HSV: Herpes Simplex Virus Micr. 401 – Virology Case Study #15

1. HSV: Herpes Simplex VirusMicr. 401 – Virology Case Study #15 By: Kevin Chau Jere Wilson Yessenia Velazco

2. Case Study #15: • A 2-year-old child with a fever for 2 days was not eating and was crying often. On examination, the physician noted that the mucous membranes of the mouth were covered with numerous shallow, pale ulcerations. A few red papules and blisters were also observed around the border of the lips. The symptoms worsened over the next five days and then slowly resolved, with complete healing after two weeks • Diagnosis: Typical HSV-1 or Congenital HSV-2

3. HSV Background: • Greek “herpein” – “to creep” • Enveloped w/glycoproteins, icosahedral capsid • ds, linear w/nicks DNA genome • Tegument proteins – between envelope & capsid

4. HSV Lytic Cycle: • 1)Attachment: • gB/gC: viral ligands binding host heparan sulfate low affinity receptor • gD: viral ligand binding to primary receptor directing envelope fusion at the plasma membrane • 2)Penetration: • pH Independent Fusion – facilitates envelope fusion at the plasma membrane • Releases nucleocapsid into cytoplasm • Does not form a vesicle or pH-dependent endosome • Releases tegument proteins: αTIF & VHS • 3)Uncoating: • Retrograde Transport – nucleocapsid transport from cell periphery to the cell nucleus along microtubules scaffolds (axonal transport) • 4)Viral Synthesis: • Immediate Early Transcription activated by αTIF • VHS (virion host shutoff) – overall mRNA degradation & inhibits host translation machinery • Circularization & amplification of genome • 5)Assembly: • Rolling concatemer synthesis & assembly • 6)Viral Release: • Virus buds from nuclear membrane through intermediate glycoproteins • Obtains tegument proteins in cytoplasm • Buds into a glycosylated vesicle from Golgi apparatus • Enveloped virion is released by exocytosis

5. HSV Latency: • Viral reservoir in sensory neurons: trigeminal & sacral ganglia • LAT gene expression increases in response to viral stress on host cell • Represses VHS via Histones, but allows minimal gene transcription for reactivation • Reactivation in differentiated neural cells must require independent viral replication machinery • Common cold • Fever/Allergy • Severe sunburn • Physical fatigue • Emotional disturbance • Trauma • Gastrointestinal disturbances • Menstruation • Pregnancy

6. HSV Immune Response: • Antibodies are present 4-8 days after primary infection • Infections stimulate MHC II pathway immune response resulting in active B cells and memory B cells • Cytolytic response via MHC I is gradually downregulated by viral glycoprotein ICP47 binding to TAP • TAP facilitates antigen processing in MHC I response

7. HSV Transmission: • Direct contact of lips or genitals when sores present • Possible transmission when sores not present (viral shedding) • Virions present in semen, saliva, & vaginal fluids • Autoinoculation • Pregnancy

8. HSV-1 Background: • Also referred to as: “cold sores” - not “canker sores” • Primarily associated with oral, pharyngeal, facial, ocular, and encephalitis infections • Mainly transmitted by oral secretions in close non-sexual contact (ex. touching, kissing, sharing food or utensils) • Lesions localize anteriorally: lips, gingiva, anterior tongue, and hard palate

9. HSV-2 Background: • Primarily associated genital infections • Mainly transmitted sexually by genital secretions • Lesions found on: vulva, vagina, cervix, urethra, perineum, penis, and rectum • Despite typical, clinical manifestations, both HSV-1 & HSV-2 can cause oral-facial and genital infections.

10. HSV Clinical Symptoms: • Swollen lymph glands • Headache • Fever • Sore mouth/genitals • Itching sensation • Lower back & leg pains – associated with genital herpes • Visualization: Yellowish-pus, epidermal lesion w/erythematous border • Incubation time is 2 to 20 days after contact; symptoms last 10-21 days • May be less severe for certain individuals (especially HSV-1) • First attack causes visible sores; asymptomatic after primary infection (latency) • Reactivation produces active lesions (secondary infections) • Examine medical/sexual history

11. HSV-2 & Pregnancy: • Vaginal tract infection associated w/ spontaneous abortion, premature birth, and 90% congenital neonatal herpes. • 5% Transplacental infection & 5% post-partum • Untreated neonatal herpes: 60% neonatal mortality or severe developmental defects

12. HSV Diagnosis: • 1) Viral isolation: • Reserved for patients with active lesions • Involves collecting viral fluid from vesicles by swabbing & incubation in growth media for cell culture • Virus can be identified by monoclonal antibodies detecting early antigens of HSV • Tissue culture for HSV is positive within 48 hours of inoculation. • Visual examination of cell culture for viral cytopathic effect Ex. ballooning of cells and rapid cell death of entire monolayer

13. HSV Diagnosis: • 2)Tzanck Smear: • Provides rapid and reliable diagnosis of an infection caused by Herpesviridae • Purpose of this technique is to observe viral-induced degenerative changes in the cells: multinucleated giant keratinocytes and ballooning degeneration. • Limitation: Cannot distinguish between type-specific herpes infection

14. HSV Diagnosis: • 3) Direct fluorescent antibody (DFA): • Used to detect viral antigens. • DFA is very specific, sensitive, fast & inexpensive method for labeling HSV antigens with monoclonal antibody, identifying type-specific HSV • Immunofluorescent staining of tissue culture cells can quickly differentiate types 1 or 2 HSV.

15. HSV Diagnosis: • 4) Viral DNA detection w/PCR: • Rapid detection of HSV DNA in clinical specimens with polymerase chain reaction (PCR) techniques. • In HSV encephalitis, PCR using cerebrospinal fluid (CSF) provides a rapid, noninvasive diagnostic technique that is as sensitive as brain biopsy. • PCR can detect viral infection during asymptomatic stages (latency)

16. HSV Diagnosis: • 5)Serological Assays: • Enzyme-linked immunosorbent assay (ELISA): • Fast and inexpensive assay • ELISA recognizes the antigenic subtype differences between glycoprotein G1 and G2 • False-positive results may occur due to the potential for cross-reactivity between the two glycoprotein • Immunoblot (Western): • Necessary for confirming positive results as it detects antibodies across all viral proteins to determine if the infection is caused by HSV1 or HSV2.

17. HSV Treatment: • There is no cure that can eradicate the virus, but there are treatments that can modify the course of the disease. • 1)Fusion Inhibitors (Docosanol): • An over-the-counter product approved for topical treatment • Prevents the fusion of the viral envelope with the cell membrane, preventing the entry of the virus into the host cell • 2) Helicase-primase Inhibitors (Non-nucleoside): • New non-nucleoside antivirals that target the helicase-primase, which is one of the most important complex involved in HSV DNA replication

18. HSV Treatment: • 3) Nucleoside Antivirals: • Acyclovir, Valacyclovir, Penciclovir: are viral agents that target the DNA polymerase & viral replication • Acyclovir and valacyclovir perform selective inhibition of DNA polymerase resulting in termination of further elongation of viral DNA • Penciclovir, on the other hand, halts viral DNA replication by competitive inhibition of DNA polymerase rather than chain termination

19. HSV Treatment: • 4) Vaccine (Herpevac): • Herpevac is a vaccine used for HSV-2 • Studies showed 70% effectiveness for women but not for men\ • 5) Unproven Remedies: • High Lysine Diet (Amino Acid) • Topical Undecylenic acid (Castor oil derivative) • Topical Butylated hydroxytoluene - (food preservative)

20. Reference: • Bloom, D.C. Experimental investigation of herpes simplex virus latency. Clin Microbiol Rev. 1997 July; 10(3):419-443 • Brown, J.C. Uncoating the herpes simplex virus genome. J. Mol. Biol. 2007 370 (4): 633-42. • Geraghty, R.J. Herpes simplex virus type 1 mediates fusion through a hemifusion intermediate by sequential activity of glycoproteins D, H, L, and B. Proc. Natl. Acad. Sci. U.S.A. 2007. 104 (8): 2903-8. • Hsia, S.C. Repressor element-1 silencing transcription factor/neuronal restrictive silencer factor (REST/NRSF) can regulate HSV-1 immediate-early transcription via histone modification. Virol. J. 2007. 4: 56. • Kimberlin D, Powell D, Gruber W, et al. 1996. Administration of oral acyclovir suppressive therapy after neonatal herpes simplex virus disease limited to the skin, eyes and mouth: results of a phase I/II trial. Pediatric Infectious Diseases Journal 15:247-54. • Kramer, Martha F. Accumulation of Viral Transcripts and DNA during Establishment of Latency by Herpes Simplex Virus. J Virol. 1998. 72:2 • Mahnaz F, Schwartz R. 2007. Human herpes simplex virus infections: Epidemiology, pathogenesis, symptomatology, diagnosis, and management. Journal of American Academy of Dermatology 57:737-763. • McIntosh E. 2005.  Paediatric infections: prevention of transmission and disease- implications for adults. Vaccine 23:2087–2089.  • Wechsler, S.L. Fine mapping of the latency-related gene of herpes simplex virus type 1: alternative splicing produces distinct latency-related RNAs containing open reading frames. J. Virol. 62:11 • Whitley, R.J. Herpesviruses. in: Baron's Medical Microbiology. 2008. 4th ed. • Antiviral Drugs For Herpes Treatment • http://ezinearticles.com/?Antiviral-Drugs-For-Herpes-Treatment&id=265576 • Herpes & Cold Sores • http://www.herpes-coldsores.com • The Tests You Take to Diagnose Genital Herpes • http://www.webmd.com/genital-herpes/guide/genital-herpes-diagnosis?src=rss_womenshealth • Herpes Diagnosis • http://www.herpesdiagnosis.com/blood.html • Herpes Viruses • http://pathmicro.med.sc.edu/virol/herpes.htm • Immune Response • http://www.brown.edu/Courses/Bio_160/Projects2000/Herpes/HSV/Immune_response.html • Dr. Edward K. Wagner’s Herpes Virus Research • http://www.dbc.uci.edu/~faculty/wagner/hsv7f.html

21. The End • Use a Prophylatic • Get Tested