Short Communication: The Distribution of Potential N-Linked Glycosylation Sites in Gp120 Differs Among Major HIV-1 Subtypes Circulating in China

Abstract

CRF01_AE shows an obvious sexual transmission advantage over other major HIV-1 subtypes circulating in China. Previous studies showed that the presence or absence of potential N-linked glycosylation sites (PNGSs) in variable loops might affect HIV-1 transmission; it is therefore of interest to compare the distribution of potential PNGSs on envelopes of different subtypes circulating in China. Compared to CRF07_BC, CRF08_BC, B, and B′ subtypes isolated in China, CRF01_AE subtypes isolated from both China and outside China had significantly fewer PNGSs in total and in V2/V4, while they had significantly more PNGSs in V5. HIV-1 subtype CRF01_AE has a unique PNGS distribution pattern in Gp120, which may contribute to its advantage in sexual transmission in China.

Introduction

HIV/AIDS remains one of the major threats to public health in China. As reported by the National Center for AIDS/STD Control and Prevention (NCAIDS) of China, by the end of October 2014, a total of 496,525 individuals were infected by HIV-1, of whom 12,145 died.¹ HIV-1 B′, CRF01_AE, CRF07_BC, and CRF08_BC are the four dominant subtypes now circulating in China; however, compared to 20 years ago, the ranking of dominance has significantly changed. The proportion of CRF01_AE significantly increased while the proportion of subtype B′ significantly decreased.² The major transmission route of CRF01_AE in China is through sex,^2,3 which is consistent with its origin.⁴ It seems that the prevalence of CRF01_AE increased to accompany the shift in the major transmission route from intravenous drug use to sexual contact, as indicated by the fact that during the same period, the proportion of transmission through injecting drug use versus sex changed from 34.1% versus 33.1% in 2006 to 17.3% versus 75.2% in 2011.⁵

HIV-1 subtype CRF01_AE originated in Africa, established a major epidemic in Thailand, and then dispersed to neighboring Asian countries.⁶ It was estimated that CRF01_AE was introduced into China in the 1990s,⁷ which is much later than the origin of HIV-1 B′ in China.⁸ However, its prevalence increased fast: 9.6% in 1998, 27.6% in 2006, and more than 40% after 2007.² The underlying mechanisms are not completely understood, especially regarding its advantage in sexual transmission.

Materials and Methods

HIV-1 env sequences contained the complete DNA sequence (CDS) of Gp120 from the Los Alamos HIV Database (www.hiv.lanl.gov/). In total, 2,276 sequences were retrieved, including 726 sequences sampled from China (230 CRF01_AE, 328 CRF07_BC, 60 CRF08_BC, 63 B′, and 45 B sequences) and 1,550 CRF01_AE sequences sampled from outside China.

HxB2 was used as the reference strain to perform multiple sequence alignment by using Bio-Edit version 7.0.9.0. The number of PNGSs and the length of the variable loops (V1–V5) were tallied by the Variable Region Characteristics tool of the Los Alamos HIV databases (www.hiv.lanl.gov/content/sequence/VAR_REG_CHAR/index.html). The percentages of PNGSs on each variable loop were calculated according to the following formula: Frequency = (the number of PNGSs in a variable loop)/(the total number of PNGSs in the same Gp120) × 100%.

All statistical analyses were performed by using GraphPad Prism version 6.01 (GraphPad Software, La Jolla, CA). Comparisons of multiple groups were performed by the Kruskal–Wallis test. p < 0.05 was considered statistical significant (two-tailed).

Results

We found that the total numbers of potential N-linked glycosylation sites (PNGSs) of both CRF01_AE-CN and CRF01_AE-non-CN were slightly lower than those of the other HIV-1 subtypes (Fig. 1A). The differences were more significant when looking at the numbers of PNGSs in V2, V4, and V5. Compared to 07_BC and 08_BC, the PNGS numbers of both CRF01_AE-CN and CRF_01 AE-non-CN were significantly lower in V2 (Fig. 1B), while they were significantly higher in V5 (Fig. 1D). In V4, the PNGS numbers of both CRF01_AE-CN and CRF01_AE-non-CN were significantly lower than all the other subtypes (Fig. 1C). The numbers of PNGSs in V1 and V3 were similar across all these subtypes (data not shown).

FIG. 1.

Comparisons of potential N-linked glycosylation site (PNGS) numbers in Gp120 and variable loops. (A) Comparisons of total PNGS numbers in Gp120. Comparisons of PNGS numbers in V2, V4, V5 are shown in (B), (C), and (D), respectively.

To further characterize the difference of PNGS distribution among these HIV-1 subtypes, we calculated and compared the percentages of total PNGSs that distributed in each variable loop of Gp120. Very similar to the above findings, we found that CRF01_AE-CN and CRF01_AE-non-CN had significantly lower percentages of PNGSs in V2 (Fig. 2A) and V4 (Fig. 2B), while they had higher percentages of PNGSs in V5 (Fig. 2C).

FIG. 2.

Percentages of total PNGSs that distributed in variable loops. Percentages of total PNGSs that distributed in V2, V4, V5 were compared and are shown in (A), (B) and (C), respectively.

Discussion

N-glycans comprise more than 40% of the mass of the HIV-1 envelope protein.⁹ While many studies focused on the roles of N-linked glycans in activities such as maintaining the tertiary structure of Gp120¹⁰ and evading the host immune attack,¹¹ several lines of evidence also indicated that N-linked glycans may impact the infectivity of HIV-1.^12
–14 In this study, we reported that the number and distribution of PNGSs on HIV-1 Gp120 were different between CRF01_AE and other major subtypes circulating in China. Of note, we found that significantly fewer PNGSs were positioned in V2 and V4 of HIV-1 CRF01_AE Env. As removing N-glycans from the V2/V4 domains was shown to significantly improve the immunogenicities of HIV-1 Env proteins^15,16 and the neutralization sensitivities of HIV-1/SIV viruses,¹⁷ it is understandable that several HIV-1 CRF01_AE isolates were found to show slightly higher neutralization sensitivity by polyclonal antibodies in plasma of HIV-1-infected subjects.¹⁸

Moreover, according to previous studies, the short V1/V2 and fewer PNGSs in V1/V2/V4^19

–22 are the major characteristics of mucosally transmitted founder viruses, hence our data emphasize the possibility that the fewer PNGSs in the V2 and V4 loops may contribute to the advantage of CRF01_AE in sexual transmission. To support this hypothesis, we compared the sexually and intravenously transmitted CRF01_AE-CN Env sequences. We found that the numbers of PNGSs in the V1, V2, V4, and V5 loops tended to be fewer in sexually transmitted sequences than in intravenous drug use (IDU) sequences (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/aid). However, no statistical significance was reached, possibly due to the relatively small sample size. We believe that meta-analysis of founder virus sequences transmitted through different routes will provide more solid evidence in the future.

CRF01_AE has resulted in an international epidemic, mainly in East and Southeast Asia.⁶ A recent study indicated that different coreceptor preferences might lead to different prevalences of HIV-1 B, CRF01_AE, and CRF07_BC in the men having sex with men (MSM) population in Beijing, China.²³ Here, our data provide another clue to understanding the CRF01_AE epidemic and emphasize the importance to obtaining greater insight into the influence of envelope glycosylation on HIV-1 infectivity.

Accession Numbers of Sequence Data

CRF01_AE-CN

AY008718, AY008714, JX112866, JX112865, JX112864, JX112863, JX112862, JX112861, JX112860, GQ845126, GQ845125, GQ845124, HM215424, HQ699987, HQ699977, EF036527, DQ859178, EF036529, EF036528, EF036535, EF036534, DQ859179, GU564229, GU564228, GU564227, GU564225, GU564224, GU564223, GU564222, GU564221, EF036530, EF036536, HM215427, HM215422, HM215415, HM215410, HQ699993, DQ859180, EF036532, EF036531, EF036533, GU475046, GU475025, GU564230, JX112858, JX112857, HM215419, HM215418, HM215409, HM215393, HM215391, HM215390, HM215389, HM215387, HM215386, HM215385, HM215383, HM215379, HM215378, HM215377, HM215376, HM215374, HM215373, HM215372, HM215371, HM215368, HM215367, HQ699988, HQ699986, HQ699956, HQ699955, GU475031, GU475015, GU475036, GU475030, GU475045, GU475043, GU475017, JX112829, JX112853, JX112830, JX112852, JX112851, JX112850, JX112831, GU475042, GU475037, GU475028, GU475020, JX112859, JX112817, JX112816, JX112815, JX112814, JX112813, JX112811, JX112809, JX112818, JX112826, JX112828, JX112855, JX112854, JX112822, JX112856, JX112840, JX112841, JX112825, JX112824, JX112823, KF835505, FJ441290, GU475040, GU475024, JX112827, JX112832, JX112833, JX112834, JX112821, JX112820, JX112819, JX112843, GU475041, GU475022, JX112842, JX112845, JX112844, KF835538, KF835536, KF835533, KF835528, KF835527, KF835523, KF835519, KF835518, KF835516, KF835513, JX112835, KF835514, JX112836, JX112837, KF835543, KF835542, KF835499, KF835504, KF835503, KF835502, JX112839, JX112838, GU475026, HM215394, GU475023, GU475034, GU475033, GU475032, GU475029, GU475044, GU475039, GU475038, GU475035, GU475027, GU475021, GU475019, GU475018, GU475016, GU475014, GU475013, JX960612, JX960617, JX960606, KC596062, KC596061, JX960630, JX960615, JX960618, JX960604, JX960627, JX960605, JX960636, JX112868, JX112867, JX960638, JX960607, JX960634, JX960628, JX960629, JX960613, HQ215555, HQ215553, JX960625, JX960626, JX112870, JX112869, JX960621, JX960631, JX960614, JX960639, JX960623, JX960603, KC596063, JX960611, JX112797, JX112796, JX112798, JX112799, JX112801, JX112800, JX112806, JX112805, JX112804, JX112803, JX112802, JX112808, JX112807, JX960616, JX960637, JX960610, JX960633, JX960609, JX960624, JX112847, JX112846, JX112849, JX112848, JX960620, JX960632, JX960608, JX960622, KC596064, KC596065

CRF01_AE-non-CN

DQ518412, AY444805, EU191614, AJ277818, U51188, AF197341, AF197340, AY713423, JQ715409, AY713419, JQ715414, FJ185252, AB253656, AY945724, AY444803, AY358063, AY945731, AY713424, JQ715407, FJ185248, JX512902, AY945721, AY358057, AB032740, AY961589, AY945727, AY945718, AF209205, AF070704, AY358052, AY945722, JQ352782, AY945732, JX512903, AY713420, KC822430, U48266, JN944661, AY945716, AY945728, JQ352783, AY358065, AY358043, FJ185251, AB032741, DQ789392, U51189, FJ185247, AY669771, AY358061, AY358051, AY358049, AY358067, AY358045, AF015921, AF015920, AF015919, AY358066, AY358046, AY358048, AY358047, AY358039, AY945725, AY945717, JN944664, FJ185260, AF070711, AY358060, AY444804, FJ185245, FJ185249, AY945712, JN944660, FJ185255, FJ185235, AF070709, U48264, AF070710, FJ185243, FJ185241, FJ185258, FJ185253, AF015918, AF015917, AF015916, FJ185246, DQ354117, FJ185237, AY444806, FJ185242, FJ185259, AF070713, AY125894, AY713421, JQ715412, FJ185239, AY945726, DQ313251, DL258716, AY713422, FJ185240, U08458, AF070707, JQ715410, AY713426, AF209206, AF209207, AF209208, AF209209, AF070703, FJ185238, FJ185257, FJ185256, FJ185234, FJ185236, FJ185229, FJ185231, FJ185230, FJ185228, FJ185233, FJ185232, AY494967, AY669779, FJ185244, U08457, AF219273, AF070708, FJ185250, AF164485, AB220946, AB220947, AB220944, FJ185254, AF070705, AF219268, U39261, U39255, L03701, JN944663, AY231158, AY669776, U08810, BD187399, AB052995, AY669772, AY669770, U39260, AY494968, U39256, AY669774, U08456, AY669773, AB253425, AB253424, AF197338, AB485653, AB485652, AY669775, L03703, AY669778, U09131, AB026050, AB070352, AB220945, AB220948, AF197339, AF219267, KC822429, AY713425, JQ715397, L14572, U54771, AY736838, JQ715396, EF553537, L03698, L03699, JN944662, AF259955, AF259954, AY736837, JQ715395, EU743781, EU743779, EU743780, EU743782, EU743783, JN248356, JX447719, JX447715, JX447717, JX447713, JX447718, JX447711, JX447709, JX447710, JX447714, JX447712, JX447716, JX447720, JX447552, JX447554, JX447550, JX447555, JX447553, JX448095, EU743763, EU743764, EU743792, EU743785, EU743787, EU743786, JN944654, JF939057, EU743790, JN248355, JX448128, JX448123, JN944655, JX448118, JX448119, JX448120, JX448121, JX448124, JX448125, JX448126, JX448127, JX448122, JX448279, JX448282, JX448280, JX448281, EU743784, JX447296, JX44729, JX447294, JX447295, JX447298, JX447299, JN248327, JX447470, JX447467, JX447469, JX447465, JX447468, JX447473, JX447471, JX447472, JX447466, JX447474, JX447529, JX447531, JX447532, JX447533, JX447534, JX447530, JX446703, JX446705, JX446707, JX446708, JX446702, JX446706, JX446700, JX446701, JX446709, JX446698, JX446699, JX446704, JX447412, JX447415, JX447416, JX447413, JX447414, EU743794, JX447925, JX447926, JX447927, JX447922, JX447924, JX447923, JX447928, JX447936, JX447921, JX447929, JN248330, JX447567, JX447564, JX447561, JX447563, JX447568, JX447562, JX447565, JX447566, JX446984, JX446982, JX446983, JX446978, JX446986, JX446981, JX446985, JX446980, JX446979, JX446976, JX446977, JX448291, JX448289, JX448293, JX448292, JX448295, JX448290, JX448294, JX447227, JX447229, JX447225, JX447221, JX447223, JX447224, JX447222, JX447220, JX447228, JX446713, JX446715, JX446711, JX446719, JX446710, JX446714, JX446718, JX446717, JX446712, JX446716, EU743789, JX448023, JX448021, JX448019, JX448027, JX448020, JX448024, JX448026, JX448022, JX447315, JX447317, JX447316, JX446910, JX446912, JX446915, JX446911, JX446913, JX446914, JX448253, JX448254, JX448261, JX448256, JX448258, JX448260, JX448259, JX448255, JX448257, JX447589, JX447594, JX447590, JX447591, JX447595, JX447593, JX447592, EU743765, EU743766, JX448149, JX448144, JX448148, JX448151, JX448150, JX448146, JN944657, JX448141, JX448142, JX448143, JX448145, JX448147, JX448152, JX447347, JX447348, JX447344, JX447345, JX447346, JF297226, JX447890, JX447885, JX447884, JX447888, JX447889, JX447891, JX447886, JX447887, AY358068, EU743770, JX447910, JX448163, JN944658, JX448170, JX448168, JX448164, JX448167, JX448172, JX448165, JX448166, JX448169, JX448171, JX447912, JX447905, JX447904, JX447911, JX447909, JX447903, JX447906, JX447913, JX447908, JX447907, JX447914, JX447521, JX447515, JX447517, JX447522, JX447516, JX447518, JX447520, JX447519, HQ691083, HQ691084, HQ691088, HQ691089, JF297233, HQ691085, HQ691086, HQ691090, HQ691087, HQ691092, HQ691082, HQ691091, JX448217, JX448220, JX448223, JX448218, JX448222, JX448224, JX448219, JX448221, JF297225, JN248328, JX447632, JX447635, JX447620, JX447629, JN944656, JX447636, JX447619, JX447633, JX447634, JX447630, JX447618, JX447631, JX447639, JX447637, JX447642, JX447646, JX447645, JX447640, JX447643, JX447641, JX447638, JX447644, EU743767, EU743768, HQ245668, HQ245672, EU743758, AY358036, AY358038, EU743788, EU743778, EU743769, EU743774, JF297224, JX448209, JX448200, JX448207, JX448203, JX448202, JX448199, JX448206, JX448201, JX448208, JX448205, JX448198, JX448204, EU668991, JN248342, JX448302, JX448304, JX448301, JX448303, JX448305, JX448306, JX447965, JX447961, JX447958, JX447966, JX447959, JX447967, JX447963, JX447960, JX447962, JX447964, JN248341, JX447700, JX447708, JX447701, JX447707, JX447706, JX447703, JX447699, JX447705, JX447702, JX447704, AY358041, JX447544, JX447536, JX447537, JX447538, JX447543, JX447539, JX447540, JX447541, JX447535, JX447542, JX447048, JX447049, JX447051, JX447047, JX447044, JX447043, JX447046, JX447050, JX447052, JX447045, EU743775, EU743776, EU743777, JX447091, JX447090, JX447089, JX447093, JX447087, JN248339, EU743772, EU743771, EU743773, JX447823, JX447814, JX447821, JX447822, JX447815, JX447819, JX447818, JX447816, JX447820, JX447825, JX447824, JX447817, JX447826, EU743791, EU743793, JN248336, JX446960, JX446961, JX446962, JX446964, JX446963, JX447202, JX447205, JX447201, JX447199, JX447206, JX447207, JX447204, JX447203, JX447209, JX447200, JX447208, HQ690964, HQ690965, JF297223, HQ690972, HQ690967, HQ690971, HQ690968, HQ690969, HQ690966, HQ690970, EU743757, JQ352782, JX448272, JX448273, JX448277, JX448276, JX448271, JX448274, JX448275, JX447313, JX447312, JX447314, JX447400, JX447398, JX447395, JX447396, JX447397, JX447399, JX447401, JX447135, JX447136, JX447137, JX447134, JX447132, JX447133, JN248324, HQ691019, JF297227, HQ691014, HQ691015, HQ691017, HQ691013, HQ691018, HQ691016, JX447448, JX447449, JX447450, JX447447, JX447451, JX447452, JX447453, JX446748, JX446747, JX446750, JX446736, JX446737, JX446753, JX446751, JX446749, JX446752, JX446754, JX447080, JX447077, JX447083, JX447085, JX447079, JX447082, JX447081, JX447078, JX447084, JX447086, JX447355, JX447351, JX447353, JX447349, JX447356, JX447354, JX447350, JX447357, JX447352, JX447358, JX446677, JX446676, JX446680, JX446678, JX446684, JX446666, JX446683, JX446682, JX446679, JX446675, JX446681, JX446899, JX446900, JX446901, JX446903, JX446902, JX448039, JX448044, JX448040, JX448041, JX448042, JX448043, JX447985, JX447978, JX447980, JX447983, JX447979, JX447987, JX447989, JX447990, JX447984, JX447988, JX447981, JX447982, JX447986, AY358040, KC748975, KC748995, KC749000, KC748998, KC748988, KC748974, KC748983, KC748976, KC748980, KC748985, KC748982, KC748996, KC748979, KC748990, KC748978, KC748997, KC748977, KC748984, KC748989, KC748991, KC748999, KC748972, KC748987, KC748981, KC748993, KC748973, KC748986, KC748992, KC748994, JF297221, JN248318, JN248338, JX448184, JX448183, JX448186, JX448185, JX448182, JN944659, JX448180, JX448181, JX446856, JX446863, JX446864, JX446865, JX446854, JX446868, JX446867, JX446855, JX446866, JX446853, AY358064, JN248334, JX448241, JX448238, JX448242, JX448239, JX448240, AY358056, AY358037, AY358062, JX447280, JX447278, JX447279, JX447266, JX447277, JX447282, JX447281, JX447276, JX447267, JX447268, AY945720, HQ245667, JX447687, JX447680, JX447682, JX447684, JX447683, JX447686, JX447679, JX447678, JX447681, JX447685, HQ245669, HQ245671, JX447583, JX447585, JX447582, JX447584, JX447579, JX447581, JX447586, JX447587, JX447588, JX447580, JX447102, JX447108, JX447109, JX447110, JX447107, JX447103, JX447104, JX447111, JX447106, JX447105, JX447664, JX447660, JX447657, JX447663, JX447666, JX447665, JX447667, JX447659, JX447658, JX447662, JX447661, AY945719, JX447025, JX447024, JX447026, JX447018, JX447021, JX447019, JX447023, JX447027, JX447020, JX447022, JX447854, JX447858, JX447860, JX447862, JX447863, JX447857, JX447853, JX447861, JX447855, JX447856, JX447859, AY358050, JX447402, JX447411, JX447403, JX447407, JX447406, JX447408, JX447409, JX447404, JX447410, AY945713, AY358051, AY358059, AY945730, JN944665, KJ541845, JX446947, JX446952, JX446944, JX446942, JX446948, JX446953, JX446946, JX446943, JX446945, JX446949, JX446951, JX446950, GQ916587, JN860767, JN388164, JN388167, JN388165, JN388166, JN388170, JN388168, JN388169, JN388163, JN388161, JN388162, JX448088, JX448091, JX448090, JX448095, JX448086, JX448089, JX448094, JX448092, JX448093, JX448087, JN388173, JN388171, JN388177, JN388178, JN388176, JN388180, JN388175, JN388179, JN388174, JF680932, KF268040, 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JX447775, JX447783, JX447781, JX447779, JX447778, JX447784, JX447780, JX512896, JX512897, JX447459, JX447462, JX447454, JX447455, JX447464, JX447463, JX447457, JX447461, JX447458, JX447460, JX447456, JX447613, JX447610, JX447615, JX447608, JX447612, JX447616, JX447617, JX447614, JX447609, JX447611, JN388135, JN388140, JN388137, JN388131, JN388138, JN388136, JN388133, JN388134, JN388132, JN388139, JN388222, JN388229, JN388224, JN388227, JN388230, JN388223, JN388226, JN388221, JN388225, JN388228, GQ916589, GQ916584, JX447061, JX447058, JX447055, JX447059, JX447057, JX447056, JX447062, JX447054, JX447060, JX512893, JN860766, JX446840, JX446842, JX446839, JX446837, JX446838, JX446841, JX446844, JX446843, JX446847, JX446845, JX446846, JX447387, JX447384, JX447381, JX447386, JX447388, JX447382, JX447383, JX447385, JX447075, JX447072, JX447076, JX447074, JX447073, GQ916569, JX448065, JX448066, JX448067, JX448062, JX448063, JX448064, JX448058, JX448059, JX448060, JX448057, JX447727, JX447731, JX447730, JX447735, JX447729, JX447732, JX447733, JX447726, JX447734, JX448001, JX448005, JX448007, JX448004, JX448006, JX448010, JX448003, JX448009, JX448002, JX448008, JX446758, JX446759, JX446761, JX446763, JX446760, JX446757, JX446755, JX446756, JX446764, JX512898, JX447499, JX447508, JX447507, JX447504, JX447505, JX447501, JX447503, JX447500, JX447506, JX447502, GQ916585, JX446874, JX446875, JX446877, JX446873, JX446876, JX446850, JX446851, JX446848, JX446849, JX446852, JN860763, JN388220, JN38821, JN388217, JN388212, JN388219, JN388215, JN388216, JN388213, JN388211, JN388214, KF859741, JX447746, JX447750, JX447751, JX447752, JX447754, JX447755, JX447749, JX447747, JX447753, JX447756, JX447748, JX447366, JX447364, JX447361, JX447359, JX447360, JX447365, JX447362, JX447363, JX447368, JX447235, JX447238, JX447237, JX447236, JX447239, JX447240, JX447722, JX447721, JX447725, JX447723, JX447724, JX446791, JX446792, JX446793, JX446794, JX446790, GQ916588, HQ691076, JF297232, HQ691081, HQ691078, HQ691080, HQ691075, HQ691073, HQ691079, HQ691077, HQ691074, KF268036, JX446895, JX446890, JX446897, JX446896, JX446891, JX446893, JX446894, JX446892, JX446888, JX446889, HQ691004, HQ691009, HQ691005, JF297226, HQ691006, HQ691011, HQ691012, HQ691007, HQ691008, HQ691010, JN388208, JN388201, JN388202, JN388205, JN388206, JN388209, JN388204, JN388210, JN388203, JN388207, GQ916579, JX448053, JX448055, JX448051, JX448054, JX448052, JX448056, JN388194, JN388198, JN388195, JN388199, JN388191, JN388193, JN388200, JN388192, JN388196, JN388197, GQ916578, HQ691039, HQ691048, HQ691042, HQ691050, HQ691043, HQ691051, HQ691047, JF297231, HQ691041, HQ691046, HQ691040, HQ691044, HQ691045, HQ691049, JX448316, JX448314, JX448313, JX448312, JX448315, JX447064, JX447071, JX447065, JX447066, JX447067, JX447063, JX447493, JX447494, JX447496, JX447498, JX447495, JX447497, GQ916591, GQ477441, GQ916590, JN860761, GQ916576, JX447485, JX447482, JX447483, JX447480, JX447481, JX447484, KF268044, HQ690988, HQ690992, HQ69098, HQ690991, HQ690976, HQ690989, HQ690979, HQ690980, HQ690998, HQ690990, HQ690974, HQ690981, HQ691002, HQ690977, HQ690997, HQ690978, HQ691001, HQ690984, HQ690985, HQ690975, HQ690973, HQ690983, HQ690987, HQ690999, HQ690993, HQ690994, HQ690996, HQ690986, HQ690995, HQ691000, JX448071, JX448072, JX448075, JX448073, JX448074, JN388158, JN388159, JN388154, JN388153, JN388155, JN388151, JN388152, JN388157, JN388156, JN388160, JN388097, JN388092, JN388091, JN388093, JN388094, JN388098, JN388100, JN388099, JN388095, JN388096, HQ691072, HQ691066, HQ691052, HQ691054, HQ691063, HQ691070, HQ691059, HQ691071, HQ691061, HQ691053, HQ691069, HQ691064, HQ691056, HQ691058, HQ691062, HQ691065, JF297230, HQ691055, HQ691068, HQ691067, HQ691057, HQ691060, GQ916574, JN388081, JN388085, JN388090, JN388084, JN388086, JN388082, JN388087, JN388088, JN388089, JX447028, JX447030, JX447031, JX447029, JX447032, JX447941, JX447942, JX447945, JX447943, JX447944, JX447946, GQ916581, GQ916572, JX512894, JX448028, JX448030, JX448029, JX448031, JX448033, JX446926, JX446925, JX446920, JX446922, JX446918, JX446921, JX446917, JX446919, JX446916, JX446923, JX446924, JX447332, JX447336, JX447331, JX447328, JX447334, JX447330, JX447333, JX447335, JX447329, JX447337, JX447118, JX447117, JX447120, JX447119, JX447121, JN860768, HQ691024, JF297228, HQ691021, HQ691020, HQ691023, HQ691026, HQ691025, HQ691022, HQ691027, JX446992, JX446991, JX446996, JX446990, JX446995, JX446993, JX446988, JX446994, JX446987, JX447260, JX447263, JX447258, JX447262, JX447259, JX447253, JX447257, JX447264, JX447254, JX447255, JX447252, JX447256, JX447261, JX447265, JX447286, JX447284, JX447283, JX447285, JX447287, JX446782, JX446781, JX446776, JX446778, JX446775, JX446783, JX446780, JX446779, JX446777, JX446784, GQ916583, KF268041, KF268046, KF268045, KF268047, KF268039, KF268035, JX447434, JX447435, JX447428, JX447439, JX447437, JX447427, JX447432, JX447429, JX447433, JX447438, JX447431, JX447430, JX447436, JX446655, JX446659, JX446660, JX446661, JX446658, JX446663, JX446662, JX446656, JX446665, JX446664, JX446657, KF268037, KF268038, GQ916577, JN388142, JN388146, JN388148, JN388141, JN388149, JN388150, JN388147, JN388143, JN388144, JN388145, JN388172, JN860764, JN388109, JN388110, JN388103, JN388101, JN388106, JN388108, JN388102, JN388107, JN388104, JN388105, JN388182, JN388187, JN388186, JN388183, JN388188, JN388185, JN388190, JN388181, JN388184, JN388189

CRF07_BC-CN

AX149898, AX149771, AX149672, AX149647, AF286226, AY669747, AF286230, EU178249, EU178246, EU178245, EU178244, EU178239, EU178235, EU178234, EU178233, EU178232, EU178231, EU178230, EU178229, EU178228, EU178227, EU178226, EU178225, EU178224, EU178223, EU178222, EU178221, EU178220, EU178219, EU178218, EU178217, EU178216, EU178215, EU178214, EU178213, EU178212, EU178211, EU178210, EU178209, EU178208, EU178207, EU178206, EU178205, EU178204, EU178203, EU178202, EU178201, EU178200, EU178199, EU178198, EU178197, EU178196, EU178195, EU178194, EU178193, EU178192, EU178191, EU178190, EU178189, EU178188, EU178187, EU178186, EU178185, EU178184, EU178183, EU178182, EU178181, EU178180, EU178179, EU178178, EU178177, EU178176, EU178175, EU178174, EU178173, EU178172, EU178171, EU178170, EU178169, EU178168, EU178167, EU178166, EU178165, EU178164, EU178163, EU178162, EU178161, EU178160, EU178159, EU178420, EU178419, EU178418, EU178417, EU178416, EU178415, EU178414, EU178413, EU178412, EU178411, EU178410, EU178409, EU178408, EU178407, EU178406, EU178405, EU178404, EU178403, EU178402, EU178401, EU178400, EU178399, EU178398, EU178397, EU178396, EU178395, EU178394, EU178393, EU178392, EU178391, EU178390, EU178389, EU178388, EU178387, EU178386, EU178385, EU178384, EU178383, EU178382, EU178381, EU178380, EU178379, EU178378, EU178377, EU178376, EU178375, EU178374, EU178373, EU178372, EU178371, EU178370, EU178369, EU178368, EU178367, EU178365, EU178364, EU178362, EU178361, EU178360, EU178359, EU178358, EU178357, EU178356, EU178355, EU178354, EU178353, EU178352, EU178351, EU178350, EU178349, EU178348, EU178347, EU178346, EU178345, EU178344, EU178343, EU178342, EU178341, EU178340, EU178339, EU178338, EU178337, EU178336, EU178335, EU178334, EU178333, EU178332, EU178331, EU178330, EU178329, EU178328, EU178327, EU178326, EU178325, EU178324, EU178323, EU178322, EU178321, EU178320, EU178319, EU178318, EU178317, EU178316, EU178315, EU178314, EU178313, EU178312, EU178311, EU178310, EU178309, EU178307, EU178306, EU178305, EU178303, EU178302, EU178301, EU178300, EU178298, EU178296, EU178294, EU178292, EU178291, EU178290, EU178289, EU178288, EU178287, EU178286, EU178285, EU178284, EU178282, EU178281, EU178280, EU178279, EU178277, EU178276, EU178275, EU178274, EU178273, EU178272, EU178271, EU178270, EU178269, EU178268, EU178267, EU178266, EU178265, EU178264, EU178263, EU178262, EU178261, EU178260, EU178259, EU178258, EU178257, EU178256, EU178255, EU178254, EU178439, EU178438, EU178437, EU178436, EU178432, EU178431, EU178430, EU178428, EU178427, EU178425, EU178424, EU178423, EU178422, DQ230841, JF346900, JF346906, JF346905, JF346904, JF346903, JF346902, JF346901, EF420986, EF368372, EF368371, EF368370, KC503855, KC503854, KC503853, KC503852, KC492738, KC492737, EU131811, HQ699994, JX392384, JX392382, JX392380, JX392378, JX392383, JX392381, JX392379, KF250376, JF346915, HM215407, HM215406, HM215405, HM215392, HM215388, HM215381, HM215380, HM215375, HM215364, HM215363, HQ699984, HQ699983, HQ699982, HQ699965, JQ423923, KF250385, KF250377, KF250375, KF250374, KF250373, KF250372, KF250371, KF250366, KF835494, KF835510, KF835511, KF835532, HQ215552, JX960601, JF346914, JF346913, JF346912, JF346911, JF346910, JF346909, JF346908, JF346907, JX960602, JX960600, KF234628

CRF08_BC-CN

AY008717, AY008716, AY008715, AY669745, AF286229, AB746345, AB746344, AB773884, AB773885, KC914396, AB746343, AB746342, HM067748, HQ700004, HQ700001, HQ699976, HQ699975, HQ699974, HQ699973, HQ699972, HQ699971, HQ699970, HQ699969, HQ699968, HQ699967, HQ699966, HQ699964, HQ699963, HQ699962, HQ699961, HQ699960, HQ699959, HQ699958, HQ699957, HQ699954, HQ699953, HQ699952, JF719819, KF250369, KF250368, KF312642, KF835493, KF835496, KF835495, KF835509, KF835507, KF835506, KF835508, KF835535, KF835534, KF835531, KF835530, KF835529, KF835512, KF835541, KF835497, KF835501, KF835500, KF835547, KF835545

B'-CN

JF932495, JF932496, GU177863, GU647198, GU647197, GU647196, EF057102, AY180905, DQ007903, DQ007902, DQ007901, JF932492, HM215400, HM215399, HM215397, HQ699997, HQ699996, HQ699990, HQ699989, HQ699979, HQ699978, HQ699951, DQ990880, HM215428, HM215420, HM215398, HQ699998, HQ699995, HM215423, HM215413, HQ699949, JF932482, HQ700000, JF932499, JF932491, JF932489, JF932487, JF932485, JF932483, JF932477, JF932475, JF932473, JF932471, JF932469, JF932500, JF932490, JF932488, JF932486, JF932484, JF932476, JF932474, JF932472, JF932470, JF932468, JF932493, JF932481, JF932479, JF932498, JF932480, JF932478, JF346917, JF932497, JF932494

B-CN

EU131808, EU131806, EU131801, EU131797, EU131796, EU131792, EU131791, EU131790, EU131789, EU131788, EU131787, AY905497, AY905496, AY905495, AY905494, EU131803, EU131810, EU131809, EU131807, EU131805, EU131804, EU131802, EU131800, EU131798, EU131794, FJ460501, FJ460500, FJ460499, HM215384, HM215382, HM215370, HM215369, HM215366, HM215365, HM215395, HQ215554, JF346918, JF346919, KC596066, JF346916, JX960599, HQ215556, JX960598, JX960597, JX140658

Footnotes

Acknowledgments

We would like thank Dr. Zhefeng Meng for critical discussion. This work is supported by the National Natural Science Foundation of China (81302605) and the National Grand Program on Key Infectious Disease Control (2013ZX10001-002).

Author Disclosure Statement

No competing financial interests exist.

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