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During the 6-year period, 8275 nasopharyngeal samples from patients(male:female = 1.03:1, age(mean SD)= 49 36 years)with acute respiratory tract infections were collected from the 2 public hospitals for the present study. HCoVs were detected in 77(0.93%)nasopharyngeal samples from patients with the median age of 79.5 years(range, 22 days to 95 years). Thirty-seven were males and 40 were females.
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The partial RdRp genes of 77 HCoV strains detected from the nasopharyngeal samples were amplified and sequenced. The phylogenetic tree was shown in Figure 1. The nucleotide sequences of the 12 HCoV strains showed 99.5% 100% nucleotide identities to HCoV-229E, those of the 6 HCoV strains showed 98.9% 100% nucleotide identities to HCoV-NL63, those of the 48 HCoV strains showed 98.6% 100% nucleotide identities to HCoV-OC43 and those of the 11 HCoV strains showed 97% 100% nucleotide identities to HCoV-HKU1. Accordingly, the most frequently detected HCoV species was HCoV-OC43(48/8275, 0.58%), followed by HCoV-229E(12/8275, 0.15%), HCoV-HKU1(11/8275, 0.13%) and HCoV-NL63(6/8275, 0.07%)by RT-PCR(Table 1).
Figure 1. Phylogenetic trees of partial RdRp gene sequences of 77 HCoV strains detected from the nasopharyngeal samples. The tree was inferred from the partial RdRp gene data by ML method, with bootstrap values calculated from 1000 trees. CCoV, canine coronavirus; FIPV, feline infectious peritonitis virus; HCoV-229E, human coronavirus 229E; HCoV-NL63, human coronavirus NL63; Mi-BatCoV HKU8, Miniopterus bat coronavirus HKU8; PEDV, porcine epidemic diarrhea virus; Rh-BatCoV HKU2, Rhinolophus bat coronavirus HKU2; Ro-BatCoV HKU10, Rousettus bat coronavirus HKU10; Sc-BatCoV 512, Scotophilus bat coronavirus 512; TGEV, porcine transmissible gastroenteritis virus; BCoV, bovine coronavirus; ChRCoV HKU24, China Rattus coronavirus HKU24; CRCoV, canine respiratory coronavirus; DcCoV UAE-HKU23, dromedary camel coronavirus UAE-HKU23; ECoV, equine coronavirus; HCoV HKU1, human coronavirus HKU1; HCoV-OC43, human coronavirus OC43; MHV, murine hepatitis virus; PHEV, porcine hemagglutinating encephalomyelitis virus; RCoV, rat coronavirus; RbCoV HKU14, rabbit coronavirus HKU14.
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Analysis of age distribution among patients positive for HCoVs showed that HCoVs mainly infected the elderly, with the highest detection frequency(36/2380, 1.51%)in those > 80 years and the majority of cases infected by HCoV-OC43(26/36, 72%)(Table 1). HCoVs also commonly infected children, with the detection rate of 0.91%(23/2529) in the age group of 0 10 years and about half of the cases infected by HCoV-OC43(12/23, 52%). Interestingly, the detection rate of HCoV-OC43 in the age group of > 80 years(26/2380, 1.09%)was significantly higher than the rates in the age groups of 0 10 years(12/2529, 0.47%) and 71 80 years(3/1237, 0.24%)(P < 0.05)(Table 2). No significant differences were noted between the detection rate of HCoV-OC43 in the age group of > 80 years and the rates in other age groups from 11 to 70 years(P > 0.05)(Table 2). None of the samples from patients aged between 11 and 30 years were positive for HCoVs. However, an increasing trend in the detection frequency of HCoVs with age was noted among other age groups, from 0.42%(1/238) in the age group of 31 40 years to 0.65%(4/617) in the age group of 61 70 years and 0.57%(7/1237) in patients aged between 71 80 years.
Age group (years) Number of samples No. (%) of positive samples HCoVs HCoV-229E HCoV-NL63 HCoV-OC43 HCoV-HKU1 0–10 2529 23 (0.91) 5 (0.2) 2 (0.08) 12 (0.47) 4 (0.16) 11–20 307 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 21–30 177 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 31–40 238 1 (0.42) 0 (0) 0 (0) 1 (0.42) 0 (0) 41–50 323 2 (0.62) 1 (0.31) 0 (0) 1 (0.31) 0 (0) 51–60 465 3 (0.65) 0 (0) 1 (0.22) 2 (0.43) 0 (0) 61–70 617 4 (0.65) 1 (0.16) 0 (0) 2 (0.32) 1 (0.16) 71–80 1237 7 (0.57) 2 (0.16) 1 (0.08) 3 (0.24) 1 (0.08) > 80 2380 36 (1.51) 3 (0.13) 2 (0.08) 26 (1.09) 5 (0.21) Unknown 2 1 - - 1 - Total 8275 77 (0.93) 12 (0.15) 6 (0.07) 48 (0.58) 11 (0.13) Table 1. Age distribution of nasopharyngeal samples positive for human coronaviruses.
Age groups (years) Number of samples No. (%) of samples positive for HCoV-OC43 P value * 0–10 2529 12 (0.47) 0.014 11–20 307 0 (0) 0.066 21–30 177 0 (0) 0.162 31–40 238 1 (0.42) 0.328 41–50 323 1 (0.31) 0.184 51–60 465 2 (0.43) 0.186 61–70 617 2 (0.32) 0.077 71–80 1237 3 (0.24) 0.007 > 80 2380 26 (1.09) - Note: * Using > 80 years old as the reference age group; P < 0.05 was considered to be statistically significant. Table 2. Comparisons between patients aged > 80 years and those of other age groups infected with HCoV-OC43
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HCoVs were detected over the 6-year study period, with the highest and lowest detection rates of HCoVs noted in the periods of September 2010 August 2011(22/1500, 1.47%) and September 2013 August 2014(4/1200, 0.33%), respectively(Figure 2). HCoVs were most frequently detected in the fall and winter during the 6-year period, but each HCoV species displayed different seasonal patterns(Figure 3). HCoV-OC43 was the most frequently detected species throughout all years, with the highest detection rate in the period of September 2010 August 2011(13/1500, 0.87%). HCoV-OC43 was predominant in the fall and winter. HCoV-HKU1 was also detected throughout all years, with the highest detection rate in the period of September 2012 August 2013(4/1300, 0.31%). HCoV-HKU1 infections mainly occurred in winter. HCoV-229E was detected in alternate years [except in September 2011 August 2012, but with very low detection rate(1/1400, 0.07%)], with the highest detection rate in the period of September 2012 August 2013(4/1300, 0.31%). HCoV-229E infections mainly occurred in winter and spring, with a few cases occurred in summer in 2011. HCoV-NL63 was the least frequently detected HCoV species, with the highest detection rate during September 2010 August 2011(3/1500, 0.2%). HCoV-NL63 was predominant in the summer and fall.