Sunday, October 13, 2013

Cytomegalovirus Overview

Historical background

early the during newborns in described first was virus The as to referred infection systemic fatal a with of children urine the in cells large described reports multiple 20th century when mid 20 disease. In the cytomegalic inclusionth cell of development century with human cytomegalovirus (HCMV) of culture methods enabled propagation.

Virus structure and genome organization

Human cytomegalovirus (HCMV) belongs to the beta herpes virus family. Beta herpes viruses are more closely related to each other than to other herpes viruses with large genomic overlapping.
Beta herpes viruses are a subfamily of human herpes viruses that share architectural features of their virion, including a core containing a linear double-stranded DNA of 235-kb, an icosahedral capsid with 162 capsomeres. The capsid is surrounded by a proteinaceous tegument and an outer lipid envelope.

Virion proteins

Purified virions of human, murine, and simian CMV consists of at least 30 readily detected polypeptides with molecular weights ranging from 20 to over 200 kd, distributed in the capsid, tegument and envelope

Figure IX: Structure of Human cytomegalovirus (HCMV)

The capsid proteins

It is composed of seven proteins. The major capsid proteins (MCP) , minor capsid proteins (mCP), minor capsid binding protein (mC-BP), the smallest capsid protein (SCP) and three assembling protein related to proteins associate with capsids.

Tegument Structure and Function

The tegument in HCMV is located between the outer lipid membrane and the icosahedral protein capsid, which contains the viral genomic double-stranded DNA.

A tegument contains at least 25 proteins, of which are phosphorylated. The basic phosphoprotein 150 (pp150) and lower matrix protein 65 (pp65) are the most abundant proteins expressed during virus replication. The other prominent tegument proteins are pp28, pp71and huge tegument protein. Several transcriptional transactivator proteins have also been localized to the tegument.

The tegument proteins comprise more than half of the total proteins found within infectious virion. Tegument proteins are phosphorylated, but the significance of this phosphorylation and other post translational modifications to these proteins remains largely unexplored.

Envelope glycoprotein:
The envelope consists of two prominent glycoprotein complexes, a complex of dimers of glycoprotein B (gB), and a complex of the gH, gL, and gO. These glycoprotein complexes are assumed to play an important role in virus entry.

HCMV genome

HCMV DNA from strain AD 169 is linear, double stranded DNA molecule of 229 to 354 base pairs. It is divided into two unique regions termed unique short ( Us) and unique long (Ul). The Us is bounded by an inverted and a terminal repeat sequence. The polarity of genetic information in either of the unique regions can be inverted, leading to four isomeric forms of virion DNA.

The HCMV genome of AD 169 strains has been completely sequenced and contains 208 predicted proteins open reading frames (ORFs) of greater than 100 amino acid.

Figure : Structure of the HCMV genome of AD 169 strain: (UL = unique long region, US = unique short region, TRL = terminal repeat sequence long, TRS = terminal repeat sequence short, IRL = inverted repeat sequence long, IRS = inverted repeat sequence short). The most abundantly expressed viral genes at immediate early (IE) time are transcribed from the major IE locus (MIE), located in UL region (MIEP = major IE promotor). The primary transcript is differentially spliced to generate two major proteins, IE1 and IE2 ( Mocarski and Courcelle, 2001).

HCMV genotypes
Although most of the HCMV genome is highly conserved among the various HCMV strains, a subset of genes exhibits a high degree of variability. A high level of genetic heterogeneity usually occurs in a limited number of distinct genotypes.

Among the most variable genes are the viral envelope glycoprotein N (gN) and gO genes and the gene encoding the predicted membrane glycoprotein UL139.

Sequence analysis of highly polymorphic regions within these three genes allows the discrimination of seven distinct gN genotypes and eight distinct gO and UL139 genotypes. The existence of such a large number of distinct genotypes provides a useful tool for investigating HCMV population diversity within a host

Viral Growth Cycle

Human cytomegalovirus exhibits a restricted host range in cell culture. In vitro the most commonly used cells are primary differentiated human fibroblasts derived from skin or lung. HCMV can also replicate in endothelial, epithelial cells, smooth muscle cells and leukocytes.
The whole replication cycle of HCMV is slow, requiring approximately 48-72 hours. The cytopathic effect in response to HCMV is characteristically cell enlargement with intranuclear inclusions.

Virions gain entry into a cell through a membrane fusion event involving the outer membrane of the cell and glycoproteins on the lipid envelope of virions. Once the fusion of these two membranes occurs, the DNA-containing protein capsid and the tegument proteins are released into the cell.

Viral entry is the result of a cascade of interactions; HCMV attach epidermal growth factor receptor (EGFR) on permissive cells and other interactions between HCMV envelope glycoproteins and cellular integrins promote receptor clustering.
Following primary infection, these viruses, as well as other herpes viruses, maintain latency in the human host by integrating in the host cell chromosomes or by maintaining subclinical low level viral replication that is adequately controlled by a functioning immune system. The viruses may reactivate during immunosuppression in transplanted patients.
Viral gene expression
The gene expression pattern follows a similar cascade as used by other herpes viruses. During the lytic infection, viral immediate-early genes are expressed.

The expression of these genes results in the production of viral immediate-early (IE) proteins that modulate the host cell environment and stimulate the expression of viral early genes. These genes produce proteins that are responsible for replicating the double-stranded viral genomic DNA; after DNA replication. IE genes turn on the expression of viral late genes which are mainly structural components of the virion that assist in the assembly and release of newly formed viral particles.

A latent infection is characterized by the minimization of viral gene expression and the inhibition of the assembly and egression of new viral progeny .

DNA replication
After penetration, the capsid passes through the cytoplasm to a nuclear pore, the DNA is released. Viral DNA replication occurs along with the expression of the late genes that produce the gamma proteins which are mainly structural proteins.

Packing of viral DNA into newly formed empty capsids occurs in the nucleus. Maturation occurs by budding from the inner nuclear membrane.

HCMV Epidemiology and Transmission

Human cytomegalovirus infects humans of all ages. Acquisition of the virus usually occurs early in life, mainly during the first two decades. The great seroprevalence and early acquisition of the virus have been associated with lower socioeconomic circumstances, developing countries and over crowded populations.

Vertical transmission of HCMV may occur transplacentally, through contact with the virus in the maternal genital tract during birth and through mother's milk.

Horizontal transmission requires direct contact with infected body fluid, including saliva, urine, breast milk, semen and blood (Britt, 2008).
Following HCMV infection the virus is excreted in body fluids for months or even years and reactivation of the virus is common, especially in immunosuppressed patients ().

Increased risk for infection is clearly associated with sexual activity. Antibodies to HCMV are highly prevalent among male homosexual and women with sexually transmitted diseases.

Human cytomegalovirus is an important hazard of blood transfusion and organ transplantation. It is associated with other infections, with episodes of acute rejection and treatment with lymphocyte-depleting regimens that induce release of inflammatory cytokines.

Pathogenesis of HCMV infection

It has been suggested that epithelial cells of the upper alimentary, respiratory and genito-urinary tracts are the main sites of primary replication after the direct contact of infectious virus and mucosal surface.

Human cytomegalovirus viremia may last for several weeks during the primary infection. Hematogenous spread typically results in the infection of ductal epithelial cells at the initial site. HCMV can infect a wide range of tissues including salivary glands, gastrointestinal tract, lung, liver, brain, kidney and other tissues. Then it replicates in diverse cell types, including fibroblasts, epithelial cells, macrophages, vascular smooth muscle cells and endothelial cells.
The pathologic changes are minor in the immunocompetent host and infection is generally a symptomatic. In rare cases HCMV mononucleosis syndrome develops with potential complication including pneumonia, hepatitis, meningitis and autoantibody production (Percivalle et al., 1993).

In immunocompromised host, HCMV infection may lead serious effects;
• Gastrointestinal tract (GIT): causes GIT lesion and even perforation of the gut.
• Respiratory system: leads to severe problems mainly pneumonia).
• In kidney: leads to renal dysfunction and graft rejection In central nervous system: leads to inflammation and possible damage to retina and cochlea.
• Human cytomegalovirus is also suggested to infect the vascular wall and play a role in the development of atherosclerosis.

There are three morphological transforming regions called mtrI, mtrII, mtrIII which are found in the HCMV genome, in one study mtrII bind to tumor suppressor protein p53 and it down regulates p53 activated transcription, indicating its ability to alter p53 directed cellular regulatory mechanisms

In another study, HCMV causes reduction in human leucokytic antigen (HLA) class II molecules on HCMV infected macrophages that inhibits T- cell proliferation by different distinct pathways.

Infected cells may be multinucleated or bear intranuclear inclusions surrounded by a clear halo given rise to the term owl's eye appearance.

HCMV and immunity

• Role of cell mediated immunity
After the primary infection, long-term cellular and humoral immunity develops. The cell mediated immunity, including both natural killer cells and cytotoxic-T lymphocytes, plays an important role in the control of HCMV infection.

• Role of humoral immunity
Humoral immune response also develops but it has been suggested to be less important in the control of HCMV infection.

• HCMV and evasion the host immune system
The immune defence is unable to clear the virus completely from the host, and the virus remains latent lifelong within the host cell. The ability of the virus to evade the host immune system may be due to various HCMV gene products, which show sequence and functional relatedness to host proteins.

• HCMV latency and persistence
Human cytomegalovirus latency-associated transcripts have also been found in myeloid cells in both natural and experimental infection.

Tropism for different tissue targets is influenced by both viral and host determinants. The salivary glands are an important target for growth and dissemination of HCMV which replicate in salivary gland acinar epithelium).

Reactivation of a latent virus by allogeneic stimulation of peripheral blood mononuclear cells has been demonstrated. Differentiations of latently infected monocytes into macrophages by activation of the immune response lead to virus reactivation.

Cytokines activation and signal transduction pathways have been suggested to play an important role in reactivation of the virus. The differentiation state of the cell seems to be critical for the silencing of IE gene expression and thus for the maintenance of latency.

Clinical presentation of HCMV infection

A - Clinical syndromes
Human cytomegalovirus infects 60%-100% of humans, with primary HCMV infection occurring most commonly during the first 2 decades of life. In immunocompetent individuals, the infected individuals are mostly asymptomatic or may present with a benign febrile infectious mononucleosis-like illness.

In immunocompromised patients such as liver transplant recipients, clinical disease with high morbidity may develop and, in some cases, may lead to death.

• Infection in the normal host
Primary infection with HCMV is usually asymptomatic but in young adults, HCMV may cause mononucleosis-like clinical symptoms. Furthermore it represents the major infectious cause of birth defects.

Persistant fever, myalagia and non specific conistitutional symptoms. Infrequent complication of HCMV mononucleosis include pneumonia, hepatitis, aseptic meningitis, Guillian- Barre syndrome and variety of complications.

Post transfusion syndrome , after blood transfusion aquired HCMV infection usually self limited , but it may cause fever and hepatitis in hospitalized patients.

• Congenital infection
Maternal infection during pregnancy are classified primary and non-primary or recurrent infection. In primary infection during pregnancy result in a fetal transmission rate of 35% - 50%. In recurrent infection, fetal transmission rate range from 0.2% to 2% suggesting maternal immunity to HCMV.

Other factors that contribute in fetal transmission include younger maternal age, lower socioeconomic status and previous history of sexually transmitted diseases (STDs).

Fewer than 5% of congenitally infected infants develop symptoms during new born period ranging from intra-uterine growth retardation, jaundice, hepatosplenomegaly, mental retardation, centeral nervous system abnormalities.
• Delayed infection
In individuals who received antiviral prophylaxis, HCMV disease occurred 3 months to 6 months after completing antiviral prophylaxis; hence, the term “delayed-onset HCMV disease”.

Human cytomegalovirus (HCMV) hepatitis and other organs such as the central nervous system and the lungs may be infected, and present themselves through headache, delirium, changes in mental function, and cough and dyspnea, respectively.

• Infection in allograft recipients
The degree of immunosuppression of the recipient can be correlated with clinically significant HCMV infection. Seronegative transplant recipient of an organ from seropositive are at risk of primary infection and thus develop more serious disease than seopositive recipients of seropositive organs.

Allograft may provide a sequestered site for latency infected cells since MHC mismatch at these sites may prevent generation of major histocompitability complex (MHC) restricted virus specific cytotoxic T- cell responses.

Prevention and treatment of HCMV disease

The pharmacologic agents used for HCMV prevention have evolved, from the use of acyclovir , immunoglobulins and IV and oral ganciclovir and valganciclovir
Antiviral prophylaxis
One major strategy is antiviral prophylaxis, where antiviral drugs such as valganciclovir or oral ganciclovir are given to patients for at least 3 months after liver transplantation. However, antiviral prophylaxis is associated with delayed-onset HCMV disease, which typically occurs soon after completion of prophylaxis

The second strategy for HCMV disease prevention is pre-emptive therapy, which relies on a close virologic follow-up through serial blood markers (such as viral load or pp65 antigenemia) as the trigger for antiviral therapy, usually with intravenous ganciclovir or valganciclovir

The recommended agents and dose for prophylaxis are ganciclovir 5 mg/kg once daily for at least 3 months after transplantation, whereas in preemptive therapy, patients are closely monitored and given ganciclovir 5 mg/kg twice daily whenever CMV viremia is detected Ganciclovir-resistant HCMV is now emerging as an important complication of prolonged antiviral drug use after transplantation
The potential drugs for the treatment of multidrug resistant HCMV include the use of immunoglobulins and leflunomide. The potential clinical utility of maribavir in the treatment of resistant HCMV is highly anticipated

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