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华南地区HGV的流行状况和同源性分析
中华微生物学和免疫学杂志 1999年第3期第19卷 病毒学
作者:李刚 廖家杰 马会慧 梁英杰 姚集鲁 姚春斓 苏永洪 汤文辉 崇雨田 陈青
单位:李刚 马会慧 姚集鲁 姚春斓 崇雨田 陈青 510630 广州 中山医科大学传染病学教研室;廖家杰 香港大学玛丽医院胃肠肝病科;梁英杰 苏永洪 粤港肝炎研究中心;汤文辉 云南省昆明医学院附属一院传染科
关键词:庚型肝炎病毒;聚合酶链反应;DNA序列分析;同源性
【 摘要 】 目的 了解华南地区庚型肝炎病毒(HGV) 的流行状况及HGV不同株和不同基因区的同源性。方法 采用逆转录聚合酶链反应技术(RT-PCR)检测来自广东、香港、云南的不同人群血清标本共1 991份。对其中20份的5端非编码区(5UTR) 238bp和3份非结构蛋白5区(NS5) 621bp进行了序列测定和同源性分析。结果 一般人群HGV RNA阳性率为(0.73~1.34)%,献血员(2.52~2.90)%,静脉吸毒者17.86%,血液透析患者14.13%,接受骨髓移植者41.67%,在非甲~戊型肝炎病人为25.30%,肝细胞癌14.48%,乙型肝炎7.22%,丙型肝炎(8.33~16.13)%。不同株5UTR同源性介乎(90.40~100)%; 不同株NS5区核苷酸同源性(93.30~94.00)%,氨基酸为(97~99.2)%。结论 接受骨髓移植、血液透析、静脉吸毒者,乙型、丙型、非甲~戊型肝炎病人及肝细胞癌患者是HGV的高感染人群。不同HGV株存在一定的地区性差异;同一地区不同人群的HGV株变异不明显;序列中存在高度保守区及较大变异区。
Prevalence of HGV infection and homology of different HGV strains in Southern China
LI Gang, George K.K.Lau, MA Huihui, et al. Department of Infectious Disease, Sun Yat-sen University of Medical Science, Guangzhou 510630【 Abstract 】 Objectives To investigate the prevalence of hepatitis G virus (HGV) infection and to analyse the homology of different HGV strains in Southern China.Methods A total of 1991 sera from different groups in Guangdong, Hong Kong and Yunnan were detected by reverse transcription polymerase chain reaction (RT-PCR). The nucleotide sequences of 5' untranslated region (5' UTR) derived from 20 strains and NS5 region from 3 strains were determined.Results The positive rate of HGV RNA was 0.78%-1.34% in society population, 2.52%-2.90% in blood donors, 17.86% in intravenous drug users (IVDUs), 14.34% in patients with hemodialysis, 14.48% in patients with hepatocellular carcinoma, 25.30% in non A-E hepatitis, 7.22% in hepatitis B, 8.33%-16.13% in hepatitis C, 41.67% in patients with bone marrow transplantation (BMT), respectively. The homology was 90.40%-100% in 5' UTR between different strains. The homology of NS5 region was 93.3%-94% in nucleotide sequence, 97%-99.2% in amino acid sequence.Conclusions The HGV infection rates among populations having accepted bone marrow transplantation, hemodialysis, and patients with hepatitis B, non A-E hepatitis, hepatitis C and hepatocellular carcinoma as well as the intravenous drug abusers were high. Sequence variation in different strains were associated with geographical factors. No apparent variation between different strains from different groups in Southern China and no mutation in same patient over a period of 3 months were observed. Some highly conserved and relatively variable areas were seen.
【 Subject words 】 Hepatitis G virus Polymerase chain reaction Prevalence DNA sequence determination Homology
HGV是近年发现的一种新型肝炎病毒〔1,2〕,主要通过非经口途径传播,流行广泛。到目前为止,全球大部分国家均发现HGV感染病例。HGV基因组结构类似黄病毒,全长约9.4kb, 由两侧的非编码区及中间的结构蛋白和非结构蛋白编码区组成。5端非编码区(5UTR)是核糖体结合位点,对HGV的翻译和复制起重要作用;非结构蛋白编码区中的NS5区是依赖RNA的RNA聚合酶的编码部位。为了解HGV的流行状况和不同株5UTR及NS5区的同源性,我们采用RT-PCR方法对华南地区不同人群的HGV感染进行了流行病学调查, 并对来自华南不同地区,不同人群, 感染后不同时间的20个HGV株的5'UTR和3个HGV株的NS5区进行了序列测定和同源性分析。
材料与方法
1.血清标本:共1 991份。采自广东、香港和云南三个省区,来自多个人群(详情见表1和表3)。血清在1994~1997年期间采集,-20℃以下保存。
表1 测序标本
Table 1. Samples for sequencing
No. | Sex | Age(y) | Resource | Diagnosis | Region |
GD1 | F | 58 | Guangdong | non-A-E hepatitis | 5UTR |
GD3027 | M | 47 | Guangdong | Hepatitis B | 5UTR, NS5 |
GD3040 | M | 76 | Guangdong | Hepatitis B | 5UTR |
GD3064 | F | 20 | Guangdong | non-A-E hepatitis | 5UTR |
GDCA | M | 26 | Guangdong | Hepatocellular carcinoma | 5UTR |
YN1 | M | 26 | Yunnan | IVDUs | 5UTR |
YN2 | M | 38 | Yunnan | IVDUs | 5UTR |
YN3 | M | 45 | Yunnan | IVDUs | 5UTR |
HKC | F | 42 | HongKong | Hepatitis C | 5UTR |
HKC16 | M | 38 | HongKong | Hepatitis C | 5UTR |
HK8 | M | 56 | HongKong | BMT | 5UTR |
HK9 | M | 42 | HongKong | BMT | 5UTR |
HK10 | M | 38 | HongKong | BMT | 5UTR |
HK11 | M | 42 | HongKong | BMT | 5UTR |
HK12 | M | 42 | HongKong | BMT | 5UTR |
HK24 | F | 42 | HongKong | BMT | 5UTR |
HK80 | M | 40 | HongKong | BMT | 5UTR |
HK108 | M | 18 | HongKong | BMT | 5UTR |
HK116 | M | 42 | HongKong | BMT | 5UTR |
HK120 | M | 40 | HongKong | BMT | 5UTR |
A132 | M | 20 | HongKong | Blood donor | NS5 |
A711 | M | 25 | HongKong | Blood donor | NS5 |
HK9 and HK12, HK11 and HK116 are samples from the same patients with a duration of 3 months respectively. IVDUs: intravenous drug abusers; BMT: bone marrow transplantation; F: female; M: male
表3 HGV在华南地区不同人群的感染状况
Table 3. Prevalence of HGV infection from different groups in Southern China
Group | Resourse | HGV RNA | ||
n | Positive(n) | Percentage(%) | ||
Non-A-E hepatitis | Guangdong | 83 | 21 | 25.30 |
IVDUs | Yunnan | 84 | 15 | 17.86 |
Blood donors | Guangdong | 69 | 2 | 2.90 |
HongKong | 437 | 11 | 2.52 | |
BMT | HongKong | 108 | 45 | 41.67 |
Hepatocellular carcinoma | Guangdong | 145 | 21 | 14.48 |
HongKong | 16 | 1 | 6.25 | |
Hepatitis B | Guangdong | 263 | 19 | 7.22 |
Hepatitis C | Guangdong | 31 | 5 | 16.13 |
HongKong | 24 | 2 | 8.33 | |
Hepatitis E | Guangdong | 29 | 2 | 6.90 |
Hepatitis B+A | Guangdong | 16 | 1 | 6.25 |
Hepatitis B+C | Guangdong | 6 | 1 | 16.67 |
Hepatitis B+E | Guangdong | 26 | 3 | 11.54 |
Normal | Guangdong | 413 | 3 | 0.73 |
HongKong | 149 | 2 | 1.34 | |
Hemodialysis | Guangdong | 92 | 13 | 14.13 |
Total | 1991 | 167 | 8.39 |
2.引物:根据GenBank中GBV-C、PNF2161及R10291株序列设计,位置及序列见表2。
表2 RT-PCR所用引物序列
Table 2. Primers used in RT-PCR
Code No | Polarity | Site | Sequence |
G1 | (+) | 117-136 | 5 ATGCGTGATGACAGGGTTGG 3 |
G2 | (-) | 451-471 | 5 TAGGTGGCCCCATGCATTTCC 3 |
G3 | (+) | 161-180 | 5 GGTAGCCACTATAGGTGGGT 3 |
G4 | (-) | 379-398 | 5 CACTGGTCCTTGTCAACTCG 3 |
33 | (+) | 6672-6697 | 5 GTTGAATTCGCGATGGAGCGCTACAC 3 |
34 | (-) | 7267-7292 | 5 CTGGGATCCGTATCATGTATGGTTCT 3 |
35 | (+) | 6573-6592 | 5 TCGATTGCTGTAGCTGAGCC 3 |
36 | (-) | 7327-7346 | 5 GGTAAGTTCATTGCCCACCA 3 |
Site:based upon strain PNF2161;G3,G4:inner primer,amplified product 238bp;33,34:inner primer,amplified product 621bp
3. RT-PCR:采用酶消化热变性方法提取血清中RNA,特异负链外引物引导逆转录反应,经巢式扩增后得到5UTR片段238bp, NS5区片段621bp。具体方法参见文献〔3〕。
4. PCR产物分子克隆: 将1例血清(YN1)的5UTR PCR产物238bp提纯后,用Klenow酶补平,克隆入pUC18的SmaⅠ位点,获得重组体pGN。将2例血清(A132, A711)的NS5区PCR产物621bp分别克隆入pUC19的SmaⅠ和BamHⅠ位点,获得重组体pUGN132及pUGN711。对另1例血清(GD3027) NS5区621bp产物用EcoRⅠ酶切后得到2个片段,分别为196bp和425bp, 将其中的 425bp片段插入pUC19的EcoRⅠ和BamHⅠ位点之间,得到重组体pUGN3027。获得的重组体用PCR法和酶切法鉴定。
5. 序列测定: 测序标本共23份,20份测5UTR,3份测NS5区;4份PCR产物克隆后测序,19份PCR产物直接测序。采用双脱氧链末端终止法,在310 DNA全自动测序仪(ABI)测定核苷酸序列,同一标本经正反方向测定,所测序列用DNASIS, PROSIS软件进行同源性分析并推导氨基酸序列。
结果
1.HGV在华南地区不同人群的感染状况:结果列于表3。
2. HGV不同株5UTR cDNA序列和NS5区cDNA和氨基酸序列的同源性分析:见图1, 表4, 表5。其中,不同株间NS5区的序列比较未在图中列出,其碱基变异主要呈散在分布, 但亦发现几个较大变异区,包括6698~6837,7080~7092,及7157~7251(按PNF2161株位置)。
图1 HGV不同地理株5UTR cDNA相应序列的比较
Fig 1. Comparison of HGV 5UTR cDNA sequences from different geographical strains
GD stands for Guangdong, YN for Yunnan, HK for Hong Kong. C964 was derived from Northern China〔4〕, PNF2161 and R10291 from United States〔1〕, GBV-C from West Africa〔2〕.Dots indicate identity with sequence of GD1
表4 HGV 不同株5’UTR 序列相互间的同源性比较(%)
Table 4 Homology analysis of HGV 5’UTR sequences from different strains
GK3027 | GD3040 | GD3064 | GDCA | YN1 | YN2 | YN3 | HKC9 | HKC16 | HK8 | HK9 | HK10 | HK11 | HK12 | HK24 | HK80 | HK108 | HK116 | HK120 | C964 | PNF2161 | R10291 | GBV-C | |
GD1 | 97.98 | 98.99 | 98.99 | 95.45 | 96.87 | 93.94 | 97.98 | 98.48 | 98.99 | 96.97 | 98.99 | 97.98 | 96.97 | 98.99 | 97.47 | 96.97 | 98.99 | 96.97 | 93.94 | 92.93 | 90.40 | 89.39 | 86.87 |
GD3027 | 98.99 | 98.99 | 96.46 | 96.97 | 93.43 | 97.98 | 98.48 | 98.99 | 96.97 | 98.99 | 97.98 | 96.97 | 98.99 | 97.47 | 96.97 | 98.99 | 96.97 | 93.94 | 92.42 | 89.90 | 88.89 | 86.36 | |
GD3040 | 100 | 96.46 | 97.98 | 94.44 | 98.99 | 99.49 | 100 | 97.98 | 100 | 98.99 | 96.97 | 100 | 98.48 | 97.98 | 100 | 96.97 | 94.95 | 93.43 | 90.91 | 89.90 | 87.37 | ||
GD3064 | 96.46 | 97.98 | 94.44 | 98.99 | 99.49 | 100 | 97.98 | 100 | 98.99 | 96.97 | 100 | 98.48 | 97.98 | 100 | 96.97 | 94.95 | 93.43 | 90.91 | 89.90 | 87.37 | |||
GDCA | 94.44 | 92.93 | 95.45 | 95.96 | 96.46 | 97.47 | 96.46 | 96.46 | 96.46 | 96.46 | 96.97 | 96.46 | 96.46 | 96.46 | 94.44 | 90.91 | 89.90 | 89.39 | 86.87 | ||||
YN1 | 94.44 | 98.99 | 98.48 | 97.98 | 95.96 | 97.98 | 96.97 | 95.96 | 97.98 | 96.46 | 95.96 | 97.98 | 95.96 | 92.93 | 92.42 | 90.91 | 89.90 | 86.36 | |||||
YN2 | 94.44 | 94.95 | 94.44 | 93.43 | 94.44 | 93.94 | 93.94 | 94.44 | 93.94 | 93.43 | 94.44 | 93.94 | 90.40 | 91.92 | 91.92 | 91.92 | 87.88 | ||||||
YN3 | 99.49 | 98.99 | 96.97 | 98.99 | 97.98 | 96.97 | 98.99 | 97.47 | 96.97 | 98.99 | 96.97 | 93.94 | 93.43 | 90.91 | 89.90 | 87.37 | |||||||
HKC9 | 99.49 | 97.47 | 99.49 | 98.48 | 97.47 | 99.49 | 97.98 | 97.47 | 99.49 | 97.47 | 94.44 | 93.94 | 91.41 | 90.40 | 87.88 | ||||||||
HKC16 | 97.98 | 100 | 98.99 | 96.97 | 100 | 98.48 | 97.98 | 100 | 96.97 | 94.95 | 93.43 | 90.91 | 89.90 | 87.37 | |||||||||
HK8 | 97.98 | 97.98 | 95.96 | 97.98 | 99.49 | 98.99 | 97.98 | 95.96 | 96.46 | 93.43 | 91.92 | 90.91 | 88.89 | ||||||||||
HK9 | 98.99 | 96.97 | 100 | 98.48 | 97.98 | 100 | 96.97 | 94.95 | 93.43 | 90.91 | 89.90 | 87.37 | |||||||||||
HK10 | 95.96 | 98.99 | 97.47 | 96.97 | 98.99 | 95.96 | 94.44 | 92.42 | 89.90 | 88.89 | 87.37 | ||||||||||||
HK11 | 96.97 | 96.46 | 95.96 | 96.97 | 100 | 92.93 | 92.93 | 90.40 | 89.39 | 86.87 | |||||||||||||
HK12 | 98.48 | 97.98 | 100 | 96.97 | 94.95 | 93.43 | 90.91 | 89.90 | 87.37 | ||||||||||||||
HK24 | 99.49 | 98.48 | 96.46 | 96.46 | 93.94 | 92.42 | 91.41 | 88.89 | |||||||||||||||
HK80 | 97.98 | 95.96 | 95.96 | 93.43 | 91.92 | 90.91 | 88.38 | ||||||||||||||||
HK108 | 96.97 | 94.95 | 93.43 | 90.91 | 89.90 | 87.37 | |||||||||||||||||
HK116 | 92.93 | 93.43 | 90.40 | 89.39 | 86.87 | ||||||||||||||||||
HK120 | 90.40 | 89.39 | 88.38 | 86.36 | |||||||||||||||||||
C964 | 93.94 | 95.96 | 86.36 | ||||||||||||||||||||
PNF2161 | 97.98 | 87.37 | |||||||||||||||||||||
R10291 | 85.86 |
表5 HGV的A132、A711、GD3027株与其它分离株之间的NS5区部分基因同源性比较
Table 5. Homology analysis of HGV NS5 sequences from different strains
NM/AA*(%) | A132 | A711 | GD3027 | PNF2161 | R10291 | GBV-C |
A711 | 93.3/97.9 | - | -
- |
|||
GD3027 | 94/99.2 | 93.8/97 | ||||
PNF2161 | 87.5/96.8 | 87.5/96.8 | 88.3/97 | - | - | - |
R10291 | 88/95.8 | 87.1/95.2 | 89.5/96.2 | 92.3/98.4 | - | - |
GBV-C | 91.4/97.9 | 93.8/97.9 | 93.3/97 | 87.9/97.9 | 88.9/95.2 | - |
C964 | 91.2/95.8 | 90/96.3 | 90/94 | 93.3/96.3 | 90.9/95.8 | 90.7/96.8 |
* NM:nucleotide monophosphate, AA: amino acid
讨论
目前,确定HGV感染的主要手段是RT-PCR,检测抗HGV抗体不能反映现症感染。我们在5UTR设计引物建立逆转录巢式PCR扩增5UTR 238bp片段,采用该法检测1 991份来自华南三个省区不同人群血清标本,发现一般人群HGV感染率广东省为0.73%, 香港特别行政区为1.34%, 献血员(2.52~2.90)%, 接受血液透析、静脉吸毒者及非甲~戊型肝炎、肝细胞癌、乙型肝炎、丙型肝炎患者HGV感染率高于一般人群(有统计学意义)。接受骨髓移植患者HGV RNA阳性率为41.67%, 明显高于其它人群, 推测与反复输注血制品可能有关。
通过测定华南地区20个不同HGV株5UTR序列, 发现相互间同源性介乎(90.40~100)%, 除YN2和HK120株外, 相互间同源性超过94.44%。YN2和HK120与其它株同源性较低,可能属于不同亚型。YN2与HK120之间同源性最低,为90.40%。华南各株与报道的中国北方株(C964)比较,同源性较低,为(90.40~93.94)%, 与国外株的同源性为(86.36~92.42)%。在NS5区的同源性有类似结果,三个华南地区HGV株NS5区核苷酸和氨基酸同源性分别为(93.3~94)%及(97~99.2)%, 与中国北方株(C964)及国外株比较同源性较低。从两个区域的比较提示,HGV核酸序列存在地区性差异。
不同碱基主要呈散在分布, 但亦发现几个较大变异区,包括187~222,299~306,6698~6837,7080~7092,及7157~7251(按PNF2161株位置)。
对2个病例感染HGV后相隔3个月采集标本, 发现5UTR序列均无任何变化, 同源性100%。分析华南地区不同感染人群(如献血员, 静脉吸毒者,非甲~戊型肝炎,肝细胞癌,乙型肝炎,丙型肝炎,骨髓移植)的序列,发现其变异与病种关系不显著。
我们对GD3027株的5UTR和NS5区均进行了序列测定, 发现在5UTR,与C964同源性最大(92.42%), 与GBV-C同源性最小(86.36%); 而在NS5区,与GBV-C同源性最大(93.3%),与PNF2161同源性最小(88.3%),与C964同源性为90%。结果提示,分析株间序列同源性与所选择区段关系密切,利用哪个区段作为基因分型标准对结果有一定影响。
志谢:感谢中山医科大学达安基因诊断中心的任秀容老师和中国预防医学科学院病毒学研究所基因工程国家重点实验室的姚二梅老师在测序方面提供的方便。
本研究受国家自然科学基金(项目号:39600130)及广东省医学科学研究基金资助
参考文献
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2 Leary TP, Muerhoff AS, Simons JN, et al. Sequence and genomic organization of GBV-C: a novel member of the Flaviridae associated with human non A-E hepatitis. J Med Virol, 1995,48∶60-67.
3 李刚, 陈青,姚集鲁,等.静脉毒瘾者84例HGV感染状况.中华传染病杂志,1998,16(1)∶26-28.
4 周育森,陈薇,赵秋敏,等.中国人庚型肝炎病毒全基因cDNA克隆及序列测定.军事医学科学院院刊,1996,20(4)∶249-254.
(收稿:1998-03-13 修回:1998-06-19)