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Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), was first identified in 1994 by Chang and colleagues [3]. KSHV has been consistently detected in four clinical epidemiological variants of Kaposi's sarcoma (KS): classic, endemic, iatrogenic and AIDS-associated KS. KSHV has also been detected in primary effusion lymphoma (PEL) and a subset of multicentric Castleman's disease (MCD), particularly in those with HIV infection [2,18].
KSHV is a gamma-2 herpesvirus related to Epstein-Barr virus (EBV). KSHV is a large double-stranded DNA virus of approximately 90 identified open reading frames, and over 60 of them show homology with other rhadinoviruses [20]. KSHV has two replication stages during viral life cycle: latency and lytic replication. During latency, the KSHV does not produce virion particles, and persists as extra-chromosomal episomal DNA circles. Only a few genes are expressed during latency, in KS endothelial cells, the genes of orfK12/kaposin, orfK13/vFLIP, orf72/v-cyclin, and orf73/LANA-1 are expressed; in PEL and MCD B-lymphoma cells, the orfK10.5/LANA-2 is also expressed [19].
The worldwide distribution of KSHV has been extensively investigated using serologic assays measuring antibodies to specific KSHV antigens. The most commonly used serologic assays detect either antibodies to the latency-associated nuclear antigen 1 (LANA-1), to an minor capsid protein encoded by open reading frame (orf) 65 by ELISA or Western blot, or to a virion glycoprotein encoded by orfK8.1 [16]. Using the serologic assays, the seroprevalence of KSHV has been found to be in the order of 40%-60% in sub-Saharan Africa and 20%-40% in South Africa. In the United States, Asia, and Western Europe, the seroprevalence of KSHV is lower than 10%. However, in Mediterranean countries such as Italy, Greece, and Spain, prevalence rates are higher.
KSHV orf73 encodes a latency-associated nuclear antigen (LANA), named LANA-1, which is the basis for several serologic assays [13]. The KSHV orf73 encoded protein is expressed during a latent infection and co-localizes with host cell chromosomes, and plays an important role in episomal maintenance by tethering viral genomes to host cell chromosomes [9]. It can be found in nearly all infected cells in KS, as well as in PEL and MCD [11,12]. And the antibodies to the latent nuclear antigen-1 are highly specific for persistent KSHV infection, for it has no homology to other known human herpesviruses [15,17]. It was also reported that antibodies to the latent nuclear antigen were detected in all 12 sera obtained from patients with classical KS by the immunofluorescence assay, and nine of them were positive in ELISA [10].
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A fragment of 651 bp was obtained by PCR with specific primers and cloned into pGMT-easy vector. The 651 bp fragment was identified as c-terminal orf73 gene by restriction endonuclease cleavage and sequence analysis. Then the orf73 gene fragment was subcloned into the pQE-80L vector to obtain the prokaryotic expression plasmid pQE80L-orf73. Plasmid pQE80L-orf73 was then transformed into BL21 (DE3) and also identified by digestion of Sal Ⅰ and BamH Ⅰ.(Fig. 1).
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Total cell proteins were attained from BL21 (DE3) transformed by pQE80L-orf73 both before and after IPTG induction. Proteins in the supernatant and the precipitate were obtained by centrifugation. Recombinant ORF73 protein was purified by Ni2+-NTA acid resin affinity chromatography. All proteins mentioned above were separated by SDS-PAGE (Fig. 2). SDS-PAGE showed that recombinant ORF73 protein was approximately 27 kDa as expected and was highly expressed in BL21 (DE3) cells after the induction of IPTG. The separation of supernatant and precipitate indicated that ORF73 had a higher concentration in the precipitate of the lysed BL21 (DE3) transformed by pQE80L-orf73 than that in the supernatant. By using bandscan software, the purity of recombinant ORF73 protein was analyzed and found to be as high as 99%.
Figure 2. Analysis of the expression of recombinant ORF73 protein. Lane 1, The lysate of BL21 (DE3) transformed with pQE80L-orf73 before IPTG induction; 2, The lysate of BL21 (DE3) transformed with pQE-80L-orf73 after IPTG induction; 3, The supernatant of the lysed BL21 (DE3) transformed with pQE-80L-orf73 induced with IPTG; 4, The precipitate of the lysed BL21 (DE3) transformed with pQE-80L-orf73 induced with IPTG; 5 and 6, Purified ORF73 protein; M, protein marker. Arrow indicates the expressed ORF73.
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Proteins of the whole BL21 (DE3) cells transformed with pQE80L-orf73 both before and after IPTG induction and the purified recombinant ORF73 were electrophoresed by SDS-PAGE and transferred to a PVDF membrane. Then, the PVDF membrane was coated with the KSHV positive serum or negative serum (Fig. 3). The Western blot results showed that a specific 27 kDa band occurred in the lanes of the total protein of lysed BL21 (DE3) transformed with pQE80L-orf73 after the induction of ITPG, and the purified recombinant ORF73, when they were incubated with KSHV positive serum; no band occurred when incubated with serum from healthy blood donors. The results above revealed that the antigenicity of recombinant ORF73 was quite strong.
Figure 3. Western blot of recombinant ORF73. Lane 1 and 4, The lysate of BL21 (DE3) transformed with pQE-80L-orf73 before IPTG induction; 2 and 5, The lysate of BL21 (DE3) transformed with pQE-80L-orf73 after IPTG induction; 3 and 6, Highly purified recombinant ORF73; 1, 2 and 3, Reacted with KSHV positive serum; 4, 5 and 6, Reacted with negative serum.
ELISA was also used to check the antigenicity of the recombinant ORF73 protein. The recombinant ORF73 was used as antigen to coat the ELISA plates; KSHV positive and negative serum were used as the primary antibody separately (Fig. 4). The results not only showed that the antigenicity of the recombinant ORF73 recombinant protein was strong, but also revealed that ELISA using the recombinant ORF73 was a good choice for screening a large amount of sera for epidemiological research.
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By using different concentrations of recombinant ORF73 protein and different dilutions of serum, the optimal condition of ELISA was investigated (Fig. 4). As the ELISA results showed, all the chosen concentration of recombinant ORF73 could show the difference between negative and positive serum; at concentrations of 10 µg/mL and 5 µg/mL, the values of OD405 were higher than 0.6; when the concentration declined to 2µg/mL, the value of OD405 was about 0.6; while at the concentration of 1 µg/mL, the OD405 value was lower than 0.3 (Fig. 4A). As to the optimal dilution of the serum in ELISA, it showed that all four different dilutions could distinguish the difference between negative and positive serum. The OD405 values of the dilution of 1:100 and 1:200 were both higher than 0.6; when the dilution became 1:500, the OD405 value declined to almost 0.6; while at the 1:1 000 dilution, the OD405 value became lower than 0.3 (Fig. 4B). Thus, 2 mg/mL recombinant ORF73 and 1:100 dilution of the serum will be used in ELISA in the future KSHV epidemiological research.
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To determine the specificity and sensitivity of the recombinant ORF73 in detecting KSHV in sera, 20 sera from KS patients collected by the Laboratory of Xinjiang Endemic and Ethnic Diseases and 50 healthy subjects which were characterized as negative for KSHV [5,6]were detected for KSHV infection. Overall, the recombinant ORF73 could detect 20 KS samples as KSHV positive, and 50 healthy subjects as negative. Thus, the recombinant ORF73 had a combined specificity of 100% and sensitivity of 100% (Table 1).
Table 1. Specificity and sensitivity of the recombinant ORF73
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Of 560 subjects from the general Han population in Hubei, 16 (2.9%) were KSHV-positive. KSHV seroprevalence in this population was compared with the general Han population in Xinjiang, a region known to have high KSHV seroprevalence. Of 430 subjects from the general Han population in Xinjiang, 29 (6.7%) were KSHV-seropositive (Table 2). Logistic regression analysis showed that the Han people in Xinjiang had 146% increase in their risk for KSHV infection compared to their counterparts in Hubei (6.7% vs 2.9%, OR: 2.46, 95%CI: 1.32-4.59, P = 0.005) (Table 2).
Table 2. Logistic regression analysis of KSHV seroprevalence in different Han population from Hubei and Xinjiang