Diversity of human rhinovirus species among children with severe or mild acute lower respiratory infection in Beijing, China, during 2016–2019


 Background Human rhinovirus (HRV) infections were confirmed in severe acute lower respiratory infections (ALRI) . Methods To evaluate the diversity and circulation pattern of HRVs species, specimens were collected from pediatric patients with ALRI during Dec 2016 to Feb 2019 and screened by RVP FAST Assay. Specimens positive for HRVs/ Enteroviruses (EVs) were then tested for HRVs and EVs by PCR. Then the capsid protein gene of HRVs was amplified and sequenced for species identification, and clinical data of HRV infections were analyzed. The chi-square (χ 2 ) test and rank sum test was used for statistical analysis using SPSS Statistics 22.0 version. In order to test and evaluate the relationship between patients positive for different HRV species and the outcome of Intensive Care Unit (ICU) (ICU group) or Department of Respiratory admission (Respiratory group), a logistic model was constructed using multiple logistic regression analysis. Results Among 1835 specimens tested, 363 (19.8%) were positive for EVs/HRVs, and 314 (86.5%, 314/363) were positive for HRVs, including 177 (56.4%, 177/314) HRV A, 29 (9.2%, 29/314) HRV B, and 108 (34.4%, 108/314) HRV C. Using HRV C as the control species, HRV A was a dangerous factor for severe clinical outcome (OR=1.983, 95% CI=1.091-3.605) ( p =0.025). Patients positive for HRVs from ICU group were significantly younger than those from Respiratory group (0.39 years: 1.80 years, p =0.000) and age was confirmed as a protective factor for severe ALRI. In August and September, HRV A and B are the dominant species, then HRV C in October and December. Among 96 types confirmed in the study, the predominant types usually showed in no consecutive years. Conclusions HRVs were important viral pathogens for ALRI in children. HRV A and C are more popular. Compared to HRV C, HRV A were associated more powerful with severe ALRI. The risk of severe ALRI by HRVs infections is decreased with the increasing of age.

To evaluate the diversity of HRVs species among children with severe or mild lower acute respiratory infection in Beijing and to reveal the circulation patterns of HRVs, clinical specimens for respiratory virus screening were collected from pediatric patients during Dec 2016 to Feb 2019 for pan-HRV detection in a universal nucleotide test in the study, and then the capsid protein gene of HRVs was sequenced for species identification and clinical data were analyzed.

Clinical specimens
In the retrospective study, nasopharyngeal aspirates (NPA) were obtained for respiratory virus screening from patients aged < 16 years who visited the Children's Hospital affiliated with the Capital Institute of Pediatrics (CIP) (Beijing, China) and were diagnosed with acute lower respiratory infections (ALRIs), including bronchitis, bronchiolitis, and pneumonia, between November 2016 to February 2019. The diagnosis of ALRI in this study was according to the Zhu Futang Textbook of Pediatrics (7 th Edition) (19).
Specimens were immediately stored at 4°C and sent to the laboratory within 12 hours.
Upon arrival at the laboratory, each of the clinical specimens was handled in a Class II biosafety cabinet (BSC II) and processed immediately in 2.5 ml of viral transport medium (VTM) (Yocon Biotechnology Co., Ltd, Beijing, China) and centrifuged at 500 × g for 10 min to obtain the supernatant for respiratory virus screening.
The study was approved by the Ethics Committee of the Capital Institute of Pediatrics.

Identification and genotyping of HRVs or EVs by RT-PCR and sequence analysis
For EVs/HRVs-positive clinical specimens, semi-nested revere-transcription PCR (RT-PCR) was performed for HRV-confirmation, and the amplified products of the second seminested PCR for a 539bp nucleotides fragment within the VP4/VP2 region were sequenced

Clinical characters of different HRV species in severe or mild acute lower respiratory tract infection
As shown in Table 1, there were 925 patients discharged from Intensive Care Unit (ICU) with severe ALRI (ICU group) and 508 patients discharged from the Department of Respiratory with mild ALRI (Respiratory group) compared to those from ICU. The HRVs positive rates are similar and HRV A and HRV C are the major pathogens in two groups.
However, the positive rates of HRVs species were significant different between the two groups (p=0.011), which may be explained by more HRV A in ICU group (63.0%: 45.2%, p=0.006), and more HRV B (15.1%:6.5%, p=0.028) in Respiratory group. However, no significant difference was shown on HRV C (39.8%: 30.5%, p=0.136) between the two groups. Compared to patients positive for HRVs from Respiratory group, patients who were positive for HRVs from ICU group were significantly younger (0.39 years: 1.80 years, p=0.000).
In the ICU group, about 44.8% (n=69) were viral co-infection, with HBoV (n=27) as the major virus followed by PIV (n=19) and RSV (n=18). In the Respiratory group, about 45.2% (n=42) were viral co-infection, with RSV as the most common viral pathogen (n=12) followed by PIV (n=11) and ADV (n=9). No significant difference was shown in the whole viral co-infection (p=0.957) and viral co-infection in different HRV species (p=0.239) between ICU group and the Respiratory group. Then a logistic model was constructed to test and verify the variables associated with severe ALRI (Table 2). By using HRV C infection as the reference group among HRV species when gender, age and co-infections were considered as factors, there is a significant difference as the odds of HRV A related to severe ALRI was OR=1.983 (95%CI=1.091-3.605) (p=0.025), while no significant difference was shown (OR=0.707, 95%CI=0.260-1.920) (p=0.496) when the odds of HRV B related to severe ALRI was evaluated. In the logistic model, gender was excluded from variables associated with severe ALRI (OR=0.710, 95% CI=0.389-1.295, p=0.264), and age (OR=0.703, 95% CI=0.607-0.813, p=0.000) was confirmed as a protective factor (OR<1, the upper limit of 95% CI<1) for severe ALRI.
When the correlation of HRV sero-or geno-types with severe or mild ALRI was evaluated (Fig 5), 39 HRV types were shown in both groups. In ICU groups, HRV A24 followed by A49, HRV B79 followed by B6, and HRV C2 were the most popular serotypes of different species, while HRV A49 followed by A18, HRV B14, B79 and B92, and HRV C5 followed by C2 were the most popular serotypes of different species in Respiratory group. However, as a result of restrictions on the numbers of specimens belonging to each sero-or geno-type, no skewing towards one type in either group was observed.

Discussion
Determined by RVP FAST Assay in the study, EVs/HRVs were the second viral agent However, their results showed no significant difference in clinical symptoms according to HRV species (14). In the study, when gender, age and co-infection were all considered and using HRV C as the control species, HRV A was a dangerous factor to cause severe clinical outcome (OR=1.983>1 ,95% CI=1.091-3.605, the lower limit of 95% CI>1) (p=0.025), and HRV B has no relationship with severity of clinical outcome. Therefore, infection of HRV A was weight higher than that of HRV C in causing severe ALRI. Patients who were positive for HRVs from ICU group were significantly younger than those from Respiratory group (0.39 years: 1.80 years, p=0.000), and age (OR=0.703, 95% CI=0.607-0.813, p=0.000) was confirmed as a protective factor (OR<1, the upper limit of 95% CI<1) for severe ALRI, which implied that the risk of severe ALRI by HRVs infections is decreased with the increasing of age. In three Sub-Saharan African countries, those with pneumonia and HRV C were older (12.1 vs. 9.4 months, P=0.033) and more likely to present with wheeze (35% vs. 25%, P=0.031) compared to HRV A cases (13). Therefore, age is an important variable associated with severe ALRI when HRVs infections were evaluated. It has been demonstrated that cellular receptor for the majority of HRV A and B (major group) is intercellular adhesion molecule 1 (ICAM-1), for a minority of isolates (minor group, ~10 HRV A), low-density lipoprotein receptor (LDLR) family members (24), for HRV C, the cadherin-related family member 3 (CDHR3) suggested (25). These considerable diversities of genetic, immunogenic, and receptor use properties might account for the differences in clinical presentation of different HRV species infection (26). was the dominant type, in HRV B, B79, in HRV C, C2. However, HRV A24, B79, and C2 were the most popular serotypes of different species, respectively, in ICU groups, while HRV A49, B14, and C5 were the most popular serotypes of different species, respectively, in Respiratory group. In the research work, although no obvious circulation pattern was shown for each sero-or geno-type, most prevalent strains were not detected in consecutive years, for example, A49 was found only between March 2017 and October 2017, B79 was detected in the winter of 2018, and C2 was detected more often in the late of 2018. We did not observe some skewing towards one type in children with severe illness. In Shanghai, 77 sero-or geno-types were detected including 43 for HRV A, 10 for HRV B, and 24 for HRV C, among which A78, A12, A89, B70, C2, C6, and C24 predominated during 2013-2015 (14). More data should be accumulated to reveal the epidemiology characters of each sero-or geno-type.
There are some limitations in the study. Co-bacterial infection was not evaluated, which maybe a variable associated with the severity of ALRI in HRVs infections. We collected specimens only in two years, and more data should be accumulated to provide more powerful information supporting the circulation patterns of HRVs concluded in the study.
In conclusion, HRVs outweigh EVs and were second only to RSV as the important viral pathogens for ALRI in children in Beijing during 2016 to 2019. HRV A and C are more popular than HRV B in two groups with mild or sever ALRI. By using HRV C as the reference species, HRV A were associated more powerful with severe ALRI, and age was a variable when association of HRV species infection with the severity of disease was evaluated. The predominant sero-or geno-types were different in two groups with mild or sever ALRI, and usually showed in no consecutive years.

Ethics approval:
The study was approved by the Ethics Committee of the Capital Institute of Pediatrics.    Figure 1 Scheme for specimens' selection