Herpesviruses Dongli Pan Herpes comes from Greek word “herpein”（to creep or crawl） Herpesviruses Dongli Pan Department of Medical Microbiology and Parasitology Zhejiang University School of Medicine pandongli@zju.edu.cn

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Classification Herpesviridae Alphaherpesvirinae HSV-1 HSV-2 VZV ..…. (animal viruses) Betaherpesvirinae HCMV HHV-6 HHV-7 …...(animal viruses) Gammaherpesvirinae EBV KSHV There are 8 currently known human herpesviruses

Baltimore classification DNA viruses RNA viruses Baltimore classification Fields Virology, 6th edition

Viruses are small Principle of Virology

Common features 1.Shape and structure：spherical, 150-200 nm in diameter, dsDNA genome (125-245kb，encoding 70-200 proteins）；Capsids have icosahedral symmetry. There is a tegument and an envelope

2. Viral replication: Replication and assembly of viral capsids occurs in the nucleus. 3.Immunity: The viruses encode multiple proteins to evade and manipulate host immunity. Host immunity can control productive infection, but cannot prevent establishment of latency, nor can eliminate latent virus. 4. Types of infection All herpesviruses a. Primary infection: viral replication and cytolysis b. Latent infection: No viral replication, DNA genome remains in host for the life time of host c. Recurrent infection: viral replication after reactivation Some herpesviruses d.Congenital infection：HSV-1, HSV-2, VZV, HCMV e.Integrated infection：HHV-6, occasionally in EBV

Replication cycle HSV-1

Biological Cycle of Herpesviruses (Including congenital infection) Primary Infection Establish latency Latent infection Productive (lytic) infection Reactivation Establish latency Recurrent Infection

Human herpesviruses Subfamily Virus Species Primary Target Cell Site of Latency Means of Spread Diseases α herpesvirinae HSV-1 (HHV-1) Mucoepithelial cells Neuron Close contact Cold sores, gingivostomatitis Herpetic keratitis, Encephalitis HSV-2 (HHV-2) Close contact ( sexual transmission) Genital herpes VZV (HHV-3) Respiratory and close contact Chicken pox, Shingles β HCMV (HHV-5) Monocyte, lymphocyte, and epithelial cells Monocyte, lymphocyte, and ? Close contact transfusions, tissue transplant, congenital Infectious mononucleosis, Retinitis, Diseases for newborn and immunocompromised HHV-6 T cells and ? Roseola, Others? HHV-7 ? γ EBV (HHV-4) B cell and epithelial cell B cell Saliva ( Kissing disease ) Infectious mononucleosis, nasopharyngeal carcinoma, Burkitt’s lymphoma malignant lymphoma KSHV (HHV-8) Lymphocyte and other cell Close contact (sexual), saliva? Kaposi’s sarcoma

Herpesviridae Herpes Simplex Virus (HSV) • HSV-1 (HHV-1)

Biological properties Spherical, 120-150 nm, 150 kb dsDNA Can be easily propagated in many cell lines (most commonly Vero cells) Form visible plaques, multinucleated giant cells and Cowdry type 1 inclusion bodies (acidophilic nuclear inclusion bodies) Animal models: mouse, rabbit, Guinea pig. multinucleate cell with dark staining inclusions Plaque

Diseases caused by HSV HSV-1 HSV-2 Cold sores Herpetic gingivostomatitis Herpetic keratitis Herpes simplex encephalitis (70% fatality rate) Neonatal herpes Genital herpes, can be caused by HSV-1 too) Herpetic meningitis Neonatal herpes 13

Pathogenesis • Primary infection Congenital infection – Transmission by direct touch or sex – cytolysis – Blisters in the skin, ulcer in the eye, or swelling of brain tissues Congenital infection Mother to baby transmission through placenta It can lead to abortion, premature delivery, stillbirth, malformation, or encephalitis

Reactivation and recurrent infection • Latent infection – The virus establishes a non-replicating state in sensory neurons – HSV-1, trigeminal ganglia – HSV-2, sacral ganglia, also HSV-1 Reactivation and recurrent infection Triggers: fever, emotional stress, fatigue, menstruation, immunosuppression, etc. The virus starts to replicate and travels back to peripheral tissues, resulting in recurrent infection.

HSV latency and reactivation Field’s Virology, 6 edition

Diagnosis Approach Test/Comment Tzanck test: Direct microscopic examination of cells from base of lesion Multinucleated giant cells and cowdry type A inclusion bodies. Cell culture (gold standard) Inoculate Vero, BHK-21 cells, etc. HSV replicates and cause identifiable cytopathic effects (CPE): see plaques. Assay of tissue biopsy, smear, cerebrospinal fluid, or vesicular fluid for HSV antigen or genome ELISA, immunofluorescent stain, in situ hybridization and PCR Serology Specific IgM indicates recent infection

HSV Thymidine Kinase (UL23) Treatment and prevention Acyclovir (or similar nucleoside analogs and their derivatives) Foscarnet No vaccine yet. N HN H2N CH2 O HO-CH2 Acyclovir, a nucleoside analog, inhibits viral DNA polymerase Acyclovir (ACV) N HN H2N CH2 O HO-CH2 HSV Thymidine Kinase (UL23) ATP N HN H2N CH2 O P-O-CH2 ATP ATP N HN H2N CH2 O P-P-P-O-CH2 Inhibit HSV DNA polymerase Cellular kinases

Herpesviridae Varicella-Zoster Virus (HHV-3)

Biological properties Similar to HSV Has only 1 serotype Replicates in fibroblast or epithelial cells Form multinucleated giant cells and Cowdry type A inclusion bodies, like HSV But no working animal model

VZV primary infection (Chicken pox) Droplets, direct contact Enter through respiratory system, conjunctiva or skin, etc Skins of whole body Second Viremia Proliferate in local lymph node Proliferate in mononuclear phagocyte system in spleen and liver First Viremia

VZV latency and reactivation: Shingles Chicken pox as a child Virus establishes latency in dorsal root ganglia or trigeminal ganglia Replicate in skin 10-20% of cases Travel along Axons of sensory neuron to skin After adulthood, reactivated VZV reactivation occurs more frequently in immuncompromised persons and in the elderly Host immunity may help maintain latency

Congenital Varicella syndrome • Caused by infection in utero during the first trimester • Leads to scarring of the skin of the limbs, damage to the lens, retina and brain

Diagnosis and intervention Symptoms are typical. Usually no need of lab tests. Tzanck test: Staining of samples scraped from the base of a vesical and look for multinucleated giant cells, Cowdry type A inclusion bodies, etc. Treatment Acyclovir Vaccine Live attenuated vaccine virus VZV-Ig for emergency prevention (like for immunocompromised patients)

Herpesviridae Human cytomegalovirus (HCMV, HHV-5)

Biological properties Spherical, 180-250 nm. HCMV only infects human cells. Fibroblast cells are used for propagation. It replicates slowly. Infected cells become enlarged and form owl’s eye inclusion bodies (big acidophilic nuclear inclusions with the appearance of an “owl’s eyes”) Resistance is weak. Long term storage requires liquid nitrogen. H&E stain of lung section showing owl’s eye inclusions

Modes of transmission Direct contact: Sexual transmission, mouth to mouth, hand to mouth Vertical transmission: transplacental, intrauterine infection, cervical secretions, milk, saliva, etc Medical procedures: Blood transfusion, organ graft 60-90% of adults are seropositive

Outcomes of HCMV infections Infection of healthy children and adults Usually a lifelong and subclinical infection (60-90% of people are seropositive) Primary infection can lead to infectious mononucleosis Congenital infection HCMV infection is the leading cause of infectious congenital disease Infection of CNS can cause neurological abnormalities and death Neonatal infection Virus reactivated during delivery Usually protected by mothers’ antibodies Infection of immunosuppressed individuals Transplant recipients and AIDS patients Severe complications including retinitis, Meningitis, pneumonia, colonitis 90% in North America HCMV retinitis

Diagnosis and intervention Laboratory diagnosis Cell culture qPCR Tests of pp65 antigen in peripheral blood leukocytes Serology: ELISA for CMV-specific IgM indicates active infection, but may give false-positives Treatment and prevention Ganciclovir Foscarnet No vaccine, but under development

Herpesviridae Epstein- Barr Virus (HHV-4)

Biological properties Typical herpesvirus morphology and structure, 180 nm in diameter. Cannot be grown in normal cell culture. Lytic infection of EBV usually occurs during primary infection of oropharyngeal epithelial cells or during reactivation from latency in B cells. Important lytic antigens include: early antigen (EA), viral capsid antigen (VCA) and membrane antigen (MA) Latent infection mostly occurs in B cells. During latent infection limited viral antigens are expressed. They include EB nuclear antigen (EBNA) and latent membrane protein (LMP) Latent infection by EBV can transform and immortalized B cells, and occasionally results in malignancies. EBNA-2, EBNA-3C and LMP-1 antigens are essential for transformation.

Pathogenesis Transmitted mainly by saliva Replication in oropharyngeal epithelial cells Infects B cells in the local lymphatic system Spread throughout the body in B cells Latent infection of B cells (lifetime) B cells are transformed resulting in malignancies.

Diseases related to EBV infection Infectious mononucleosis (mostly caused by primary infection, fever, sore throat, enlarged lymph nodes in the neck, and feeling tired) Nasopharyngeal carcinoma (Guangdong, China; areas in southeast Asia) Burkitt's lymphoma (Africa) Malignant lymphoma A conjunctival hemorrhage of the right eye of this patient with infectious mononucleosis. Burkit’s lymphoma.

Diagnosis and intervention Heterophile antibody test: rapid test for antibodies induced by EBV infection. It’s diagnostic for infectious mononucleosis. EBV-specific antibody test PCR test for viral DNA No drugs or vaccines are available for Epstein-Barr virus.

Pathogens of vertically transmitted infections