| Objective: To establish a nested reverse transcription-polymerase chain reaction (nested RT-PCR) assay for detectioning specific fragment of human Rhinovirus (HRV) RNA,and to analyze the sequence of amplicons.To understand viral etiology of acute low respiratory tract infection (ALRTI) in children in Shanghai area, other virus tested by direct immunofluorescence assays (DFA).Subjects and Mothods:1. We collected 342 nasopharyngeal secretions (NPS) samples from ALRTI cases who were hospitalized during January 2005-December 2005.2. Nested RT-PCR techniques was used to detect HRV-specific RNA.Fifteen of the 46 positive PCR products were sequenced, and the sequence of nucleotides were analyzed by using diological software and compared with those in the GeneBank. DFA was applied to identification nearly 7 different virus, such as respiratory syncytial virus (RSV), influenza virus (IFV) type A and type B, parainfluenza virus (PIV) type 1, 2, 3 and adenovirus (ADV).3. Viral epidemiological data and seasonal variations were analyzed. The correlation of virus infections and clinical characteristics were investigated.Results:1. Forty-six (13.45%) of 342 samples were HRV positive on nested RT-PCR. Compared the sequences of HRV viruses in the GeneBank. The sequence of 15 amplicons shared high homology of 83%-97%. Within the 15 amplicons, nucleotides identity varied from 64.4% to 98.4%, and the ratio of genetic variationwas from 1.6% to 48.3%. The sequences of 15 amplicons were highly varied, single nucleotide mutation and several nearby nucleotides mutations were found. We also found ribonucleotide deletion and insertion in the nucleotide sequence.2. HRV positive rates were detected 14.7% in boys and 10.9% in girls respectively. The ratio of infection cases between boys and girls was 2.5:1. But there was no significant difference of positive rate between boys and girls (X~2=1, P>0.05). Of 46 HRV infections, 27 (58.7%) of the children were less than 12 months of age and 38 (82.6%) less than 3 years old. Amount of HRV infectious cases and prevalence of HRV infectious peak were from March to May. There were another small highness of infectious cases and positive rate from September to November. The positive rate of HRV infections was relatively higher in July.3. Out of 342 cases, RSV positive cases accounted for 64 (18.70%). RSV positive rates detected 20.60% of boys and 15.10% of girls respectively. The ratio of infection cases between boys and girls was 2.6:1. But there was no significant difference of positive rate between boys and girls (X~2=2.41, P>0.05).46 of 64 RSV infectious cases (71.8%) in the children were less than 1 year old. The positive rate of children less than 1 year old was higher than those older than 12 months of age (47.90%, 12.30% respectively, X~2=5.05, p<0.05). Amount of RSV infectious cases predominated from January to April and from September to December. On December, the amount of RSV infectious cases got to climax. So the rate of RSV infections increased From August and peak on December.4. Out of 342 cases, ADV positive cases accounted for 9 (2.63%), PIV for 8 (2.3%), IFV for 7 (2.0%), respectively. There were 7 cases of PIV type 1 positive specimens and 1 PIV type 2 positive among all the 342 cases. 5 IFV type A positive cases and 2 IFV type B positive cases were also found.5. The overlap infection of 2 kinds of virus was found in 4 cases. One case of RSV plus HRV infections, 1 case of RSV plus ADV infections, 1 case of RSV plus PIV type 1 infections and 1 case of HRV plus IFV type A infections were found respectively.6. 130 cases (38%) showed positive in virus etiology among all these 342 cases. The positive rate of males (43%) was remarkably higher than that of famles (28.6%) (X~2=6.9, p<0.01). Of all the 130 cases, 88 (67.7%) of the children were less than 12 months of age. The positive rate of children less than 6 years old was evidently higher than those older than 6 years of age (40.3%, 8.3% respectively; X~2=9.6,p<0.01). There were remarkable seasonal varieties of viral infections. The positive rate from January to March, from April to June, from July to September and from October to December were 37.20%, 30.10%, 32.30% and 53.40%, respectively. There was significant difference of positive rate from October to December and from January to September (53.40%, 33.8% respectively; X~2=9.35, p<0.01).Conclusions:1. This nested RT-PCR techniques was highly specific, rapid and convenient for the detection of HRV RNA in NPS of patients with ALRTI and the genome of HRV viruses was highly variable.2. Viral infections occupied an important status in children with ALRTI in Shanghai. ALRTI are predominance in boys. Viral low respiratory infections were common in children less than 6 years old and much more in those younger than 12 months of age. There were remarkable seasonal distributions of viral low respiratory infections in Shanghai area. Viral low respiratory infections predominated in the fourth season.3. RSV and HRV were the two main causes of viral low respiratory infections. There were a few ADV, PIV and IFV in children with ALRTI in Shanghai.Mixed viral infections were quite a few.4. The incidence of HRV infections predominates in children less than 3 years old and peak in children younger than 12 months. HRV infections occur predominantly in the spring and autumn.5. Children less than 1 year old were the most common age group affected by RSV. RSV was prevalent in spring, fall and winter when the air temperature was low in Shanghai.
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