Characterization of Human Coronavirus Etiology in Chinese Adults with Acute Upper Respiratory Tract Infection by Real-Time RT-PCR Assays

Background In addition to SARS associated coronaviruses, 4 non-SARS related human coronaviruses (HCoVs) are recognized as common respiratory pathogens. The etiology and clinical impact of HCoVs in Chinese adults with acute upper respiratory tract infection (URTI) needs to be characterized systematically by molecular detection with excellent sensitivity. Methodology/Principal Findings In this study, we detected 4 non-SARS related HCoV species by real-time RT-PCR in 981 nasopharyngeal swabs collected from March 2009 to February 2011. All specimens were also tested for the presence of other common respiratory viruses and newly identified viruses, human metapneumovirus (hMPV) and human bocavirus (HBoV). 157 of the 981 (16.0%) nasopharyngeal swabs were positive for HCoVs. The species detected were 229E (96 cases, 9.8%), OC43 (42 cases, 4.3%), HKU1 (16 cases, 1.6%) and NL63 (11 cases, 1.1%). HCoV-229E was circulated in 21 of the 24 months of surveillance. The detection rates for both OC43 and NL63 were showed significantly year-to-year variation between 2009/10 and 2010/11, respectively (P<0.001 and P = 0.003), and there was a higher detection frequency of HKU1 in patients aged over 60 years (P = 0.03). 48 of 157(30.57%) HCoV positive patients were co-infected. Undifferentiated human rhinoviruses and influenza (Flu) A were the most common viruses detected (more than 35%) in HCoV co-infections. Respiratory syncytial virus (RSV), human parainfluenza virus (PIV) and HBoV were detected in very low rate (less than 1%) among adult patients with URTI. Conclusions/Significance All 4 non-SARS-associated HCoVs were more frequently detected by real-time RT-PCR assay in adults with URTI in Beijing and HCoV-229E led to the most prevalent infection. Our study also suggested that all non-SARS-associated HCoVs contribute significantly to URTI in adult patients in China.

Upper respiratory tract infections (URTI) or ''the common cold'' are a significant health burden, especially among children.

Ethics Issues
All aspects of the study were performed in accordance with the national ethics regulations and approved by the Institutional Review Boards of the Centre for Disease Control and Prevention of China, as well as the Ethics Committee of Peking Union Medical College Hospital. Participants were received "Written Informed Consent" on the study's purpose and of their right to keep information confidential. Written consent was obtained from all participants or their guardians.

Patients and Specimens
Nasopharyngeal swabs were collected from adults presenting with URTI to the Peking Union Medical College Hospital, Beijing, China, between March 2009 and February 2011. Patients provided informed consent for specimen collection and testing. All patients over 14 years of age were selected according to a set of criteria that included respiratory symptoms, a body temperature above 37.5uC, and a normal or low leukocyte count(#10 10 /L), but not LRTI identified by pulmonary abnormalities on radiography and clinical descriptions(without bronchitis, bronchiolitis and pneumonia). Demographic data and clinical findings at the time of diagnosis were recorded on a standard form. All swabs were collected into viral transport medium and transported for immediate testing to the National Institute for Viral Disease Control and Prevention (NIVDC), China Center for Disease Control and Prevention.

Nucleic Acid Extraction and cDNA Synthesis
Viral RNA was extracted from 200 ml of sample using QIAamp MinElute Virus Spin kits (Qiagen, Germany). cDNA was synthesized with AMV reverse transcriptase and random hexamer primers (Promega, USA) as described previously [19,23,25].

Real-time RT-PCR for HCoV Detection
Real-time RT-PCR amplification was performed using Taq-ManH One-Step RT-PCR Master Mix Reagents Kit (Applied Biosystems, USA) [19,23,26]. A real-time reverse transcription (RT)-PCR technique was used to screen for each of the 4 non-SARS HCoVs (Table 1). PCR was performed under the following conditions: 48uC for 30 min, 95uC for 15 min, followed by 40 cycles at 95uC for 15s, 68uC for 1min.The lower limit of detection of each HCoV real-time RT-PCR assay was 50-100 copies/ 25uL.The assay sensitivity, specificity and coefficients of variability(CVs) were evaluated and validated as previously described [19,26].

Statistical Analysis
Statistical analysis was performed using the predictive analysis software (PASW) statistics 18 package with X 2 -test and Fisher's exact test.

Epidemiology of HcoVs
The seasonal distribution of HCoVs is shown in Fig.1. HCoV-229E was the most common species, appearing in most months and without an obvious seasonal distribution. Peaks in 229E detections occurred in May 2009, December 2009 and August 2010, corresponding with spring, winter and summer, respectively, in Beijing. HCoV-OC43 detections peaked in April 2009. This virus continued to circulate for the next 10 months but only sporadic infections were detected in the second half of the study. In 2009, when most coronavirus infections occurred, 229E and  OC43 were detected in every evaluable month, with the exception of 229E in September. A peak of NL63 activity occurred in March and April 2009 with little or no subsequent activity. HCoV-HKU1 activity was sporadic throughout, with no obvious seasonal distribution.
HCoVs were detected across all age groups, although NL63 cases were absent in the 30-39 years age group (Fig.2). A significantly higher detection frequency of HKU1 occurred in patients aged over 60 years (P = 0.03), but there were no significant differences in age group distribution for 229E, OC43 and NL63.

Clinical Characteristics of Patients with HCoVs Infection
Approximately equal numbers of males and females were infected with OC43, HKU1 and NL63; 229E infected significantly more female than males (62.5% versus 37.5%, P = 0.009). The clinical characteristics are summarized in Table 3. All HCoV positive patients presented with URTI. Fever, sore throat and headache were the most common symptoms at presentation. Gastrointestinal tract associated symptoms were rare. Overall there were no statistically significant differences in the clinical symptoms between speciesspecific HCoVs except for rhinorrhea, which was observed less in 229E-infected patients than in the other cases (P = 0.025).

Co-detections of other Respiratory Virus in HCoV-positive Cases
High rates of co-infection with non-HCoV respiratory viruses were observed (Table 4). Of the 157 HCoV positive patients, 48 (30.57%) were co-infected, 38 of them with one other virus and 10 with 2 other viruses. Most HCoV-associated co-infections involved human rhinoviruses (all picornaviruses positive samples were confirmed to be rhinoviruses by DNA sequencing) and Flu A virus (more than 30%). Compared to single HCoV infections, there was no evidence to indicate that patients with co-infections had more serious illnesses (results not shown).
HCoVs exhibit variable circulation approximately every 2-3 years [1,17,24,[34][35], and this was confirmed by our observation that detection rates for both OC43 and NL63 were statistically significant between 2009/10 and 2010/11. Nevertheless, 229E strains circulated throughout the 2 years of study, often in high numbers and without discernible seasonality, in contrast to that reported in the UK [23]. A previous study in Beijing reported higher detection rates for HCoVs in individuals over 65 years of age [24]. In contrast, in this study HCoVs were detected essentially in all adult age groups. Of note, however, HKU1 infection predominated in patients aged over 60 years, suggesting that adults in this category may be at higher risk for infection with this species.
URTI occurs commonly in both children and adults and is a major cause of mild morbidity which have a high cost to society. URTI among adults are responsible for missed work and unnecessary medical care (antibiotic prescriptions), also occasionally associated serious sequelae. The clinical diagnosis of all HCoVs positive patients we studied was URTI. The main clinical signs at presentation were fever, cough, sore throat and headache, in accord with several previous studies [1,7,22]. Some cases also showed gastrointestinal symptoms except in HCoV-HKU1 infection patients [1,32]. Rhinorrhea was observed in significantly fewer patients infected with 229E. Many previous reports have indicated that HCoVs have high rates of co-infection with other respiratory viruses [1,19,23,26], including rhinoviruses, influenza, HMPV, ADV and RSV. In this study, co-infections were associated with each of the non-SARS HCoVs at rates similar to previous studies [1,18,23,26]. Human rhinoviruses and influenza A viruses were the most common respiratory viruses detected in HCoVs-positive patients, possibly reflect the level of their circulation at the time, and which were consistent with rhinoviruses being the most common respiratory viruses for common cold [1,36]. In contrast with virus infection among children with URTI [1,7,23,36], RSV, PIV and HBoV were detected in very low rate(less than 1%) among adults with URTI in this study. It was consistent with the previous reports [26,37]. Comparison of clinical symptoms experienced by patients with and without coinfections indicated that co-infection with HCoVs did not affect illness severity.
In summary, we found that the four non-SARS HCoVs are regularly associated with URTI in adult patients in China, although distinct seasonal patterns of circulation are not obvious. Our observation that elderly adults may be are at high risk of HKU1 infection requires further study. Our results suggest that testing algorithms that include sensitive HCoVs detection contribute to the likelihood of obtaining a laboratory diagnosis when respiratory virus infection is suspected [26,[37][38]. To the best of our knowledge, this is the first comprehensive analysis of the potential impact of four non-SARS HCoVs (HCoV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HKU1) as causes of URTIs in adults admitted to hospital in China by real time RT-PCR assays. To identify the role of HCoVs infection in URTI of adults, more specimens include control subjects with clear clinical and epidemiological profiles warrant be investigated.