The index case was a one-year-old female from Xixiang County, Hanzhong, Shaanxi Province. She had an onset on 15 January with an admission to Hanzhong Central Hospital with complaints of cough, expectoration asthma with a fever for 5 days. The clinical diagnosis was acute bronchitis with heart failure and toxic encephalopathy where the patient died on 30 January. Thirty-two cases of severe acute lower respiratory tract infections were reported through 9 February 2009; and another thirty-eight cases were found in four hospitals in Hanzhong city of Shaanxi Province with the definition of severe pneumonia:
1. Prolonged fever continuing at 37.5 C - 40 C.
2. Iconographic pneumonia with apparent respiratory symptoms.
3. Normal or lower total WBC.
4. No apparent improvement or became more severe after 3-day treatment with antibiotics.
During the outbreak of the disease, 21 pharynx swab specimens and 21 acute-phase sera samples were collected from 21 patients; and 12 returning patients gave convalescent-phase sera. The pharynx swab specimens were collected and transferred to 1 ml viral transport medium.
The 21 pharynx swab specimens collected from the patients were inoculated onto HEp-2 cells and were cultured in a maintenance medium (Minimal Essential Medium containing 2% fetal calf serum, 100 U/ml penicillin G and 100 mug/ml streptomycin) at 37 C in a closed system without added CO2. Cultures exhibiting an adenovirus-like CPE were passed again to confirm the presence of the virus. Primary identification of positive isolates was performed using PCR with adenovirus-specific primers.
The stored serum samples were inactivated at 56 C for 30 min; diluted eight times with the maintenance medium; and filtered through a 0.22 mum filter membrane. Dilutions of the serum samples (1:8 to 1:1,024) were prepared and 50 mul of each dilution was added to four wells of a 96-well microplate. The entire virion of the HAdV strain isolated and identified was used as the neutralization virus. The 50% cell culture infective dose (CCID50) per 50 mul was calculated using the formula of Karber.
An ELISA Classic adenovirus IgA kit (Institute Virion/Serion GmbH, W.rzburg, Germany) that enables the detection of serum antibodies against all serotypes of HAdV pathogenic for humans was used to detect HAdV immunoglobulin A (IgA) from the 21 acute phase sera samples from 21 patients.
The viral nucleic acid was directly extracted from the clinical specimens using a QIAamp mini-viral RNA extraction kit or a QIAamp DNA mini kit (Qiagen, Valencia, CA). Reverse transcription-PCR (RT-PCR) or PCR was performed using the Seeplex RV Detection Set I (RV6C00Y, Seegene, USA) that is designed to detect 11 types of RNA viruses and one type of DNA virus responsible for most respiratory diseases. The 11 RNA respiratory viruses include influenza A and B virus, human respiratory syncytial virus A and B, human metapneumovirus, human parainfluenzavirus, human rhinovirus A, and human coronavirus 229E/NL63 and OC43. The DNA respiratory virus was human adenovirus. We also used the adenovirus species-specific PCR that can distinguish the six species of adenovirus and type-specific PCR of species B described by Pring-.kerblom. The PCR was performed using primer pairs ADSD/AD52 as described by Zhen. The PCR reaction was performed using a GeneAmp 9700 thermal cycler (Applied Biosystems). The amplification products were analyzed by electrophoresis of the samples in 1% agarose gels; and they were visualized with ethidium bromide under UV light.
The PCR products were sequenced directly after purification (QIA gel extraction kit; Qiagen, KK, Japan) using the dye terminator method (Big Dye Terminator, version3.1, cycle sequencing kit; Applied Biosystems) with an ABI Prism 3100 genetic analyzer (Applied Biosystems). The primers of adenovirus used for sequencing are shown in Table 2. The primers of human respiratory syncytial virus, human parainfluenzavirus, human rhinovirus and enterovirus were donated by colleagues in other laboratories in the Institute.
The sequence data were stored as standard chromatogram format files (.ab1) and were analyzed using Sequencer soft ware (version 4.0.5; Gene Codes, Ann Arbor, MI). The nucleotide sequence homology was inferred from the identity scores obtained using the BLASTn program (National Center for Biotechnology Information, Bethesda, MD). Sequence alignments were created with BioEdit Sequence alignment editor software (version 5.0.9; Tom Hall, North Carolina State University); and a phylogenetic dendrogram was constructed using the neighbor-joining method with the MEGA program (Sudhir Kumar, Arizona State University); and the reliability of the tree was estimated with 1,000 bootstrap pseudo-replicates.
The nucleotide sequence of the entire hexon gene for strain HAdV7-HZ/SHX/CHN/2009 determined in this study was deposited in the GenBank nucleotide sequence database under accession number GU230898.