2002;99:63-70.

3. Lee MS, Chang PC, Shien JH, Cheng MC, Shieh HK. Identification and
subtyping of avian influenza viruses by reverse transcription-PCR. J
Virol Methods 2001;97:13-22.

4. Chen H, Smith GJD, Li KS, et al. Establishment of multiple
sublineages of H5N1 influenza virus in Asia: implications for
pandemic control. Proc Natl Acad Sci U S A 2006;103:2845-2850.

5. Treanor JJ, Campbell JD, Zangwill KM, Rowe T, Wolff M. Safety and
immunogenicity of an inactivated subvirion influenza A (H5N1)
vaccine. N Engl J Med 2006;354:1343-1351.