• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Comparative Genom ic Analysis of Enterovirus 71 Revealed Six New Potential Neurovirulence-associated Sites*

    2016-12-05 00:34:00JIAQingJunCHENXinYuLIDeZhouXUJuanJuanXUZhiGangDUANZhiLiangandWENJinSheng
    Biomedical and Environmental Sciences 2016年10期

    JIA Qing Jun, CHEN Xin Yu, LI De Zhou, XU Juan Juan, XU Zhi Gang, DUAN Zhi Liang, and WEN Jin Sheng,#

    Comparative Genom ic Analysis of Enterovirus 71 Revealed Six New Potential Neurovirulence-associated Sites*

    JIA Qing Jun1,?, CHEN Xin Yu2,?, LI De Zhou3, XU Juan Juan1, XU Zhi Gang1, DUAN Zhi Liang2,#, and WEN Jin Sheng1,#

    In the present study, the complete genomes of four common (4/EV71/Wenzhou/CHN/2014, 15/ EV71/Wenzhou/CHN/2014, 116/EV71/Wenzhou/ CHN/2014, and 120/EV71/Wenzhou/CHN/2014) and two virulent (11/EV71/Wenzhou/CHN/2014 and 109/EV71/Wenzhou/CHN/2014) enterovirus 71 (EV71) isolates were sequenced and described. They are 7405 bp in length and belong to EV71 sub-genotype C4 (C4a cluster). Nucleotide sequence alignment revealed six nucleotide variations (GP151→TP151, GP199→AP199, GP261→TP261, AP328→CP328, GP422→AP422, and GP437→TP437) in the two virulent isolates w ithin the 5'UTR of the IRES element. RNA secondary structure predictions of IRES and FCE indicated that the common isolates shared sim ilar structures, which were different from those of the virulent isolates. Moreover, the GP114→CP114and GP151→TP151mutations in the virulent isolates contributed to the formation of the unique RNA secondary structures in SL II. Furthermore, nucleotide/am ino acid sequence alignments of 82 EV71 isolates indicated that six sites (TP488and CP577in the 5'UTR; AsnP57in 2A; IleP56in 3C; CP10and AP47in the 3'UTR) are potentially associated w ith the neurovirulence of EV71. Finally, the 3D structures of 2A were analogous, whereas the structures of VP1 and 3C were variable.

    Enterovirus 71 (EV71) is a non-enveloped virus containing a positive-sense single-stranded RNA genome (approximately 7.4 kb) and belongs to the Enterovirus genus of the fam ily Picornaviridae. Structurally, six stem loop (SL) structures, SL I to SL VI, are contained in the EV71 5'UTR. Functionally, the 5'UTR is divided into three regions: a 5' term inal cloverleaf (1 to 89-101 bp), an internal ribosome entry site (IRES) element (123-126 bp to 602-605 bp, ranging from SL II to SL VI), and a hypervariable region (the last 120 bp)[1]. This type of positive-sense single-stranded RNA virus utilizes a cap-independent translation initiation mechanism to regulate the expression of its viral genome[2]. Jerrey et al. found that the functional cis-acting element (FCE, spanning SL IV to SL VI w ithin the 5'UTR) enabled Poliovirus mRNA to translate in a cap-independent fashion and that the major determ inant of this FCE is located in a domain(nts320-631)[3].Unlikeother hand-foot-mouth disease (HFMD) pathogens [such as Coxsackie virus A (CVA)], EV71 infection is more likely to cause severe neurological complications, includingbrainstemencephalitis,aseptic encephalitis, meningitis, poliomyelitis-like paralysis, neurogenic pulmonary edema, and even death[4]. Some studies have reported that the nucleotide mutations w ithin the 5'UTR (such as the stem loop II, SL II, nts 105-179) and some am ino acid variations are associated w ith EV71 neurovirulence[5-6]. Generally, common EV71 isolates cause m ild and self-lim ited herpes outbreaks on the skin and mucosae, whereas virulent EV71 isolates (‘virulent' particularly refers to neurovirulent) lead to severe neurological complications and even death[7].

    In the present study, patients diagnosed with either m ild HFMD (with herpes only on the skin and mucosa) or severe HFMD (w ith both herpes and meningitis) were recruited from the Second Affiliated Hospital of Wenzhou Medical University. Throat swab samples or cerebrospinal fluid sampleswere filtered through a 0.22-μm sterile filter, and the filtered suspension was added to either RD cells or Vero cells, which were maintained in RPMI-1640 medium supplemented with 10% FBS, 100 U/m L penicillin, and 100 μg/m L streptomycin. After an infection period of 1.5 h in a 37 °C/5% CO2incubator, the cell supernatant was discarded, and fresh RPMI-1640 medium w ithout FBS was added to the cells. After a 4-d incubation, the supernatant was blind inoculated into freshly prepared cells several times until the cells exhibited a cytopathic effect (CPE), as scored by ‘+++' (the majority of cells had shrunk and become dislodged from the bottom of the culture bottles). Based on EV71-specific primers (TableS1inthewebsiteofBES, www.besjournal.com),RT-PCRassayswere conducted to amplify six overlapping gene segments (638 bp, 1575 bp, 1433 bp, 1624 bp, 2286 bp, and 786 bp), which span the complete genome of EV71, and the PCR products were sequenced. The complete genome sequences and deduced am ino acid sequences of six EV71 isolates were deposited in the GenBank database. The GenBank Accession Numbers of four common isolates (4/EV71/ Wenzhou/CHN/2014, 15/EV71/Wenzhou/CHN/2014, 116/EV71/Wenzhou/CHN/2014, and 120/EV71/ Wenzhou/CHN/2014) were KT008669, KT345960, KT008672, and KT345959, respectively. Two virulent isolates(11/EV71/Wenzhou/CHN/2014and 109/EV71/Wenzhou/CHN/2014) were given the following accession numbers: KT008670 and KT008671, respectively. In this study, six nucleotide variations (GP151→TP151, GP199→AP199, GP261→TP261, AP328→CP328, GP422→AP422, and GP437→TP437) within the IRES region were observed simultaneously in the two virulent EV71 isolates when compared w ith the four common isolates (Figure 1A). RNA secondary structure predictions for IRES, which includes domain I to domain V, were carried out. The results showed that the four common isolates shared sim ilar RNA structures, whereas the two virulent isolates had a large circular stem loop structure between domain II and domain III. This structural difference between the common and virulent isolates may have an impact on RNA translation and neurovirulence (Figure 2). In order to learn more about the subtle RNA structure variations, we also performed RNA secondary structure predictions for two specific functional regions: SL II and FCE. The results indicated that the variation of GP114→CP114in virulentisolate11/EV71/Wenzhou/CHN/2014 contributed to the formation of an additional stem loop structure and that the mutation of GP151→TP151in both virulent isolates produced an enhanced stem loop structure (w ith two more nucleotides) (Figure 3A). Moreover, the variations of GP114→CP114and GP151→TP151resulted in higher m inimum free energy (-12.4 kcal/mol and -17 kcal/mol, respectively) and contributed to the formation of relatively stable secondary structures (Figure 3A). The predicted secondary structures of the FCE of the four common isolates were sim ilar, whereas the structures of two virulent isolates were variable (Figure 3B). Wen et al. demonstrated that the 5'UTR RNA secondary structures of virulent EV71 isolates differed from those of common isolates[8]. However, they did not perform a detailed analysis of additional subtle variations in key regions w ithin the 5'UTR. In this study, instead of repeating previous studies on predictions of the RNA secondary structure of the whole 5'UTR, we selected the IRES for RNA secondary structures analysis and specifically studied two special regions (SL II and FCE) that were previously reported to be associated w ith neurovirulence and viral mRNA translational efficiency, respectively. In agreement w ith previous studies on the structure of the whole 5'UTR, the secondary structures of SL II and FCE were sim ilar among the four common isolates but differed from those of the two virulent isolates. Furthermore, sequence alignment of the 3'UTR (84 nts) suggested that the nucleotides in the 3'UTR were relatively conserved and that only four nucleotide positions were variable. The variation of TP10→CP10or AP10was found in two virulent isolates when compared w ith four common isolates (Figure 1B).

    Next, 76 EV71 isolates along w ith the previous six EV71 isolates were divided into ‘Virulent' and‘Common' groups (Table S2 in the website of BES, www.besjournal.com). The nucleotides/am ino acids that exhibited large differences between the‘Virulent' and ‘Common' groups were picked and subjected to Chi-square tests. For example, in the 488th position of the 5'UTR, there were two ‘T's in the ‘Common' group but seven ‘T's in the ‘Virulent' group. The results of the Chi-square tests showed that this difference was statistically significant, and it was concluded that this difference was associated with the neurovirulence of EV71 (χ2=4.204, P= 0.04, α=0.05) (Table 1). As a result, two alternativenucleotides (TP488and CP577) in the 5'UTR, two alternative nucleotides (CP10and AP47) in the 3'UTR, and three alternative am ino acids (GlnP22in VP1; AsnP57in 2A; IleP56in 3C) were considered to be relevant to neurovirulence. To expound the impact of these neurovirulence-related am ino acids on the structures of the corresponding proteins, 3D structurepredictionsoftwareforproteins (https://sw issmodel.expasy.org/interactive/xTem Lr/ models/) was used to predict the structures of VP1, 2A, and 3C. We wanted to determ ine whether, like the predicted RNA structures of the 5'UTR, the 3D structures of these isolates differed between the common and virulent isolates. Unfortunately, the 3D structures were not noticeably different between the common and virulent isolates. 2A had almost the same structure for all six isolates, whereas both VP1 and 3C were variable among the six isolates. The on-line software (ProMod3 Version 1.0.0.) used for the analysis is the most commonly used software for the 3D structure analysis of proteins, and all sequence identities between our am ino acid sequences and the models built on-line were above 90%, w ith several sequence identities reaching 100% (seq identities >50% were conclusive; for more information, please refer to the SWISS-MODEL Homology Modeling Reports in the supplementary materials) (Figure S2 in the website of BES, www.besjournal.com). As a result, these 3D results are probably attributed to the following factors: (1) the lim ited number of samples on 3D structure predictions; (2) a single am ino acid change in a pivotal site may contribute more to EV71 neurovirulencethanthestructureofthe corresponding protein; and (3) a crucial am ino acid may determ ine EV71 vitality when binding to host cells. In summary, further studies are needed to determ ine how these variations affect the neurovirulence of EV71.

    Researchers have explored the association of certainnucleotides/am inoacidswiththe neurovirulence of EV71. For example, Liu et al. reported four neurovirulence-related am ino acid substitutions,HisP22→GlnP22,GluP98→LysP98, GluP145→GlyP145, and AlaP289→ThrP289, in VP1[9]. ValP263→IleP263in 3D of virulent isolates was demonstrated to be a unique am ino acid variation that distinguished the virulent isolates from common isolates[10]. Among the seven nucleotides/am ino acids identified in the present study, GlnP22in VP1 has already been described[9]. However, neither the four common isolates nor the two virulent isolates had this neurovirulence-associated am ino acid. Of the six new ly identified neurovirulence-associated nucleotides/am ino acids, five variations were unique to the two virulent isolates. It should be emphasized that we searched and analyzed the whole genome of almost all virulent EV71 isolates (C4 subgenotype) that had been described in either published papers or the GenBank database. Moreover, all analyzed EV71 isolates were carefully chosen. All common isolates were from patients w ith herpes only on the skin and mucosae and w ithout any neurological symptoms. Virulent isolates were isolated from patients w ith not only herpes but also at least one of the follow ing severe neurological complications: brain stem encephalitis, aseptic encephalitis, meningitis,poliomyelitis-likeparalysis,and neurogenic pulmonary edema. Most importantly, the number of EV71 isolates (either common or virulent isolates) utilized in the present study was higher than in any previous EV71 genome sequence analysis of C4. Therefore, all of these factor make our findings more convincing.

    Taken together, we demonstrated the genetic characteristics of six native EV71 isolates and identified six novel nucleotide/am ino acid sites (TP488and CP577in the 5'UTR; AsnP57in 2A; IleP56in 3C; CP10and AP47in the 3'UTR) that were concluded to be associated with the neurovirulence of EV71. Furthermore, the RNA structure predictions of IRES, SL II, and FCE suggested that there may be a relationship between EV71 neurovirulence and the RNA secondary structures of IRES, SL II, and FCE in the 5'UTR. The results of the present study add to the know ledge on EV71 origin, evolution, and neurovirulence and will aid in developing safe and effective vaccines. In the future, further studies should be conducted to determ ine whether and how these am ino acid/nucleotide variations, as well as changes to the corresponding RNA and protein structures, affect neurovirulence using cell culture systems, animal models, and molecular biology techniques.

    ACKNOWLEDGEMENT

    We would like to thank the participants for their cooperation in this study.

    ?These authors contributed equally to this work.

    #Correspondence should be addressed to WEN Jin Sheng, E-mail: w js78@wmu.edu.cn, Tel: 86-577-86689910; DUAN Zhi Liang, E-mail: dzl3032@163.com, Tel: 86-577-88816276.

    Accepted: October 1, 2016

    REFERENCES

    1. Skinner MA, Racaniello VR, Dunn G, et al. New model for the secondary structure of the 5' non-coding RNA of poliovirus is supported by biochem ical and genetic data that also show that RNA secondary structure is important in neurovirulence. J Mol Biol, 1989; 207, 379-92.

    2. Bedard KM, Sem ler BL. Regulation of picornavirus gene expression. M icrobes Infect, 2004; 6, 702-13.

    3. Pelletier J, Kaplan G, Racaniello VR, et al. Cap-independent translation of poliovirus mRNA is conferred by sequence elements w ithin the 5' noncoding region. Mol Cell Biol, 1988; 8, 1103-12.

    4. Mou J, Dawes M, Li Y, et al. Severe hand, foot and mouth disease in Shenzhen, South China: What matters most? Epidem iol Infect, 2014; 142, 776-88.

    5. Yeh MT, Wang SW, Yu CK, et al. A single nucleotide in stem loop II of 5'-untranslated region contributes to virulence of enterovirus 71 in m ice. PLoS ONE, 2011; 6, e27082.

    6. Kataoka C, Suzuki T, Kotani O, et al. The role of VP1 am ino acid residue 145 of enterovirus 71 in viral fitness and pathogenesis in a cynomolgus monkey model. PLoS Pathog, 2015; 11, e1005033.

    7. Wang Y, Zhu QQ, Zeng M, et al. Complete genome sequence of a human enterovirus 71 strain isolated from a fatal case in Shanghai, China, in 2012. Genome Announc, 2014; 2. pii: e00457-14.

    8. Wen HL, Si LY, Yuan XJ, et al. Complete genome sequencing and analysis of six enterovirus 71 strains w ith different clinical phenotypes. Virol J, 2013; 10, 115.

    9. Liu YJ, Fu C, Wu SY, et al. A novel finding for enterovirus virulence from the capsid protein VP1 of EV71 circulating in mainland China. Virus Genes, 2014; 48, 260-72.

    10.Chang GH, Lin L, Luo YJ, et al. Sequence analysis of six enterovirus 71 strains w ith different virulence in humans. Virus Res, 2010; 151, 66-73.

    d

    *oi: 10.3967/bes2016.103 This study was funded by Natural Science Foundation of Zhejiang (LQ14C010006); National Natural Science Foundation of China (81501363); and Planned Science and Technology Project of Zhejiang (2014C33261).

    1. Institute of Arboviruses, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou 325000, Zhejiang, China; 2. Department of Clinical Laboratory, The Second Affiliated Hospital & Yuying Children's Hospital of Wenzhou Medical University, Wenzhou 325000, Zhejiang, China; 3. Department of Liver, The Second Hospital of Ningbo, Ningbo 315010, Zhejiang, China

    Biographical notes of the s: JIA Qing Jun, male, born in 1990, Master's degree candidate, majoring in pathogen biology; CHEN Xin Yu, male, born in 1976, associate chief technician, Master's degree candidate, majoring in biomedical engineering.

    May 13, 2016;

    亚洲欧美成人综合另类久久久| a级毛片黄视频| 久热久热在线精品观看| 深夜精品福利| 青草久久国产| 制服人妻中文乱码| 韩国av在线不卡| 涩涩av久久男人的天堂| 成人亚洲欧美一区二区av| 午夜av观看不卡| 中文字幕最新亚洲高清| 在线免费观看不下载黄p国产| 免费少妇av软件| 最近中文字幕2019免费版| 欧美bdsm另类| 天堂8中文在线网| 亚洲欧美中文字幕日韩二区| 成年动漫av网址| 国产精品av久久久久免费| 免费播放大片免费观看视频在线观看| av国产精品久久久久影院| 在线观看www视频免费| 久久这里有精品视频免费| 成年美女黄网站色视频大全免费| 久久久久视频综合| 国产精品久久久久久精品电影小说| 国产野战对白在线观看| 久久人妻熟女aⅴ| 成人国产av品久久久| 不卡av一区二区三区| 亚洲伊人色综图| 国产精品.久久久| 哪个播放器可以免费观看大片| 国产视频首页在线观看| 激情视频va一区二区三区| 热99久久久久精品小说推荐| 久久精品亚洲av国产电影网| 久久青草综合色| 亚洲精品中文字幕在线视频| 看十八女毛片水多多多| 一级毛片 在线播放| 国产精品久久久久久av不卡| 91成人精品电影| 欧美日韩综合久久久久久| 一区二区av电影网| 一本大道久久a久久精品| 97在线人人人人妻| 满18在线观看网站| 伦理电影免费视频| 国产成人欧美| 亚洲人成77777在线视频| 日韩精品有码人妻一区| 一区二区三区乱码不卡18| 男人舔女人的私密视频| 亚洲成色77777| 久久久精品国产亚洲av高清涩受| 超碰成人久久| 午夜免费男女啪啪视频观看| 男人操女人黄网站| 日本av手机在线免费观看| 久久久久久免费高清国产稀缺| 欧美老熟妇乱子伦牲交| 69精品国产乱码久久久| 久久午夜福利片| 黑人巨大精品欧美一区二区蜜桃| 精品国产乱码久久久久久男人| 日本vs欧美在线观看视频| 咕卡用的链子| 国产亚洲精品第一综合不卡| 久久免费观看电影| 校园人妻丝袜中文字幕| 久久精品国产a三级三级三级| 女性生殖器流出的白浆| 久久久久视频综合| 欧美日韩精品网址| 免费黄网站久久成人精品| 另类精品久久| 一区二区三区精品91| 日韩人妻精品一区2区三区| 视频区图区小说| 99香蕉大伊视频| 一级片免费观看大全| 母亲3免费完整高清在线观看 | 电影成人av| 最黄视频免费看| 欧美另类一区| 亚洲欧美中文字幕日韩二区| 丝袜在线中文字幕| 久久久国产欧美日韩av| 99re6热这里在线精品视频| 久久久久精品人妻al黑| 天堂中文最新版在线下载| 欧美日韩视频精品一区| 少妇的丰满在线观看| 校园人妻丝袜中文字幕| 亚洲国产av新网站| 激情五月婷婷亚洲| 一二三四在线观看免费中文在| 久久久久久久国产电影| 国产精品麻豆人妻色哟哟久久| 大陆偷拍与自拍| 中文字幕人妻熟女乱码| 国产男女内射视频| 国产 精品1| 亚洲欧美色中文字幕在线| 午夜免费男女啪啪视频观看| 免费在线观看完整版高清| 亚洲成色77777| 91aial.com中文字幕在线观看| 成人国语在线视频| 制服人妻中文乱码| 啦啦啦在线免费观看视频4| 国语对白做爰xxxⅹ性视频网站| 五月天丁香电影| 亚洲av综合色区一区| 韩国av在线不卡| 女人高潮潮喷娇喘18禁视频| av视频免费观看在线观看| 精品人妻在线不人妻| 18+在线观看网站| 国产福利在线免费观看视频| 久久久久精品久久久久真实原创| 国产一区亚洲一区在线观看| 久久久国产一区二区| 色吧在线观看| av一本久久久久| av国产精品久久久久影院| 国产精品久久久久久av不卡| 麻豆精品久久久久久蜜桃| videos熟女内射| 一区二区日韩欧美中文字幕| 伊人久久大香线蕉亚洲五| av视频免费观看在线观看| 欧美激情高清一区二区三区 | 久久久久久伊人网av| 免费av中文字幕在线| 中文字幕精品免费在线观看视频| 麻豆乱淫一区二区| av在线老鸭窝| 国产成人午夜福利电影在线观看| 欧美精品高潮呻吟av久久| 日本欧美视频一区| 高清视频免费观看一区二区| 中文字幕另类日韩欧美亚洲嫩草| 18在线观看网站| 777米奇影视久久| 日本猛色少妇xxxxx猛交久久| 精品卡一卡二卡四卡免费| 一级a爱视频在线免费观看| 涩涩av久久男人的天堂| 成年人免费黄色播放视频| 婷婷色麻豆天堂久久| 免费久久久久久久精品成人欧美视频| 91精品伊人久久大香线蕉| 咕卡用的链子| 久久久久久人妻| 久久国产精品男人的天堂亚洲| 精品人妻熟女毛片av久久网站| 亚洲精品日韩在线中文字幕| 免费观看a级毛片全部| 欧美日韩精品成人综合77777| 免费av中文字幕在线| 精品久久蜜臀av无| 精品少妇久久久久久888优播| 看免费av毛片| 久久人人爽人人片av| 久久婷婷青草| 久久国产精品男人的天堂亚洲| 久久这里有精品视频免费| 亚洲欧美精品自产自拍| 免费观看在线日韩| 成人手机av| 丰满迷人的少妇在线观看| 男人爽女人下面视频在线观看| 成年av动漫网址| 不卡视频在线观看欧美| 宅男免费午夜| 成年女人在线观看亚洲视频| 波野结衣二区三区在线| 亚洲人成网站在线观看播放| 国产又色又爽无遮挡免| 人人妻人人爽人人添夜夜欢视频| 亚洲欧美精品自产自拍| 爱豆传媒免费全集在线观看| 最近中文字幕高清免费大全6| 国产精品99久久99久久久不卡 | 黑人巨大精品欧美一区二区蜜桃| 日日啪夜夜爽| 人妻一区二区av| 这个男人来自地球电影免费观看 | 18禁裸乳无遮挡动漫免费视频| 少妇猛男粗大的猛烈进出视频| 亚洲精品第二区| 18禁动态无遮挡网站| 国产精品二区激情视频| 久久人妻熟女aⅴ| 国产精品免费大片| 一本久久精品| 欧美成人午夜精品| 婷婷色av中文字幕| 亚洲国产精品成人久久小说| 黄色 视频免费看| 侵犯人妻中文字幕一二三四区| 欧美 亚洲 国产 日韩一| 一本久久精品| av在线app专区| 亚洲久久久国产精品| 国产熟女欧美一区二区| 日本色播在线视频| 卡戴珊不雅视频在线播放| 久久韩国三级中文字幕| 日本欧美国产在线视频| 欧美 日韩 精品 国产| 免费在线观看完整版高清| videosex国产| 免费人妻精品一区二区三区视频| 久久午夜综合久久蜜桃| 观看美女的网站| 欧美日韩国产mv在线观看视频| 亚洲婷婷狠狠爱综合网| 男男h啪啪无遮挡| 啦啦啦中文免费视频观看日本| 日韩欧美精品免费久久| 精品国产一区二区三区四区第35| 美女大奶头黄色视频| 日韩伦理黄色片| 丰满少妇做爰视频| 国产爽快片一区二区三区| 91精品三级在线观看| 日本色播在线视频| 97精品久久久久久久久久精品| 国产白丝娇喘喷水9色精品| √禁漫天堂资源中文www| 国产精品蜜桃在线观看| 免费高清在线观看视频在线观看| 国产精品久久久久成人av| 亚洲精品美女久久久久99蜜臀 | 亚洲美女视频黄频| 国产一级毛片在线| 欧美日韩成人在线一区二区| 毛片一级片免费看久久久久| 亚洲在久久综合| 日韩av免费高清视频| 伊人久久大香线蕉亚洲五| 亚洲精品视频女| 午夜福利影视在线免费观看| 欧美日本中文国产一区发布| 免费久久久久久久精品成人欧美视频| av在线老鸭窝| 性少妇av在线| 老汉色∧v一级毛片| 交换朋友夫妻互换小说| 我要看黄色一级片免费的| 高清视频免费观看一区二区| 一区在线观看完整版| 人人澡人人妻人| 婷婷成人精品国产| 午夜免费观看性视频| av.在线天堂| 中国三级夫妇交换| 日日爽夜夜爽网站| 侵犯人妻中文字幕一二三四区| 美女中出高潮动态图| 人体艺术视频欧美日本| 日韩一卡2卡3卡4卡2021年| 欧美日韩成人在线一区二区| 久久99一区二区三区| 99热网站在线观看| av一本久久久久| 亚洲国产色片| 午夜免费观看性视频| 国产成人免费观看mmmm| 久久人人97超碰香蕉20202| 一区二区三区精品91| 久久久久网色| 亚洲av电影在线进入| 日韩成人av中文字幕在线观看| 精品人妻在线不人妻| 中国三级夫妇交换| 精品亚洲成a人片在线观看| 国产一区二区三区av在线| 国产精品久久久久久精品电影小说| 久久国产精品大桥未久av| 国产综合精华液| 日韩欧美一区视频在线观看| 色婷婷av一区二区三区视频| www.自偷自拍.com| 99香蕉大伊视频| 麻豆精品久久久久久蜜桃| 纵有疾风起免费观看全集完整版| 我要看黄色一级片免费的| 色94色欧美一区二区| 天堂8中文在线网| 少妇被粗大的猛进出69影院| 夫妻午夜视频| 美女中出高潮动态图| 99热网站在线观看| 三上悠亚av全集在线观看| 久久99精品国语久久久| 一二三四在线观看免费中文在| 天堂中文最新版在线下载| 午夜日韩欧美国产| 亚洲精品美女久久av网站| 亚洲av电影在线观看一区二区三区| 国产精品香港三级国产av潘金莲 | 人成视频在线观看免费观看| 99久国产av精品国产电影| 色94色欧美一区二区| 日韩成人av中文字幕在线观看| 另类亚洲欧美激情| 中文精品一卡2卡3卡4更新| 国产深夜福利视频在线观看| 26uuu在线亚洲综合色| 在线观看www视频免费| 亚洲av中文av极速乱| 亚洲av免费高清在线观看| 久久女婷五月综合色啪小说| 国产av国产精品国产| 欧美人与善性xxx| 男女边吃奶边做爰视频| 国产淫语在线视频| 2018国产大陆天天弄谢| 午夜免费观看性视频| 国产成人精品婷婷| 少妇精品久久久久久久| 在线观看三级黄色| 青春草视频在线免费观看| 免费不卡的大黄色大毛片视频在线观看| 国产深夜福利视频在线观看| 色婷婷av一区二区三区视频| videos熟女内射| 亚洲国产av新网站| 国产一区二区 视频在线| 国产淫语在线视频| 亚洲欧洲日产国产| 日韩一区二区视频免费看| 中文字幕色久视频| 国产免费又黄又爽又色| 777米奇影视久久| 天天躁狠狠躁夜夜躁狠狠躁| 国产精品人妻久久久影院| 高清在线视频一区二区三区| 国产成人免费观看mmmm| 国产一区有黄有色的免费视频| 美女国产视频在线观看| 国产野战对白在线观看| 日韩三级伦理在线观看| 日韩视频在线欧美| 在线观看三级黄色| 秋霞伦理黄片| 97在线视频观看| 久久这里有精品视频免费| 熟女少妇亚洲综合色aaa.| 一级爰片在线观看| 一级片'在线观看视频| 亚洲欧美色中文字幕在线| 一本色道久久久久久精品综合| videos熟女内射| 日日撸夜夜添| 久久久国产欧美日韩av| 国产男人的电影天堂91| 精品人妻在线不人妻| 国产亚洲午夜精品一区二区久久| 精品人妻在线不人妻| 色哟哟·www| 精品一区二区三卡| 免费av中文字幕在线| 赤兔流量卡办理| 国产亚洲最大av| 80岁老熟妇乱子伦牲交| 久久热在线av| 精品人妻熟女毛片av久久网站| 日韩成人av中文字幕在线观看| 91精品三级在线观看| 亚洲精品久久午夜乱码| 成人免费观看视频高清| 人妻人人澡人人爽人人| 亚洲欧美一区二区三区黑人 | 91aial.com中文字幕在线观看| 热re99久久精品国产66热6| 国产精品无大码| 啦啦啦啦在线视频资源| 观看av在线不卡| 亚洲成人一二三区av| 欧美人与性动交α欧美精品济南到 | 两个人免费观看高清视频| 亚洲欧洲日产国产| 久久亚洲国产成人精品v| 黑人欧美特级aaaaaa片| 国产在线免费精品| 一区二区三区乱码不卡18| av.在线天堂| www日本在线高清视频| 高清黄色对白视频在线免费看| 老司机亚洲免费影院| 人成视频在线观看免费观看| 国产精品国产三级国产专区5o| 街头女战士在线观看网站| 黄色 视频免费看| 久久精品久久久久久噜噜老黄| 亚洲美女搞黄在线观看| 桃花免费在线播放| 国产xxxxx性猛交| 啦啦啦在线观看免费高清www| 青春草视频在线免费观看| 国产精品 欧美亚洲| 人妻 亚洲 视频| 亚洲欧洲精品一区二区精品久久久 | 日本91视频免费播放| xxx大片免费视频| 男人添女人高潮全过程视频| 国产视频首页在线观看| 久久鲁丝午夜福利片| 91精品国产国语对白视频| 国产精品一区二区在线观看99| 最近手机中文字幕大全| 天天躁夜夜躁狠狠久久av| 亚洲欧美日韩另类电影网站| 午夜av观看不卡| 成年av动漫网址| av免费在线看不卡| 亚洲国产看品久久| 亚洲av电影在线观看一区二区三区| 亚洲在久久综合| 午夜福利视频精品| 寂寞人妻少妇视频99o| 黄色视频在线播放观看不卡| 亚洲美女黄色视频免费看| 18禁动态无遮挡网站| 国产成人91sexporn| 国产又爽黄色视频| 丰满乱子伦码专区| 亚洲国产日韩一区二区| 一本久久精品| 久久久亚洲精品成人影院| 亚洲精品在线美女| 精品一区二区三区四区五区乱码 | 日本欧美国产在线视频| 1024视频免费在线观看| 女人被躁到高潮嗷嗷叫费观| 亚洲一级一片aⅴ在线观看| 精品一品国产午夜福利视频| 精品第一国产精品| 五月开心婷婷网| 午夜91福利影院| 永久免费av网站大全| 亚洲精品第二区| 国产熟女午夜一区二区三区| 日日啪夜夜爽| 国产精品三级大全| 久久久久精品人妻al黑| 亚洲综合精品二区| 男女边摸边吃奶| 另类精品久久| 一级毛片 在线播放| 久久精品熟女亚洲av麻豆精品| 国产高清国产精品国产三级| 九草在线视频观看| 2021少妇久久久久久久久久久| a级毛片在线看网站| 2021少妇久久久久久久久久久| 亚洲av日韩在线播放| 精品少妇一区二区三区视频日本电影 | 男女下面插进去视频免费观看| 久久人人爽人人片av| 观看av在线不卡| 国产亚洲精品第一综合不卡| av一本久久久久| 国产欧美亚洲国产| 日本欧美国产在线视频| 91国产中文字幕| 亚洲伊人色综图| 亚洲精品国产一区二区精华液| 亚洲第一青青草原| 国产免费一区二区三区四区乱码| 永久免费av网站大全| 性色av一级| 菩萨蛮人人尽说江南好唐韦庄| 久久狼人影院| 欧美少妇被猛烈插入视频| 国产成人一区二区在线| 天天躁日日躁夜夜躁夜夜| 边亲边吃奶的免费视频| 在线免费观看不下载黄p国产| 中文字幕人妻熟女乱码| 国产色婷婷99| 亚洲精品久久午夜乱码| 美女高潮到喷水免费观看| 国产1区2区3区精品| kizo精华| 亚洲成色77777| 黄片播放在线免费| 午夜免费观看性视频| 久久久久久伊人网av| 波多野结衣av一区二区av| xxx大片免费视频| 久久久精品免费免费高清| 丰满迷人的少妇在线观看| 九草在线视频观看| 免费观看无遮挡的男女| 亚洲av电影在线进入| 2021少妇久久久久久久久久久| 成人毛片a级毛片在线播放| 亚洲av免费高清在线观看| 电影成人av| 狂野欧美激情性bbbbbb| 91国产中文字幕| 天天躁夜夜躁狠狠躁躁| 国产成人91sexporn| 涩涩av久久男人的天堂| 亚洲av男天堂| 欧美精品高潮呻吟av久久| 一边亲一边摸免费视频| 精品人妻在线不人妻| 国产高清国产精品国产三级| 欧美黄色片欧美黄色片| 日本av手机在线免费观看| 亚洲精品美女久久av网站| 欧美日韩视频高清一区二区三区二| av视频免费观看在线观看| av一本久久久久| 赤兔流量卡办理| 久久久精品94久久精品| 国产精品国产av在线观看| www.精华液| 亚洲精品久久成人aⅴ小说| av电影中文网址| 精品国产露脸久久av麻豆| 丰满少妇做爰视频| 精品国产乱码久久久久久男人| 亚洲欧美一区二区三区久久| 熟女电影av网| 王馨瑶露胸无遮挡在线观看| 国产高清不卡午夜福利| 国产1区2区3区精品| 欧美老熟妇乱子伦牲交| 激情五月婷婷亚洲| 亚洲,欧美精品.| 黄片播放在线免费| 亚洲经典国产精华液单| 免费在线观看视频国产中文字幕亚洲 | 亚洲男人天堂网一区| 国产一区二区 视频在线| av有码第一页| 国产在视频线精品| 亚洲精品aⅴ在线观看| 国产精品国产三级国产专区5o| 欧美日韩精品成人综合77777| 在线观看人妻少妇| 夫妻午夜视频| 国产在线视频一区二区| 日韩欧美精品免费久久| 久久99一区二区三区| 99热全是精品| 一区在线观看完整版| 精品人妻一区二区三区麻豆| 色哟哟·www| 老汉色av国产亚洲站长工具| 色播在线永久视频| 国产成人精品在线电影| 一级毛片我不卡| 中文天堂在线官网| 在线免费观看不下载黄p国产| 中文字幕人妻熟女乱码| av线在线观看网站| 自拍欧美九色日韩亚洲蝌蚪91| 久久毛片免费看一区二区三区| av在线播放精品| 老司机影院毛片| 99久国产av精品国产电影| 1024视频免费在线观看| 国产精品国产三级国产专区5o| 免费人妻精品一区二区三区视频| 黄色视频在线播放观看不卡| 只有这里有精品99| 久久精品久久久久久久性| 国产精品无大码| 在线亚洲精品国产二区图片欧美| 日本午夜av视频| 亚洲伊人色综图| 爱豆传媒免费全集在线观看| 熟女av电影| 国产精品熟女久久久久浪| 国产欧美日韩综合在线一区二区| 赤兔流量卡办理| 一本大道久久a久久精品| 欧美日韩精品成人综合77777| 两个人免费观看高清视频| 一区二区三区乱码不卡18| 男人操女人黄网站| 成人午夜精彩视频在线观看| 美国免费a级毛片| 亚洲天堂av无毛| 成人午夜精彩视频在线观看| 亚洲综合精品二区| av免费观看日本| 久久毛片免费看一区二区三区| 一本大道久久a久久精品| 欧美日韩一级在线毛片| 久久久欧美国产精品| 又黄又粗又硬又大视频| 美女主播在线视频| 免费看不卡的av| 老司机亚洲免费影院| 国产精品秋霞免费鲁丝片| 国产成人一区二区在线| 亚洲欧美中文字幕日韩二区| 久久久久久伊人网av| 国产在线视频一区二区| 久久久久国产精品人妻一区二区| 一本色道久久久久久精品综合| 精品酒店卫生间| 国产亚洲一区二区精品| 有码 亚洲区| 色网站视频免费|