Detection and gene characteristics of human bocavirus in acute respiratory tract infection cases in Luohe, Henan
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摘要:
目的 分析河南省漯河市急性呼吸道感染(ARI)病例中人博卡病毒(HBoV)的检出情况及病毒基因特征。 方法 使用实时荧光定量聚合酶链式反应(qRT-PCR)对2018年6月至2020年12月收集的1 185例ARI病例咽拭子标本进行呼吸道多病毒检测。 对HBoV核酸阳性的标本进行VP1基因巢式PCR扩增和序列测定,同时结合GenBank下载的中国地区HBoV1 VP1基因序列,进行病毒基因特征分析。 结果 在1 185例ARI病例标本中,共有32例(2.70%)为HBoV阳性,其中17例(53.13%)同时检出≥2种病毒。 96.88%(31/32)HBoV阳性病例为5岁以下的儿童,其中以12~23月龄患儿的检出率最高(5.52%),其次是6~11月龄(3.39%)。 HBoV阳性病例主要集中在夏、秋、冬3个季节,但三者间差异无统计学意义(P>0.05)。 HBoV的单一感染和混合感染均可引起上呼吸道或下呼吸道感染,但差异无统计学意义(P>0.05)。 本研究所获得的21条HBoV序列均属于HBoV1。 Ⅰb分支是2018—2020年河南省漯河市HBoV1主要优势流行株,与我国其他地区一致。 VP1基因平均分子进化速率约为2.59×10−4替换/位点每年,在影响病毒抗原的主要区域中存在着多个氨基酸变异。 结论 HBoV是河南省漯河市ARI病例中检出的重要病毒之一。 5岁以下儿童,尤其是2岁以下的婴幼儿是HBoV感染的主要人群。 包括河南漯河株在内我国流行HBoV1 VP1基因序列相对保守,但有个别抗原相关位点发生了变异。 Abstract:Objective To understand the detection and genetic characteristics of human bocavirus (HBoV) in acute respiratory infection cases in Luohe, Henan province. Methods Throat swabs collected from 1185 acute respiratory infection cases in Luohe from June 2018 to December 2020 were used for the nucleic acid detections of multiple common respiratory viruses with quantitative real-time polymerase chain reaction (qRT-PCR). Nest PCR amplification and sequencing of VP1 gene were performed in HBoV nucleic acid positive samples, and the viral gene characteristics were analyzed in combination with Chinese HBoV VP1 gene sequences downloaded from Genbank. Results Among 1185 acute respiratory infection cases, 32 (2.70%) were positive for HBoV; 17 (53.13%) were positive for two or more viruses. Most HBoV-positive cases were children under 5 years old (96.88%, 31/32), the detection rate was highest in children aged 12−23 months (5.52%), followed by infants aged 6−11 months (3.39%). HBoV-positive cases mainly occurred in summer, autumn and winter, the differences in positive rate were not significant among three seasons (P>0.05). HBoV can cause upper or lower respiratory tract infections in both single infection cases and co-infection cases, but the difference in case number was not significant (P>0.05). The 21 HBoV sequences obtained in this study belonged to HBoV1. The strains in Ib branch were predominant in Luohe from 2018 to 2020, consistent with other regions in China. The average molecular evolutionary rate of the VP1 gene was approximately 2.5×10−4 substitutions/site/year, and there were multiple amino acid variations in the main regions of antigen of HBoV. Conclusion HBoV is one of the important viruses detected in acute respiratory infection cases in Luohe. Children under 5 years old, especially infants/young children under 2 years old, are the susceptible population of HBoV infection. The VP1 gene sequence of HBoV1 in China, including Luohe strain, is relatively conservative, but some amino acid variations have occurred in antigen-related sites. -
Key words:
- Acute respiratory infection /
- Human bocavirus /
- Detection /
- Genetic characteristic
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表 1 人博卡病毒VP1基因巢式聚合酶链式反应扩增引物
Table 1. Primers of nest PCR to amplify HBoV VP1 gene
引物
名称a序列(5'~3') 位置
(核苷酸)b长度(bp) 4FW TGGCAGACAACTCATCACAGGA 2 491~2 512 1 000 4RW TGTCTGAAAAGTGAGAACCTCCG 3 574~3 596 4FN ACACAGTGGGGAGAGAGGCT 2 535~2 554 4RN CCTCCTCCAATACTTCCTGTTCC 3 512~3 534 5FW TTTAAAATAAAGCGCGCCGTGG 3 308~3 329 953 5RW GCTTGTTGGTTTTGAGTATGGC 4 309~4 330 5FN TCTGGGAAATAAAGAGAGAGCC 3 337~3 358 5RN CTGCTGTGCTTCCGTTTTGTCT 4 268~4 289 6FW TCGCAGATCTTGATGGAAATGC 4 038~4 059 1 031 6RW TGTACAACAACAACACATTAAAAGA 5 275~5 299 6FN TTGAAAACAGTGACCACCAAGT 4 116~4 137 6RN TACAGTCACCCTTCACTTTCCG 5 125~5 146 注:a. 引物名称:F. 正向引物;R. 反向引物;W. 第一轮引物;N. 第二轮引物;b. 位置(核苷酸):参考基因组 DQ000496.1 表 2 人博卡病毒阳性病例流行病学特征
Table 2. Epidemiological characteristics of HBoV positive cases
分析变量 HBoV阳性
病例数(例)总例数
(例)检出率
(%)χ2值 P值 性别 0.486 0.589 男性 16 664 2.41 女性 16 521 3.07 年龄组 16.865 0.009a < 6月龄 0 22 0.00 6~11月龄 2 59 3.39 12~23月龄 19 344 5.52 2~4岁 10 437 2.29 5~17岁 0 203 0.00 18~39岁 0 27 0.00 40~59岁 0 32 0.00 ≥ 60岁 1 61 1.64 季节 9.589 0.018a 春季(3-5月) 0 158 0.00 夏季(6-8月) 6 159 3.77 秋季(9-11月) 15 363 4.13 冬季(12至次年2月) 11 505 2.18 总计 32 1 185 2.70 注:a. 该组内HBoV阳性检出率差异具有统计学意义;HBoV. 人博卡病毒 表 3 人博卡病毒阳性病例病毒感染情况
Table 3. Co-viral infection in HBoV-positive cases
检出类型 病毒 病例数
(上感/下感)构成比
(%)a单一病毒 HBoV 15(9/6) 46.87 混合感染 17(8/9) 53.13 2种病毒 HBoV+HMPV 5(1/4) 15.62 HBoV+HPIV 3(2/1) 9.37 HBoV+HRSV 2(1/1) 6.25 HBoV+HAdV 1(0/1) 3.13 HBoV+EV 1(0/1) 3.13 3种病毒 HBoV+HRV+EV 2(2/0) 6.25 HBoV+Flu+HAdV 1(1/0) 3.13 HBoV+HAdV+HMPV 1(0/1) 3.13 4种病毒 HBoV+HRV+EV+HRSV 1(1/0) 3.13 合计 32(17/15) 100.00 注:上感. 上呼吸道感染;下感. 下呼吸道感染;a. 不同病毒检出类型阳性病例数占总阳性病例数的构成比;HBoV. 人博卡病毒; HMPV. 人偏肺病毒; EV. 人肠道病毒;HRV. 人鼻病毒;HAdV. 人腺病毒;HRSV. 人呼吸道合胞病毒;HPIV. 人副流感病毒;Flu. 人流感病毒 表 4 人博卡病毒VP1基因抗原主要区域氨基酸变异情况分析
Table 4. Amino acid variations in antigen main regions of HBoV VP1 gene
VP1u(1~129 aa) VP3的N端区域(130~165 aa) βG-βH 插入序列区域(374~576 aa) aa ST1 ST2 中国株(n=51) aa ST1 ST2 中国株(n=51) aa ST1 ST2 中国株(n=51) 17 R R K(7) 135 I I F(1) 415 G G S(3) 24 P P S(1) 137 D D N(1) 425 S S R(1) 26 D D N(1) 143 V V G(1) 427 M M I(1) 40 L L S(3) 147 Q Q R(1) 468 S S T(1) 47 E E K(2) 149 A T N(1) 474 S N S(51) 68 D D N(3) 151 G G R(1) 529 I I L(1) 72 D D N(1) 533 P P L(1) 74 W W R(1) 538 D D G(1) 84 F F V(1) 540 F F Y(2) 109 A A V(1) 560 I I V(2) 128 S S L(1) 注:括号内的数字代表该氨基酸位点变异的数量;表格内大写字母代表氨基酸名称:R. 精氨酸;K. 赖氨酸;P. 脯氨酸;S. 丝氨酸;D. 天冬氨酸;N. 天冬酰胺;L. 亮氨酸;E. 谷氨酸;W. 色氨酸;F. 苯丙氨酸;V. 缬氨酸;A. 丙氨酸;I. 异亮氨酸;G. 甘氨酸;Q. 谷氨酰胺;T. 苏氨酸;M. 蛋氨酸;Y. 酪氨酸;aa,氨基酸位点 -
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