VIRUS - HOST

INTERACTIONS
basics
Dr.T.V.Rao MD

Dr.T.V.Rao MD

Agents That Cause Disease.

Pathogens

Viruses

Bacteria

Parasites

Fungi

Dr.T.V.Rao MD

The Immune Response to Infectious Disease

Dr.T.V.Rao MD

Important General Features of Immunity to Pathogens.

Defense against pathogens is mediated by both innate and specific immunity. The innate immune response to pathogens plays an important role in determining the nature of the specific immune response. The immune response is capable of responding in distinct and specialized ways to different pathogens in order to combat these infectious agents most effectively.
Dr.T.V.Rao MD 4

Viruses
Obligatory intercellular pathogens that replicate within cells. Use the nucleic acid and protein synthetic machineries of the host cell. Infect a variety of cell populations by utilizing normal cell surface molecules as receptors to enter cell.
Dr.T.V.Rao MD 5

Virus - Host
Virus effect Host can cause No effect Cell damage or Death

Polio cell death paralysis proliferation in Moll scum contagiosum

Malignant transformation oncogenic virus
Dr.T.V.Rao MD 6

Humans react to Viral Infections

Dr.T.V.Rao MD

Newton's

rd 3

Law works

Dr.T.V.Rao MD

Dr.T.V.Rao MD

Non Immunological responses

Phagocytosis Body Temperature. Hormones Malnutrition Age Young Old are more prone for viral Infections
Dr.T.V.Rao MD 10

BARRIERS TO INFECTION
Inherent Barriers The host has a number of barriers to infection that are inherent to the organism. These represent the first line of defense which function to prevent or limit infection. Skin The skin acts a formidable barrier to most viruses and only after this barrier is breached will viruses be able to infect the host. Lack of Membrane Receptors Viruses gain entry into host cells by first binding to specific receptors on cells
Dr.T.V.Rao MD 11

Mucus
The mucus covering an epithelium acts as a barrier to prevent infection of host cells. In some instances the mucus simply acts as a barrier but in other cases the mucus can prevent infection by competing with virus receptors on cells. For example, orthomyxo- and paramyxovirus families infect the host cells by binding to sialic acid receptors.

Dr.T.V.Rao MD

12

Ciliated epithelium
The ciliated epithelium which drives the mucociliary elevator can help diminish infectivity of certain viruses. This system has been shown to be important in respiratory infections since, when the activity of this system is inhibited by drugs or infection, there is an increased infection rate with a given inoculum of virus.
Dr.T.V.Rao MD 13

Low pH
Low pH The low pH of gastric secretions inactivate most viruses. However, enteroviruses are resistant to gastric secretions and thus can survive and replicate in the gut.
Dr.T.V.Rao MD 14

The Immune System

The principal function of the immune system is to protect the host against pathogenic microbes. Immunity may be innate or specific.

Dr.T.V.Rao MD

15

Components of Human Immune System

Dr.T.V.Rao MD

16

Virus can cause Chromosomal Damage

Chromosomal Injury damage Measles Mumps, Adenovirus,CMV Varicella virus Damage to chromosomes of the Host C 17
Dr.T.V.Rao MD 17

Pathogens & Disease

Pathogens are defined as microbes capable of causing host damage. When host damage reaches a certain threshold, it can manifest itself as a disease.
The evolution of an infectious disease in an individual involves complex interactions between the pathogen and the host.
Dr.T.V.Rao MD 18

Evasion of Immune Mechanisms by Viruses

Viruses can also escape immune attack by changing their antigens. A large number of viruses evade the immune response by causing generalized immunosuppression.
Dr.T.V.Rao MD 19

Inclusion Bodies
Inclusion bodies are nuclear or cytoplasmic aggregates of stainable substances, usually proteins. They typically represent sites of viral multiplication in a bacterium or a eukaryotic cell and usually consist of viral capsid proteins.

Dr.T.V.Rao MD

20

Inclusion Bodies ( Elementary Bodies )

Inclusion bodies differ size, shape, Location, staining ,properties, Some are seen under microscope In Cytoplasm Pox virus Nucleus is affected Herpes virus

Dr.T.V.Rao MD

21

Negril Bodies

Dr.T.V.Rao MD

22

Staining the Virus in Inclusion bodies

Giemsa staining can produce Acidophilic / Basophilic inclusions Esinophilic inclusions Negril bodies in brain cells in Rabies Mollusca Bodies moll scum contagiosum
Dr.T.V.Rao MD 23

Pathogenesis of Viral Infection

In apparent ( Sub clinical ) Apparent ( Clinical ) Acute Sub acute Chronic Can produce latency Herpes zoster virus in nerve toot ganglion, Kuru Human slow virus infection.
Dr.T.V.Rao MD 24

How Virus enter the Host

Through Respiratory tract Gastro Intestinal tract Skin, Conjunctivae By sex contact
Dr.T.V.Rao MD 25

Respiratory tract
Small pox Chicken pox Influenza Rhinovirus Rhinovirus

Dr.T.V.Rao MD

26

Gastro Intestinal tract

Enterovirus, Adenovirus Reovirus Hepatitis A E Rota virus
Dr.T.V.Rao MD 27

Skin
Papilloma virus Cow pox Molloscom contagiosum
Animal Bite Rabies Injections Hepatitis B infections.
Dr.T.V.Rao MD 28

Most common Viral Infections

Arbovirus Mosquito bite Dengue

Chikungunya
Dr.T.V.Rao MD 29

Other Routes of Entry

Conjunctiva Adenovirus, Genital tract sexual contact HIV Congenital Infection Rubella and CMV

Dr.T.V.Rao MD

30

Spread of Virus
Spread from various sources Lymph nodes, Blood stream, Reach target organs. Viremia locate to various organs.

Dr.T.V.Rao MD

31

Incubation period
May be short, long Variable. Depend on site of entry and site of lesions. Common cold very rapid onset. Chicken pox and Poliomyelitis 10 20 days Arbovirus 5 6 days Hepatitis B 2 6 months AIDS ? Dr.T.V.Rao MD Slow Virus many years

32

How Host Responds

Non Specific Immunity Humoral Cell mediated.
Dr.T.V.Rao MD 33

Specific Immune Response to Viruses

Mediated by a combination of humoral and cell mediated immune mechanisms. Humoral mediated immune response.
Antibodies specific for viral surface antigens are

often crucial in containing the spread of a virus during acute infection and in protecting against reinfection. Specific antibodies are important in defense against viruses early in the course of infection and in defense against cytopathic viruses that are liberated from lysed infected cells.
Dr.T.V.Rao MD 34

Dr.T.V.Rao MD

35

Specific Immune Response to Viruses

Cell-mediated immune responses.
Most important in host defense, once a viral infection is established. CD8+ Tc cells (Cytotoxic T lymphocytes; CTLs) and CD4+ th1 cells (helper T lymphocytes) are the main components of cell mediated antiviral defense.
Dr.T.V.Rao MD 36

CD8+ T and CD4+ T

Dr.T.V.Rao MD

37

Humoral Immunity
Immunoglobulin Ig G Ig M Ig A

Ig A Mucosal surface secretary immunoglobulin Antibodies neutralize with help of complement.

Dr.T.V.Rao MD 38

HIV destroys CD 4 cells

Cell Mediated Immunity Delayed Hypersensitivity

HIV destroys CD 4 Lymphocytes.

Dr.T.V.Rao MD 39

Evasion of Immune Mechanisms by Viruses

Viruses have evolved numerous mechanisms for evading host immunity. A number of viruses have strategies to evade complementmediated destruction.
Dr.T.V.Rao MD 40

Evasion of Immune Mechanisms by Viruses

Viruses can also escape immune attack by changing their antigens. A large number of viruses evade the immune response by causing generalized immunosuppression.
Dr.T.V.Rao MD 41

HUMORAL COMPONENTS INVOLVED IN RESISTANCE TO VIRAL INFECTIONS

Nonspecific

A number of humoral components of the nonspecific immune system function in resistance to viral infection. Some of theses are constitutively present while others are induced by infection.
Dr.T.V.Rao MD 42

Interferon (IFN)
Interferon (IFN)
IFN was discovered over 40 years ago by Issacs and Lindemann who showed that supernatant fractions from virus-infected cells contained a protein that could confer resistance to infection to other cells. This substance did not act directly on the virus, rather it acted on the cells to make them resistant to infection (Figure 1).
Dr.T.V.Rao MD 43

Types of Interferon's

Alpha Beta Gamma

Dr.T.V.Rao MD

44

Interferon's
There are three types of interferon, IFNalpha (also known as leukocyte interferon), IFN-beta (also known as fibroblast interferon) and IFN-gamma (also known as immune interferon). IFN-alpha and IFN-beta are also referred to as Type I interferon and IFN-gamma as Type II. There are approximately 20 subtypes of IFN-alpha but only one IFN-beta and IFNgamma. Molecular wt. MD Dr.T.V.Rao 17,000 45

Interferon's
Isaacs Linder Mann

Interferon protection against viral infection Contain Host coded proteins Produced by viral and Non viral inducer On exposure to Interferon cell produce translation inhibiting proteins TIP Inhibits translation of m RNA But no effect on cell mRNA Viral transcription inhibited .
Dr.T.V.Rao MD 46

Biological effects of Interferon's

Antiviral effects. Antimicrobial effects. Cellular effect ts Inhibition of cell growth and proliferation. and of DNA and protein synthesis Expansion MHC antigens Immunoregulatory effects Enhanced cytotoxic activity of NK, K and T Suppression of DTH.
Dr.T.V.Rao MD 47

Interferon's
Hum Interferon alpha leukocyte interferon Produced by leukocytes 16 subspecies Beta Interferon Inf Produced by Fibroblast and epithelial cells. Gamma Interferon Inf produce by lymphocytes, Produce Immuno modulation and ant proliferative function Resist heating at 56 - 600 c for 30 6o mt
Dr.T.V.Rao MD 48

Interferon's
Interferon's are species specific RNA viral infections are better inducers than DNA virus Potent are Toga viridae Vesicular stomatitis virus and Sendai virus. Production starts in > 1 hour and increases in 6 12 hours.
Dr.T.V.Rao MD 49

Uses of Interferon's
Molecular wt. 17,000 Used in Prophylaxis and treatment Non toxic and Non antigenic Anti cancer agent lymphomas Used in Hepatitis B and C infections.
Dr.T.V.Rao MD 50

Complement
Most viruses do not fix complement by the alternative route. However, the interaction of a complement-fixing antibody with a virus infected cell or with an enveloped virus can result in the lysis of the cell or virus. Thus, by interfacing with the specific immune system, complement also plays a role in resistance to viral infections.
Dr.T.V.Rao MD 51

Cytokines
Cytokines other than IFN also may play a role in resistance to virus infection. Tumor necrosis factor alpha (TNF-), interleukin1 (IL-1) and IL-6 have been shown to have antiviral activities in vitro. These cytokines are produced by activated macrophages but their contribution to resistance in vivo has not been fully elucidated.
Dr.T.V.Rao MD 52

Specific
Antibody produce by the specific immune system is involved primarily in the recovery from viral infection and in resistance to subsequent challenge with the virus. IgG, IgM and IgA antibodies can all play a role in immunity to virus infection but the relative contributions of the different classes depends on the virus and the portal of entry. For example, IgA will be more important in viruses that infect the mucosa while IgG antibodies will be more important in infections in which viremia is a prominent feature. Antibodies can have both beneficial and harmful effects for the host.
Dr.T.V.Rao MD 53

Immunity Notsustanbale in Influenza Infection Need Vaccination to New strains

Dr.T.V.Rao MD

54

Laboratory Diagnosis of Viral Diseases

Different from Bacterial Infections

Dr.T.V.Rao MD

55

Virological Tests
An Overview

Dr.T.V.Rao MD

56

Why Difficult
In the past Growth of Virus was not rapid. Diagnosis becomes routine today due to availability of Rapid method. Important in HBV and HIV infections. Rubella in pregnant women. Simple methods Microscopy and detection of inclusion bodies.
Dr.T.V.Rao MD 57

Diagnostic Methods in Virology

1. Direct Examination

2. Indirect Examination (Virus Isolation) 3. Serology

Dr.T.V.Rao MD 58

Microscopy
Light Microscopy elementary bodies Electron Microscopy Rota viral detection Florescent Microscopy Direct / Indirect

Dr.T.V.Rao MD

59

Direct Examination
1. Antigen Detection 2. Electron Microscopy immunofluorescence, ELISA etc. morphology of virus particles immune electron microscopy histological appearance inclusion bodies hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
Dr.T.V.Rao MD 60

3. Light Microscopy

4. Viral Genome Detection

Indirect Examination
1. Cell Culture cytopathic effect (CPE)
haemabsorption immunofluorescence 2. Eggs pocks on CAM haemagglutination inclusion bodies disease or death

3. Animals

Dr.T.V.Rao MD

61

Serology
Detection of rising titers of antibody between acute and convalescent stages of infection, or the detection of IgM in primary infection.
Classical Techniques 1. 2. 3. 4. 5. Complement fixation tests (CFT) Haemagglutination inhibition tests Immunofluorescence techniques (IF) Neutralization tests Counter-immunoelectrophoresis Newer Techniques 1. 2. 3. 4. 5. Radioimmunoassay (RIA) Enzyme linked immunosorbent assay (EIA) Particle agglutination Western Blot (WB) RIBA, Line immunoassay

Dr.T.V.Rao MD

62

Virus Isolation
Cell Cultures are most widely used for virus isolation, there are 3 types of cell cultures:
1. Primary cells - Monkey Kidney 2. Semi-continuous cells - Human embryonic kidney and skin fibroblasts 3. Continuous cells - HeLa, Vero, Hep2, LLC-MK2, MDCK Primary cell culture are widely acknowledged as the best cell culture systems available since they support the widest range of viruses. However, they are very expensive and it is often difficult to obtain a reliable supply. Continuous cells are the most easy to handle but the range of viruses supported is often limited.
Dr.T.V.Rao MD 63

Cell Cultures
Growing virus may produce
1. Cytopathic Effect (CPE) - such as the ballooning of cells or syncytia formation, may be specific or non-specific. 2. Haemabsorption - cells acquire the ability to stick to mammalian red blood cells. Confirmation of the identity of the virus may be carried out using neutralization, haemabsorption-inhibition or immunofluorescence tests.

Dr.T.V.Rao MD

64

Detection of Viral antigen

Use of Immunofluorescence
Imunoelectrphoresis Radio immunoassay RIA ELISA
Dr.T.V.Rao MD 65

Serology
Since the isolation and identification of viruses is not commonly done in the clinical laboratory, the clinical picture and serology plays a greater role in the diagnosis of viral disease. The major types of antibodies that are assayed for are neutralizing, haemagglutination inhibiting and complement fixing antibodies. Complement fixing antibodies follow the kinetics of IgM and are most useful in indicating a current or recent infection
Dr.T.V.Rao MD 66

Serology
The development of antibodies to different components of the virus is used in staging the disease. For example in hepatitis B and HIV infections this approach is used.
Dr.T.V.Rao MD 67

Serological Diagnosis
Detection of Immunologlublins Ig G. Ig M Ig A Raise of titers Ist sample later sample (convalescent sample) tested after 10 14 days Raise of titer is diagnostic
Dr.T.V.Rao MD 68

ELISA for HIV antibody

Microplate ELISA for HIV antibody: coloured wells indicate reactivity

Dr.T.V.Rao MD 69

Western Blot
HIV-1 Western Blot
Lane1: Positive Control Lane 2: Negative Control Sample A: Negative Sample B: Indeterminate Sample C: Positive

Dr.T.V.Rao MD

70

Polymerase Chain Reaction

Advantages of PCR:
Extremely high sensitivity, may detect down to one viral genome per sample volume Easy to set up Fast turnaround time

Disadvantages of PCR
Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay.

A positive result may be difficult to interpret, especially with latent viruses such as CMV, where any seropositive person will have virus present in their blood irrespective whether they have disease or not.
.
Dr.T.V.Rao MD 71

Schematic of PCR

Each cycle doubles the copy number of the target

Dr.T.V.Rao MD

72

Growing Technologies
Molecular methods Probes Polymerase chain reaction Can produce Rapid Highly scientific Specific
Dr.T.V.Rao MD 73

Regular Methods in Use

Egg inoculation Pox virus, Influenza Into tissue culture
Dr.T.V.Rao MD 74

Advantages of Molecular Methods Increases Sensitivity and Specificity. PCR RT PCR

Dr.T.V.Rao MD

75

Antiviral Agents
Restricted spectrum No standardized in-vitro susceptibility tests Most inhibit replication. Cure depends on host immune system to eradicate. If patients are immunocompromized, may have recurrences. Many need to be activated by viral and cellular enzymes before exerting antiviral effect. Activity of enzymes and concentration of substrates will influence the efficacy.

Dr.T.V.Rao MD

76

Nucleoside Analogues
General Mechanism of Action 1. Taken up by cells 2. Converted by viral and cellular enzymes to the triphosphate form 3. The triphosphate form inhibits:
1. DNA polymerase 2. Reverse transcriptase 3. RNA polymerase

4. Or it may get incorporated into growing DNA leading to abnormal proteins or breakage.

Dr.T.V.Rao MD

77

Ganciclovir
Mechanism like Acyclovir Active against all Herpes viruses including CMV Low oral bioavailability given I.V. Most common adverse effect: bone marrow suppression (leukopenia 40%, thrombocytopenia (20%) and CNS effects (headache, behavioral, psychosis, coma, convulsions). Drug of choice for CMV infections: retinitis, pneumonia, colitis
Dr.T.V.Rao MD 78

Anti-retroviral Agents
Zidovudine (AZT) Cellular enzyme phosphorylate to the triphosphate form which inhibits RT and causes chain termination Adverse effect: Granulocytopenia and anemia: 45% in AIDS but 5% if asymptomatic HIV Severe headache, nausea, insomnia, myalgia mortality & opportunistic infections, gain weight, better quality of life, delays signs and symptoms of AIDS

Dr.T.V.Rao MD

79

Protease Inhibitors
Produce non-infectious particles or virions Reduces the number of new rounds of infection in susceptible cells To be effective must be prolonged, profound and constant. Pharmacokinetics important to maintain constant concentrations within the effective range. Metabolic adverse effects (DM, hyperglycemia) and GI (diarrhea, pain vomiting).

Dr.T.V.Rao MD

80

Drug Examples
Tamiflu Prevents the mature viruses from leaving the cell It is a neuraminidase inhibitor, it works on both influenza A and B

Neuraminidase is an enzyme found on the virus which cleaves sialic acid from cell membrane, leading to a more effective release of viruses. mechanism
Dr.T.V.Rao MD

Other Drugs
Amantadine Interferon's
Prevents uncoating (?) Antiviral, anticancer and &/or assembly immunomodulating CNS Toxicity due to Several sites of action in dopaminergic action viral cycle but mainly Prophylaxis of Influenza A inhibit translation of viral during epidemics. proteins If used within 48 hours Toxicity: flu-like may help cure Influenza syndrome, BM infection suppression; CNS Rimantadine: analog with less CNS toxicity Hepatitis B and C Dr.T.V.Rao MD 82

BEWARE VIRAL INFECTIONS CAN INFECT ANY ONE

Dr.T.V.Rao MD

83

Programme created by Dr.T.V.Rao MD for Medical Students in the Developing World

Email

doctortvrao@gmail.com

Dr.T.V.Rao MD

84