COMPARATIVE ANALYSIS OF HUMAN CYTOMEGALOVIRUS CLINICAL ISOLATES FROM KIDNEY TRANSPLANT RECIPIENTS

Background: Human cytomegalovirus (HCMV) is an opportunistic, ubiquitous DNA virus that causes severe diseases in patients with immature or impaired immune systems, such as kidney transplant recipients (KTRs). Understanding the immune parameters that control HCMV infection, and the strategies the viruses use to evade immune recognition, is fundamental to unveil how immunological dysfunction in transplant patients can lead to graft failure, and to develop novel immunological or virological strategies for preventing and controlling HCMV-associated diseases. Although it is known that HCMV can extensively hijack cellular genes that may contribute to its immune evasion capability and that the virus is polymorphic among hosts, the source of genomic variability remains unresolved. Study design: The main objectives of this study can be summarized as follows: 1. isolation of HCMV strains from urine of KTRs; 2. quantitative analysis of in-vitro growth properties of HCMV clinical isolates by focus expansion assay (FEA); 3. evaluation of IE-, E-, and L-viral gene expression by Western Blot and immunofluorescence analysis; 4. analysis of genome variability among the different HCMV isolates by high-throughput sequencing; 5. comparison of the viral DNA sensor signature triggered by different HCMV clinical isolates, focusing on the nuclear DNA sensor IFI16- associated pattern. Results: Several HCMV strains have been isolated from the urines of KTR patients of the Universital Hospitals of Turin, Novara and Ulm (Germany). Growth analysis on fibroblasts has demonstrated that viral isolates differ in their replication capability and can be grouped in three categories: fast-, intermediate-, or slowreplicating strains. Moreover we have observed that the viral growth was not always restricted to well defined foci but there was a combination of very large foci and homogeneous dissemination; in some cases the foci had a characteristic comet-like shape. Thus, a genetic variability may exist among HCMV clinical isolates, which may reflect a different biology with a consequent diversified impact on HCMV-associated immunopathology. Conclusions: We have observed that HCMV isolates from KTRs display different pattern of in vitro replication capability. A detailed genetic characterization of HCMV clinical isolates is thus needed in order to characterize their interplay with host immune response, and consequently the graft fate. Moreover, correlations between the virus genetic make-up and biology, the antiviral innate response and clinical outcome will be determined with the ultimate goal of defining new biomarkers and eventually new interventions for a better management of KTRs.