Isolation, Identification And Molecular Epidemiology Of Respiratory Adenovirus

1. Background and ObjectiveHuman Adenoviruses (HAdV) are nonenveloped, dsDNA genome and icosahedral viruses which capsids are consisted of hexon, penton and fiber. Hexon is a homologous trimer and carrying the pathogenic epitope. HAdV now have been totally recognized more than 68 types and grouped into 7 species (A-G). HAdV cuase a large spetrum of diseases, such as conjunctivitis, cystitis and gastroenteritis, and the HAdV-B species were associated with Acute Respiratory Disease (ARD) among infants and children.Generally, cell culture and ELISA method are used for HAdV typing. Based on hexon epitope and fiber, haemagglutination inhibitions and neutralization tests were performed to classify adenovirus and analyze their biochemical and biological properties. Due to the ambiguous classification of serotypes, ICTV carried out a new genotyping methodology that provided a tool to facilitate the adenovirus research and avoid the inaccurating of serological test.Naive population, especially infant and children with underlying conditions were more likely to have severe outcome after infection. Both HAdV-B7 and HAdV-B3 were associated with lung damage and even death. Besides high transimission, HAdV-7 is relevant to a higher mortality than HAdV-3. HAdV-B14, a member of HAdV-B2, has been identificated as an acute respiratory pathogen that causing several contagious outbreaks of ARD. Until 2010, HAdV-14 appeared in several provinces of China.Universal primers and type-specific primers of HAdV-3,-4, and-7 were designed to identify and type adenovirus. A hundred throat swab specimens of bronchopneumonia were collected to investigate the predominant adenoviruses of Guangzhou, southern China. Moreover, an isolate from a child with sever bronchopneumonia in Guangzhou was identified and typed. Complete hexon gene was sequended for further analysis.During the epidemic investigation period, we got the first case HAdV-14 of China in 2010 unexpectedly, which was collected from a 17-month-old infant with tonsillitis and named GZ01 strain. Genomic comparasion and phylogenetic analysis of GZ01 strain against 303600 strain and deWit strain widen the horizen of HAdV-14 distribution, epidemiology and evolution, and provided the molecular evolutional data to HAdV-14.On the other hand, DG01 strain was collected among the specimens of an ARD outbreak of Dongguan, China in March 2011 and subsequently identified, full-genome sequenced, annotated and analyzed. The DG01 strain belongs to HAdV-7 genome type 7d, which had been reported in China during 1990 to 2009. The re-emeregence of this HAdV-7d calls for nationwide surveillance. Availability of the genome sequence of the HAdV DG01 in China will facilitate the epidemic reseach of HAdV-7 for disease prevention and surveillance.In China, a large naive population was susceptive to HAdV-3,-7 and-14. The rare herd immunity against these adenovirus can result in a severe outbreak, especially in children. All in all, molecular epidemiology, genomic bioinformatitis and phylogenetic analyis of adenovirus provide and progress a deep insight of adenovirus as well as the virus reseaches and put forward significant evidence toward HAdVs surveillance.2. Methods(1) Identification and typing of Human adenovirus. Post-infected A549 cells were cultured in 37℃ incubator. Viruses were harvested after CPE. On the other hand, viral DNA was extracted and amplified with universal primers and typing primers. As for a survey of the predominant adenovirus types in southern China, a hundred clinical specimens were tested by this method.(2) HAdV-3 isolation and identification. HeLa cells were infected with a specimen and cultured in condition of 37℃ 5% CO2. Viruses named GZ13 were collected after CPE occurence. The epidemiological method we built above was used to identify and classify GZ13. SeqMan and Bioedit 7.0 softwares were performed to character and analyze the GZ13 hexon sequences. The phylogenetic trees were built by MEGA5.0 to provide an insight to the HAdV-3 molecular epidemiology in southern China.(3) HAdV-14 isolation, identification and genome analysis. A549 cells were infected with a specimen named GZ01 and cultured in 37℃ with 5% CO2. Viruses were harvested when CPE occured. Genome DNA was extracted by an optimized method. The Primer Premier 5.0 software was used to design walking primers to sequence the genome DNA. SeqMan, Sequencher 4.01 and BioEdit 7.0 softwares were used to assemble, analyze, annotate genome sequence and build phylogenetic trees. The genome sequence was submitted into GenBank. DNA restriction endonucleases analysis was carried out with Vector NTI 10.3.0 software to elucidate the genome type of GZ01.(4) HAdV-7 isolation, identification and genome analysis. Post-infected with a specimen named DG01, A549 cells were cultured in condition of 37℃ with 5% CO2. Viruses were harvest after CPE and viral DNA was extracted and amplified with universal primers and type-specific primers. Genome DNA was extracted by using an optimized method as above. Designed by Primer Premier 5.0, walking primers were used to sequence the whole genome DNA. SeqMan, Sequencher 4.01, BioEdit 7.0 softwares were used to assemble genome, analyze, annotate and built phylogenetic trees. The whole genome sequence was submitted into GenBank. DNA restriction endonucleases analysis was carried out with Vector NTI 10.0 software to illustrate the genome type of DG01.3. Results(1) A rapid and efficient method of identifying and typing respiratory adenovirus in China. According to alignment of archived HAdV-3,-4, and-7 sequences, universal primers HexF and HexR reveal only 2-3 mutants at the 5’ terminal, while specific-primers show 3-9 deletions and 2-4 substitutions at the 3’ terminal. Only adenovirus reference strain, not other viruses, displayed a single 300bp band when amplified with universal primers. According to type-specific test, the 300bp bands appeared only in specific lanes of HAdV-3,-4, and-7 specifc-primers respectively.(2) Respiratory HAdVs molecular epidemiology in Guangzhou. Fifty five of one hundred specimens were identified as human adenovirus infection and amounted 92.7% for HAdV-3,3.6% for HAdV-7 and 1.8% for HAdV-14 in percentage. According to tradictional virus isolation method which was less accurated than our method, only 44 specimens were adenovirus-positive. HAdV-B (98.2%), especially HAdV-B3 (92.7%) is the predominant type circulating in Guangzhou, Southern China during Oct 2010 and Dec 2011.(3) Identification, classification and phylogeny results of GZ13 strain. Identification and classification results showed GZ13 belongs to HAdV-B3. The hexon sequence re-affirmed GZ13 to HAdV-3 again and phylogenetic trees indicated a high homology between GZ13 strain and TW strains.(4) Identification, classification, genomic and phylogeny analysis of HAdV-14 GZ01 strain. Virus DNA was extracted from A549 cells infected with an isolate from a 17-month-old infant dignosed as tonsilitis. The virus was named HAdV-B/CHN/GZ01/2010/14[P14H14F14]. The GenBank assession number is JQ824845.1. Genome length of GZ01 is 34,767bp and consists of 26.08% base A, 24.4% base C,24.42% base G and 25.08% base T. The GC content is about 48.83% and approaches to the mean 48.9% of B2 subspecies. The HAdV-B14/CHN/GZ01 genome contains early, intermediate and late transcription regions, thirteen genes and thirty-seven putative protein-coding sequences. The Inverted Terminal Repeat (ITR) is 133 bp. In-silico restriction enzyme analysis classified GZ01 into HAdV-14p1. GZ01 strain reveals a high indentity (99.92%) to strain 303600, higher than 99.6% identity to the prototype strain deWit. Consequently, Chinese HAdV-14 might derive from oversea.(5) Identification, classification, genomic and phylogeny analysis of HAdV-7 DG01 strain. Virus DNA was extracted until 80%-90% CPE appeared. Genome DNA was sequenced, annotated, submitted into GenBank under the GenBank accession no. KC440171 and named HADV-DG01/2011/7/P7H7F7. Identification and classification results show that DG01 belongs to HAdV-7. Restriction enzyme analysis classified it into HAdV-7d. DG01 genome DNA is 35,240 bp in length, consisting of 25.32% base A,25.81% base C,25.27% base G, and 23.60% base T. The relative molecular weight of the genomic DNA is about 2.1×10. It contains 13 predicted genes, three transcription regions (E region, middle region and L region), the same as other mammalian adenoviruses, and 39 DNA coding sequences and 2 RNA coding sequences from our prediction. Phylogenetic trees displayed a high homology of 99.9% between DG01 and HAdV7-0901HZ/ShX (GenBank accession number is JF800905.1) which contains only 8 mutants and a conserved organization of hexon and fiber.4. Conclusion(1) In this study, universal primers and specific-primers were designed to detect and classify HAdVs. Six speices of adenovirus can be detected by PCR with these universal primers, producing single bands, while no band was produced from other respiratory pathogens. Specific PCR bands were produced only by corresponding type-specific primers, indicating a higher sensitivity of our method than cell culture. For advantages of our method, the single pair of primers in single PCR reaction make it easier to optimize the PCR system. Furthermore, three simultaneous ampified reactions save much time. Finally, it is accessible and economized. The epidimology result showed that HAdV-B3 has been circulating in Guangzhou even in China for many years.(2) Identification and typing analysis of a bronchpneumonia isolate named GZ13 suggested it belongs to HAdV-B. Hexon sequence and phylogenetic tree elucidated GZ13 strain might derive from TW strain. Our method will provide the etiological evidence for disease prevention and surveillance.(3) The first isolate of HAdV-14 in Guangzhou China alarmed that the HAdV-14 may emerge and cause outbreaks in future. The genome analysis and phylogenetic tree of GZO1 strain show a high identity (99.92%) to USA strains, suggesting Chinese HAdV-14 strains might be derive from USA. The molecular epidemiology of HAdV-14 provides strong foundation for adeno virus surveillance in China and even all over the world.(4) Genome analysis, bioinformatics analyses and restriction enzyme analysis identitied HAdV-DGO1 as HAdV-7d. Phylogenetic trees demonstrated that HAdV-7d and HAdV-7d2 are two predominant types that circulating in China, hinting a high transmissible re-emergence of HAdV-7 that calls for emerging surveillance. The strategies and softwares used in this study are important for viral diseases surveillance in the future and helpful for adenovirus epidemic research and vaccine development.