| ObjectiveThe present paper attempts to establish a method with nested reverse transcription-polymerase chain reaction (nested RT-PCR) for detecting specific fragment of human parainfluenza virus3(HPIV3) RNA and to analyze the sequence of amplicons in the hope that a better understanding viral etiology of acute respiratory tract infection (ARI) in children in Lanzhou area will be achieved.Subjects and Methods1. Specimens were collected from 300 children with acute respiratory infections from July to December in 2005 and from October in 2006 to April in 2007 in Lanzhou .2.Nested RT-PCR was used to amplify the Hemagglutinin (HN) gene fragment of HPIV3 from clinical specimens. Primer pairs derived from a conserved region of the HN genes of HPIV3 were used to develop the nested RT-PCR for detecting HPIV-3. Eleven of the positive PCR products randomly selected were sequenced, and the sequence of the nucleotides were analyzed by using biological software and compared with those in the Gene Bank.3.A preliminary research was made on the features of HPIV3' s evolution in Lanzhou from the angle of the genetic evolution.4.Viral epidemiological data and seasonal variations were analyzed. The correlation of HPIV3 infections and clinical characteristics were investigated.Results1.Forty-six(15.33%) of 300 samples were HPIV-3 positive on nested RT-PCR.2.HPIV3 positive rates were 16.04% in boys and 14.49% in girls respectively, and the positive rates in URI were 16.67% ,in LRI were 12.96%. Of 46 HPIV-3 infections, 17(36.96%)of the children were under 12 months of age and 42 (91.30%) under 3 years old. 3. Positve specimens can be detected in each month of every year. They were collected in July to December, 2005. October 2006 to April, 2007. The amount of HPIV-3 infectious cases and the peak of its prevalence were November, 2005 as well as January, and February in 2007.4. Through a comparative study with the sequence of the HPIV3 in the Gene Bank, the sequence of 11 amplicons shared high homology with them to the degree of 91～99.6%. Phylogenetic analysis for those 11 amplicons' sequences indicated that those specimens were HPIV3 positive.5.The analysis of Phylogenetic trees reveals that the sequence of the eleven samples belongs to the same branch with Japanese isolated strains. The sequence of the samples collected from August to December, 2005 are from the same branch except 145SEQ. Moreover, the sequences of the samples collected from October, 2006 to April, 2007 are from the same branch except 1630SEQ..Conclusion1. HPIV3 infections occupied an important status in children with ARI in Lanzhou. .HPIV3 infection were common in children under 3 years old and much more in those younger than 12 months of age. There is no significant difference of the infectious rate between the boys and the girls(x~2=0.3856 P>0.05),as well as URI and LRI(x~2=0.7303 P>0.05).2. Positve specimens can be detected in each month of the year.but the peak of its prevalence were in spring and winter. Obvious seasonal variations were not detected due to the impossibility of keeping the samples long enough to observe for more research.3. This nested RT-PCR techniques were highly specific, and convenient for the detection of HPIV3 RNA in NPS of patients with ARI.4. The analysis of Phylogenetic trees reveals that the sequence of the eleven samples belongs to the same of Japanese isolated strains. So a close relationship of the evolution between them is shown from the research. The sequence of the samples collected from July to December 2005 are from the same branch except 145SEQ. Therefore, they also demonstrate the close relationship. Moreover, the sequences of the samples collected from October, 2006 to April, 2007 are from the same branch except 1630SEQ. From these data, HPIV3 between these two periods in Lanzhou are different in the sequence of the HN gene high conserved region. It might be assumed that prevalence of HPIV3 should be related to areas.
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