Detection of sequences of endogenous retroviruses was done with the PCR approach. Genomic DNA of examined people was isolated from peripheral EDTA-anticoagulated blood. DNA was extracted making use of protease K digestion and purification on QIAamp silica-gel columns (QIAampH DNA Blood Mini Package Qiagen, Germany). Then, PCRs for detection of HERV-K113 (according to Moyes et al., 2005) [19] and HERVK115 (based mostly on Burmeister et al., 2004) had been done [26]. PCRs were being performed in 10 ml reaction volume in a T3000 thermocycler (Whatman Biometra, Germany) making use of Taq polymerase (Polgen, Poland). PCR primers utilised in described reactions (Symbios, Straszyn, Poland) are offered in Table 2. 3 independent reactions have been expected for detection of HERVK113 for just about every sample. The first response (A), working with the pair of primers K113-F and K113-R (Desk 2), created a 300 bp fragment, corresponding to the insertion website of the endogenous retrovirus (no insertion). The next one (B), working with the pair of primers K113-F and K113-LTR-R (Desk 2), was utilized to detect the 59LTR sequence and HERV-K113 provirus fragment (1253 bp fragment). The amplification with primers pair LTRK113-F and K113-R (Desk 2) (reaction C) was employed to get a 483 bp fragment corresponding to the 39LTR of HERV-K113. A positive end result of the past two reactions confirmed the presence of HERV-K113 in the genome of analyzed persons. The PCR circumstances for HERV-K113 detection ended up as follows: 3 min at 94uC forty cycles: 1 min at 94uC, one min at 55uC (for reactions A and C) or 1 min at 59uC (for reaction B) 1 min at 72uC and the closing extension stage of five min at 72uC. PCR items ended up divided in two% agarose (AppliChem, Darmstadt, Germany) gel stained with ethidium bromide (.five mg/ml) and visualised in UV light (Fig. 1). 4 individual reactions were being carried out for detection of HERVK115 for every sample. The 1st just one (A), with the pair of primers K115-F and K115-R (Table two), was executed to detect the “wildtype” sequence (without HERV-K115 in the genome, 557 bp). The next a single (B), with K115-F and K115-LTR-R primers (Table 2), allow us locate a fragment of 59LTR (380 bp). The third 1 (C) with K115-F and K115-PROV-R primer set (Desk 2) served in detection of provirus presence in the examined genome (1269 bp band). The fourth reaction (D) necessary amplification of K115LTR-F and K115-R primers and gave PCR fragment for 39LTR (436 bp). A good consequence of the final a few reactions indicated the existence of HERV-K115 in the genome of examined individuals. PCRs ended up done as follows: 3 min at 94uC thirty cycles: thirty s at 94uC, 30 s at 54uC, 30 s at 72uC and a last extension set of ten min at 72uC. Amplification solutions were analysed in two% ethidium bromide stained agarose gel and visualised in UV light (Fig. two).
The distribution of HERV-K113 and HERV-K115 was calculated in control (n = 303), EU (n = 121) and HIV(+) (n = 470) groups. Variances among groups had been analysed employing two-sided Fisher specific test. A p price of a lot less than .05 was taken to be significant. A conceivable relation involving studied endogenous retroviruses and HIV an infection was evaluated making use of logistic regression in the normal linear model’s plan. The comparison of genetic and non-genetic factors in EU (n = 114) and HIV(+) (n = 452) teams was carried out. Beside the impression of HERVK113 and HERV-K115, the role of route of publicity to HIV (homosexual, heterosexual and intravenous drug use), sexual intercourse and age of individuals, and HCV carrying have been also tested. Variables were being provided in the product with p,.05. All statistical analyses ended up executed making use of the platform R-CRAN model 2.eight.1
Prevalence of HERV-K113 and HERV-K115 in the Decrease Silesia inhabitants of Poland was eleven.eight% and 7.ninety two%, respectively (manage team, n = 303 Desk three). No homozygous individual or solo LTR was detected in any of the analyzed teams. Genotype distributions of both equally HERVs in control as properly as in EU and HIV(+) teams have been compatible with the Hardy-Weinberg basic principle. Prevalence of HERV-K113 and HERV-K115 in the EU group (exposed uninfected men and women n = 121) was 8.26% and five.71% respectively. In the HIV(+) team (n = 470) these sequences were detected in twelve.ninety eight% for HERV-K113 and seven.23% for HERV-K115. There were being no statistically major variations amongst talked about groups and the manage team (p..05). To analyse the prospective relationship in between researched endogenous retroviral factors and HIV an infection we in comparison HERVK113 and K115 distributions in the group of HIV-infected people (n = 452) and in the group of sufferers exposed frequently for a very long time to HIV, but seronegative (EU, n = 114). We also took into account other factors: age, sex, type of exposure to HIV and HCV carrying. All stated variables were being analysed by logistic regression. The final results of endogenous retroviruses detection in EU and HIV(+) teams with reference to intercourse, manner of exposure and HCV coinfection are presented in Desk 4. Between examined agents only HCV co-an infection (OR = twelve.90 CI95% four.sixty nine?35.48 p = .00002) and homosexual exposure (OR = seven.69 CI95% one.88?one.48 p = .007017) have been identified to be factors rising susceptibility to HIV infection, as we described beforehand [40]. The homosexual EU team was very confined (n = 2) so it demands inclusion of far more patients to validate that conclusion. We discovered no relation among examined HERVs and HIV infection (p..05).