Molecular Characterization of Human Cytomegalovirus Strains causing Infections in Immuno-compromised Pakistani population

Abstract

Human cytomegalovirus (HCMV) is a global pathogen with ability to establish a persistent infection in the host for many years. Diseases are more serious in congenitally infected infants and immunocompromised individuals such as AIDS patients and transplant recipients. It rarely causes disease among immunocompetent individuals. The envelope glycoproteins of HCMV (gB, gH and gN) are essential for viral infectivity as they are involved in attachment and penetration of the host cell, cell to cell viral transmission and fusion of infected cells. Also, they are known to be important targets for humoral and cell mediated immune responses against the virus. A hypothesis states that HCMV genotypes are constantly mutating and evolving in immuno-compromised patients especially in congenitally infected infants and transplant patients where virus able to evade the immune system and genotypes become more pathogenic. Some seroprevalence studies have been carried out in Pakistan among individuals at risk of blood donors and pregnant women. However, no study has been conducted previously, to investigate the etiology of Human cytomegalovirus in congenital CMV (cCMV) infection and in kidney transplant and dialytic patients. In routine there is no Laboratory diagnosis in Pakistan due to lack of research data about the consequences of this virus and necessary laboratory facilities. In the present study, a total of 631 samples were collected from pregnant women, cCMV infected infants, kidney transplant patients and renal failure dialytic patients showing symptoms of HCMV disease, and tested by ELISA and nested xxv PCR for the presence of HCMV. Actively infected pregnancies were followed until delivery, to detect the outcome of overt cCMV infection in neonates. PCR positive HCMV samples were screened for glycoproteins (gB, gH and gN) and then glycogenotyped by nested PCR and sequencing. Genetic characterization of CMV strains was performed by sequence analysis of envelope glycoproteins: gB, gN and gH to detect the circulating genotypes. In pregnant women the seroprevalence of anti-CMV IgG and IgM was 97.5% (399 out of 409) and 12.7% (52 out of 409), respectively, while 20% (82/409) pregnant women were found positive for CMV DNA by PCR. Logistic regression analysis showed a significant association of active infection with parity [OR= 2.56, 95% CI=1.82-2.62, p=0.04], febrile illness [OR=1.84, 95% CI=1.76-3.65, p=0.01] and jaundice [OR=22.5, 95% CI=4.5385.02, p=0.002]. We were able to isolate virus in 41 out of 70 neonates; 36.6% (15 out of 41) of them were symptomatic at birth while 63.4% (26 out of 41) were asymptomatic. The most prominent clinical feature observed in symptomatic neonates was hepatosplenomegaly (26.6%; 4 out of 15). CMV strains belonged to gB, gN, gH genotypes, where gB1 genotype was mainly found in 75% infants with hepatic damage. Seroprevalence of kidney transplant patients of this study was IgG 100% and IgM 44.4% while 52.7% found to be positive for HCMV DNA when tested by PCR. Samples from renal failure dialysis patients found to be 98% IgG positive, 35.4% were IgM antibodies positive while 55% CMV DNA positive. Most prevalent genotype among renal patients was gN followed by gH and gB respectively however no significant association have been observed in these patients between genotypes and disease severity. Phylogenetic analysis of Pakistani strains showed 96 to 100% of their prototype strains are available in GenBank. In conclusion, Active CMV infection during pregnancy is a major cause of congenital CMV infection and distribution of gB, gN and gH genotypes was similar in symptomatic and asymptomatic neonates. Our findings emphasize to conduct a comprehensive large scale survey and introduction of country wide routine screening at maternity clinics for early diagnosis of CMV to reduce its associated devastating outcomes. The investigation of different glycoprotein genotypes in different samples has shown no significant correlation between single glycoprotein and disease outcome. Recombination between HCMV strains xxvi may lead to progression of disease. However, studying a large number of isolates positive for HCMV from immunocompromised patients may clarify the correlation between glycoprotein types and disease outcome and lead to improved strategies for treatment and prevention of HCMV associated disease.