云南省狂犬病病人和犬类感染狂犬病病毒及M基因序列分析

丁继超，章域震，李　浩，杨卫红，冯　云，陶晓燕，申辛欣，唐　青，张海林



云南省狂犬病病人和犬类感染狂犬病病毒及M基因序列分析

丁继超1,2，章域震2，李浩3，杨卫红2，冯云2，陶晓燕3，申辛欣3，唐青3，张海林2

目的调查云南省部分地区狂犬病病人和疫点犬群携带狂犬病病毒(rabies virus, RABV)状况及RABV膜基质蛋白(matrixprotein, M)基因序列分析，为狂犬病防控提供科学依据。方法2008-2009年在云南省采集犬脑组织标本606份，狂犬病病人唾液8份，脑脊液1份，用直接免疫荧光试验检测RABV抗原，用RT-PCR检测RABV核酸，对阳性标本进行M基因序列测定和分析。结果所有标本经检测，RABV抗原和/或核酸阳性16份，其中疫点扑杀的貌似健康犬脑组织3.10%(10/323)，狂犬病病人唾液5份、脑脊液1份。狗肉餐馆屠宰犬的脑组织283份均为阴性。云南16株RABV的M基因核苷酸和氨基酸同源性分别为88.5%～100%和85.2%～99.5%。它们与中国人用疫苗株aG核苷酸和氨基酸同源性分别为83.9%～85.7%和82.3%～93.6%；与人用疫苗株CTN181核苷酸和氨基酸同源性分别为99%～99.7%和98.5%～99%。系统进化分析表明，云南16株RABV均属基因Ⅰ型并可分为进化Ⅰ和Ⅱ群并分别与泰国等东南亚国家和相邻省份流行株具有较近的亲缘关系。结论云南省狂犬病流行与周边省份和东南亚地区的狂犬病传播扩散有一定关系；狂犬病疫点部分貌似健康犬携带RABV并具有传染源意义；云南狂犬病病毒株M基因与我国人用狂犬病疫苗CTN株的同源性和亲缘关系较近，但与aG株同源性存在。

狂犬病病毒；膜基质蛋白基因；同源性；遗传进化；流行病学

Supported by grants from the National Department Public Benefit Research Foundation (No. 201103032), and the Key Laboratory for Medical Virology, Ministry of Health(No:open project 2011-2012).(Ding Ji-chao and Zhang Yu-zhen contributed equally to this work)

Corresponding authors: Zhang Hai-lin, Email: zhangHL715@163.com

狂犬病是由狂犬病病毒(Rabies virus，RABV)引起的急性致死性中枢神经系统感染的人兽共患病。本病在亚洲和非洲流行较为严重[1-2]，我国是狂犬病高发国家[3-4]，但近几年疫情有下降趋势[5]。云南省1956年开始有人狂犬病记载，20世纪80年代出现较大范围的流行，90年代中期得到有效控制[6-8]，近十多年来，流行范围不断扩大[9-11]，成为日趋严重的公共卫生问题。RABV基因组为单股负链不分节段的RNA，由基因组的3′端至5′端依次排列着N、P、M、G、L 5个结构基因，分别编码N、P、M、G和L蛋白。此前，我们对RABV核蛋白(nucleoprotein, N)基因序列分析发现，云南省RABV有4个进化群，具有多样性的特点[9]。膜基质蛋白(matrixprotein, M)是狂犬病病毒最小的结构蛋白，对病毒的基因转录和复制水平具有调控作用，在疾病的致病性方面也起到重要作用[12]。为掌握云南狂犬病病人和疫点扑杀犬群RABV感染状况及流行株的M蛋白基因的分子特征，本研究对云南省疫点犬群和狂犬病病人标本进行RABV抗原、核酸检测和M基因序列分析，探讨云南省RABV进化特点及其与流行的关系，为狂犬病防控提供科学依据。

1　材料与方法

1.1标本采集本研究于2008-2009年在云南省狂犬病流行区共采集到犬脑组织标本606份，其中疫点扑杀犬323份(曲靖市100份，昭通市95份，西双版纳州128份)，狗肉餐馆屠宰犬283份(文山州200份，德宏州83份)。采集到狂犬病病人唾液8份(玉溪市4份，红河州3份，西双版纳州1份)，西双版纳州病人脑脊液1份。

犬脑标本用头颅解剖或塑料管插入枕骨大孔的方法获取大脑、中脑和小脑组织标本，病人唾液用棉签收集。标本存放入2 mL或15 mL螺旋盖冻存管，登记和编号后，立即保存于当地CDC -20 ℃冰箱，用液氮罐运输到云南省地方病防治所直至检测。

1.2直接免疫荧光试验(DFA)犬脑组织用DFA法检查RABV抗原。分别取大脑、中脑和小脑组织进行印片，干燥后冷丙酮(4 ℃)固定10 min，用荧光素标记抗RABV单克隆抗体染色，37 ℃湿盒30 min，PBS振洗2次，蒸馏水振洗1次，90%甘油封片，Nikon荧光显微镜观察结果。

1.3RT-PCR扩增及测序经DFA检测阳性的犬脑和病人唾液和脑脊液标本用该法检测RABV核酸。以TRIzol试剂(Invitrogen公司)提取细胞总RNA，经Ready-To-Go You-Prime First-Strand Beads(Amersham Bioscience公司)试剂盒逆转录得到cDNA。巢式PCR法扩增RABV特异M基因，引物见文献[12]，1%琼脂糖凝胶检测扩增产物。PCR产物纯化(QIAquick PCR Purification Kit，QIAGEN公司)后送测序公司双向测序，使用ATGC软件完成核苷酸序列的拼接。

1.4序列分析用BioEdit、Clustalx 1.8对获得的核苷酸序列进行编辑比较，DNAStar中MegAlign进行序列同源性分析，用MEGA 3.1软件邻接法(Neighbor-Joining)进行系统进化树分析。用于比较分析的RABV M基因序列来自GenBank(表1)。

表1本研究引用的狂犬病病毒M基因序列的背景资料

Tab.1Background information of rabies virus M gene used in this study

名称Strainname基因型Genetype宿主Host国家Country年代Year基因库号GenBankno.CTN181I人Human中国山东Shandong/China1956ABU52976CTNI疫苗株Vaccine中国China-DQ4900768743THAI犬Dog泰国Thailand1983AY540348CYN0701HI犬Dog中国云南Yunnan/China2007EU004752CYN0601HI犬Dog中国云南Yunnan/China2006EU004751CNX8511I人Human中国China-DQ490074CNX8611I人Human中国China-AB490075CGZ0619DI犬Dog中国贵州Guizhou/China-EU004723CGX0622DI犬Dog中国广西Guangxi/China-EU004703CGX0521DI犬Dog中国广西Guangxi/China-EU004702CGZ0514DI犬Dog中国贵州Guizhou/China-EU004720CAH0512DI犬Dog中国安徽Anhui/China-EU004686CJS0622DI犬Dog中国江苏Jiangsu/China-EU004743CGZ0509DI犬Dog中国贵州Guizhou/China-EU004715CHN0615DI犬Dog中国河南Henan/China-EU004732CHN0614DI犬Dog中国河南Henan/China-EUOO473291RABN0783I狐狸Fox加拿大Canada2001AF360852CVSI疫苗株Vaccine法国French-D10499PVI疫苗株Vaccine法国French1989M1321592RABL0867I臭鼬Skunk加拿大Canada-AF360848NY516I浣熊Raccoon加拿大Canada-AF360857NishigaharaI疫苗株Vaccine日本Japan-AB044824aGI疫苗株Vaccine中国China-DQ490077LogosII果蝠Fruitbat尼日利亚Nigeria-AY540349MokolaIII不详Unknown津巴布韦Zimbabwe-AY540347

2　结　果

2.1RABV抗原和核酸检测对采集到的606份犬脑标本进行DFA检测，其中323份疫点扑杀犬群中RABV抗原阳性10份，阳性率为3.10%，其中昭通市威信县疫点犬群阳性率高达6.32%(6/95)，西双版纳州景洪市和曲靖市富源县疫点犬群阳性率分别为2.34%(3/128)和1.00%(1/100)。狗肉餐馆屠宰犬283份均为阴性。经RT-PCR检测，上述10份RABV抗原阳性标本均为RABV核酸阳性。检测病人唾液8份，RABV核酸阳性5份(玉溪市2份，红河州2份，西双版纳州1份)，其中西双版纳州病人的脑脊液标本也为RABV核酸阳性(表2)。上述16份RABV核酸阳性样本经巢式PCR扩增，经序列测定，获得RABV的M基因序列(609 bp)，所有序列均已提交GenBank(表2)。

2.2M基因核苷酸和氨基酸序列同源性分析云南16株RABV的M基因核苷酸序列同源性为88.5%～100%，氨基酸序列同源性为85.2%～99.5%。这16株RABV的M基因与中国的人用疫苗株aG核苷酸同源性为83.9%～85.7%，氨基酸同源性为82.3%～93.6%，具有一定的差异；与人用疫苗株CTN核苷酸和氨基酸同源性分别为99%～99.7%和98.5%～99%，CTN株同源性明显高于aG株；与此前云南保山市狂犬病病人脑组织分离株CYN0701H和CYN0601H株[9]核苷酸和氨基酸同源性分别为89.7%～91%和84.7%～96.6%；与PV、CVS、Nishigahara、91RABN0783(国外固定毒株)核苷酸同源性为83.6%～88.3%，氨基酸同源性为80.3%～93.6%(表3)。此外，CYN0901D的M基因与其它云南株同源性较低，特别是与国外株和aG疫苗株更低(80.3%～82.3%)。

表2RABV DFA和RT-PCR检测阳性标本背景信息

Tab.2Background information of M gene of 16 rabies virus strains isolated from Yunnan Province

编号Number基因库株名Strainname基因库号GenBankno.标本种类Sampletype采集地(县/州市)Location(County/Prefecture)采集时间DateDFART-PCRCYN0916DCYN0810DJF819630犬脑Dogbrain曲靖市富源县Fuyuan/Qujing2008.5.5++CYN0907DCYN0813DJF819629犬脑Dogbrain昭通市威信县Weixin/Zhaotong2008.10.23++CYN0913DCYN0814DJF819625犬脑Dogbrain昭通市威信县Weixin/Zhaotong2008.10.23++CYN0902DCYN0815DJF819626犬脑Dogbrain昭通市威信县Weixin/Zhaotong2008.10.23++CYN0915DCYN0816DJF819640犬脑Dogbrain昭通市威信县Weixin/Zhaotong2008.10.23++CYN0906DCYN0817DJF819627犬脑Dogbrain昭通市威信县Weixin/Zhaotong2008.10.23++CYN0901DCYN0818DJF819628犬脑Dogbrain昭通市威信县Weixin/Zhaotong2008.10.23++CYN0903DCYN0811DJF819631犬脑Dogbrain版纳州景洪市Jinghong/Banna2008.11.1++CYN0908DCYN0812DJF819632犬脑Dogbrain版纳州景洪市Jinghong/Banna2008.11.1++CYN0909DCYN0919DJF819639犬脑Dogbrain版纳州景洪市Jinghong/Banna2009.6.17++CYN0905HCYN0920HJF819637人唾液Humansaliva玉溪市华宁县Huaning/Yuxi2009.3.20ND+CYN0904HCYN0922HJF819638人唾液Humansaliva玉溪市江川县Huaning/Yuxi2009.7.19ND+CYN0914HCYN0923HJF819636人唾液Humansaliva红河州弥勒县Mile/Honghe2009.8.22ND+CYN0910HCYN0924HJF819635人唾液Humansaliva红河州弥勒县Mile/Honghe2009.8.27ND+CYN0911HCYN0921HTJF819634人唾液Humansaliva版纳州景洪市Jinghong/Banna2009.4.13ND+CYN0912HCYN0921HNJF819633人脑脊液HumanCSF版纳州景洪市Jinghong/Banna2009.4.13ND+

+：阳性Positive；ND：未做Not done；CSF：脑脊液Cerebrospinal fluid。

表3云南株与参考株狂犬病病毒M基因核苷酸和推导氨基酸序列同源性分析

Tab.3Homology comparisons of the M sequences of the rabies virus and isolates from Yunnan Province Strain

株名Strain123456789101112131415161718192021222324251aG***97.492.191.187.884.284.284.284.284.284.284.284.184.184.284.184.283.983.98786.985.285.285.771.62Nishigahara97.5***91.390.687.883.983.983.983.983.983.983.983.783.783.983.783.983.683.686.486.285.485.485.671.43PV91.191.1***90.888.384.284.284.284.284.284.284.284.184.184.184.184.283.984.285.685.486.486.587.469.14CVS91.191.690.1***88.385.785.785.785.785.785.785.785.685.685.685.485.785.485.786.786.585.785.986.769.5591RABN078393.693.693.692.6***85.785.785.785.785.785.785.785.685.985.685.485.785.486.287.28787.787.888.369.56CYN0911H93.693.692.692.196.1***10010010010010010099.899.899.899.399.799.79991.1919090.190.671.67CYN0905H93.693.692.692.196.199.5***10010010010010099.899.899.899.399.799.79991.1919090.190.671.68CYN0912H93.693.692.692.196.199.599.5***10010010010099.899.899.899.399.799.79991.1919090.190.671.69CYN0908D93.693.692.692.196.199.599.599.5***10010010099.899.899.899.399.799.79991.1919090.190.671.610CYN0913D93.693.692.692.196.199.599.599.599.5***10010099.899.899.899.399.799.79991.1919090.190.671.611CYN0907D93.693.692.692.196.199.599.599.599.599.5***10099.899.899.899.399.799.79991.1919090.190.671.612CYN0916D93.693.692.692.196.199.599.599.599.599.599.5***99.899.899.899.399.799.79991.1919090.190.671.613CYN0902D93.193.192.191.695.699.099.099.099.099.099.099.0***99.799.799.299.599.598.99190.889.89090.571.614CYN0914H93.693.692.692.196.199.599.599.599.599.599.599.599***99.799.299.599.598.99190.889.89090.571.615CYN0915D93.693.692.692.196.199.599.599.599.599.599.599.59999.5***99.299.599.598.99190.890.19090.571.616CYN0906D92.692.691.691.195.198.598.598.598.598.598.598.59898.598.5***99.39998.490.590.389.789.890.371.317CYN0903D93.193.192.191.695.69999999999999998.5999998.5***99.398.790.890.69090.190.671.418CTN93.193.192.191.695.69999999999999998.599999898.5***98.790.89189.789.890.371.619CYN0901D82.382.380.880.384.786.786.786.786.786.786.786.786.286.786.786.786.786.2***90.890.690.690.891.371.620CYN0701H93.193.192.693.196.196.696.696.696.696.696.696.696.196.696.695.696.196.184.7***99.888.788.889.770.821CYN0601H93.193.192.693.196.196.696.696.696.696.696.696.696.196.696.695.696.196.184.799.5***88.588.789.570.922CYN0904H93.194.192.692.196.696.196.196.196.196.196.196.195.696.196.195.195.695.685.795.695.6***99.798.771.423CYN0909D93.194.192.692.196.696.196.196.196.196.196.196.195.696.196.195.195.695.685.795.695.699.5***9971.424CYN0910H92.693.692.191.696.195.695.695.695.695.695.695.695.195.695.694.695.195.185.295.195.19999***71.825Mokola77.377.875.975.478.878.878.878.878.878.878.878.878.878.878.877.878.378.371.478.878.879.879.879.3***

注：右上三角数据为核苷酸同源性百分率，左下三角数据为氨基酸同源性百分率。

Notes: The percent nucleotide sequence identities of the genome are presented at the upper right. The percent amino acid sequence identities of the genome are presented at the lower left.

2.3M基因系统进化树分析云南16株RABV 的M基因序列与来自GenBank中25株(基因I型23株，基因II和III型各1株；表1)的相应序列构建种系发生树。图1显示，云南16个RABV标本与基因I型RABV参考株同在一个大分支，与其他国家的固定毒株如91RABN0783、CVS、PV、Nishigahara间亲缘关系较远，与基因II和III型亲缘关系较远，表明包括云南16株在内的39株RABV同属基因Ⅰ型。

聚集了包括本次16株云南RABV在内的中国RABV的分支进一步可分为2个进化群(图1)，其中云南13株(CYN0903D、CYN0912H、CYN0913D、CYN0905H、CYN0911H、CYN0901D、CYN0902D、CYN0915D、CYN0908D、CYN0914H、CYN0916D、CYN0907D和CYN0906D)处在Ⅰ群中，并与中国疫苗株CTN和CTN181以及此前分离自云南保山市狂犬病病人的 CYN0701H和CYN0601H株[7]及泰国流行株8743THA株有较近的亲缘关系；Ⅱ群中的云南3株(CYN0910H、CYN0904H和CYN0909D)与中国广西、广东、贵州、江苏、安徽和河南的RABV亲缘关系较近。

图1　云南株与参考株狂犬病病毒M基因种系发生树Fig.1　Phylogenetic trees based on the M gene from Yunnan Province and reference strains of rabies virus

3　讨　论

云南省位于我国西南边境地区，东部和北部与我国狂犬病流行较为严重的广西、贵州和四川省[3-5]相连，南部和西部与狂犬病地方性流行区的越南、老挝、缅甸和泰国相邻，地理位置较为特殊。流行病学分析提示，云南省狂犬病流行与周边省份和东南亚地区的狂犬病传播扩散有一定关系。张永振等[13]将中国2003-2008年的37株RABV分为2个进化群。最近，陶晓燕等[14-15]和郭振洋等[16]将中国1949-2010年RABV分为6个进化群。本研究云南16株RABV可分为进化Ⅰ群和Ⅱ群并可能分别来源于东南亚国家和相邻省份。此外，我们曾于2006和2007年从云南省保山市病人脑组织分离到2株RABV[11]，2010年从云南省德宏州梁河县一只狂犬的脑组织分离到1株RABV[17]，它们均与泰国株具有较近的亲缘关系。综合分析认为，云南省存在这2种进化群RABV的共同流行与东南亚国家和周边省份的RABV通过犬类流动而传播扩散有关。

最近，解庭波等[18]发现我国安徽和浙江等省10株RABV的M基因序列与中国人用疫苗株CTN株亲缘性较近。本研究证实，云南16株RABV的M基因核苷酸和氨基酸序列与中国人用疫苗株aG株具有较为明显的差异，但与中国人用疫苗株CTN同源性较高，提示CTN株可能对云南流行株的保护性优于aG株。值得注意的是，CYN0901D的M基因与其它云南株同源性较低，特别是与国外株和aG疫苗株更低(80.3%～82.3%)，提示云南存在与该疫苗株差异稍大的流行株，应深入研究CYN0901D株及其它云南株的生物学和分子特征以及疫苗对这类病毒株的保护效果。然而，关于疫苗保护性问题有待于进行相关抗原性和免疫原性的研究，尤其是糖蛋白结构及功能研究。

本研究中的犬标本采自有动物或人狂犬病疫点的扑杀犬群，这些犬中可能包含有处在狂犬病潜伏期即将发病的犬。张永振等[19]和李浩等[20]证实，我国贵州、广西和湖南狂犬病流行区貌似健康犬群中存在RABV感染犬。本次从云南省疫点扑杀的貌似健康犬群中也检测到该病毒，而且不同地区3个疫点犬群均有阳性犬，其中昭通市威信县疫点犬群感染率高达6.32%，由此证明云南疫点貌似健康犬群中仍有犬只携带RABV，如不及时捕杀，一旦它们发病将会传播狂犬病。因此，疫点中处于狂犬病潜伏期的犬只在该病毒保存和传播中的作用不能忽视。扑杀犬群是一种古老而残忍的方法，对其在疫情控制中的效果也有争议，但在犬类管理和免疫措施得不到全面落实的情况下，云南省一些地区对疫点犬群实施扑杀，在局部地区疫情控制中可能有一定效果。

综上所述，云南省狂犬病防控中仍然存在一些亟待解决的问题，如制定犬类管理的法规或相关规定；建立犬狂犬病疫情监测体系，及时发现犬间疫情，及时扑杀病犬、可疑犬；控制犬类在境内外、省内外和省内地区之间的流动；进一步评价现行使用疫苗的免疫效果，广泛使用安全有效的疫苗，全面提高犬类的免疫覆盖率。总之，为实现我国2025年消除人间狂犬病的目标，全面落实犬类管理和免疫是控制和消除人间狂犬病的关键措施。

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Survey on rabies viruses in rabies patients and dog population from endemic sites of Yunnan Province, China and the viral M gene sequence analysis

DING Ji-chao1, 2, ZHANG Yu-zhen2, LI Hao3, YANG Wei-hong2, FENG Yun2,TAO Xiao-Yan3, SHEN Xin-xin3, TANG Qing3, ZHANG Hai-lin2

(1.SchoolofPublicHealth,DaliUniversity,Dali671000,China;2.YunnanInstituteofEndemicDiseasesControlandPrevention/YunnanProvincialKeyLaboratoryforZoonosisControlandPrevention,Dali671000,China;3.NationalInstituteforViralDiseaseControlandPrevention,ChineseCenterforDiseaseControlandPrevention,Beijing102206,China)

We conducted investigation on the rabies virus (RABV) infected from rabies patients and dog population in endemic sites, Yunnan Province, China and the viral matrixprotein (M) gene sequence analysis, providing references for control and prevention of rabies. A total of 606 brain tissue samples of dogs collected from endemic sites, as well as 8 saliva and 1 cerebrospinal fluid samples of rabies patients collected from Yunnan Province in 2008-2009. RABV antigen and nucleic acid were detected by direct immunofluorescence assay and RT-PCR respectively, and positive samples were sequenced for the M gene.According to the nucleotide and amino acid of target gene, the genetic homology and the phylogeny were analyzed. Above specimens tested, 16 specimens were RABV antigen and/or nucleic acid positive. Among them, 3.10% (10/323) were from apparently healthy dog brain specimens of endemic sites, 5 saliva and 1 cerebrospinal fluid were from human rabies cases. In addition, 283 dog brain specimens collected from dog meat restaurant were negative. The M gene sequence of the 16 strains have been acquired, the rate of their nucleotide homology and deduced amino acid homology were 88.5%-100% and 85.2%-99.5% respectively. Compared with aG and CTN181 strains (vaccine strains for humans in China) nucleotide (deduced amino acid) homology were 83.9%-85.7% (82.3%-93.6%) and 99%-99.7% (98.5%-99%) respectively. The phylogenic analysis indicated that the 16 isolates from Yunnan Province divided into I and II phylogenic subgroups. These 2 phylogenic subgroups of RABV from Yunnan Province have a closer genetic relationship with the street strains isolated from Southeast Asia and neighboring provinces of China. Rabies epidemic in Yunnan Province might be attributed to rabies spread from Southeast Asia and neighboring provinces. Some apparently healthy dogs can infected RABV. The M gene nucleotide and amino acid of Yunnan strains of rabies virus have a higher homology and a closer genetic relationship with CTN strain of human rabies vaccine used in China, but they with aG strains of human rabies vaccine used in China homologous exist obvious difference.

rabies virus; matrixprotein gene; homology; phyletic evolution; epidemiology

张海林，Email: zhangHL715@163.com

1.大理大学公共卫生学院，大理671000；2.云南省地方病防治所/云南省自然疫源性疾病防控技术重点实验室,大理671000；3.中国疾病预防控制中心病毒病预防控制所，北京102206

R373.9

A

1002-2694(2016)06-0518-07

2015-10-08；

2016-02-29

DOI：10.3969/j.issn.1002-2694.2016.06.003

公益性行业(农业)科研专项基金(No.201103032)；中国卫生和计划生育委员会医学病毒学重点实验室开放课题(No.开放课题2011—2012)丁继超和章域震有同等贡献。