A total of 1294 research subjects were randomly selected from two regions of Xinjiang between January and December 2015 using a stratified, multistage sampling. Blood samples obtained from the research subjects were divided into two groups. Group 1 included 700 plasma samples and whole blood samples from individuals in Urumqi. Group 2 comprised plasma samples from 594 individuals in Kashgar. Written informed consent was obtained from all participants of the study. Additionally, KSHV-seronegative plasma samples from anonymous healthy blood donors from a local blood bank in Lincoln, NE, USA, were used for assay controls.
Blood samples were collected in EDTA tubes, and plasma was separated. Specimens were coded by a unique identification number. All specimens were stored at –70 °C until testing.
Two KSHV serological tests were used. First, all plasma samples were diluted 1:40 in PBS and tested using monoclonal- enhanced immunofluorescence assays (mIFAs), as described by Minhas et al. (2008) . Briefly, BC3 cells were stimulated with tetradecanoyl phorbol acetate, then fixed with 4% paraformaldehyde and incubated with patient plasma. Mouse monoclonal antihuman IgG (CRL-1786; American Type Culture Collection, Manassas, VA, USA) was used as a secondary antibody and DyLight488-conjugated donkey anti-mouse IgG (Jackson Immuno Research, West Grove, PA, USA) was used as the tertiary antibody. Second, Spodoptera frugiperda clone 9, expressing three viral recombinant proteins, ORF73, ORF65, and K8.1, was used (Fu et al., 2009). The procedure was similar to the BC-3 immunofluorescence assay. A sample was considered to be HHV-8-seropositive only if it was positive at a standard serum dilution of 1:40 with both the BC-3 and the S. frugiperda clone 9 assay. Each slide was read independently by two experienced laboratory workers. To determine the geometric mean titer (GMT) of KSHV antibody, all positive plasma was diluted further and tested by mIFA to estimate the KSHV antibody titer of each sample.
DNA was extracted from the whole blood samples using a Gentra Puregene Blood kit (Qiagen, Germany) according to manufacturer’s protocol. Extracted DNA was subjected to polymerase chain reaction (PCR); the sequences of primers are listed in Supplementary Table 1. PCR-positive samples were analyzed by nested PCR for the presence of KSHV DNA. Each PCR reaction was performed in a total volume of 25 μL, using 0.4 μmol/L primers and a TaKaRa Ex Taq DNA polymerase kit (TaKaRa Biotechnology) according to the manufacturer’s protocol, with the exception that 2.5 units enzyme was used. For control β-actin reactions, 1 μL genomic DNA was used. For first- and second-round KSHV ORF26 PCR, 2 μL genomic DNA and 2 μL PCR product were used, respectively. All reactions were performed under the following conditions: 95 °C for 5 min, 35 cycles of 95 °C for 30 sec, 58 °C for 30 sec, 72 °C for 30 sec, and one cycle of 72 °C for 7 min. Samples positive for the ORF26 gene were then tested for presence of the KSHV gB gene by nested PCR amplification. Positive results for DNA detection were defined as those samples positive for both ORF26 and gB genes.
Characteristic Group 1 (Urumqi) Group 2 (Kashgar) n (%) n (%) Total Number 700 594 Ethnicity Han 373 (53.3) 282 (47.5) Uygur 327 (46.7) 312 (52.5) Gender Male 364 (52.0) 244 (41.1) Female 336 (48.0) 350 (58.9) Median Age (years) 43 48 Education Elementary or below 273 (39.0) 330 (55.6) Junior high or beyond 427 (61.0) 264 (44.4) Population stationary population 437 (62.4) 507 (85.4) immigrated population 263 (37.6) 87 (14.6)
Table 1. Characteristics of the study population
Neutralizing antibodies against KSHV were detected for all seropositive samples by a flow cytometry based neutralization assay as described previously with minor modifications (Kimball et al., 2004). Plasma was diluted and mixed with recombinant KSHV expressing green fluorescent protein (GFP; rKSHV.219) and incubated for 1 h at 37 °C. The virus and plasma mixture was added into a 96-well plate seeded with 293 cells. After incubation for 72 h, green cells were counted by flow cytometry. Results were confirmed by counting at least 100 cells under a fluorescence microscope. A positive neutralizing antibody outcome was defined as ≥ 50% reduction in infectivity as compared with the seronegative control serum. Positive plasma samples were diluted further to determine the neutralizing antibody titer.
Original questionnaire data and laboratory results were entered and managed with EpiData 3.0 (Odense, Denmark), and then transferred to SPSS 17.0 (SPSS Inc., Chicago, IL, USA) for further analysis. Differences in KSHV seroprevalence were measured by chi-square test or Fisher’s exact test. The Mann-Whitney U-test was used to assess the difference in GMTs of KSHV between Uygur and Han ethnic groups. Correlation analysis was evaluated by Pearson’s correlation coefficient. Power and sample size estimation was done using Sample Power 2 (SPSS). GraphPad Prism 5.0 (GraphPad, La Jolla, CA) was used to construct figures. A P value of less than 0.05 was considered statistically significant.
The characteristics of the two groups in the study population (N = 1294) are summarized in Table 1. Group 1 participants (N = 700) aged 16–82 years were recruited from Urumqi. Among them, 373 participants (53.3%) were of Han ethnicity and 327 participants (46.7%) were of Uygur ethnicity. Group 2 participants (N = 594) aged 15–86 years were recruited from Kashgar. comprising 282 participants (47.5%) of Han ethnicity and 312 participants (52.5%) of Uygur ethnicity.
The overall KSHV seroprevalence of Uygur participants was 40.4%, which was significantly higher than that of Han ethnicity participants (31.1%) in Urumqi (P < 0.05) ( Figure 1A). The end-point titer of all KSHV-positive Han and Uygur plasma samples was determined, and we observed that the GMT of Uygur ethnicity individuals was significantly higher than that of the Han ethnicity individuals (P < 0.001) ( Figure 1B). The KSHV seroprevalence of Uygur participants in Kashgar was 59.9%, which was also significantly higher than that of Han participants (21.3%) (P < 0.001) ( Figure 1C), and the GMT of all KSHV-positive Uygur ethnicity individuals was significantly higher than that of all KSHV-positive Han ethnicity individuals (P < 0.001) ( Figure 1D).
Figure 1. KSHV seroprevalence and antibody titers in Han and Uygur groups in the study population of Urumqi and Kashgar, Xinjiang. (A, C) Prevalence of KSHV infection in Han and Uygur populations of Urumqi and Kashgar. (B, D) Comparison of KSHV antibody titers in Han and Uygur populations of Urumqi and Kashgar.
The association between KSHV seroprevalence and the characteristics of the 1294 participants stratified by geographic region is presented in Table 2. In the Urumqi group of 700 study participants, 116 Han (31.1%) and 132 (40.4%) Uygur were KSHV seropositive. KSHV infection was correlated with advancing age (P = 0.005) and education level (P = 0.047). Interestingly, participants in the stationary population exhibited high serum prevalence of KSHV (38.4%). In the Kashgar group of 594 study participants, 60 Han (21.3%) and 187 (59.9%) Uygur were KSHV seropositive. Again, KSHV infection was correlated with advancing age (P = 0.047) and education level (P = 0.003). The stationary population showed higher KSHV seroprevalence (44.4%) than the immigrated population (25.3%). No clear correlation was observed between KSHV infection and gender in either Urumqi or Kashgar participants.
All peripheral blood DNA samples from Urumqi were analyzed using nested PCR for the presence of KSHV DNA based on primers for the ORF26 and gB genes. Reaction products of ORF26 and gB by nested PCR in the second round showed that the target gene fragments were amplified in a position-specific manner, and the product size was 174 bp and 383 bp, respectively (Supplemenaty Figure S1A, 1B). Samples scored positive for KSHV DNA detection were positive for amplification of both the ORF26 and gB genes. Table 3 presents the KSHV DNA positive rate associations with ethnicity, gender, age, and education, and stratified by population status (stationary or immigrated). There were 26.9% (88/327) of Uygur participants and 17.4% (65/373) of Han participants whose peripheral blood DNA showed KSHV-positive results, which indicated a significant difference (P = 0.002). Among all participants, the KSHV DNA positive rate increased significantly with age from 18.9% in those aged < 50 years to 25.7% in those aged ≥ 50 years ( P = 0.03). Furthermore, there was a significant difference in the KSHV DNA positive rate with respect to population status (P = 0.018), from 17.1% in the immigrated population to 24.7% in the stationary population. A correlation of borderline significance was detected between KSHV DNA positive rate and education level (P = 0.052), while no associations were observed between KSHV infection and gender.
Characteristic Cases (n) Urumqi χ2 P Cases (n) Kashgar χ2 P KSHV + (%) KSHV - (%) KSHV + (%) KSHV - (%) Ethnicity Han 373 116 (31.1) 257 (68.9) 6.542 0.011 282 60 (21.3) 222 (78.7) 91.132 0.000 Uygur 327 132 (40.4) 195 (59.6) 312 187 (59.9) 125 (40.1) Gender Male 364 136 (37.4) 228 (62.6) 1.240 0.265 244 105 (43.0) 139 (57.0) 0.359 0.549 Female 336 112 (33.3) 224 (66.7) 350 142 (40.6) 208 (59.4) Age < 50 397 123 (31.0) 274 (69.0) 7.926 0.005 286 107 (37.4) 179 (62.6) 3.948 0.047 ≥ 50 303 125 (41.3) 178 (58.7) 308 140 (45.5) 168 (54.5) Education Elementary or below 273 109 (39.9) 164 (60.1) 3.958 0.047 330 155 (47.0) 175 (53.0) 8.871 0.003 Junior high or beyond 427 139 (32.6) 288 (67.4) 264 92 (34.8) 172 (65.2) Population Stationary population 437 168 (38.4) 269 (61.6) 4.623 0.032 507 225 (44.4) 282 (55.6) 11.142 0.001 Immigrated population 263 80 (30.4) 183 (69.6) 87 22 (25.3) 65 (74.7)
Table 2. The associations of KSHV seroprevalence with population characteristics stratified by geographic region from Xinjiang, China
Given the high prevalence of KSHV infection in people of both Uygur and Han ethnicity in Xinjiang, and the high KSHV antibody titer in those of Uygur ethnicity, we investigated the prevalence of neutralizing antibodies in all KSHV-positive participants in Urumqi. We found that 17/132 (12.9%) seropositive Uygur participants had detectable levels of neutralizing antibodies. In contrast, only 5/116 (4.3%) KSHV-seropositive Han participants had detectable neutralizing antibody (Figure 2A). Since there was a significant difference (P = 0.018) in the prevalence of neutralizing antibodies between samples from individuals of Uygur and Han ethnicity, all neutralizing antibody positive samples were further titrated to investigate whether there were differences in the neutralizing antibody titers within these groups. Figure 2B compares the GMT of neutralizing antibodies of all samples that had detectable neutralizing antibodies between Han and Uygur participants. The result clearly demonstrated that the GMT of neutralizing antibodies in those of Uygur ethnicity was significantly higher than that in those of Han ethnicity (P = 0.026).
Figure 2. Prevalence and titer of KSHV neutralizing antibodies in Han and Uygur groups in the study population in Urumqi, Xinjiang. (A) Percentage prevalence of neutralizing antibodies in individuals of Han and Uygur ethnicity. (B) KSHV neutralizing antibody titers (log2 transformed) in samples from Han and Uygur participants. GMT of neutralizing antibodies in each group is also marked.
Characteristic Cases (n) KSHV + (%) KSHV - (%) χ2 P Ethnicity Han 373 65 (17.4) 308 (82.6) 9.178 0.002 Uygur 327 88 (26.9) 239 (73.1) Gender Male 364 72 (19.8) 292 (80.2) 1.915 0.166 Female 336 81 (24.1) 255 (75.9) Age < 50 397 75 (18.9) 322 (81.1) 4.722 0.030 ≥ 50 303 78 (25.7) 225 (74.3) Education Elementary or below 354 88 (24.9) 266 (75.1) 3.778 0.052 Junior high or beyond 346 65 (18.8) 281 (81.2) Population Stationary population 437 108 (24.7) 329 (75.3) 5.558 0.018 Immigrated population 263 45 (17.1) 218 (82.9)
Table 3. The associations of KSHV DNA positive rate with population characteristics in Urumqi region of Xinjiang.
Table S1. Primer sequences
Figure S1. Reaction products of ORF26 and gB by nested PCR in the study population in Urumqi, Xinjiang. (A) Reaction products of ORF26 by nested PCR in the second round. (B) Reaction products of gB by nested PCR in the second round. M: DNA marker; 1–8: reaction products of KSHV serum positive samples; 9: negative control (double-distilled H2O); 10: positive control (DNA of KSHV)
Prevalence of Kaposi’s sarcoma-associated herpesvirus in Uygur and Han populations from the Urumqi and Kashgar regions of Xinjiang, China
- Received Date: 29 July 2017
- Accepted Date: 18 September 2017
- Published Date: 25 October 2017
Abstract: Kaposi’s sarcoma-associated herpesvirus (KSHV) is the infectious etiologic agent associated with Kaposi’s sarcoma (KS), primary effusion lymphoma, and multicentric Castleman disease. It has been shown that high KSHV prevalence and high incidence of both classic KS and AIDS associated KS are found mostly among people of Uygur ethnicity in Xinjiang, while people of Han ethnicity in Xinjiang have a higher KSHV seroprevalence than those of other Han populations in mainland China. However, it is still unclear why there is such geographical and population variation in KSHV distribution in China. In this work, we focused on the populations in the Kashgar region and Urumqi area, where a total of 1294 research subjects were randomly selected to investigate the potential correlation between KSHV prevalence and different ethnicities in endemic areas of Xinjiang, and to determine risk factors that may affect KSHV infection rates or KS incidence. We identified a high seroprevalence of KSHV and high peripheral blood DNA infection in the general Uygur and Han populations in both Urumqi and Kashgar regions of Xinjiang, and determined that advancing age, low education level, and stationary population status affect KSHV infection rates. Further, KSHV-positive Uygur participants were shown to have higher prevalence of neutralizing antibodies and neutralizing antibody titers than KSHV-positive Han participants.