viruses in periodontics

VIRUSES IN PERIODONTAL INFECTIONS

CONTENTS• Introduction• Virus classification • Viral components and structure• Replication of viruses • Host response to viral infections• Human immunodeficiency virus• Structure • Pathogenesis • Periodontal conditions• Herpes Viruses in periodontal disease• Structure • Herpesviral–bacterial model of periodontitis• Evidence for the viral etiology• Challenges for the viral hypothesis • Summary • References

INTRODUCTION • Virus -

is an infective agent that typically consists of a nucleic acid

molecule in a protein coat, is too small to be seen by light microscopy,

and is able to multiply only within the living cells of a host.

• Smallest living unit

• Viruses – first described – filterable agents

• “Living chemicals” – crystallised like chemicals- Stanley 1935

extraction of “infectious nucleic acid” - Geirer & Schramm 1956

• Organisms at the edge of life

• The etiopathogenesis of periodontal disease is a complex process• Bacterial etiology alone has not been able to substantiate…

• Recently, various HHVs have emerged as putative pathogens in destructive periodontal disease

Slots et al. 2006• HHV-bacterial pathogen model

Slots et al 2007

1. Rapid periodontal tissue breakdown with minimal plaque 2. Phase of disease activity & quiescence 3. Site specificity in periodontal disease 4. Progression to advanced periodontal destruction in a

fraction of given population

EVOLUTION OF VIRUSES

BACTERIA VIRUSIntercellular organisms Intracellular organisn

Size Larger (1000nm) Smaller (20 - 400nm)

Living attributes

Living organismOpinions differ on whether viruses

are a form of life or organic structures that interact with living

organisms.

Structures

DNA and RNA floating freely in cytoplasm. Has cell wall

and cell membrane.

DNA or RNA enclosed inside a coat of protein.

Ribosomes Present Absent

Reproduction Fission- a form of asexual reproduction

Invades a host cell and takes over the cell causing it to make copies of the viral DNA/RNA. Destroys the

host cell releasing new viruses.

Treatment AntibioticsVaccines prevent the spread and

antiviral medications help to slow reproduction but can not stop it

completely.

VIRAL NOMENCLATURE

• Picornavirus - pico + rna

• Papovavirus - papilloma , polyoma , vacuolating viruses

• Retrovirus - reverse – virus directed synthesis of DNA from

RNA template

• Poxviruses - symptoms caused by one of its members , smallpox

BALTIMORE CLASSIFICATION (1971)

Enveloped

Herpesvirusespoxvirus

Nonenveloped

AdenovirusPapillomavirus

Nonenveloped

parvovirus

Enveloped

Retrovirus HIV

Enveloped

Hepandavirus

Enveloped

Togavirus

Nonenveloped

Picornavirus

Nonenveloped

Reovirus Enveloped

Orthomyxovirus, paramyxovirus

VIRAL COMPONENTS AND STRUCTURE

• Virion –is a complete virus particle consisting of RNA or DNA surrounded by a protein shell, constituting the infective form of virus

i. Genome - either DNA or RNA but not bothii. Capsid iii. Envelope iv. Peplomers v. Enzymes

CAPSID

• Shell made of protein building blocks or promoters

• Self assemble into larger capsomere – then into virion capsid

• Simplest structures - symmetrical

i. Helical – form rods

ii. Icosahedral – approximation of a sphere

ENVELOPE

• Similar to cellular membranes

• Consists of lipids , proteins and glycoproteins

• Glycoproteins – extend from surface , act as virus attachment

proteins (VAP) , are major antigens for protective immunity

VIRUS REPLICATION

VIRUS REPLICATION

HOST RESPONSE

I] ANTIBODY MEDIATED ANTIVIRAL IMMUNITY

a) Viral Antibodies :

• IgG – block adsorption and penetration into host cells

• Enhance ingestion by PMN’s and macrophages

• Agglutinate the virus – reduce viral load

• Ag – Ab complex - increase size to trigger phagocytosis

b) Antibodies with activation of complement system• Generation of C3 –viral Ab complex allows binding of C3

receptor – phagocytosis

• Ab and Complement – lysis of virus with envelope – damage lipid membrane

- Lysis of infected cells• Complement alone – activate alternate pathway – lysis or

increased phagocytosis of coated material

c) Ig bound to virus infected cell interacts with Fc receptor bearing lymphocytes (K cells) – cell lysis

II] CELL MEDIATED ANTIVIRAL IMMUNITY

III] NON SPECIFIC FACTORS1. Fever2. Interferon Production3. Natural Killer Cell Activity

ACUTE INFECTIONS

• Events that contribute to cytopathic changes & cell death1) Inhibition of host cell DNA , RNA and protein synthesis2) Virion proteins3) Activation of lysosomal enzymes4) Inhibition of Na K pump

PERSISTENT INFECTIONS

INTERFERON

• Innate mechanism

• Group of peptides – bind to receptors of neighbouring cells -

resistant to infection - limiting focus of infection

• Regulate cell differentiation and immune reactivity

• IFN α – T lymphocytes

• IFN β – virus stimulated fibroblasts

• IFN γ- most potent , Ag activated T cells

IFN

Degrade viral RNA

Cytotoxic T cell

activity

Expression of

adhesion proteins

Production of

cytotoxic factorsMitogenes

is of B cells

APC function

Effects on macropha

ge

NK cell activity

1. synthesis of lysosomal enzymes

2. Expression of receptors for Fc

portion of Ig

3. Decreased migration of

macrophages – expression of MIF

4. Production of IL 1 , TNF alpha

– augment lymphocyte activation

5.Contribute to T cell response -

enhance APC function of

activated macrophagess

HUMAN IMMUNODEFICIENCY VIRUS

• Retrovirus

• HIV 1

• HIV 2

• 1981 – first indication of AIDS in NY and LA

• 1983 – Luc Montagnier et al isolated retovirus and called it

LAV – lymphadenopathy associated virus

• 1984 – Robert Gallo – HTLV III – Human T cell lymphotropic

virus III

• 1986 – International committee of Virus Nomenclature - HIV

STRUCTURE

VIRAL GENES AND ANTIGENS

PATHOGENESIS

• Receptor for the virus is CD4• Primary CD4+ - T lymphocytes (helper/inducer)

5-10% of B lymphocytes 10-20% of monocytes and macrophages• Specific binding to CD4 receptor - envelope glycoprotein gp120.• Cell fusion - transmembrane gp41.• CXCR4 for T cell tropic HIV ,

CCR5 for macrophages –coreceptors

VIRUS ATTACHMENT AND PENETRATION

PATHOGENIC MECHANISM This damage is caused to CD4+ T cells. T4 cells and T4:T8 ratio is reversed.suppress function of infected cells without causing structural damage.T4 cells do not release IL2, IFN, and other lymphokines

•Helper T cell activity is essential for optimal B cell function•Hypergammaglobulinemia is more a hindrance than help because of ‘USELESS Ig’ to irrelevant Ags and also autoantibodies.

• Affected due to lack of secretion of activating factors by T4 lymphocytes.•So chemotaxis, antigen presentation and intracellular killing is diminshed.•Activity of NK cells and cytotoxic T cells is affected

MONOCYTE –MACROPHAGE FUNCTION

HUMORAL MECHANISM

LINEAR GINGIVAL ERYTHEMA

• A persistent , linear , easily bleeding, erythematous gingivitis

• Possible etiology – candida dubliniensis

• Prevalence in HIV patients – 0-49%

NECROTISING ULCERATIVE GINGIVITIS

PERIODONTITIS , STOMATITIS• NUG – destruction of 1 or more interdental papilla ,

confined to marginal gingiva

• NUP- involves PDL and alveolar bone. Bone is exposed

– necrosis – sequestration

• NUS – extends past MGJ into mucosa and osseous

tissues

• Higher prevalence in HIV patients

• NUP – marker of immune deterioration with 95%

predictive value

• Prevalence of Chronic periodontitis –

5-69%

HIV latently infecteted cells

Activation of membrane receptors by pathogens and cytokines

Turns on down stream signaling pathways

Activation of transcription factors like NF-kB

Activation of proinflammatory genes

Production of proinflammatory cytokine

HIV LTR transactivation

Co infection

PROPOSED MODEL OF HIV REACTIVATION DURING CO-INFECTION

HERPES VIRUSES

• Membership in the family herpesviridae is based on four layered structure of virion.

CLASSIFICATION

αHSV1HSV2VZV

βHCMVHHV6HHV7

γEBV HHV8

VIRUS REPLICATION

Capsid Nonstructural proteinsDNA

Attachment andpenetration by fusion

Nucleus DNAgenome

mRNA

DNA

ProteinImmediate earlyProtein synthesis

LATENT

EarlyProtein synthesisand genome replication

ACTIVE

LateProtein synthesis(structural protein)

Exocytosis and release

Assembly and release

Lysis and release

MECHANISMS OF PERIODONTOPATHIC POTENTIAL OF HERPES VIRUSES

1. Direct cytopathic effects

2. Impair cells involved in host defense

3. Promote subgingival attachment and colonization

4. Altered Inflammatory mediator

5. Reducing the cell surface expression of MHC class I

molecules

HERPES SIMPLEX VIRUSES

• HSV1,2 - usually affect skin and mucosa

• HSV1- shed principally in saliva- Orofacial Infections

• HSV2- transmitted sexually- genital infection

• Primary infection - childhood.

• At sites of epithelial infection - viral Ags induce cell

mediated immunity which is the key of recovery and

latency.

Replication in epithelial cells

Viral nucleocapsids ascend local sensory neurons by reterograde

axonal transport

Establish lifelong latency in corresponding spinal or cerebral

ganglion

Reactivation occurs at any time

Virus replication in infected neurons

Virus transproted down the axon to original position

• Primary herpetic gingival disease of viral origin is one of the most common infection(Kuzushima 1991)

• Asymptomatic in childhood.• Clinical features- gingivitis, vesicles that leave ulcerations,

lymphadenopathy and fever.• Healing - 10-14 days.• Recurrence - mucocutaneous junction of lips,palate or gingiva

(recurrent herpetic gingivostomatitis).

VARICELLA ZOSTER VIRUS

• Varicella (chickenpox) - childhood

• Herpes zoster- aged and immunocompromised

• Enters by inhalation - replicates in mucosa of respiratory tract.

• Dissemination - bloodstream and lymphactics.

• Virus multiplies in mononuclear leukocytes and capillary

endothelial cells.

VARICELLA

• Occurs during first 5-10 years of life.• Vesicles and ulcers first appear in mouth on palate, tongue,

gingiva followed by cutaneous rash that spreads centrifugally from head and trunk.

• Oral lesions-painful• Cutaneous lesions-painless but itchy and lead to secondary

infection and permanent scars

HERPES ZOSTER

• Herpes zoster- from reactivation of virus that remain latent in sensory ganglia.

• Lesions similar to varicella but remain confined to single dermatome and are unilateral.

• Intraorally lesions found if 2nd and 3rd branch of trigeminal nerve is involved, which may lead to alveolar bone necrosis.

EPSTEIN BARR VIRUS

• Transmitted by oral secretions or blood.• Virus replicates in epithelial cells or B cells of oropharynx.• All seropositive patients-actively shed virus in saliva.• Resting memory B cells –main site of persistence of EBV.• EBV infection - in children is subclinical,

in adults-infectious mononucleosis.• Symtoms of infectious mononucleosis- fever,

lymphadenopathy, pharyngitis, oral ulcers, palatal petechiae, less commonly gingival ulcerations

Oral hairy leukoplakia• Main lesion of EBV .• Non malignant hyperpalstic lesion of epithelial cells which shows

noncytolytic EBV replication.• OHL-appears as white corrugated lesion on ventral –lateral aspect of

tongue and may be unilateral/bilateral.

HUMAN CYTOMEGALOVIRUS

• Most common cause of congenital and perinatal infections.

• Infants infected through placenta, during delivery or breast

feeding.

• Infects epithelial cells, endothelial cells, smooth muscle cells,

mesenchymal, hepatocytes, granulocytes, macrophages.

• Found in saliva, urine, semen, breast milk.

• In HIV infected patient-oral ulcers as well as gingival

hyperplasia is noted.

HERPESVIRAL–BACTERIAL MODEL OF PERIODONTITIS

Healthy gingiva

Bacterial biolfilm

Gingivitis

Herpes viruses activation

Periodontopathic properties

1. Inflammation2. Collagen degradation

3. Bone resorption

Sufficient time span

Destructive periodontal disease

•Macrophages with latent HSV & HCMV•B cells with latent EBV•T cells with latent HSV & CMV

1. Immunosuppression from infection or cytotoxic therapy2. Inflammation3. Psychosocial or nutritional stress4. Hormonal changes/pregnancy5. Physical or chemical tissue injury6. Tobacco usage7. Aging8. Others

Cytokines or Enzymes

1. IL-1β2. TNF-α3. PGE24. MMPs

Immunosuppression & upgrowth of pathogenic

bacteria

1.P .gingivalis 2. T. forsythia3.A. actinomycetemcomitans 4. D. pneumosintes

Cytotoxicity/Tissue necrosis with severe

immunosuppression

1. HIV-infection2. Nutritionally stressed children/adolescents

HERPESVIRUS : AN ETIOLOGIC FACTOR FOR PERIODONTAL DISEASE?

1. Virus detection in gingival tissue• Cultured epithelial cells and fibroblasts – healthy gingiva – susceptible

to HSV infection – may act as reservoir of latent virusZakay 1982

• Indirect immunofluroscence assay – HSV 1 Ag detected in PD diseased patient’s gingival biopsiesEhrlich et al 1983

• HSV Ag – healthy gingiva Amit et al 1992

• HSV DNA in intact gingival cells – virus present in latent state• In periodontitis patient by using nested PCR –i. Monocytes and macrophages - HCMV , HSVii. T cells – HCMV , HSViii. B cells - EBV

2) Higher frequency of virus detection in the gingival tissue of periodontitis sites than in healthy sites

•In 20 patients with periodontitis HCMV DNA – 13 biopsies, EBV DNA – 10 biopsies HSV DNA-7 biopsies Healthy gingiva – HSV in1/3

Contreras et al , 1999

• Herpes viral DNA in gingival tissues could be detected in periodontally healthy or diseased subjects by nested PCR.

3. Higher frequency of herpes virus detection in GCF from periodontaly diseased sites than from gingivitis/healthy sites

• Parra and Slots (1996)– 78% of advanced periodontitis patients were positive for at least

1/5– HCMV (60%) >EBV (30%)> HSV (20%) HPV (17%) and HIV

(7%).– Only 31% of the gingivitis patients were virus positive

• 89% of the patients yielded at least 1/5 (HCMV) deep pockets, whereas only 56% yielded viral DNA from shallow periodontal sites

4. Higher frequency of virus detection in subgingival plaque from periodontaly diseased than from healthy sites

• Saygun et al 2002...

• Frequency of detection lower compared to previous studies

VIRUS CHRONIC PERIODONTITIS

HEALTHY

HCMV DNA 44 %

14.3%

EBV DNA 17 % 14.3%

HSV DNA 6.7% -

5. Detection of activated herpes virus in the GCF of periodontal lesions

Contreras & Slots 1998 –• Reverse transcriptase-PCR to examine mRNA transcription of

subgingival HCMV.• HCMV major capsid proteins - in deep periodontal pockets but

not in any shallow pocket• Active HCMV replication could occur in periodontal sites

Croen et al 1991• Several risk factors for periodontal disease – have potential to

reactivate Herpesvirus

6. Interaction of herpesviruses with periodontal pathogens

• The subgingival detection of EBV, HCMV could be associated with an increased presence of periodontal pathogens(A.a ,P g)

Contreras et al 1991• LJP – Aa associated with active HCMV infection • HCMV , EBV , HSV along with Pg , D pneumosintes – active

periodontitis• And immunosuppressive properties of herpesvirus –

overgrowth of subgingival periodontal bacteria

• Their can be a bidirectional interaction between herpesviruses and bacteria

• Bacterial enzymes or other inflammation-inducing factors have the potential to activate periodontal herpesviruses

• In addition ,P. gingivalis suppress the interferon-gamma antiviral host response

• Herpesvirus infection predisposes periodontal tissue to the bacterial superinfection

CHALLENGES FOR THE VIRAL HYPOTHESIS

1. Investigators

• Most clinical association studies have been carried out by the same group of investigators

• Samples were analysed in the same laboratory

• Confirmation by other independent researchers is lacking

2. Method

• The nested PCR works with two primer pairs and two different

amplification tests, one after the other

• Display higher specificity& sensitivity

• It is very susceptible to contamination, and can produce false

positive results

3. Sample population

• A higher frequency of co-infection and occurrence of EBV and

HHV-6 has been noted in HIV-positive

Contreras et al, 2001• HIV status was not investigated

• The doubt remains whether the subjects included were true

representative

4. Inferences of causality• One difficulty directly relates to the sampling procedure in

diseased sites• Samples from diseased sites are more likely to contain viruses

present in blood.

• Active HCMV replication has been demonstrated in periodontal sites

• The reactivation - periodontal disease activity, -just the opposite may be the case:

-periodontal disease activity caused by bacterial infection - trigger virus reactivation.

SUMMARY

• Contemporary Oral Microbiology and Immunology- Slots,

Taubman.

• Textbook of Microbiology 7th Edition Ananthnarayan and Paniker

• Pushpa S P, Soumya B G .Herpesviruses in Human Periodontal

disease.Reality or Myth…? J. Int Oral Health 2010,2(2)59-64• Jorgen slots Herpes viruses in periodontal Disease Perio 2000, Vol.

38, 2005, 33–62• I Cappuyns, P Gugerli, A Mombelli . Viruses in periodontal disease

– a review Oral Diseases 2005, 11, 219–229• Jorgen slots. Human viruses in periodontitis Perio 2000, Vol. 53,

2010, 89–110• Uppoor and Nayak HIV and Periodontal Disease: Redemption or Resurrection, J AIDS Clinic Res 2012,