Trinity University of Asia College of Medical Technology

MIC112/SIM100 Lecture Handouts Prof. Jude Anthony Trinidad, RMT, MSMT, ASCPi CM)
de Guzman, Angelo Christian O. | 3MT01

RNA VIRUSES  Laboratory Methods

HEPATITIS VIRUSES o Indirect ELISA – Detect Anti-HEV IgM/IgG
 Inflammation of the liver o Real Time PCR – HEV RNA
 Jaundice
 Historical Perspective of Viral Hepatitis HEPATITIS B VIRUS S
 The only DNA Hepatitis
 Family: Hepadnaviridae
 Genotype: A to H (8)
 MOT: Parenteral, Sexual, and Vertical
 Chronic Infection
 Liver Cirrhosis/Liver Cancer
 Markers:
o HBsAg – Marker for Acute Infection; Aka Australian
Antigen
 Hepatitis B – First Hepatitis discovered (1963) by Blumberg
 The envelope protein consisting of 3
 Hepatitis A – Discovered in 1973
Polypeptides
 Hepatitis C – Most common NANB
o Anti-HBs – Marker of Immunity
 Types of Hepatitis
 Immunity to Hepatitis B Surface Antigen
HEPATITIS A B C D E
o Hepatitis B Core Antigen – Antigen targeted by Anti-HBc
Blood
Source Feces Blood-Borne Feces  Found in infected Hepatocytes (Liver Biopsy)
Body Fluids o Anti-HBc – Best marker against WINDOW PERIOD/Core
Permucosal Period (False (-))
MOT Fecal-Oral Fecal-Oral
Percutaneous
Chronic
o HBeAg – Marker of becoming very infectious
NO YES YES YES NO
Infection o Anti-HBe – Marker of Convalescence
Pre/Post
Pre/Post Pre/Post Blood Donor Ensure  HBV-DNA – Tested by Reverse Transcriptase PCR
Exposure,
Prevention
Exposure Exposure Screening,
Immunization,
safe  Tests for HBV
Immunizatio Immunizatio Risk Behavior drinking
Risk Behavior o 1st Generation – Ouchterlony Technique
n n Modification water
Modification o 2nd Generation
 Counterelectrophoresis
HEPATITIS A VIRUS S
 Rheophoresis
 Family: Picornaviridae  Complement Fixation
 Genus: Hepatovirus
o 3rd Generation – Most sensitive
 MOT: Fecal-Oral
 Reverse Passive Latex Agglutination
 Virus: Shed in the Feces
 ELISA
o Antigen is shed in the feces
 Reverse Passive Hemagglutination
 Self-limiting – the infection does not become chronic  Radioimmunoassay
 Aka infectious/short-incubation Hepatitis
 Diagnosis HEPATITIS D VIRUS S
o Hard to culture  Defective Virus – For Hepatitis D to survive/replicate, it needs
o Detection of Anti-HAV IgG/IgM Hepatitis B.
o Laboratory: RIA/EIA detect the presence of specific HAV  Delta Hepatitis
Antibodies  MOT: Parenteral, Sexual
o HAV RNA – Tested commonly from food and water  Co-infection and Superinfection
o Laboratory procedure to detect HAV Antigen or HAV RNA: o Co-infection: B and D simultaneously
 Real Time PCR o Superinfection: B first then D after
o Specimen: o Differentiated through Anti-HBc IgM
 Serum – Antibody will be detected (Anti-HAV)  Co-infection: (+)
 Feces: Antigen will be detected (HAV RNA)  Superinfection: (-)
 Laboratory Diagnosis
HEPATITIS E VIRUS S o Indirect Diagnosis
 Family: Herpeviridae  Indirect ELISA – Anti-HDV IgG/IgM
 Genus: Herpevirus  RT-PCR – HDV RNA
 Ingestion of contaminated water
 Fatal to pregnant women HEPATITIS C VIRUS S
 MOT: Fecal-Oral Route  Most common NANB
 Self-limiting  Post-transfusion Hepatitis
 Diagnosis:  Family: Flaviviridae
o Anti-HEV IgG/IgM  Genus: Hepacivirus
o HEV RNA  MOT: Parenteral (Blood Transfusion)

BS Medical Technology | Third Year, Second Semester | April, 2015 | Clinical Microbiology 112 and Clinical Immunology and Serology Final Period |Page 1 of 6
 Trinity University of Asia College of Medical Technology
MIC112/SIM100 Lecture Handouts Prof. Jude Anthony Trinidad, RMT, MSMT, ASCPi CM)
de Guzman, Angelo Christian O. | 3MT01

 Tests for HCV o GP 160

o Serologic Tests for Anti-HCV: EIA/RIA
o RIBA (Recombinant Immunoblot Assay)
 HCV RNA (Persistent HCV Infection)
Icosahedral Capsid
o TMA (Transcription Mediated Amplification)
P17 (17 represents Molecular weight in kilo
 Provide earlier results than serological methods Dalton)
3-Layered structure
VIRUSES WITH UNCERTAIN ASSOCIATION WITH HEPATITIS S
Enzyme – Reverse Transcriptase
 Torquetenovirus (TTV)
o ssDNA, Naked
o Genus Annellovirus
o Diagnosis: PCR
 Hepatitis G (Aka G8 Virus type, Hepatitis G Virus, GBV-C)  Main Structural Genes
o Family: Flaviviridae o ENV (Envelope)
o Enveloped ssRNA  GP 120 aid in the fusion and attachment of HIV
o MOT: Bloodborne, Perinatal, Sexual to CDH (+) Cells
o Diagnosis: RT-PCR, Detects HGV RNA o GAG Gene (Group Antigen)
 Retroviridae  P15, P17, P24 (found in Nucleocapsid)
o Has the Reverse Transcriptase Enzyme (capable of RNA  o POL (Polymerase)
DNA)  RT – Transcribe RNA to DNA
o Sub Families:  Integrase – Inserts viral DNA to host DNA
 Lentivirinae (HIV)  Protease – Cleaves Structural Protein located in
 Oncovirinae (HTLV-I, II, III) the core near the nucleic acid
 Other Facts
RETROVIRIDAE o Proenzyme
 Oncovirinae  Viral Genetic Material that is incorporated to
o HTLV-I the DNA of host cell
o HTLV-II o Spikes
o HTLV-III  Attaches to the hosts
 Lentivirinae  GP120 – Virus Receptor
o HIV  CD4 – Host Receptor
 T-Helper Cell
HUMAN IMMUNODEFICIENCY VIRUS S o T-Tropic
 Also known as  Or X4 Strain
o HTLV III (Human T-Lymphotropic Virus III)  Infects T-Cells
o LAV (Lymphadenopathy-associated virus) o M-Tropic
o ARV (AIDS-Associated Retrovirus)  Or R5 Strain
 Types of HIV  Infects Macrophages and T-Cells
o HIV-I (Global; Pandemic)  Mode of Transmission
o HIV-II (West Africa) o Sexual Intercourse (MSM)
 HIV-I o Parenteral (Needle)
o Pandemic Infection o Perinatal
 Luc Montagnier of France  RA 8504
 Robert Gallo and Jay Levy of USA o AIDS Awareness Act of 1994
o HIV-I subgroups o February 13, 1994
 Group M (Main/Major) o Information Dissemination
 Majority of HIV-I Infection worldwide  HIV targets T-Helper Cells
 Subtypes: A-D, F-H, J-K  Acquired Immunodeficiency Syndrome – end stage result of HIV
 Subtype C – Most predominant infection
subtype worldwide  Clinical Symptoms of HIV
 Subtype B – Most prevalent subtype o Primary Infection
in USA and Europe o Clinical Latency
 Group N (Non-M or Non-O; New Group) o AIDS
 Group O (Outlier Group)  Medicine for HIV (Combination of Drugs)
 Group N and O – Central Africa o HAART – High Active Anti-Retroviral Therapy
 Diagnostically-Important Proteins in HIV Markers o HIV – Prone to resistance
o P 24  Testing:
o GP 41 (Glycoprotein with 40 kilo Dalton) o CD4 Counting:
o GP 120  Gold Standard to count CD4

BS Medical Technology | Third Year, Second Semester | April, 2015 | Clinical Microbiology 112 and Clinical Immunology and Serology Final Period |Page 2 of 6
 Trinity University of Asia College of Medical Technology
MIC112/SIM100 Lecture Handouts Prof. Jude Anthony Trinidad, RMT, MSMT, ASCPi CM)
de Guzman, Angelo Christian O. | 3MT01

 Flow Cytometry o RT-PCR – Detect RNA

o Antigen and Antibody Detection
 Anti P24 SINGLE STRANDED RNA VIRUSES
 First Antibody to reproduce ARENAVIRIDAE
o Nucleic Acid Detection  “Arena” – sand
 Polymerase Chain Reaction o Sandy appearance
 Agents of Hemorrhagic fever (Platelets are affected)
 Zoonotic transmission (Contact and Inhalation)
 Diagnosis: ELISA (IgG/IgM)
 Reservoir: Rats, Hamsters
 Prevention: Vector Control
OLD WORLD NEW WORLD
Tacariber, Junin, Machupo,
Amapari, Cupixi, Parana, Latino,
Pichinide, Tamiami, Flexal,
 Tests for HIV Lymphocytic Choriomeningitis
Guanarito, Sabia, Oliveros,
o Screening test (LCM), Lassa virus (including
Whitewater Arroyo, Pirital, Bear
 ELISA – Most common Mopeia, Mobala, Ippy)
Canyon, Allpahuayo, Catarina,
 Radio Immunoassay Pampa, Skinner Tank, Chapare
 Agglutination test Virus
o Confirmation test  Junin – 1st virus that can cause Hemorrhagic fever
 Western Blot (+)  LCM – Causes influenza-like
 AKA Immunoblot  Lassa virus – most known Arenaviridae
 RITM – (+) Donor’s Blood testing
 SLH (SACCL – STD/AIDS Cooperative BUNYAVIRIDAE
Central Laboratory) – (+) Patient’s  Genus: Orthobunyavirus, Phlebovirus, Nairovirus
Blood Screening  MOT: Vector-borne (Mosquito)
 Notes  Agents of Hemorrhagic Fever, Encephalitis
o Testing is often done at 6 weeks, 3 months, and 5 months  Diagnosis: EIA to detect IgG/IgM, Immunohistochemistry for
after exposure to find out if a person is infected with HIV. Hantavirus (Antigen)
o The USA-CDC defines a positive test for HIV as a o Hantavirus not carried by Arthropods but by Rodents
“Repeatedly positive ELISA followed by a positive  Diseases:
Western Blot” test o Riff Valle Fever Virus
o No marker on Window Period (False Positive) o LaCrosse Virus (LCV) and California Encephalitis Virus
o High Risk Individual – Bisexual Male  Causes Summer flu/cold
 To Inactivate HIV and HBV  Common among children
o Bleach 1:10 o Crimean-Congo Hemorrhagic Fever
o 10 minutes for HBV  Most deadly
o 2 minutes for HIV  Targets the vascular endothelium and liver
o Hantavirus
DOUBLE STRANDED RNA VIRUSES  Urine, Stool, Saliva carries the infection, not the rodent itself.
REOVIRIDAE
 The only Double Stranded RNA Virus Hantavirus S
 Generally causes Hemorrhagic Fever with Renal Syndrome (HFRS)
GENUS Rotavirus S  Hantaan virus
 Most common cause of viral Gastroenteritis in infants and  Seoul virus
children  Puumala Virus (Nephropathia epidemica)
 MOT: Oral-Fecal  Dobrara virus
 Virus is shed in the stool  Sin Nombre virus (“No name”) – Hantavirus Pulmonary Syndrome
 Diagnosis
o Serologic tests CALICIVIRIDAE
 ELISA  General (Sapovirus, Norovirus, Lagovirus, Verivirus)
 Latex Agglutination Test  Newly proposed genera: Berovirus, Recovirus
 Species: Sapporovirus, Norwalk virus (Most common cause of
GENUS Coltivirus S Adult Encephalitis), Rabbit Hemorrhagic disease virus, Feline
 Dengue-like infection calicivirus
 Arbovirus – Arthropod-borne  Agent of Gastroenteritis
 MOT: Tick-borne (Dermacentor andersoni)  MOT: Food/Water-borne
 Diagnosis:  Diagnosis: Electron Microscope, RT-PCR
o Recombinant Immunoblot Assay (RIBA) – IgG

BS Medical Technology | Third Year, Second Semester | April, 2015 | Clinical Microbiology 112 and Clinical Immunology and Serology Final Period |Page 3 of 6
 Trinity University of Asia College of Medical Technology
MIC112/SIM100 Lecture Handouts Prof. Jude Anthony Trinidad, RMT, MSMT, ASCPi CM)
de Guzman, Angelo Christian O. | 3MT01

o Two ways:
 Genetic Reassortment
CORONAVIRUS  Adaptive mutation
 Genus Coronavirus INFLUENZA VIRUS S
 Looks like a crown  Types
 Cold-like infection in adults, Pandemic Respiratory Disease o H1N1- Spanish Flu; Swine Flu
o SARS-CoV – Severe Acute Respiratory Syndrome- o H2N2
Associated Coronavirus (Started in Asia [Hong Kong]) o H3N2 – Hong Kong Flu
o MERS-CoV – Middle East Respiratory Syndrome-Associated o H5N1 – Avian Flu; Bird Flu; Highly Pathogenic Avian
Coronavirus Influenza (HPAI)
 Reservoir: Bats  MOT: Aerosol Inhalation
 MOT: Direct contact, Droplet, Airborne Routes  Samples: Nasopharyngeal Swab, Washes, Aspirates
 Diagnosis: RT-PCR, Immunofluorescence, EIA  Diagnosis; EIA, Immunoassay, Grows in the Amniotic Cavity of the
Embryonated Chicken Eggs and various Mammalian cells
FILOVIRIDAE
 Genus: Marburgvirus (Aka Lake Victoria Marburg Virus), PARAMYXOVIRIDAE
Ebolavirus (Zaire [most virulent], Sudan, Reston, Bundibugyo, Tai  Genera: Morbilivirus, Paramyxovirus, Pneumovirus, Rubulavirus
Forest Strain)  2 Surface Antigens; Hemagglutinin-Neuramidase (HN) and Fusion
o Marburgvirus – first isolated in Marburg, Germany (F)
 Unknown reservoir  Agents of Repsiratory Disease in Children; Croup
o But scientists believe that it came from Monkeys (Tracheolaryngobronchitis)
 Agent of Hemorrhagic Feevr  MOT: Aerosol Inhalation; Direct Contact
 Diagnosis: PCR, Immunohistochemistry, IgM capture ELISA  Sample for culture: Aspirated Secretions, Nasopharyngeal Washes
 Type 1, 3 – Paramyxovirus
FLAVIVIRIDAE  Type 2, 4 – Rubulavirus
 Arbovirus (Mosquito)
 Genus: MUMPS S
o Japanese Encephalitis virus (most prevalent in Asia)  Genus: Rubulavirus
o Dengue virus  MOT: Droplets of infected saliva
o Yellow virus (Causes Jaundice)  Swelling of the Parotid Glands (Testes, Ovaries, Pancreas)
o St. Louis Encephalitis virus (by Culex spp.)  Immunization: MMRV
o West Nile Virus (Host: Birds; Dead-End hosts: Humans)  Causes Infertility
 Agents of Encephalitis and Hemorrhagic Fever
 Diagnosis: EIA, IFA, RT-PCR MEASLES (RUBEOLAVIRUS) S
 Transmission cycles  Genus: Morbilivirus
o Sylvatic – Mosquito to Monkeys  MOT: Direct contact with Aerosolized droplets
o Urban – Mosquito to Human  Presence of Koplik Spots (spots in the mouth)
o Intermediate – Human, Mosquito, Monkeys  Diagnosis: Measles-specific IgM, RT-PCR

Dengue virus S RESPIRATORY SYNCTIAL VIRUS (RSV) S

 Causes Dengue fever (DF), Dengue Hemorrhagic Fever  Genus: Pneumovirus
(DHF), and Dengue Shock Syndrome (DSS)  Causes: Croup, Bronchitis, Pneumonia
 Vector: Aedes aegypti, Aedes albopticus  Identified from Nasopharyngeal Swabs and Washes
 4 Serotypes: 1,2,3,4  CPE: Synctia (Clumping)
 Treatment: Supportive treatment
HUMAN METAPNEUMOVIRUS S
 Low Platelet counts
 Respiratory Infection similar to RSV
 Prevention: Vector control (Removal of Stagnant Waters)
 Also known as Break-bone fever
PICORNAVIRIDAE
o Patients suffer from Bone Pain
 Smallest RNA viruses
 Significant genera: Enterovirus, Rhinovirus, Hepatovirus,
ORTHOMYXOVIRIDAE
Parechovirus
 Genera: Influenza A, B, C
 2 Major Structural Proteins: Matrix (M), Nucleoprotein (N)
ENTEROVIRUS S
 H Antigen (Hemagglutinin): Used to bind host cells
 Species:
 N Antigen (Neuraminidase): Cleaves budding
o Poliovirus 1 to 3 (Patient is suffering from Atrophy)
 Antigenic Drifts: Minor change in Antigenic Structures
o Coxsackievirus A1 to A23
o Common among Influenza A, B, C
o Coxsackievirus B1 to B6
 Antigenic Shifts: Drastic change
o Enterovirus 68 to 104 (HEV A, HEV B, HEV C, HEV D)
o Only Influenza A can do this
o Echovirus 1 to 33

BS Medical Technology | Third Year, Second Semester | April, 2015 | Clinical Microbiology 112 and Clinical Immunology and Serology Final Period |Page 4 of 6
 Trinity University of Asia College of Medical Technology
MIC112/SIM100 Lecture Handouts Prof. Jude Anthony Trinidad, RMT, MSMT, ASCPi CM)
de Guzman, Angelo Christian O. | 3MT01

o Parechovirus 1 to 4 o Arenaviridae
 MOT: Inhalation, Fecal-Oral Routes, Fomites o Deltavirus
o Paramyxoviridae
RHINOVIRUS S o Rhabdoviridae
 Resistant to: Detergents, Lipid Solvents, and Temperature o Orthomyxoviridae
Extremes o Filoviridae
 Sensitive to: pH lower than 6 EPSTEIN BARR VIRUS
 Major cause of Common Cold  Heterophile Antibody – Refers to Antibodies the body produces as
part of an immune response to an infection but that are not
RHABDOVIRIDAE related to the causative agent.
 Genus: Lyssavirus (Bullet-shaped virus) o Types:
 Agent of Rabies  Heterophile Antibody in Infectious
 MOT: Bitten or Scratched by Rabid Animals (Dogs, Mononucleosis – Significant
Raccoons, Skunks, Foxes, Bats)  Produced in response to EBV infection
 Targets the Central Nervous System  Heterophile Antibody in Forssman – Produced
 Direct IF technique in response to incidental contact with
 If bitten, chop off the head of the Rabid Animal and send to Heterophile Antigens such as Salmonella,
a reference laboratory (RITM, SLH) Shigella, and other Bacterial spp.
 CPE: Negri Bodies  Heterophile Antibody in Serum Sickness –
Produced in response to parenteral injection of
TOGAVIRIDAE
Horse Serum/Products from Horse during
 Genus: Alphavirus, Rubivirus, Arterivirus
immunization
 Agents of Encephalitis
o Heterophile Antibody to IM
o Eastern Equine Encephalitis (EEE)
 Reacts with Sheep Cells, Ox (Beef) Cells, and
o Western Equine Encephalitis (WEE)
Horse Cells but not with Guinea Pigs
o Venezuelan Equine Encephalitis (VEE) o Heterophile Antibody to Forssman
 Reacts with Sheep Cells, Horse Cells, Guinea Pig
RUBELLA VIRUS S
Cells but not with Ox (Beef) Cells
 Genus: Rubivirus
o Heterophile Antibody to Serum Sickness
 Agent of Rubella/German Measles/Congenital Rubella
 Reacts with Sheep Cells, Ox (Beef) Cells, Horse,
Syndrome
and Guinea Pig Cells
o German Measles – AKA 3-day Measles
o Rashes – most common manifestation of vital infection TESTS FOR INFECTIOUS MONONUCLEOSIS S
 MOT: Respiratory Droplets, Transplacental Infection  Paul Bunnel Test
 Diagnosis: Hemagglutinin Inhibition, Complement Fixation o Screening/General Test
test, Immunoassays (ELISA) o Principle: Hemagglutination
o Reagent: 2% Suspension of Sheep RBCs
o (+) Result: Agglutination
 Davidson Differential Test
o Principle: Absorption-Hemagglutination
o Antigen: Guinea Pigs Cells and Beef RBCs
 IM will React (Guinea Pig)
 Forssman will React (Beef)
o Indicator Cells: Sheep RBCs
o Confirmatory Test/Identification Test
Absorption Pattern
Heterophil Beef RBCs Guinea Pig Cells
Forssman No Yes
IM Yes No
Serum Sickness Yes Ni
Agglutination Pattern
After absorption with
After absorption with
Heterophil Guinea Pig, Kidney
Beef Cells
Cells
Forssman High Titer Low Titer
SUMMARY IM Low Titer High Titer
 ALL are RNA Positive Strand except: BAD PROF! Serum Sickness Low Titer Low Titer
o Bunyaviridae  Monospot (Slide Method)

BS Medical Technology | Third Year, Second Semester | April, 2015 | Clinical Microbiology 112 and Clinical Immunology and Serology Final Period |Page 5 of 6
 Trinity University of Asia College of Medical Technology
MIC112/SIM100 Lecture Handouts Prof. Jude Anthony Trinidad, RMT, MSMT, ASCPi CM)
de Guzman, Angelo Christian O. | 3MT01

o Principle: Absorption-Hemagglutination
o Indicator Cells: Horse RBC (More sensitive indicator if
Antibodies found in IM)

BS Medical Technology | Third Year, Second Semester | April, 2015 | Clinical Microbiology 112 and Clinical Immunology and Serology Final Period |Page 6 of 6