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Abstract
Human herpesvirus 8 (HHV8) or human herpesvirus associated to Kaposi’s sarcoma (KSHV) is the aetiological agent of Kaposi's sarcoma (KS), an angioproliferative neoplasia and of two rare lymphoproliferative disorders: the primary effusion lymphoma (PEL / BCBL) and the Castelmann’s disease.
There are 4 variants of KS: endemic, iatrogenic, epidemic, chronic or classical, characterized by a typical aggressiveness, geographical distribution, incidence in age groups and individuals male and female.
HHV8 seroprevalence has an extremely heterogeneous geographic distribution, low in America and Asia (2-5%), it ranges from 2% to 40% in some Mediterranean areas, it reaches 40-50% in Africa. In Italy, HHV8 seroprevalence increases from the North to the South from 10% to 30% and it reaches 35% in Sicily and Sardinia.
Different ways of transmission of the infection have been described, sexual, through saliva, after transfusion or organ transplant, through infected needles, rarely vertically or through breast milk.
Phylogenetic-epidemiological studies of the ipervariable region ORF-K1 of HHV8 genome identified 5 viral genotypes (A, B, C, D, E) today associated only with a different geographical distribution.
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Aims of this thesis work were:
1. the seroepidemiological and molecular analysis of the infection in different biological fluids collected from KS patients
2. the study of the seropositivity of these subjects for antibodies against HHV8 LANA and K8.1 antigens to associate the DNA positivity with seroconversion,
3. the analysis of viral subtypes detected in different biological fluids and at different times,
4. the identification of specific characteristics of virus strains circulating in the Mediterranean,
The analysis of the HHV8 siroepidemiological and molecular incidence and the study of the seropositivity for antibodies against LANA and K8.1 antigens were performed on 30 KS patients all residents in Sassari. The results showed a consistent seropositivity for antibodies against both LANA and K8.1 antigens in the, 100% of KS. Viral typing, successfully performed in 23 KS, revealed that 10 KS were infected with the viral strain A1, 4 with the viral strain A3, 1 with the viral strain A4, 7 with the viral strain C2 and 1 with the viral strain C3.
The statistical analysis didn’t reveal any correlation between the viral load in saliva and blood of the 30 KS (R=0.239, Spearman’s correlation). Considering all patients the average viral load in saliva (68.0678 copies/ml) was significantly higher than that in blood (736 copies/ml) (p=0.02, Wilcoxon’s test for samples pairs). The analysis performed separately on groups of patients infected by different viral genotypes showed that the viral load in saliva was significantly higher in those patients infected with strain A (p=0.004 tests Wilcoxon); only in this patients the viral load in saliva was significantly higher than in blood (p=0.029, Mann-Whitney’s test), moreover setting a
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cut-off at 1000 copies/ml, the 80.0% of KS infected with the viral strain A and only the 14.3% of those infected with the viral strain C had a viral load in saliva significantly above the cut-off (p=0.007, Fisher’s tests). The same statistical analysis conducted on viral loads in blood didn’t show statistically significant results.
In 2 KS we investigated the serorpositivity separately for antibodies against LANA and K8.1 antigens, the viral load in serum, blood and saliva and the infecting genotype. Samples were collected at different times of drug treatment. Results showed that only antibodies anti-LANA were related to the stage of the disease, that the viral load in saliva was inversely proportional to that in blood and serum and that an increase of the viral replication in the oral cavity was associated with an improvement of clinical conditions. Viral typing carried out on all available biological fluids showed that both KS were infected by the viral strain A1, that is invariable during the time and in different body fluids.
Since seroepidemiological and molecular data showed a higher tropism of viral strain A for epithelial cells, such as those of the oral cavity, viral replication was reactivated by treatment with phorbol ester (TPA) in two cell lines, BCBL1 and BC3, latently infected with the A1 and C3 HHV8 strains. The virus released in cell colture surnatant, was concentrated and purified and used to infect one epithelial (HEK293) and one lymphoid (PBMCs) cell line. Viral replication and infectivity of the two strains investigated by indirect IFA and real-time PCR showed that only epithelial cells infected with the A1 strain showed a virion production higher than 103 copies/ml and a high antigenic expression.
The results of the phylogenetic study and the quantification of the viral load confirmed that saliva is the fluid where viral DNA is more detectable, the presence in the Mediterranean of both viral strains A and C, that the infectious virus strain remains
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constant during the time and in different part of the body, but especially highlighted for the first time a greater tropism and replicative rate of A strains in the epithelial cells, confirmed by the in vitro experiments.
