Adenovirus is an important human pathogen that is known to cause acute respiratory infection and infection in multiple other organ systems, such as the gastrointestinal tract, the urinary tract, eyes, liver, and bladder.Reference Lenaerts, De Clercq and Naesens 1 Adenovirus infection often causes disease outbreaks in schools, military bases, and other public places.Reference Lenaerts, De Clercq and Naesens 1
Adenoviruses are nonenveloped particles with linear double-stranded DNA. Thus far, 65 serotypes have been classified within 7 species of the virus (A-G) based on genomics.Reference Seto, Chodosh and Brister 2 Human adenovirus type 55 (HAdV-55) is thought to be an uncommon re-emergent pathogen that belongs to Adv species B. It was first identified as AdV-B11a from an outbreak during a military training exercise (Spain, 1969)Reference Hierholzer, Pumarola and Rodriguez-Torres 3 and appeared again in another military training exercise years later (Turkey, 2004).Reference Chmielewicz, Benzler and Pauli 4 In 2009, Walsh et al named AdV-55 as a hexon recombination between AdV-B11 and AdV-B14 (97.4% AdV-B14), and the sporadic appearances of the virus and its misidentification as “AdV-B11a” could be due to this partial hexon.Reference Walsh, Seto and Jones 5
AdV species B has been further classified into 2 subspecies, B1 and B2. The B1 viruses are usually associated with respiratory tract infections, whereas the B2 viruses, except for AdV-14 and Adv-55, are associated with kidney and urinary tract infections.Reference Zhu, Zhang and Xu 6 HAdV-55 has recently caused several disease outbreaks in China. HAdV-55 first appeared in China (Shanxi Province) in 2006Reference Zhu, Zhang and Xu 6 and re-emerged in Beijing 5 years later.Reference Gu, Liu and Li 7 In 2012, this pathogen caused outbreaks in both Baoding and Tianjin.Reference Chen, Nie and Xu 8 , Reference Xie, Tu and Chen 9 The typical clinical characteristics of HAdV-55 infection include cough, nasal obstruction, and pharyngalgia, which are accompanied by fever, chills, headache, and muscle pain. Viral pneumonia can develop in 20% to 50% of people infected with HAdV-55.Reference Chen, Nie and Xu 8 , Reference Xie, Tu and Chen 9
The host response to virus infection involves both receptors for viral entry and the innate immune system. Many adenovirus receptors, including coxsackievirus-adenovirus receptor (CAR), CD46, and integrins, were thought to regulate the cell-mediated recognition of the adenovirus.Reference Chen and Lee 10 , Reference Fleischli, Sirena and Lesage 11 In this study, we aimed to examine the gene polymorphisms of CD46 that are involved in the occurrence and progression of disease in people with HAdV-55 infection.
MATERIALS AND METHODS
Subjects
An outbreak of HAdV-55 infection happened in a training base from December 20, 2012, to February 25, 2013. In this outbreak, a total of 856 cases were diagnosed. According to clinical and laboratory parameters, 214 samples were randomly chosen and divided into silent infections (n=91), minor infections (n=85), and severe infections (n=38). A minor infection was defined by one of the following conditions: upper respiratory tract infection, fever with dry throat or sore throat, or dry cough and throat congestion. A severe infection was defined by the same conditions in addition to nodular, patchy, or large areas of consolidations in lung imaging.Reference Chen, Nie and Xu 8 Silent infection was defined as the absence of clinical symptoms but positive results for HAdV-55 DNA detection. This study was reviewed and approved by the ethics committees in the Affiliated Hospital of the Logistics University of the People’s Armed Police Force.
Determination of HAdV-55 Infection
HAdV-55 DNA from throat swabs was detected by sequencing of PCR products according to a previous study.Reference Chen, Nie and Xu 8
Genomic DNA Extraction and Polymerase Chain Reaction
Genomic DNA from peripheral blood leukocytes was extracted from 200 μL of whole blood by using a TIANamp Blood DNA Kit (TIANGEN Biotech, Beijing, China) according to the manufacturer’s instructions. After extraction, genomic DNA was diluted to a final concentration of 15−20 ng/μL for genotyping assays. Genotyping of each participant was completed by using a MassARRAY compact analyzer on the chip-based matrix-assisted laser desorption ionization time-of-flight mass spectrometry platform (Sequenom, San Diego, CA, USA).
Statistical Analysis
Differences between groups were evaluated by using chi-squared analysis with Fisher’s exact test. Analyses were performed by using SPSS 11.5 software (SPSS Inc, Chicago, IL, USA), and P<0.05 was considered statistically significant.
RESULTS
Demographic Characteristics of the Study Population
A total of 214 HAdV-55-infected people were used in our study. All of the populations were male and of Chinese Han ethnicity. The patients’ mean age was 21.7 years. Patients with severe infection developed adenovirus pneumonia (Table 1).
Table 1 Demographic Characteristics of the Groups
CD46 Polymorphisms and HAdV-55 Infection
To evaluate the roles of CD46 polymorphisms in the occurrence of disease in patients with HAdV-55 infection, we compared the CD46 genotype frequencies between the groups of silent infections and minor or severe infections. There were no significant differences in the CD46 allele distribution for rs10449303 (A/G), rs2724385 (A/T), rs6671947 (C/G), rs7144 (T/C), rs7545126 (C/T), rs2796275 (T/C), rs2796269 (C/T), rs2796278 (A/C), or rs14374 (T/C). Although there was no observable difference in the alleles of the minor and severe group compared with the silent infection group at rs2724385 (P=0.116), the TT genotype at rs2724385 showed a protective effect against the occurrence of disease, with an odds ratio of 0.20 (95% confidence interval: 0.04-0.97) (P=0.038) compared with the AA genotype (Table 2). Moreover, we compared the differences in the frequencies of these SNPs in HAdV-55-infected patients with minor or severe infection and found that none of these SNPs was involved in the progression of HAdV-55-infected patients with clinical symptoms (data not shown).
Table 2 Genotype and Allele Frequencies of SNPs in CD46 Gene According to Occurrence of Disease in Patients with HAdV-55 InfectionFootnote a
a Abbreviations: CI, confidence interval; OR, odds ratio; SNP, single nucleotide polymorphism.
b Fisher’s exact test.
DISCUSSION
Adenoviruses were first isolated in 1953 from human adenoids that had been obtained from an adenoidectomy.Reference Rowe, Huebner and Gilmore 12 More than 60 human Adv serotypes have now been identified and classified into 6 species (A–F) based on biological, physiochemical, and genetic properties.Reference Zhu, Zhang and Xu 6 - Reference Chen, Nie and Xu 8 Human Advs are generally not considered to be highly pathogenic viruses because they are primarily associated with self-contained respiratory infections, hemorrhagic cystitis, and gastroenteritis and generally affect infants and young children.Reference Lenaerts, De Clercq and Naesens 1 , Reference Horwitz 13 , Reference Hong, Lee and Piedra 14 However, as a member of adenovirus species B, AdV-55 may cause severe acute respiratory disease, and 20% to 50% of AdV-55 infections can develop into viral pneumonia.Reference Zhu, Zhang and Xu 6 , Reference Chen, Nie and Xu 8 , Reference Xie, Tu and Chen 9 In our study, 17.76% (38/214) of the AdV-55-infected patients developed pneumonia, which is slightly lower than the previously reported rates of disease progression.
There has been little research on the association of gene polymorphisms and susceptibility to infection by adenovirus. In this study, we evaluated the gene polymorphisms of CD46 in people with different severity of HAdV-55 infection. Our data showed the TT genotype at rs2724385 to be associated with protective effects. There were no significant associations between the other SNP sites and disease occurrence in HAdV-55 infections. We evaluated whether these SNPs were associated with the progression to pneumonia in HAdV-55-infected patients with clinical symptoms but found no significant linkage.
Adenovirus serotypes are divided into 7 species (A to G), and all serotypes, except those from groups B and G, have been shown to use the CAR as a primary cellular attachment receptor.Reference Bergelson, Cunningham and Droguett 15 , Reference Bewley, Springer and Zhang 16 CD46, a ubiquitously expressed membrane-linked glycoprotein, belongs to a family of complement activation regulators. In addition to its role in preventing complement damage to normal cells, CD46 has been described as a cellular receptor for a variety of pathogens, including Neisseria and Streptococcus strains, measles virus (particularly the Edmonston strain), human herpesvirus 6, bovine viral diarrhea virus, and several adenovirus serotypes.Reference Seya, Hirano and Matsumoto 17 It has been shown that CD46 is a major cellular receptor for all species B adenoviruses and for the species D serotype 37.Reference Fleischli, Sirena and Lesage 11 , Reference Sirena, Lilienfeld and Eisenhut 18 Our data show that rs2724385 of CD46 was associated with the occurrence of disease in people with HAdV-55 infection. These findings are consistent with the notion that CD46 is a major receptor of species B adenoviruses.
In summary, as the major receptor of species B adenovirus, one SNP of CD46 (rs2724385) may be related to disease occurrence in young men with HAdV-55 infection. However, this study was limited in that the subjects were mainly young men and thus the results do not represent the entire population. Furthermore, we only examined one outbreak. More studies of other outbreaks of HAdV-55 infection need be done to confirm our findings.
Acknowledgments
The authors thank all the individuals who participated in this study for their support.
Funding
Supported by grants from Major Projects of the National Health and Family Planning Commission of China (201302003), Tianjin Key Technology Research and Development Program (12ZCZDSF00700), and Key Program of National Natural Science Foundation of China (71533008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
