Causative Agents of Neuroviral Infections: Herpesviruses Rubella Virus

(Prepared by Inzhevatkina S.M., Department of Microbiology and Virology of of Russian National Research Medical University NI Pirogov) Causative Agents of Neuroviral Infections Viruses that infect the nervous system belong to the following families : Herpesviridae Togaviridae Picornaviridae Rhabdoviridae Flaviviridae Herpesviruses Currently more than 100 different herpesviruses are isolated. 9 herpesviruses can cause infection in humans (8 viruses cause antropotic diseases, and only one of them is typical zoonotic virus). Taxonomy Family Herpesviridae Subfamily Alphaherpesvirinae Genus Simplexvirus : types Human herpesvirus 1 и 2 (HSV-1 и HSV-2); В virus (zoonotic herpesvirus: the source of infection are monkeys) Genus Varicellovirus : type Human herpesvirus 3 (herpes zoster – causative agent of varicella (chickenpox) ) Herpesviruses Taxonomy Family Herpesviridae Subfamily Betaherpesvirinae Genus Cytomegalovirus : type Human herpesvirus 5 (Cytomegalovirus (CMV)) Genus Roseolovirus : types Human herpesvirus 6 и 7 (They replicate in T lymphocytes) Subfamily Gammaherpesvirinae Genus Lymphocryptovirus : type Human herpesvirus 4 (Epstein- Barr Virus (EBV) Genus Rhadinovirus : type Human herpesvirus 8 (Kaposi's sarcoma associated herpes virus (KSHV) ) Electron Microscopy of Herpesviruses Structure of Herpes Simplex Virus The herpes virion consists of four components: 1) a relatively large double-stranded, linear DNA genome 2) encased within an icosachedral protein cage called the capsid. 3) Next layer is called tegument. It has protein nature and differs greatly in width. In many cases it is assymenrical. 4) The envelope encloses tegument. It has peplomers (or spikes of 8 nm long). Size of virion varies greatly from 120 to 300 nm because of tegument layer. Herpesviruses have large genomes (up to 235kbp DNA), and are complex viruses containing ~35 virion proteins. All encode a variety of enzymes involved in nucleic acid metabolism, DNA synthesis and protein processing (protein kinase). Electron Microscopy of Herpesviruses

The assymetrical tegument is obviously seen Model of Herpes Simplex Virus Structure of Herpes Simplex Virus Immunofluorescent method: cells, infected by Herpes Simplex Virus (HSV) The first stages of interaction between herpesvirus and susceptible cell (through glycoprotein C) Replication of Herpesviruses The replication cycle of Herpes Simplex virus 1. Specific proteins in the viral envelope attach to host cell receptors on the cell membrane. 2. Penetration is achieved when the viral envelope fuses with the cell membrane releasing the nucleocapsid directly into the cytoplasm. 3. The virion is uncoated and the viral DNA is transported into the nucleus. 4. In the nucleus, the viral DNA is transcribed into early mRNAs which are transported to the cytoplasm for the translation of early proteins. These early proteins are brought back into the nucleus and participate in the replication of the virus DNA into many copies. The viral DNA is then transcribed into the late mRNAs which exit to the cytoplasm for translation into the late (nucleocapsid and envelope) proteins. 5. The capsid proteins encapsidate the newly replicated genomes. The envelope proteins are imbedded in the nuclear membrane. 6. The nucleocapsids are enveloped by budding through the nuclear membrane, and the mature viruses are released from the cell through cytoplasmic channels. Replication of Herpesviruses Pathogenesis of HSV-1 and HSV-2 Infections The site of the initial infection is usually the oral or genital mucosa, depending on the way in which the person acquires the virus ("above the waist or below the waist“) . It is often noted that HSV-1 penetrates by repiratory or contact mode of transmission and HSV-2 penetrates by sexual and transplacental (vertical) mode of transmission. Both types of HSV can also persistently infect macrophages and lymphocytes. The virus gains entry to the human host via skin and mucous membranes. Once inside it multiplies and causes vesicle formation in the skin, and the formation of shallow ulcers in mucous membranes. Local replication is then followed by spread to regional lymph nodes and then by viraemia. Occasionally meningitis or encephalitis may ensue, but this is rare. The virions eventually travel to the root ganglia via sensory nerves. Primary infections of these with HSV-1 or HSV-2 involve either the trigeminal or lumbosacral dorsal root ganglia respectively. Pathogenesis of HSV-1 and HSV-2 Infections Once mucosal epithelial cells are infected, the virus replicates around the lesion and enters into the innervating neuron. The virus travels along the neuron (by a process called retrograde axoplasmic flow) to the ganglion. In the case of herpes infections of the oral mucosa, the virus goes to the trigeminal ganglia, whereas in genital infections the virus invades the sacral ganglia. When the virus infects neurons it enters into latency. The virus may subsequently reactivate over intervals of weeks, months or years in response to certain stimuli that may include: colds, febrile illnesses, sunlight, menstruation, irradiation, or surgical intervention to the affected nerve. If breakage of latency occurs in these cells, the virus travels back down the nerve axon and recurrence of infection (and therefore symptoms) occurs at the same site as the initial infection. Vesicles containing infectious virus are formed on the mucosa and the virus spreads. As long as the virus is kept moist it can remain infectious. The vesicle heals and there is usually no scar as a result. Pathogenesis of HSV-1 and HSV-2 Infections

Recurrent infections are usually less pronounced than the primary infection and resolve more rapidly. These recurrences are commonly self-limiting and resolve within 1-2 weeks. HSV-1 and 2 infections are life -long and although latency is soon set up, the infected patient can infect others as a result of recurrence. The virus is found in the lesions on the skin but can also be present in a variety of body fluids including saliva and vaginal secretions. Immunity for HSV infection HSV infection induces formation of humoral and cellular immune response. Humoral immunity is presented by serum IgM and later IgG and secretory IgA (especially in respiratory tract). Also cellular immunity is induced, but both types of immunity are ineffective because there is no presence of viral proteins during replication or latency of HSV into susceptible host cell. A large proportion of the population has evidence of HSV-1 infection as judged by presence of antibodies. HSV-1 antibodies are found in more than 90% of population in some countries. Clinical manifestations of HSV infection • HSV-1: • Herpetic gingivostomatitis • Herpes labialis ('cold sores')- most common manifestation • Hepatic whitlow of the skin • Keratoconjunctivitis • Encephalitis • Erythema multiforme • HSV-2: • Genital herpes • Herpes of neonates It is proved the role of HSV-2 in development of cancer occurrence in cervix and prostata. Cold Sores ( Herpes labialis) Skin lesions Herpetic Gingivostomatitis Keratoconjunctivitis HSV Infection of Neonates Laboratory diagnosis of HSV Viroscopical (microscopical) method : a) electron microscopy of clinical specimen (especially vesicular rash) and b) light microscopy of smears stained by Romanowsky-Giemsa method (detection of multinuclear infected susceptible cells containing Cowdry bodies inside their nucleus)) Virological method : inoculation of clinical specimen into cell cultures (HeLa and Hep-2) or chicken’s embryos). Identification is based on neutralization tests with type -specific serum. Serological method (passive agglutination test, ELISA, RIA, CFT ) uses paired serums, detecting 4-fold or more increase of antibodies to prove the HSV infection. Immunofluorescent method applies monoclonal antibodies. Molecular-genetic method reveals the presence of HSV DNA by PCR, DNA hybridization and restrictase endonuclease analysis. Treatment of HSV infections Basic therapy is based on application of nucleoside analog drugs (acyclovir, famciclovir, valacyclovir etc.). These drugs act against the replicating virus (they are incorporated into the DNA as it is copied because HSV thymidine-kinase is 1000-times more active in comparison with the same host cellular enzyme). They cause defect DNA and termination of viral DNA elongation. Also nucleoside analogs inhibit viral DNA - polymerase. But they are ineffective against latent virus and cause low damage to host DNA replication. Inactivated herpetic vaccine is used for treatment of chronic HSV infections. Immunomodulators are also applied. Mechanism of action of nucleoside analogs for treatment of HSV infection Varicella-Zoster Virus (VZV) causative agent of varicella (chikenpox) and shingles Transmission of Varicella Chickenpox is a highly infectious disease that spreads from person to person by direct contact or by air from an infected person's coughing or sneezing. Touching the fluid from a chickenpox blister can also spread the disease. A person with chickenpox is infectious from one to five days before the rash. Infected persons can spread chickenpox before they know they have the disease, i.e. before any rash develops. They can infect others from about two days before the rash develops until all the sores have crusted over, usually four or five days after the rash starts. The contagious period for patient continues until all blisters have formed scabs, which may take 5 to 10 days. Incubation period is 10-21 days after contact with an infected person. Pathogenesis of Varicella The chicken pox lesions (blisters) start as a two to four millimeter red papule which develops an irregular outline (a rose petal ). A thin-walled, clear vesicle ( dew drop ) develops on top of the area of redness. This "dew drop on a rose petal" lesion is very characteristic of chickenpox. After about 8 to 12 hours the fluid in the vesicle becomes cloudy and the vesicle breaks leaving a crust. The fluid is highly contagious, but once the lesion crusts over, it is not considered contagious. The crust usually falls off after seven days sometimes leaving a crater-like scar. Although one lesion goes through this complete cycle in about seven days, another hallmark of chickenpox is that new lesions crop up every day for several days. Therefore it may be a week before new lesions stop appearing and existing lesions crust over. Rash elements of varicella vary by their shape and size: macule, papule, vesicle, pastule and scab. Infection in Pregnancy and Neonates Varicella infection in pregnant women can lead to viral transmission via the placenta and infection of the fetus. If infection occurs during the first 28 weeks of gestation, this can lead to fetal varicella syndrome (also known as congenital varicella syndrome). Effects on the fetus can range in severe malformation of brain, eye, bladder etc. Infection late in gestation or immediately following birth is referred to as neonatal varicella. Maternal infection is associated with premature delivery. The risk of the baby developing the disease is greatest following exposure to infection in the period 7 days prior to delivery and up to 7 days following the birth. The baby may also be exposed to the virus, but this is of less concern if the mother is immune. Newborns who develop symptoms are at a high risk of pneumonia and other serious complications of the disease. Neonatal Varicella Immunity after Chickenpox

• In a normal, healthy child antiviral therapy is not indicated in cases of chickenpox as the illness is self-limiting. Infection results in lifelong immunity. Laboratory Diagnosis of Varicella Diagnosis can be sought through either examination of the fluid within the vesicles, or by testing blood for evidence of an acute immunologic response. Viroscopical (microscopical) method : vesicle fluid can be examined with a Tsanck smear. Immunofluorescent method with examination for direct fluorescent antibody . Virological method. The fluid can also be "cultured", whereby attempts are made to grow the virus from a fluid sample. Serological method can be used to identify a response to acute infection (IgM) or previous infection and subsequent immunity (IgG). Molecular-genetic method : PCR of the mother's amniotic fluid can also be performed for prenatal diagnosis. Vesicles typical for chickenpox and shingles are histologically identical and differ from poxviral vesicules because varicella vesicles have giant multinuclear cells. The varicella-infected cells demonstrate the characteristic inclusions (arrow). Prevention of Varicella A varicella vaccine is prepared from the Oka strain. It is attenuated live vaccine. Protection is not lifelong and further vaccination is necessary five years after the initial immunization. Passive prophylaxis against infection may be achieved by early administration of zoster immune globulin (ZIG) during the first 3 days after contact with patient suffering chickenpox. Chickenpox attenuated live vaccine prepared from the Oka strain Shingles After the infectious period of chickenpox, the virus may migrate to the ganglia associated with areas in which the virus is actively replicated. The virus may then be reactivated under stress or with immune suppression. This usually occurs later in life. The recurrence of varicella replication is accompanied by severe radicular pain in areas innervated by the nerve in which latent infection has occurred. A few days later, chickenpox -like lesions occur in restricted areas (dermatome) that are innervated by a single ganglion. The skin lesions are somewhat different from chickenpox, being restricted to small areas of the skin. They are small and close together and usually heal in about two weeks. Chronic burning or itching pain called post-herpetic neuralgia may occur in the elderly. The pain may last well after the rash has healed, sometimes months or years. Shingles (skin lesion) Treatment of Shingles

Administration of acyclovir , famciclovir and valaciclovir are effective treatments of herpes zoster infection. Structure of Cytomegalovirus (CMV) CMV Infection Cytomegalovirus infection is found in s significant proportion of the population (40-60 in adults). The virus is spread in most secretions, particularly saliva, urine, vaginal secretions and semen. Cytomegalovirus infection is therefore transmitted by sexual contact, as well as kissing . It can also spread to a fetus in a pregnant woman and to the newborn via lactation. In the hospital, the virus can spread via blood transfusions and transplants. The CMV first infects the upper respiratory tract and then local lymphocytes. Circulating lymphocytes then spread the virus to other lymphocytes and monocytes in spleen and lymph nodes. The virus finally spreads to a variety of epithelial cells including those of salivary glands, kidney tubules, testes, epididymis and cervix. CMV Infection CMV infection is usually asymptomatic (sub-clinical) but glandular fever is sometimes seen in young adults. The virus can inhibit T cell responses. The virus elicits both humoral antibodies and cell-mediated immunity but the infection is not cleared. Although suppressed, the virus may later reactivate, particularly in cases of immunosuppression. There are two instances in which cytomegalovirus can cause serious disease. 1. During a primary infection of the mother, the virus can spread via the placenta to the fetus and congenital abnormalities can occur; in fact, this virus is the most common viral cause of congenital disease. 2. In immunosuppressed patients who have received an organ transplant or have an immunosuppressive disease (e.g. AIDS), cytomegalovirus can be a major problem. Particularly important is cytomegalovirus-retinitis in the eye, which occurs in up to 15% of all AIDS patients. In addition, interstitial pneumonia, colitis, esophagitis and encephalitis are seen in some patients. The main feature of CMV infection is occurrence of giant cells (diameter 25–40 μm) with intranuclear and cytoplasmic inclusions, resembling 'owl eye' . Detection of such cells in urine, saliva and other secretions allows to diagnose CMV infection. 'owl eye' Treatment of CMV Infection Ganciclovir, which inhibits the replication of all human herpes viruses, is usually used in treatment, especially for retinitis. Because of it's biotoxicity it is contraindicated in neonatal CMV infection. Foscarnet is also approved, but acyclovir is not effective. Specific hyperimmune immunoglobulin with high titers of specific anti-CMV antibodies IgG and IgA class is used for passive immunization “Zimeven” of patients with an organ transplant or immunosuppressive disease and neonates with CMV infection. Epstein- Barr Virus (EBV) (electron microphotography) Types of EBV infection Epstein-Barr virus is the causative agent of • Infectious mononucleosis (glandular fever) • Burkitt's lymphoma • Nasopharyngeal carcinoma in China • B-cell lymphoma . It was first discovered as the causative agent of Burkitt's lymphoma and it was later found that patients with infectious mononucleosis have antibodies that react with Burkitt's lymphoma cells. Pathogenesis of EBV Infection EBV only infects a small number of cell types that express the receptor for complement C3d component (CR2 or CD21). These are certain epithelial cells (oro- and naso-pharynx) and B lymphocytes. This explains the cellular tropism of the virus. The virus is replicated in pharyngeal epithelial cells, shed into the saliva and is taken up by CD21+ B lymphocytes. These cells are normally short -lived. Although B cells do not show any histological alterations as a result of EBV infection, they are stimulated to divide and are protected from undergoing apoptosis (cell death). They become transformed cells and they begin to appear in high levels as monocytes in the bloodstream. T cell responses are inhibited while growth of B cells and antibody secretion are stimulated. The virus also causes the cells to produce other cytokines including IL-5 and IL-6. Transmission of EBV infection Infectious mononucleosis (also known as kissing disease) is a disease seen most commonly in adolescents and young adults, characterized by fever, sore throat and fatigue. It is caused by the Epstein-Barr virus (EBV). It is typically transmitted through saliva or blood, often through kissing, or by respiratory mode of transmission. The virus is also found in the mucus of the infected person, so it is also easily spread through coughing or sneezing. It is estimated that 95% of adults in the world have been infected with the Epstein-Barr virus at some point in their lives. The virus infects B cells (B-lymphocytes), producing a reactive lymphocytosis and the atypical T cells (T- lymphocytes) which give the disease its name. Blood smear from patient suffering from infectious mononucleosis Pathogenesis of Infectious Mononucleosis Clinical features of infectious mononucleosis Symptoms usually begin to appear 4-7 weeks after infection. The first signs of mononucliosis can easily be confused with cold and flu symptoms. The typical symptoms and signs of mononucleosis are: 1. Fever - this varies from mild to severe, but is seen in nearly all cases. 2. Enlarged lymph nodes - particularly the posterior cervical lymph nodes, on both sides of the neck. 3. Sore throat (throat infection) - nearly all patients with EBV-mononucleosis have tonsillitis, usually accompanied by thick exudate. 4. Fatigue Lymphoadenopathy during Infectious Mononucleosis Pathogenesis of EBV infection Molecular Mechanism of Tumor Transformation by EBV

The most important molecular mechanism associated with African Burkitt's lymphoma is the increased transcription of the proto-oncogene c-myc , which is translocated from chromosome 8 to chromosome 14, where it may be more actively transcribed under control of the heavy chain of the immunoglobulin gene. Other translocations into chromosomes 2 and 22 and mutated p53 anti- oncogenes have been found. Cofactors: Immune suppression at an early age by infection with malaria, HIV, as well as medication, may influence the pathogenesis of EBV infection where BL is prevalent in young children. Translocation of c-myc may also be considered to be a cofactor in African BL but is not reported in the immune suppressed. Changes in differentiation of B-cells infected by EBV Immunofluorescent method (cells, infected by EBV) Rubella Virus

Taxonomy Family: Togaviridae Species: Rubivirus Type : rubella virus Structure of Rubella Virus Structure of Rubella Virus

Rubella virus is a spherical, 40- to 80- nm, positive-sense, single-stranded RNA virus with spike-like, hemagglutinin- containing surface projections. An electron-dense 30 to 35 nm core is surrounded by a lipoprotein envelope. Only one serotype has been identified. It contains three major structural polypeptides. Electron Microscopy of Rubella Virus Replication of Rubella Virus 1. Virus attaches to host receptors though E glycoprotein and is endocytosed into vesicles in the host cell. 2. Fusion of virus membrane with the vesicle membrane occurs; RNA genome is released into the cytoplasm. 3. The positive-sense genomic ssRNA is translated into a polyprotein, which is cleaved into non structural proteins. 4. Replication takes place at the surface of endoplasmic reticulum. A negative-sense complementary ssRNA is synthesized using the genomic RNA as a template. 5. New genomic RNA as well as subgenomic RNA are synthesized using the negative-sense RNA as a template. Subgenomic RNA is translated in structurals proteins. 6. Virus assembly occurs at the endoplasmic reticulum. The virion buds at the endoplasmic reticulum, is transported to the Golgi apparatus, and then bud from the cell membrane. Replication of Rubella Virus Rubella: Transmission, Pathogenesis and Immunity The disease is transmitted via direct or droplet contact with respiratory secretions. Rubella virus multiplies in cells of the respiratory system; this is followed by viremic spread to target organs. Congenital infection is transmitted transplacentally. Neutralizing and hemagglutination-inhibiting antibodies and cell-mediated immunity develop promptly. Reinfection (usually asymptomatic) can occur. Pathogenesis of Rubella Characteristic Features of Immunity against Rubella Rubella (Rash) Congenital Rubella Infection Rubella infection acquired during pregnancy can result in stillbirth, spontaneous abortion, or several anomalies associated with the congenital rubella syndrome. During fetal infection, the virus can multiply in and damage virtually any organ system. Cell culture studies show that the virus produces chromosomal abnormalities, slows cellular growth rates, and causes cell lysis and death in some cell types; these effects appear capable of producing the characteristic abnormalities of cell structure and function. Congenital Rubella Infection The clinical features of congenital rubella vary and depend on the organ system(s) involved and the gestational age at the time of maternal infection. The classic triad of congenital rubella syndrome includes cataracts, heart defects, and deafness , although many other abnormalities (growth retardation, endocrine dysfunction, hepatosplenomegaly etc.) may be seen. Defects may occur alone or in combination and may be temporary or permanent. The risk of rubella-associated congenital defects is greatest during the first trimester of pregnancy. Some defects have been reported after maternal infections in the second trimester. Congenital Rubella Infection Laboratory Diagnosis of Rubella

Clinical specimen: nasopharyngeal secretions and blood, collected before appearance of rash; congenital rubella is diagnosed by studying of urine, feces and autopsy material Virological method. Rubella virus can be cultivated in primary cells prepared from human amnion, primates and rabbit kidney. Rubella virus causes cytopathic effect (formation of eosinophilic inclusions, containing RNA). Rubella virus can’t cause cytopathic effect in cell cultures of another origin. Virus cause hemagglutination of pigeon erythrocytes. Identification of virus is based on hemagglutination and cytopathic effect neutralization. Immunofluorescent method (indirect immunofluorescence for detection of rubella virus in clinical specimen). Serological method can be used to identify a response to acute infection (IgM) or previous infection (IgG) in serum. Paired serums are used for detection of increase in titer by CFT, ELISA and RIA. Molecular-genetic method : PCR is used for analysis of autopsy material. cytopathic effect. Specific Prophylaxis of Rubella Rubella infections are prevented by active immunization using live virus vaccine. Two live attenuated virus vaccines, RA 27/3 and Cendehill strains, were effective in the prevention of adult disease. The vaccine is now given as part of the MMR vaccine. The WHO recommends the first dose is given at 12 to 18 months of age with a second dose at 36 months (in Russia the first booster immunization is by MMR for all kids at 6 year old and second booster immunization by live rubella vaccine for girls at 13 years old). Pregnant women are usually tested for immunity to rubella early on. Women found to be susceptible are not vaccinated until after the baby is born because the vaccine contains live virus.