VIROLOGY
General characteristics
Acellular
Ultramicroscopic
Obligate intracellular parasites
Unable to replicate (multiply) on their own
Lack the genes and enzymes
Depend on the ribosomes, enzymes, and metabolites
Filterable
Virus and bacteria table for comparison
Host range is determined by – “viruses ability to interact with host cell
Binding sites on viral capsid or envelope combine with receptor sites on host
cell membrane
1. Nucleic acid
2. Capsid (protein coat) [composed of many small protein
units called capsomeres]
Nucleic acid + capsid = the nucleocapsid
Some capsids are surrounded by envelope
Virions are complete, fully developed viral particles composed of nucleic acid
surrounded by a coat.
Three types
1- Helical
2- Polyhedral
3- Complex
VIRAL CAPSID: composed of small protein subunits called capsomers. The
arrangement of the capsomers determines virus symmetry.
FUNCTIONS :
1. It protects the viral genome
2. It is responsible for the structural symmetry of the virions
3. It participates in attachment of the virions to susceptible cells
4. Capsid proteins are important antigens. (immunity)
Basic structural forms
1. Naked icosahedral [e.g. poliovirus, adenovirus, hepatitis A virus]
2. Naked helical [e.g. tobacco mosaic virus ]
3. Enveloped icosahedral [e.g. herpes virus, yellow fever virus, rubella
virus]
4. Enveloped helical [e.g. rabies virus, influenza virus, parainfluenza virus,
mumps virus, measles virus]
5. Complex [e.g. poxvirus]
Bacteriophage : Viruses that use bacterial cell as a host
- Area of lyses called a plaque on the surface of the agar. Titer: concentration of the virus in the suspension
Three methods used for culturing animal viruses: 1. Living animal
2. Embryonated eggs 3. Cell culture
Specimens are are mostly contaminated 1. Stool
2. Sputum 3. Urine
LEC 2
Western blot : identification of antibodies in patient serum. Negri bodies : Found in rabies infection.
Rota virus and Adeno virus, sample is taken from stool. HSV, VZV, sample is taken from vesicle fluid
Papiloma virus, sample is taken from skin scrapings
VIRUS SEROLOGY:
Used to confirm the diagnosis when the virus cannot be cultivated. We use IgM, but is undetectable 1-4 months after acute infection resolves
Criteria for diagnosing primary infection.
-Presence of igm and sero conversion.
Criteria for diagnosing re infection
-Absence or slight increase in IgM -Extremely high IgG.
VIRAL HEMAGGLUTINATOIN:
Idea of test : works without antigen antibody reaction, so its natural. Main function is to detect the presence of viral particles. The test does not
discriminate between viral particles that are infectious and particles that are degraded and no longer able to infect cells.
Viruses that can be detected this way : Mumps, measles and influenza Influenza has viral hemagglutinatin protein. This binds to receptors on RBC
surface on sialic acid.
Very sensitive and can detect very small amount of antigen
NEUTRALISATION REACTIONS
Is an antigen antibody reaction that involves formation of specific antibodies ( called antitoxins) to neutralize the harmful effect on bacterial exotoxins.
Complements are a group of serum proteins that bind to antigen antibody complex, they are either used up or fixed..
If they are fixed : Complement fixation reactions.
They are based on depletion of a fixed amount of complement in the presence of antigen antibody reaction.
Useful in two conditions
1: Very small amounts of antibody
2: when amount of antibody is too low to cause precipitation or hemagglutination reactions.
Now a days it is used for diagnosis of viral, fungal and rickettsia diseases
FLORESCENT ANTIBODY TECHNIQUES ( LABLED REACTIONS )
To identify of antigens in clinical specimens or detect the presence of specific antibody in serum.
The procedure is quick, sensitive, and very specific.
They use antibodies labeled with florescent dyes ( isothiocynate ) to visualize them under UV light.
Florescent antibody test are two types:
1: Direct florescent antibody test
Eg: rabies virus ( LYSSA VIRUS ) (rhabdo virus family) 2: indirect florescent antibody test
Eg: identification of tripoima palladium
ENZYME LINKED IMMMUNO SORBANT ESSAY (ELISA)
IDEA: Use antibodies linked to an enzyme. The enzyme is use to detect antigen antibody reactions.substrate is added to determine if the enzyme is linked to antibody. If yes the substrate is conmverted to a product that
causes a colour change.
Enzymes used
1: horse radish perixodase\ 2: alkaline phosphatase.
Two basic methods
1: direct ELISA: for detection of antigens 2: indirect ELISA: for detection of antibodies.
VIRAL IDENTIFICATION BY MOLECULAR METHODS
1: Nucleic acid detection.: uses nuclic acid probes – short segments of DNA
complementary to viral DNA or RNA. Best used when the amount of virus used is abundant.
EG: Papilloma virus in cervical cells.
2: Nucleic acid amplification (pcr): used for amplification of short sequence of
a target DNA or RNA
Lec 3
DOUBLE STRANDED ENVELOPED DNA VIRUS
General
Herpes viruses : -
Have the ability to establish latent infection.
Simplexvirus (HHV1 and HHV 2), (HSV) type 1 & type2
Varicella –Zoster virus (HHV 3)
Epstein-Barr virus (HHV 4)
Cytomegalovirus (HHV 5)
Roseolovirus (HHV 6), (SIX DISEASE)
HHV 7 (T-lymphotropic virus)
Kaposi's sarcoma (HHV 8)
Subfamilies:
α- Herpesviridae: Herpes simplex 1 & 2 Varicella –Zoster virus
β- Herpesviridae: Cytomegalovirus & HHV 6 & 7
δ Herpesviridae: Epstein-Barr virus & HHV 8
Herpes Viruses
Morphology
:
Double strand DNA
Icosahedral
Nucleocapsid is surrounded by a lipid envelop derived from cell membrane
Contain glycoprotein spikes.
Replication and assembly of capsid takes place within the nucleus
Recurrence can be triggered by
:
• Exposure to ultraviolet radiation
• Emotional upset
• Hormonal change associated with menstruation
• Heat
• Fever
HERPES VIRUSES
HSV-1
• Mainly transmitted by contact with the HSV-1 virus found in cold sores, saliva, and
surfaces in or around the mouth and lips.
• Also be transmitted through oral sex to cause genital herpes.
• Those affected by HSV-1 are unlikely to be affected by HSV-2 in the genital area.
• Mostly occur during childhood. Infection is lifelong.
• NOTE: A person with herpes dosent have to have symptoms to spread the virus to
someone else.
• Infection: usually in infancy.
ORAL FACIAL
HERPES:-1: Acute gingivistomatitis:
most common manifestation of primary herpetic
infection.
• Pain and bleeding in gums.
• Ulcers with necrotic base present.
• Enlarged neck glands.
• Self limiting that lasts 13 days.
2: Herpes labialis (cold sore):
following primary infection, 45% experiences
reactivation.
• Is a recurrence of oral HSV.
SKIN
INFECTION:-• Herpetic whitlow: HSV-1 can be transmitted by skin contact
EYE
INFECTION:-• Herpetic Keratitis
HERPES
ENCEPHALITIS:-• HSV-2 could be more serious
• Virus may reach the brain during viraemia
• Olfactory tract and Trigeminal ganglia affected.
HERPES VIRUSES
HSV-2
• Differentiated from HSV-1 by it’s antigenic makeup, and by it’s effect on cells in
tissue culture, serology (anti -2 antibodies), PCR.
• Latent in the sacral nerve ganglia found near the base of the spine.
• Transmitted primarily by sexual contact
• Cause genital herpes
• Often has no symptoms, or mild symptoms that go unrecognized
• Incubation is one week
• Pregnant women who have genital herpes can transmit hsv-2 or hsv-1 to their
infant, through contact with the virus during delivery.
• The lesions of genital herpes are particularly prone to secondary bacterial infection
eg. S.aureus, streptococcus, trichomonas and candida albicans.
• Dysuria is a common complaint, in severe cases, there may be urinary retention.
• Vesicles contain infectious fluid.
• Semen may contain the virus.
• The virus enters a lifelong latent state in nerve cells.
NEONATAL HERPES SIMPLEX (1)
• The baby is usually infected perinatally during passage through the birth canal.
• Premature rupturing of the membranes is a major risk factor.
• The risk of perinatal transmission is greatest when there is a florid primary infection in
the mother.
• Smaller risk from recurrent lesions in the mother, probably because of the lower viral
load and the presence of specific antibody
• The virus can cross the placental barrier and affect the fetus.
• Spontaneous abortion or serious fetal damage
• Particularly dangerous in premature infants
• HSV infection varies from a mild disease localized to the skin to a fatal
disseminated infection.Organs most commonly involved are the liver, adrenals and
the brain.
• Brain is involved, the prognosis is particularly severe. The encephalitis is global
and of such severity that the brain may be liquefied.
• Survivors of neonatal HSV infection have residual disabilities.
• Acyclovir should be promptly given in all suspected cases
• Prevention :- Caesarean section to mothers with florid genital HSV lesions.
DIAGNOSIS:-• Laboratory Diagnosis
• Specimen: vesicular fluid, swab from base of the ulcer.
• Electron microscope
• Immunofluorescence of skin scrapings - can distinguish between HSV and
VZV
• Virus isolation
• Serology
• ELISA
• PCR
PREVENTION:-• Should abstain from sexual activity whilst experiencing symptoms of genital herpes
• Hsv-2 is most contagious during an outbreak of sores, but can also be transmitted
when no symptoms are felt or visible.
• Use of condoms
• Medical male circumcision can provide men life-long partial protection against hsv,
hiv, and human papillomavirus (hpv)
TREATMENT:-• Antivirals, such as acyclovir, famciclovir, and valacyclovir are the most
effective medications
• help to reduce the severity and frequency of symptoms but they cannot cure
the infection.
VARICELLA ZOSTER
CHICKENPOX (VARICELLA) &
SHINGLES (HERPES ZOSTER)
• Infection with vzv presents in two forms
• The primary infection varicella (chickenpox), is a generalized eruption.
• The reactivated infection zoster (shingles) is localized to one or few dermatomas
• Most common in childhood, highest prevalence occurring in the 4 - 10 years old
age group.
• Highly communicable
VARICELLA ( CHICKENPOX )
• Varicella-virus is transmitted by the respiratory route and is localized in skin
cells, causing a vesicular rash 3-4 days.
• Is a primary infection
• Incubation 18-21 days
• Cause chickenpox.
• The virus then moved to the dorsal root ganglion near the spine, where it
remains latent indefinitely.
• The disease is usually mild
• Complications include encephalitis, pneumonia, and Reye's syndrome.
• Later , usually in late adulthood, the latent virus becomes reactivated, causing
shingles.
• Reactivation can be caused by stress or weakening of the immune system
SHINGLES (HERPES ZOSTER)
• Secondary infection.
• Transmission, re-infection, re-activation
• Virus can remain latent in nerve cells and subsequently activate as shingles.
• Characterized by
• Band of rose or band of hell
• Very painful
• Treated with Acyclovir AND An attenuated live vaccine is available
CONGENITAL VZV INFECTION
• Most pregnant women already immune so primary infection is rare during
pregnancy.
• Primary infection during pregnancy carries a greater risk of severe disease, in
particular pneumonia.
• Mostly occurs during the first 20 weeks of Pregnancy
Clinical signs:
• Scarring of skin
• Hypoplasia of limbs
• CNS and eye defects
• Death in infancy normal
NEONATAL VARICELLA
• VZV can cross the placenta in the late stages of pregnancy to infect the fetus
congenitally
• Vary from a mild disease to a fatal disseminated infection.
• If rash in mother occurs more than 1 week before delivery, then sufficient
immunity would have been transferred to the fetus.
• Zoster immunoglobulin should be given to susceptible pregnant women
• Zoster immunoglobulin should also be given to infants whose mothers develop
varicella during the last 7 days of pregnancy or the first 14 days after delivery
LABORATORY DIAGNOSIS
• Clinical presentations are so characteristic that laboratory confirmation is rarely
required.
• Laboratory diagnosis is required only for atypical presentations, particularly in
the immunocompromised.
• Virus isolation: requires 2-3 weeks for a results.
• Direct detection: electron microscopy may be used for vesicle fluids but cannot
distinguish between HSV and VZV
• Immunofluorescence: On skin scrappings can distinguish between the two.
• Serology: The presence of VZV IgG is indicative of past infection and
immunity.
Lec 5 HEPATITIS •
•
• 5 different viruses = Hepatitis A, B, C, D, & E
• Hepatitis may result from infection with other viruses, Epstein-Barr virus
(EBV), and cytomegalovirus (CMV).
•
HEPATITIS A
Hepatitis A virus is the causative agent
HAV=Picornaviruses family, non envelope, ss -RNA icosahedra virus, it can be grown in cell culture.
HAV is able to survive the body’s highly acidic digestive tract and can live outside the body for months.
High temperatures kill the virus, although freezing temperatures do not. Its resistant to chlorine disinfectants.
Oysters are also a source of infection
HAV can survive for several days on surfaces (cutting boards.)
Transmission: from subclinical infected persons
the initial symptoms are : Anorexia,, nausea , diarrhea ,abdominal
discomfort, fever, chills and in some cases there is also jaundice ,dark urine and clay-colored stool.
No chronic Hepatitis A.
HAV may be shed in feces for 10 days before clinical symptoms appear.
the virus is not linked to liver cancer.
Diagnosis is based on tests for IgM antibodies, (ELISA) They appear about 4 weeks after infection
Recovery results in lifelong Immunity
Transmission : Oro-fecal HEPATITIS
Diagnosis: Three serologic markers available: 1. Hepatitis A Total (IgG and IgM) antibody
2. Hepatitis A IgM 3. Hepatitis A IgG
Detecting IgM anti-HAV in the serum of a patient
Liver biopsy is not indicated
Testing for anti-HAV IgG is not helpful in the diagnosis
HAV antigen can be detected in the stool or body fluids
Treatment: HAV – no specific treatment as it will often resolve itself spontaneously
Passive immunization (immunoglobulin) can provide temporary protection
All HAV vaccines contain inactivated (killed) virus = HAVRIX
Havrix is recommended as 2 injections 6-12 months apart Hepatitis B
Hepatitis B (Serum Hepatitis)
Hepatitis B virus (HBV) is the causative agent of hepatitis. HBV is classified as
hepadna virus.
HBV is large double-strand DNA envelope virus.
It passes through an intermediate RNA stage using viral reverse
transcriptase resembling retrovirus.(HBV uses reverse transcriptase to produce its DNA from m RNA)
Hepatitis B virus can survive outside the body at least 7 days
Transmitted by blood, and body fluids.
The serum from patient with HBV contain three distinct particles:
o 1- Dane particles (largest) is the complete virion, it is infectious and capable of replicating.
o 2- Spherical particles (smaller). [enveloped particle] o 3- Filamentous particles (tubular enveloped particle]
Spherical & filamentous particles are unassembled component without
nucleic acid, (non-infectious). They contain hepatitis B surface antigen
(HBSAg),{envelope}.
The antibody tests make convenient screening of blood for HBV.
Transmission : by contaminated syringes, and semen (donated for artificial insemination).
HBV is not spread through food or water, sharing eating utensils, breastfeeding, hugging, kissing, hand holding, coughing, or sneezing
HBV is transmitted by blood transfusions through sharing of razors and toothbrushes.
Intravenous drug users. Blood may contain billion viruses per/ milliliter. (body fluids)
Mothers positive for (HBSAg), may transmit the disease to her infant, usually
at birth
After entering the blood the virus infect hepatocytes (specific receptors).
The average incubation period is 3 months
recovery is usually complete, but some patients develop a chronic infection or become carriers.
If (HBSAg), persist for more than about 6 months , it is an indication of
chronic HBV hepatitis
Loss of appetite, fever, joint pains and jaundice.
90% of the acute end in complete recovery.
10% become chronic carriers of HBV.
Approximately 90% of infected infants will develop chronic infection.
25-50 % in children
Diagnosis , detection of hepatitis markers antigens, (Ag) and antibodies in the blood by ELISA.
Carriers are reservoir for transmission of the virus, and they also have a high rate of liver diseases.
Liver cancer / chronic HBV infection.
Chronic carriers are 200 times more likely to develop liver cancer
Serological testing:
Hepatitis B surface antigen (HBsAg): can be detected in the serum
HBsAg is present in serum during acute infections and persists in chronic infections.
The presence of HBsAg indicates that the person is potentially infectious.
HBsAg is the antigen used to make hepatitis B vaccine.
HBcAg is not detected in the blood. It is detected only in the nuclei of liver cell
HBeAg appears during I.P. shortly after appearance of HBsAg. Its presence indicates that person is highly infectious
Hepatitis B serologic testing involves measurement of several hepatitis B virus (HBV)-specific antigens and antibodies.
•
2- Anti (HBe)
•
Begins to rise at end of acute stage, appears after anti-HBc
and its presence correlates to a decreased infectivity
•
3-Antibody to HBsAg (anti-HBs)
- resolve acute HBV infection = indicates immunity.
The persistence of HBsAg for 6 months after the diagnosis of
acute HBV is indicative of progression to chronic HBV infection}.
Anti - HB
c(Antibody to HBV core antigen):
Total - indicates past or active infection; present whether
person is immune or chronic carrier
No antigen test
HB
eAg (Hepatitis B
eantigen):
indicates person is highly infectious
Anti-HB
e(Antibody to HBV
eantigen):
prognostic for resolution of infection; less infectious
Acute patients = rest. Chronic patients may be given interferon Viral DNA polymerase inhibitors or pegylated alpha interferon.
PEG : (polyethylene glycol) to make interferon last long in the body
Liver transplantation is often a final option in treatment.
Prevention
1-Precautions. 2-Screening of blood. 3- Vaccine against HBsAg is available. A recombinant subunit vaccine has been made for the hepatitis B virus.
Scientists inserted hepatitis B genes that code for important antigens into
common baker’s yeast.
HEPATITIS C:
Hepatitis C virus (HCV) is a member of Flaviviridae family Single stranded RNA virus, enveloped.
(HCV) is transmitted via blood, or body fluids.
Sharing needles IDU (80% ) infected with HCV.
appearance of detectable HCV antibodies takes 70-80 days
The virus does not kill the cell , but immune reaction can damage the liver cell.
Six different genotypes (genetic variation), besides it is difficult to be cultured
Hepatitis (HCV) causes both acute and chronic infection. Acute HCV infection is usually asymptomatic,
About 15–45% of infected persons spontaneously clear
The remaining 55–85% of persons will develop chronic
chronic HCV infection, the risk of cirrhosis or cancer
Symptoms may take 20 years to appear.
HCV can survive outside the body at room temp. for up to 3 weeks
Diagnosis of hepatitis is made by biochemical assessment of liver function
HCV infection is diagnosed serologically in 2 steps:
Screening for anti-HCV antibodies with a serological test identifies people who have been infected with the virus.
If the test is positive for anti-HCV antibodies
needed to confirm chronic HCV infection
After a person has been diagnosed with chronic hepatitis C infection, they should have an assessment of the degree of liver damage (fibrosis and cirrhosis).
Treatment: Interferon & ribavirin.( Guanine inhibitor)
oral directly acting antiviral agent (DAAs) therapies (targeted against viral protein, (protease)
major reason for Liver transplantation
Prevention
minimizing exposure to sharing of items such as razors, toothbrushes, & nail clippers.
Person with HCV should be vaccinated against HAV,HBV (Twinrix). Havrix is a HAV vaccine.
Having had hepatitis C once does not make you "immune" from getting hepatitis C again No vaccine
HEPATITIS D
Hepatitis D virus (HDV). discover in carriers of HBV. Hepatitis D virus (HDV) has a circular single strand of RNA, and is not able to cause an infection.
It becomes infectious when an external envelop HBsAg,(HBV)
whose formation is controlled by the genome of HBV , cover the HDV protein core (the delta antigen).
Can only infect individuals with HBV. Transmission is via blood or body fluids
Can occur as either acute (coinfection form) or chronic (superinfection form).
In chronic HBV, chronic HDV was often accompanied by progressive liver damage
HEPATITIS E
Hepatitis E virus (HEV).
Calciviridae.
Non-enveloped single strand RNA virus.
Similar to HAV, but not related serologically.
Hepatitis E virus (HEV) is spread by the
fecal-
oral route.
Does not cause chronic liver disease.
Hepatitis G (HGV)
1) Similar to HCV
2) About 20% of HCV patients have HGV
3) Is more prevalent than HCV.
•
Molecular assays
o Commercial assays for HBV DNA and HCV RNA
(must be separated from cells)
o In-house assays for HAV RNA & HDV RNA
o No molecular assay for HEV RNA
Lec 6:
Viral Diseases of the Respiratory System
1.
Influenza v.
2.
Parainfluenza v.
Ciliary escalator of the lower respiratory keeps it STERILE !
Microbes in the lungs can be phagocytized by alveolar macrophages.
Respiratory mucus contains IgA antibodies
Common cold is cause by : Rhinovirus or Coronavirus
Rhinovirus = Picornaviridae, single-strand RNA, non-enveloped virus. Grow best slightly below
body temperature.
A sever complication of bronchitis is pneumonia
Respiratory Synctial Virus:
Paramyxovirus
The most common viral respiratory disease in infants; 4,500 deaths annually.
Causes cell fusion (syncytium) in cell culture
Symptoms: Pneumonia in infants
Sample nasopharyngeal swab/wash
Diagnosis: Serological test for viruses and antibodies. CPE in C.C, E.M
No haemagglutinin, No heamolysin, No growth in E.E
Treatment: Ribavirin. •
Influenza virus
• Influenza viruses are members of the family Orthomyxoviridae. • Three types of influenza virus are known; A, B & C Influenza.
• A- causes worldwide Influenza epidemics every 10-12 years (pandemics) and outbreaks every year.
• B- causes outbreaks, but less often than A. • C- cause mild R.D.
• The influenza virus is an infectious disease of birds and mammals caused by RNA viruses.
• Commonly confused with a cold, the flu is a much more severe disease. • incubation time of 2-3 days.
• mortality, very young and very old
Illness Influenza Common cold
Clinical spectrum Often systemic Mostly local
Speed of onset Abrupt Gradual
Fever Usually high Usually low-grade Presentation Chills, , malaise, sore
throat
Sneezing, sore throat,
nasal congestion
Fatigue Marked Mild
Course Unwell for 1-2 weeks, chest problems
common, severe malaise
Complications More common and often severe -
pneumonia
Uncommon
Occurrence Seasonal All year round
• numerous projections that characterize the virus. (Spikes) There are two types of projections:
Hemagglutinin (HA) spikes. Neuraminidase (NA) spikes.
Helical nucleocapsid
Haemagglutinin
about 500 on each virus
allow the virus to:
Recognize, and attach to body cells
HA binds to cell surface receptor (sialic
acid) to initiate infection
responsible for pathogenicity of the
virus).
Main determinant of immunity.
Neuraminidase
about 100 per virus
allow the virus to:
Separate from the infected cell as the virus exit after
intracellular reproduction.
Shift (only for influenza A).
Drift (both influenza A and B)
Viral strains are identified by variation in HA & NA antigens.
Change in HA and NA determine the antigenicity of the virus.
Influenza A- virus ared found circulating in birds, human, swine & horses
antigenic shifts: Each number change represents a substantial alteration in the protein makeup of the spike.
Antigenic shifts are probably caused by: Major genetic recombination
Recombination is likely in infections caused by more than one strain.
genetic change process is called Reassortment.
Swine is a good mixing vessels in which recombination, reassortment can occur.
Antigenic shift: Probably due to genetic recombination between different strains infecting the same cell.
Changes in HA and NA spikes so is the cause of recurring epidemics
Antigenic shift, however, occurs only in influenza virus A because it infects more than just humans.
Virus strains preferentially bind to sialic acids alpha (2,3) linkage. This is the major sialic acid on epithelial cells of the duck gut.
major type of sialic acid present on human respiratory epithelial cells.
efficient human to human transmission requires that the avian viruses recognize sialic acids with alpha(2,6) linkages.
Epithelial cells of the pig trachea produce both alpha(2,3) and alpha(2,6) linked sialic acids. This is believed to be the reason why pigs can be infected with both avian and human
Global herd immunity to the new virus is usually very low and this can result in a new flu pandemic.
This phenomenon only occurs with influenza A.
Only H1, 2 and 3 and N1 and N2 subtypes circulate widely in human.
Epidemic: human-to-human spread of the virus into at least two countries in one WHO region
Pandemic: human-to-human spread of the virus with community level outbreaks in at least one other country in a different WHO region than initial epidemic.
