UNIVERSITY OF SANTO TOMAS

FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

CLINICAL VIROLOGY

TERMINOLOGIES AND BASIC PRINCIPLES

֍ VIRUS
 Obligate intracellular organism that contain only kind of nucleic acid as their genome
- The nucleic acid is encased in a protein shell (CAPSID) which may be surrounded by a
lipid membrane (ENVELOPE)
 Smallest infectious agent (from 20 to 300 nm in diameter)
- Smallest Virus : Picornaviridae
- Largest Virus : Poxviridae
 Highly Dependent on hosts
- Viruses do not have a metabolic system
֍ VIRION
 Complete viral particle
 A virus with its genome enclosed by the capsid (NUCLEOCAPSID) and its envelope
 In certain instances, synonymous to NUCLEOCAPSID
- Applicable to NAKED Viruses such as the Papillomavirus and the Picornavirus
֍ GENOME
 Genetic material composed of DNA or RNA
 May be Single Stranded (in most RNA virus) or Double Stranded (in most DNA virus)
֍ POLARITY
 The genome possessed by the virus may be of the messenger type (Sense or + strand) or a
complimentary strand (antisense or – strand)
 Sense / + Strand
- Nucleic Acid runs from 5’ to 3’ hence +ssRNA can readily be translated into viral proteins
 Antisense / - sense
- Nucleic Acid runs from 3’ to 5’ (complimentary to a mRNA) which cannot be translated
immediately by the host cell
- Needs RNA-dependent RNA polymerase packaged inside the virion
- The RNA polymerase will transcibe the antisense into a sense strand before it can be
translated by the host cell
֍ PEPLOMERS
 Viral-encoded glycoproteins found as projections on the surface of envelopes
֍ HELICAL VIRUS
 Shaped like a hollow protein cylinder which may be rigid or flexible
֍ ICOSAHEDRAL VIRUS
 Polyhedron with 20 triangular sides (capsomeres)
֍ COMPLEX VIRUS
 Capsid symmetry but not purely icosahedral nor helical
 Example are Bacteriophages and Poxviruses
֍ ECLIPSE PERIOD
 Infectious virion is not seen

VIRAL REPLICATION

֍ For replication to happen, the viral genome must be able to produce a functional mRNA to be able to
synthesize viral proteins by the host cell protein-synthesizing machinery.
֍ Outcomes
 Productive infection – successful production of PROGENY
 Lytic Infection – progeny released via cell lysis
 Persistent infection – either no lysis involved or there is a reservoir cell
 Nonproductive Infection
 Latent infection – maintains viral genomes stably in host cell; no progeny produced
 Transforming infection – no progeny but the viral genome becomes chromosomally
integrated; infected cells become cancerous cells
 Abortive infection – defective virus / no replication occurred
֍ ADSORPTION
 First step in the viral replcition cycle
 Virus recognizes a suitable host cell
 Specific binding with capsid proteins and carbohydrate receptor of host cell
 List of Host Receptors used by Specific Viruses
 Parvovirus B19 : P-antigen (globoside)
 Adenovirus : Coxsackie-Adenovirus Receptor (CAR)
 EBV : C3D (cr2 or CD21)
 HIV : CD4 receptor
 HSV1 : Fibroblast Growth Factor
 Influenza 1 : Sialic Acid
 Rubeola : CD46; Signaling Lymphocytic Activation Molecule (SLAM)
 Rhabdovirus : Acetylcholine Receptor; CD56
 Polio : CD155 [Poliovirus Receptor (PVR)]
 Rhinovirus : ICAM-1
 Vaccinia : Epidermal Growth Factor
֍ PENETRATION

VIROLOGY | 1  
 
UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

Entry of Virus into the Host Cell


Mechanisms of Entry

 Direct Fusion
 One of the mechanisms used by enveloped viruses
 The viral envelope and the cell membrane of the host fuse causing the viral
nucleocapsid to be directly delivered in the cytoplasm leaving the viral envelope
on the plasma membrane
 Ex. RETROVIRUS
 Receptor-Mediated Endocytosis
 Used by either naked or enveloped viruses
 Following the engagement of viral particles on the receptor, the virus particle-
receptor complex triggers the endocytosis by forming a coated pit on the plasma
membrane, leading to endosome formation. As a result, the virus particle
becomes located inside the endosome.
 Enveloped viruses will have its envelope fused with the endosomal membrane.
 Naked viruses will trigger endosomal membrane lysis via its capsid proteins
 Translocation
 Injection of Viral Material (use several tail fibers and lysozyme)
֍ UNCOATING
 Disassembly of the virion
 Exposure of the viral genome
֍ TRANSCRIPTION and TRANSLATION
 Transcription is the biosynthesis of mRNA from a DNA template using DNA-dependent RNA
polymerase (synthesizes RNA in 5’-3’ direction from the template DNA strand which runs in 3’-5’
direction)
 RNA viruses no longer need a DNA Template especially the +/sense ssRNA which serve as
messenger RNA and are ready for translation
 -/antisense RNA needs RNA-dependent RNA polymerase to convert it into sense strand
(enzyme packed in the virion)
 Translation is the biosynthesis of amino acids from codons leading to synthesis of peptides/proteins
 Formation of viral proteins
 Transcription and Translation occurs first before replication.
 Production of viral proteins that are necessary for replication to occur
֍ DNA REPLICATION
 Most dsDNA are able to use host enzymes to express viral genes and replicate viral DNA
 Replicate via dsDNA (ssDNA need to synthesize a complementary strand to convert the genome
into dsDNA)
 Require DNA-dependent DNA polymerase
 Each viral DNA has a specific origin of replication
 Proteins that initiate DNA replication bind to the origin
 Helicase – unwind the double helix
 ssDNA binding protein – keep the strands apart
 DNA polymerase – for transcription
 One of the daughter strands is the leading strand and the other is the lagging strand, synthesized
as Okazaki fragments, which become joined by a DNA ligase.

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 After a dsDNA molecule has been copied each of the daughter molecules contains a strand of the
original molecule. This mode of replication is known as semi-conservative, in contrast to the
conservative replication of some dsRNA viruses.
֍ dsRNA replication
 similar to dsDNA in which the helix is unwind
֍ ssRNA REPLICATION
 Translation occurs first to produce the enzyme replicase which is needed for replication
 - sense are transcribed to + sense
 ssRNA are replicated by synthesis of complementary strands of RNA that are then used as
templates for synthesis of new copies of the genome
֍ ASSEMBLY
 Progeny and Viral proteins form virion
֍ EXIT
 Virion exit the host cell then infect a new host cell

SPECIMEN COLLECTION

֍ Specimens should be collected as soon as possible after the onset of symptoms

 Likelihood of obtaining + results : first 3 days after onset of symptoms
֍ Viral Transport Media (VTM)
 Required for Respiratory samples, swabs, and tissue samples because they dry out easily.
 Formulations typically consist of a buffered salt solution such as Hanks’ balanced salt solution
buffered with HEPES to maintain a neutral pH, protein-stabilizing agents such as bovine serum
albumin or gelatin, and antimicrobials to prevent bacterial and/or fungal overgrowth.
 The inclusion of sucrose in the media serves as a cryoprotectant to maintain the integrity of viruses
if specimens are frozen (−70°C or lower) for prolonged periods.
 A sample collection and transport device that contains an absorbent matrix for drying of
specimens, ViveST (ViveBio, Norcross, GA), has been used primarily for preservation of NA at
ambient temperature, although some viruses remain viable for up to 7 days with the addition of
fetal bovine serum
֍ Swabs that are not used include calcium alginate, charcoal, and those with wooden shafts
֍ Dried samples for Nucleic Acid Testing are acceptable
֍ Transport Conditions
 Typically, specimens for viral culture should be transported to the laboratory promptly, ideally
within 2 to 4 hrs of collection
 The preferred transport temperature is 2 to 8°C, except for blood specimens, which can be
transported at ambient temperature.
 Viability is not a requirement for Ag or NA detection methods; therefore, transport time may be
less significant, unless degradation of intact cells or viral NA is a consideration.
֍ Selected Specimens
 Amniotic Fluid
 NA detection is the most commonly used method
 NAs from samples should be extracted as soon as possible and stored at 2 to 8°C for up
to 48 h or frozen at −70°C or lower if testing is delayed beyond 48 h
 Congenital CMV : 21-23 wks of gestation for most accurate prenatal dx
 Maternal VZV during 1st/2nd Trimester – VZV DNA is detectable
 Blood
 EDTA and ACD are the preferred anticoagulants for obtaining plasma for NA testing, as
heparin is inhibitory to many NA amplification chemistries
 Whole blood used for NA amplification must be processed to remove inhibitors of DNA
and RNA polymerases such as heme and metabolic precursors of heme.
 When serum is required for serologic or molecular testing, 4 to 8 ml of blood is collected
in a serum separator tube
 For virus isolation, 5 to 10 ml of anticoagulated blood (EDTA or heparin) should be
collected by venipuncture and transported to the laboratory at room temperature.
 Processing of the specimen for leukocyte fractionation should occur within 8 h.
 Whole blood should not be used to inoculate cell cultures because of toxicity
caused by red blood cells. The buffy coat fraction may also contain erythrocytes.
 For recovery of CMV in culture, the shell vial technique is more rapid and
sensitive than conventional tube culture
 Bone Marrow
 Bone marrow specimens should be stored at 2 to 8°C prior to NA extraction. Freezing and
thawing of bone marrow lyses red cells, causing release of heme, a known inhibitor of
PCR
 CSF
 Transport and store for up to 48 h at 2 to 8°C, and freeze at −70°C or lower for
longerterm storage.
 NAs should typically be extracted prior to testing since CSF contains globulins, cell-derived
proteins, and other uncharacterized substances that inhibit the activity of thermostable
polymerases used in PCR
 Eye
 swabs or scrapings of the conjunctiva, corneal scrapings, and vitreous and aqueous fluids.
 Corneal scrapings and swabs of the cornea are best collected and placed in VTM
 Direct testing of these surgically obtained fluids has resulted in PCR inhibition; therefore,
the original specimen must be extracted.
 Genital
 The most common viral causes of genital lesions are HSV-1 and -2, which are easily
detected by direct Ag (DFA) methods, NATs, or culture
 Oral
 Respiratory

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 The primary site of replication for many respiratory viruses is the ciliated epithelial cells of
the posterior nasopharynx and, to a lesser extent, the anterior nares and oropharynx.
 Nasal washes can yield high rates of respiratory virus detection by NAT with minimal
patient discomfort compared with swab, aspirate, and brush sampling
 Swabs for respiratory virus testing should be polyester, Dacron, or rayon with plastic or
aluminum shafts. A swab of the posterior nasopharynx typically yields more virus than a
swab of the anterior nares or throat.
 Woodenshaft swabs may contain substances that are toxic to cultured cells.
 Calcium alginate swabs should not be used since they may impair recovery of
enveloped viruses, may interfere with fluorescent-antibody tests, and are
inhibitory to some NATs.
 Flocked swabs, made from nylon fiber using a proprietary spray-on technology,
are designed for optimum specimen absorption and release and have been
shown to collect more respiratory epithelial cells than conventional rayon swabs
for DFA testing of respiratory viruses.
 Polyurethane foam-tipped swabs provide an alternative to nylon or Dacron swabs
for sampling of the anterior nares in patients who might be at risk for bleeding.
 Skin
 Vesicular lesions from HSV
 Stool and Rectal Swabs
 Stool - optimal specimen for identification of viruses causing gastroenteritis such as
rotavirus.
 Fresh stool specimens can be stored at 4°C for 2 to 3 days if they are not tested
immediately after collection. For prolonged storage, specimens should be kept frozen,
preferably at −70°C or lower.
 Tissue / Biopsy
 Formalin-fixed tissue is unsuitable for viral isolation and may affect the performance of
NATs
 For viral isolation from tissue, it is recommended to prepare a 10 to 20% (wt/vol)
homogenate using VTM as a diluent from small or minced tissue fragments that have
been aseptically ground in a tissue grinder
 Urine
 Collect urine specimens as soon as possible after the onset of illness or when congenital
or perinatal infection is first suspected.
 For NATs, ambient storage of fresh, unprocessed urine should be minimized since the low
pH and high urea content rapidly denature DNA and RNA
 Prior to inoculating cell culture, urine can be filtered (0.45-μm pore size) or centrifuged
(1,000 × g for 10 min) to remove bacteria and debris; the pH can be neutralized with
sodium bicarbonate (7.5% solution) to reduce toxicity

TECHNIQUES

֍ Light Microscopy – visualzie cytopathic effects

 CMV : Owl’s Eye Inclusion Body
 HPV : Koilocytosis, perinuclear halo ; wrinkled-prune nucleus
 Molluscum contagiosum : Henderson-Paerson Bodies
 Rhabdovirus : Negri Bodies
 HSV 1 : Cowdry Type A
 Adenovirus : Cowdry Type B; grape-like clusters
 Poxviruses : Guarnieri Bodies
 Rubeola : Dawson Bodies / Warthin Finkeldey/ Multinucleated giant cells with syncytia
 Yellow Fever : Councilman Body
֍ Cell Culture – gold standard
 Maintained and incubated for 1-4 weeks
 Primary Cell Line (Human Embryonic Kidney / Rabbit Kidney / Monkey Kidney)
 Sensitive to influenza, parainfluenza, mumps, enterovirus, and adenovirus
 Diploid Cell Lines (Human Diploud fibroblasts/ W1-38 / mrc-5)
 20-25 passages
 Continous/ Heteroploid / immortal cell lines
 HeLa – cervical carcinoma
 Hep-2 – laryngeal ca
 KB – nasopharyngeal
 A-549 – lung ca
֍ Shell Vial – modified conventional cell culture

TAXONOMY AND CLASSIFICATION

֍ BALTIMORE CLASSIFICATION

Group Classification Examples

I dsDNA Herpesviridae, Poxviridae, Adenoviridae, Papovaviridae
II ssDNA Parvoviridae
III dsRNA Reoviridae
Caliciviridae, Flaviviridae, Coronaviridae, Astroviridae,
IV (+) ssRNA
Picornaviridae, Togaviridae
Bunyaviridae, Arenaviridae, Paramyxoviridae, Orthomyxoviridae,
V (–) ssRNA
Rhabdoviridae, Flioviridae
VI RNA RT Retroviridae
VII DNA RT Hepadnaviridae

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

֍ DNA VIRUSES – replicate in the nucleus

 NAKED / NON-ENVELOPED
 PARVOVIRUS – single-stranded DNAV; linear NA
 Smallest DNA virus
 ADENOVIRUS – the only naked dsDNA with a linear NA
 PAPILLOMAVIRUS
 POLYOMAVIRUS
 ENVELOPED
 HEPADNAVIRUS
 HERPESVIRUS – linear NA
 POXVIRUS – largest virus; brick-shaped; replicates in cytoplasm
֍ RNA VIRUSES – replicates in cytoplasm
 Sense
 CALICIVIRUS
 CORONAVIRUS
 PICORNAVIRUS
 FLAVIVIVIRUS
 ASTROVIRUS
 TOGAVIRUS
 HEPEVIRUS
 Antisense
 FILOVIRUS
 BUNYAVIRUS
 ARENAVIRUS
 PARAMYXOVIRUS
 ORTHOMYXOVIRUS
 RHABDOVIRUS
 RETROVIRUS

DNA VIRUSES
GENERALITIES

 All are linear except Papovaviridae and Hepadnaviridae

 All are double-stranded except Parvoviridae
 All are naked except Herpesviridae, Hepadnaviridae and Poxviridae
 All are icosahedral except Poxviridae
 All replicate in the nucleus except Poxviridae
 DNA Tumor Viruses: Ex. Human Papillomavirus, EBV, HBV, HHV8, Polyomavirus, SV40 virus,
 Smallest DNA Virus = Parvoviridae
 Largest DNA Virus = Poxviridae

NAKED DNA VIRUSES

֍ PARVOVIRUSES
 Two subfamilies : Parvovirinae and Densovirinae
 Parvovirinae infect vertebrates
 Densovirinae infect insects
 Smallest DNA animal virus
 Pathology
 Erythema infectiosum / fifth disease
 Original 6 Exanthematous Disease
 1. Measles
 2. Scarlet Fever
 3. Rubella
 4. Dukes
 5. Erythema Infectiosum
 6. Erythema Subitum
 Childhood exanthem
 Polyarthralgia-arthritis syndrome in adult
 Aplastic crisis in patients with hemolytic disease
 Chronic anemia among the immonocompromised
 Fetal death / hydrops fetalis
 Icosahedral; nonenveloped
 Possess 2 core proteins
 VP2 : major protein
 VP1
 HUMAN PARVOVIRUS B19
 Erythrovirus genus
 Receptor : P antigen (globoside)
 Target : erythroid progenitors
 Alpha-5-Beta-1 Integrin : coreceptor
 NS1 : required for viral replication
 MOT
 Respiratory route
 Parenteral such as Blood transfusions
 Vertically
 Bocavirus
 Dependovirus
 Defective
 Needs adenovirus or herpesvirus

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

֍ ADENOVIRUS
 Genus Mastadenovirus
 Hexon Antigenicity
 Possess penton bases that carry toxin-like activity
 Occur at 12 vertices of capsid and have hemagglutinating fibers protruding from them
 Penton base causes marked rounding enlargement of cells that aggregate in a grape-like
cluster but without syncitia which is characteristic of CMV
 Target : Cells of epithelial origin
 Receptor : Coxsackie-Adenovirus Receptor
 Grouping : A-G
 Group A : lowest GC content; most oncogenic type (12, 18, 31)
 Pathology
 Respiratory disease
 Types 1, 2, 5 and 6  children
 Types 3, 4, 7  adolescents / adults
 Types 3, 7, 21  pneumonia in children
 Types 3, 4, 7  Acute Respiratory disease in military recruits
 Types 8, 19, 37  Keratoconjunctivitis
 Type 8  Shipyard Eye
 Type 37  Epidemic Keratoconjunctivitis
 Types 3, 7  Pharyngoconjunctival Fever / Swimming Pool Conjunctivitis
 Types 40, 41  Infantile Gastroenteritis
 Types 11, 21  Acute Hemorrhagic Cystitis
֍ PAPILLOMAVIRUS
 Oncogenic; linked to Cervical Cancer
 Target : Keratinocytes
 Once invasion occurred, it goes down to the basal layer of epidermis for replication
 MOT
 Direct epithelium-epitelium contact
 Sexual Contact
 Fomites
 Classification
 Mucosal Type
 HPV – α
 HPV 6, 11
 Invades mucosal epithelia of genital or respiratory tract
 Causes genital wart, papilloma of larynx
 HPV 16, 18
 Associated with Cervical Carcinoma and Oropharyngeal
carcinoma
 Cutaneous Type
 HPV- β
 HPV 5, 8
 Invades skin epithelia
 Associated with skin cancer
 HPV – µ
 HPV 1
 Causes warts
 HPV 38
 Associated with warts and skin cancer
 Cytopathic Effect : Koilocytosis
 Koilocytes : cells with perinuclear clearing with an increase in the density of the
surrounding
 Pathology
 Cutaneous Warts
 Verrucae vulgaris – common warts
 Seen in hands, feet, knees
 Serotypes 2, 4
 Plantar Warts – inward growth
 Seen on hands, and soles of feet
 Serotype 1
 Verricae Planae – flat warts
 Seen on skin, forehead, arms, and face
 Butcher’s Warts
 Seen on hands
 Serotype 7
 Genital Warts
 Venereal Warts / Condyloma acuminata
 May be flat or keratotic
 Easily transmitted to sexual partners
 Carcinoma
 Due to E6 and E7 viral genes
 E6 inactivate p53
 E7 inactivate retinoblastoma
 Prevention
 Gardasil Quadrivalent Vaccine
 Immunization against serotypes 6, 11, 16, and 18
 For feamles aged 9 – 26
֍ POLYOMAVIRUS

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Simian Virus 40 (SV40) and SV-40-like Viruses

 Also a tumor-associated virus
 Three Human Polyomaviruses
 JCV
 Causes progressive multifocal leukoencephalopathy among immunocompromised
aptients
 BKV
 Associated with transplant patients
 Assocated with cystitis and nephropathy
 MCV
 First polyomavirus known to cause cancer
 Merkel Cell Carcinoma
 True Polyomaviruses : carcinogenic murine polyomavirus
 Site of Latency : Kidney and B Cells

ENVELOPED DNA VIRUSES

֍ HERPESVIRUS

Type Genus Common Name Target Cell Site of Latency MOT

ALPHA-HERPESVIRINAE
HHV1 Simplexvirus HSV1 Mucoepithelial Neuron Close Contact,
(trigeminal resp secretions
ganglion) and saliva
HHV2 Simplexvirus HSV2 Mucoepithelial Neuron Close Contact,
(sacral ganglion) sexual contact,
perinatal infection
HHV3 Varicellovirus VZV Mucoepithelial Neuron Respiratory and
(dorsal root Close Contact
ganglion)
BETA HERPESVIRINAE
HHV5 Cytomegalovirus CMV Monocytes, Monocyte, Saliva, Urine,
Lymphocytes, Epithelial Lymphocyte Breast Milk
Cells
HHV6 Roseolovirus Roseolovirus, Herpes T-cells T cells Respiratory and
Lymphotropic Virus Close Contact
HHV7 Roseolovirus Pityriasis Rosea T cells T cells
GAMMA HERPESVIRINAE
HHV4 Lymphocryptovirus EBV B cell and epithelial B cells Close Contact,
cells kissing
HHV8 Rhadinovirus Kaposi’s Sarcoma Lymphocytes and other B cell Close contact
Associated cells (sexual), Saliva
Herpesvirus
 Herpes-Simplex Virus
 Very Common ; Most Infected Patients are Asymptomatic
 2 Categories
 Primary – initial infection
 Recurrent
 Mode of Transmission: Direct Contact of the infected tissue lesions (oral, genital, mucosal)
 Causes direct cytopathology
 HSV 1 – generally, transmitted orally
 HSV 2 - transmitted by sexual contact
 People at High Risk
 Children
 Sexually Active Individuals
 Medical Professionals who are in contact with oral and genital secretions for infection of
fingers
 Immunocompromised and Neonates are at risk for disseminating disease
 Pathogenesis
 Latency occurs and the virus evades the immune system by hiding in the neurons
 HSV1 = trigeminal ganglion
 HSV 2 = lumbar and sacral ganglion
 Reactivation can occur during periods of stress and immunosuppression
 Causes a lifelong infection
 Asymptomatic shedding of the virus can occur
 Clinical Manifestations of HSV1
 Oral Herpes – usually caused but not exclusive to HSV1
 Appear as intraoral mucosal vesicle or ulceration
 Affected areas : buccal, mucosa, posterior pharynx, gingival and palatal mucosa
 Other Clinical Findings
 Keratoconjunctivitis
 Encephalitis – necrotic lesion in one temporal lobe
 Herpetic Whitlow
 Herpetic Gladiatorum
 Disseminated Infections
 Clinical Manifestations of HSV 2
 Genital Herpes – usually caused by HSV2 (80-90% of cases)
 Herpes Genitalis
 Painful vesicular lesions
 Females – vesicles appear on the mucosa of the labia, vagina, or both
 Males – shaft, glans and prepuce of penis

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Neonatal Herpes – acquired by neonates from infected mothers during utero, during birth,
or after birth
 Clinical Manifestations
 Erythema multiforme
 Caused by both HSV 1 and HSV 2 but can also be caused by other agents
 Rash: Target or Bull’s Eye Lesion
 Areas: Trunk, hands and feet
 Diagnosis
 Direct Microscopic Exam
 Preparation of Tzanck Smear to Visualize Cytopathic Effects
 Multinucleated Giant Cells and Cowdry’s type A inclusion bodies
 Cell Culture – “Gold Standard”
 CPE can be seen within 1-2 days
 Serology – EIA, ICT
 Molecular Methods (DNA detection using PCR)
 NAT
 Treatment : Acyclovir
 Varicella-Zoster Virus
 Causes chickenpox and shingles
 MOT : droplet inhalation / direct contact with lesions
 Site of Infection : Respiratory Tract
 Latency : Dorsal Root or Cranial Nerve Ganglia
 Chickenpox
 2-3 weeks incubation period
 Rash: papules > vesicles > pustules and crusts
 Lesions dry, crust over, and heal in 1-2 weeks
 Elicits lifelong immunity
 Complications
 Severe if acquired in later age
 Reye’s syndrome
 Shingles
 Reactivated VZV
 Rash followed by vesicular lesions in a UNILATERAL DERMATOME PATTERN
 Epstein-Barr Virus
 Causes Infectious Mononucleosis
 Mono, Kissing Disease, Pfeiffer’s Disease, Glandular Fever
 MOT : Close oral contact
 Pathogenesis
 Virus is found in saliva and infects oral epithelial cells
 Spreading occurs to B cells
 Once it infects the B cells, the infected B cell is transformed
 The infected B cells produce many copies of the EBV DNA and is passed on to the B cell
progeny
 Infected B cells are destroyed by T cells
 Some EBV hide in the B cells causing latency
 Immunocompromised states can reactivate the virus
 Uncontrolled Growth of B-cells leading to Burkitt’s Lymphoma
 Diagnosis
 Lymphocytosis with Atypical Lymphocytes (Downey Cells)
 Culture with B cells
 EBV Nuclear Antigen
 Heterophile Antibodies
 Monospot test
 Paul Bunnell Test
 Antibody Profile
 Anti-VCA IgM
 Anti-VCA IgG – persist for life
 Anti-EA/D – appear in acute stage and indicate current or recent infection
 Anti-EA/R – appear in acute phase and persist for up to 2 years
 Anti-EBNA – 1 month after infection; peak in 6-12 months
 Cytomegalovirus
 Heterophile negative mononucleosis
 Associated Diseases: Cytomegalic Inclusion Disease, Mono-like disease, diseases affecting
immunocompromised and transplant patients
 Mode of Transmission: Acquired orally, sexually, from blood transfusions, tissue transplants, in
utero, at birth, by nursing
 Target Host Cell: Epithelial Cells, Lymphocytes,
 Site of Latency: monocytes, T cells, macrophages
 People at Risk: Neonates, Sexually Active Indvls, Immunosuppressed, Organ Transplant Patient
 Can cross placenta
 Shed in the urine
 Causes owl’s eye Cytopathic Effect
 Diagnosis
 Shell vial technique : dx w/n 72 hrs
 Culture of urine / secretions
 Serology
 Molecular methods
 HHV 6 AND 7
 Lymphotropic Viruses that target T and B cells

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Viral Receptor : CD46 for HHV6 | CD4 for HHV 7

 MOT : inhalation of aerosols/ close contact
 Shed in the saliva
 HHV 6 variatn B causes roseola infantum or exanthem subitum (sixth disease)
 Variant A are those that infect immunocompromised patients
HHV 8
 Targets B CELLS
 MOT : sexual contact, transplantation
 Causes Kaposi Carcoma among AIDS patients
֍ Hepadnavirus
 Hepatits B / Dane Particle
 3 mos incubation period
 MOT : Parenteral Route
 HBsAg surface antigen
 HBeAg indicates viral replication ; associated with infectivity
 HBcAg core antigen
 Anti-HBs indicates past infection / immunization in the absence of anti-HBc
 Anti-HBe suggest presence of HBV
 Anti-HBc indicates past infection ; first ab to be produced and detected
 Pathology
 Acute Hepatitis
 Fulminant Hepatitis
 Chronic Hepatitis
 Priamry Hepatocellular Carcinoma
֍ Poxvirus
 Largest and Most Complex Viruses
 Linear double stranded DNA
 Brick-shaped complex virus with a disk/dumb bell shaped core
 Multilayered and possess a lipoprotein envelope
 Replicates in the host cell cytoplasm
 Possess DNA-dependent RNA polymerase
 Virus is assembled in its inclusion bodies called Guarneri Bodies
 MOT: Inhalation (respiratory infection) of aerosol droplets, face to face contact and spread mainly in
the lymphatics; direct contact; through fomites
 Viruses of Medical Importance
 Smallpox Virus
 Vaccinia Virus
 Molluscum Contagiosum Virus
 Human Disease: VARIOLA or SMALLPOX
 Acute contagious disease of the reitculoendothelial, vascular endothelial and epithelial cells
 Variola major – severe form, more deadly, causes more disfigurement
 Variola minor – “Alastrim”; milder form of variola
 Synchronous Progressive Rash with Fever
 Result of Viral Replication in the skin, followed by damage caused by T cytotoxic cells
attacking the infected cells
 Now Eradicated/Extinct
 Vaccinia Virus – used to vaccinate against smallpox
 Causes a localized exanthem through epithelial cell infection
 MOT: sexually transmitted; skin contact
 1st Reservoir Host: Rabbits
 Clinical: Lesions are larger than small pox with necrotic centers
 Recombinant smallpox-cowpox
 Molluscum Contagiosum
 Disease of children and young adults
 Disease: Molluscum Contagiosum/ Water warts
 MOT: Direct Skin Contact, intimate contact, indirect contact
 Manifestation: presence of small papules on the skin or mucus membrane
 “Cup-shaped Crater”
 Seen in Immunocompromised patients, AIDS patients
 Treatment: Removal of lesion

RNA VIRUSES
GENERALITIES

 All are single except Reoviridae

 All possess a helical structure except
o Caliciviridae, Hepeviridae, Togaviridae
o Reoviridae, Retroviridae,
o Flaviviridae, Picornaviridae,
 All are enveloped viruses except HCRP
o Hepeviridae = + sense
o Caliciviridae = + sense
o Reoviridae = retro
o Picornaviridae = + sense
 All replicate in the cytoplasm except Orthomyxoviridae, Retroviridae
 Segmented RNA Viruses
o Retroviridae
o Arenaviridae = - sense
o Bunyaviridae = - sense

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

o Orthomyxoviridae = - sense
o Reoviridae
 The Largest RNA Virus: Paramyxoviridae
 The Smallest RNA Virus: Picornaviridae
 Arthropod Borne Viruses (ARBOVIRUSES)
o Bunyaviridae
o Flaviviridae
o Togaviridae
o Reoviridae
֍ Family Picornaviridae

Generalities

 ssRNA (+ strand) viruses

 Naked, Icosahedral
 Capsid: made up of 4 proteins (VP1-VP4); VPg (Viral protein genome-linked)
 Diverse Viral Family
 Two medically important groups of Picornaviridae
 Enteroviruses
 Enterovirus
 Parechovirus
 Hepatovirus
 Rhinoviruses
 Rhinovirus
 Enteroviruses are transmitted primarily via
 Aerosol inhalation, oral-fecal route, fomites
 Rhinoviruses are transmitted via
 Aerosol inhalation, contact with secretions and fomites
 Some Unique Properties:
 Enteroviruses are resistant to pH 3 to pH 9, detergents, mild sewage treatment and heat.
 Rhinoviruses are labile at acidic pH; optimum growth temperature is 33ºC.
֍ Enterovirus

 Infect intestinal epithelial cells and lymphoid tissue

 Produce a variety of clinical manifestations such as Fever of Unknown Origin, Aseptic Meningitis, Paralysis,
Sepsis like illness, Myopericarditis, Pleurodynia, Conjunctivitis, exanthemas, pharyngitis, and pneumonia
 Disease Mechanisms
 Lymphoid Tissue: site of initial viral replication
 Viremia occurs, viral dissemination to different organs (CNS, muscles, heart, liver) via bloodstream
 Most patients are asymptomatic or have mild symptoms
 Some may have more clinical manifestations depending on the immunocompetency of the patient
 Antibodies can block spread if patient was previously exposed
 Patients develop immunity even in the absence of symptoms

1. POLIOVIRUS – causative of poliomyelitis (infantile paralysis)

 Greek Word meaning polio (gray) and myelon (matter/spinal cord)

 Serotypes 1,2,3
 MOT: oral-fecal
 Virus is rapidly inactivated by heat, UV light, formalin, chlorine
 Site of Initial Infection: Oropharynx, Lymphoid Tissue and small intestine
 The virus is present in the pharynx and stool before the onset of illness
 Hematologic Spread to Lymphatics and CNS
 Viral Spread along nerve fibers
 Destruction of Nerve Fibers (anterior horn and brain stem)
 Flaccid Paralysis and Bulbar Poliomyelitis
 Clinical Findings
 Inapparent/Asymptomatic
 No clinical symptoms; Viral infection is limited to the oropharynx and gut
 90% of infections
 Abortive – minor illness; nonspecific febrile disease. Fever, headache, malaise, sore throat,
and vomiting occur in such people within 3 to 4 days of exposure
 5% are affected

 Non-Paralytic – AKA Aseptic Meningitis

 Occurs in 1% to 2% of patients with poliovirus infections.

 Virus progresses into the central nervous system and the meninges,
 Back pain and muscle spasms (stiffness of neck, back and legs) in addition to the
symptoms of the minor illness
 Paralytic – Major Illness affecting less than 1% of infections
 Resulting in Flaccid Paralysis
 It appears 1-7 days after the minor illness has subsided, thereby producing a biphasic
illness.
 Virus spread via the blood to the CNS affecting the motor cortex
 Can also lead to bulbar poliomyelitis (respiratory paralysis) and meningomyeloencephalitis
 Degree of Paralysis depends on the extent of nerve infection
 Post Polio Syndrome – sequelae of polio infection; occurs many years after the initial infection
 Deterioration of the muscles affected by the poliovirus due to loss of nerves
 Cause of deteriation is still unknown

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 PREVENTION of POLIO INFECTIONS

 Passive Immunization
 Vaccination
 Salk – use of formalin killed/inactivated vaccine
 eIPV (Enhanced polio vaccine)
 Trivalent Inactivated Polio Vaccine
 Given IM injection
 Effective; Vaccine is stable; Can be administered among immunodefficient
patients
 Booster Needed
 4 Doses (given at 2mos, 4mos, 6-18mos, and at 4-6 yrs)
 Sabin – administration of live attenuated polio virus
 Oral Administration (3 doses)
 Stimulates production of IgA (Gut Immunity)
 Effective; longer Immunity
 Disadvantages: May revert to Wildtype Polio; Cannot be given among
immunodeficent persons, vaccine should be refrigerated, immunization can be
impaired by other enteroviruses
 Laboratory Diagnosis of Polio
 Use of Cell Cultures to Isolate the Virus
 Specimens: CSF, Stool, Oropharynx Secretions
 Molecular Methods
 Serology: ex. IFA, ELISA
 Treatment: No Antiviral Therapy

2. COXSACKIE Virus

 2 Major Groups (Classification based on Murine Studies)

 Coxsackie A and Coxsackie B
 MOT: fecal-oral route; respiratory aerosols
 COXSACKIEVIRUS A – 24 Serotypes
 Affected Sites: Skin and Mucus Membrane
 Diseases:
 Myocytis in skeletal muscles of newborn leading to flaccid paralysis
 Herpangina – vesicular pharyngitis; painful eruption of vesicle in muscle and
throat
 Hand Foot and Mouth Disease (CAV Serotype 5,10,16)
o Children Affected; Malaise, headache, abdominal pain
o Appearance of Maculopapular Rashes
 Aseptic Meningitis
 Colds
 COXSACKIE B Virus
 Affected Organs: Heart, Pleura, Pancreas, Liver, CNS,
 Diseases:
 Pleurodynia - Bornholm Disease, Devil’s Grip;
o Painful inflammation of internal muscles; Severe thoracic pain on
breathing
 Pericarditis
 Myocarditis
 Spastic Paralysis
 Aseptic Meningitis
 Diabetes (CBV-4)
 Diagnosis: Cell Cultures, Serology (Serum Neutralization Tests);
 No treatment and no vaccine available

3. ECHOVIRUS – Enteric, Cytopathic, Human Orphan Virus

 Target: Children
 Disease: Infantile Diarrhea, Aseptic Meningitis, Upper RTI, febrile illness with or without rash
 Echovirus 9 – outbreaks of aseptic meningitis
4. Other Enteroviruses
 Enterovirus 70 – main cause of acute hemorrhagic conjunctivitis
 Enterovirus 71 – leading cause of viral CNS disease (meningitis, encephalitis, paralysis), HFMD

֍ Parechovirus

 Parechovirus A – causes Summer Diarrhea; Respiratory infection, meningitis, neonatal sepsis

 Parechovirus B – Ljungan Virus
 Zoonotic; may cause intrauterine fetal death and diabetes

֍ Hepatovirus

 Hepatitis A
 AKA Enterovirus 72
 MOT: Oral-Fecal; Ingestion of Contaminated Food and Water
 Disease: Epidemic or Infectious hepatitis
 Virus replicates in the GI and spreads to the liver
 Virus is shed in large amounts in feces
 Incubation period: 1 month

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Children – most frequently affected

 Manifestations: Fever, anorexia, nausea, vomiting and jaundice; pale stool, dark urine
 Diagnostics: IgM Anti-HAV and IgG Anti-HAV; Elevated Liver Enzymes; Jaundice

֍ Rhinovirus

 causes Common Cold

 113 Serotypes
 MOT: Direct Contact (Person-Person; Respiratory Droplets) / Indirect Contact
 Infects nose and throat; doesn’t cause generalized infections
 Symptoms Worsen if there is bacterial superinfection
 Symptoms cannot be differentiated from other respiratory viruses
 Viral Receptor: ICAM-1 which is expressed on epithelial fibroblast, and lymphoblastoid cells
 Rhinoviruses are unable to replicate in the gastrointestinal tract
 Diagnostics: Cell Culture (CPE), Demonstration of acid lability

֍ Family Arenaviridae

 Arena = “sand”
 EM: Appear Sandy and Granular
 Single Stranded RNA Virus; Ambisense Virus
 Segmented Virus with a Helical Structure; Enveloped
 Possess T-shaped spikes on its envelope
 MOT: Inhalation of rodent excrements, direct contact with infected rodents
 Zoonotic in Nature (Rodents)
 2 Groups:
 Old World – LCM (Lymphocytic Choriomeningitis Virus) and Lassa Virus
 New World – Tacaribe Complex Virus (Tacaribe, Guanarito, Junin and Machupo Viruses)
 Natural Hosts: Rodents (Rodent Borne Infection)
 Zoonotic; Humans are considered as dead end hosts
 Clinical Manifestations
 LCM –Lymphocytic Choriomeningitis Virus
 Natural Host: Mastomys muscularis/ House Mouse
 Flu-like illness and might lead to Aseptic Meningitis
 Lassa Fever – usually asymptomatic, some patients develop fever, headache, pharyngitis, myalgia,
diarrhea and vomiting. Hemorrhaging and CNS involvement may also occur
 Pantropic – Hemorrhage occurs in all internal organs. High Mortality.
 Natural Host: Mastomys natalensis “multimammate rat”
 Diagnosis: Isolation of Virus in Blood and Liver (Postmortem); HIGHLY INFECTIOUS; Cell Cultures;
Serology; Molecular Methods (RT-PCR)
o BSL 3 and BSL 4 should be observed for LCM virus and Lassa Virus respectively

֍ Family Caliciviridae

 Icosahedral, non-enveloped, ssRNA (+) sense virus

 With VPg at the 5’ end of the genetic material
 Structure has 32 calyxlike concavities giving the virus a “Star of David” Appearance of Capsid
 MOT: fecal-oral route
 Affects both humans and animals
 Four Genera:
 Sapovirus
 Sapporo Virus
 Norovirus
 Norwalk Virus
 Lagovirus
 Rabbit Hemorrhagic Disease Virus
 Vesivirus
 Feline Calicivirus
 Norwalk Virus/Norovirus/Small Rounded Structured Virus –MOST IMPT EPIDEMIC VIRAL GASTROENTERITIS
among ADULTS
 “Winter Vomiting Disease”
 MOT: Food borne, waterborne, person-person transmission
 Incubation: 1-2 days
 Symptoms: Nausea, low grade fever, diarrhea, vomiting
 Diagnostics: Cannot be cultured; Direct Methods (EM of Stool Material); Molecular Methods
 Sapovirus – causes gastroenteritis among infants, young children and elderly;
 Prevention: symptomatic & supportive
 careful disposal of stools
 effective hand washing
 careful processing of food
 purification of drinking water & swimming pool water
 no vaccine is available

֍ Family Hepeviridae

 Genus Orthohepevirus
 Hepatitis E Virus – formerly under Caliciviridae
 MOT: Oral – Fecal
 Similar to Hepatitis A; no chronic state
 Common in Asia, Central America and Africa

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 High Mortality among Pregnant Women (3rd Trimester)

֍ Family Astroviridae

 ssRNA;
 Non-segmented; Naked; Icosahedral Structure
 “Star-Like” Surface Appearance
 Spikes: 5 or 6 pointed spikes
 Very small, size similar to poliovirus
 MOT: Oral-Fecal Route
 Disease Manifestation: Gastroenteritis (Osmotic/Watery Diarrhea) among elderly and the young.

֍ Family Coronaviridae

 Enveloped Helical Viruses (ssRNA)

 Possess the longest known sense RNA genome
 Under EM: appear Solar Crown
 Possess club shaped/pearl shape/petal shaped projections
 Infect a variety of hosts such as humans, and other vertebrates such as dogs, cats, rodents, bats, poultry
 Two Genera
 Torovirus and Coronavirus
 Disease Manifestations: Colds, Rhinitis, Pediatric Diarrhea, Novel Diseases (SARS-CoV and MERS-CoV)
 MOT: Person to person contact via direct contact, aerosol or droplet

SARS: Severe Acute Respiratory Syndrome

 Caused by a novel coronavirus (SARS-CoV)
 Viral Pneumonia that affects the Lower Respiratory Tract
 Mean Incubation Period – 5 days
 Emerged in Guandong China and HK in 2002
 WorldWide Pandemic
 Target Receptor of SARS: Angiotensin Converting Enzyme 2 (ACE2)
 Currently, the suspected reservoir is the horseshoe bat

MERS-CoV – Middle Eastern Respiratory Syndrome Coronavirus

 Causes Middle Eastern Respiratory Syndrome
 First Reported in Saudi Arabia in 2012 and subsequent reports in the Arabian Peninsula
 Severe Respiratory Illness with symptoms of Fever, Cough, Shortness of Breath;
 50% Mortality Rate
 Risk: Elderly, immunosuppresed, patients with chronic illness
 Diagnosis
 Culture (VeroCell Line)
 Serology: ELISA, IFAT
 Molecular: RT-PCR
 EM
 Treatment: Usually Supportive

֍ Family Filoviridae

 Thread-like or Filamentous RNA viruses

 Pleomorphic virus that may exhibit branching and may appear as a “shepherd’s crook”, U or 6
 Enveloped, helical virus with protruding peplomers
 2 Genera
 Marburgvirus – Lake Victoria marburgvirus
 Ebolavirus – Ebola Virus Zaire, Sudan, Reston, Tai Forest Strains
 Both Genera causes
 Hemorrhagic Fever, Rare Occurrence, Unknown Reservoirs
 MOT: Direct Contact with monkeys and other infected persons, nosocomial transmission, sexual contact.

Lake Marburg Victoria Virus

 Hemorrhagic Fever
 Fever, Maculopapular Rash, Nausea, Vomiting, Diarrhea
 Bleeding from nose, gums, GI tract occur at the latter part; Hemorrhage of vital organs occurs leading
to death
Ebola Virus
 Ebola Zaire – more virulent
 Ebola Sudan
 Ebola Reston – contact with monkeys; less severe; no development of disease
 Symptoms: Fever, Chills, Myalgia, Anorexia, Sore Throat, Abdominal Pain, Diarrhea, Vomiting, Bleeding
 Virulence Factor: glycoprotein, proteins that inhibit the induction of IFN
 Probable Natural Host of Ebola Virus: fruit bats of the Pteropodidae family
 Diagnosis: PCR, IF, ELISA, Culture, EM
 Most Recent Outbreak of Ebolavirus Occurred in: West Africa

֍ Family Paramyxoviridae

 Nonsegmented, Negative Stranded ssRNA

 Helical nucleocapsid; Pleomorphic
 Enveloped containing surface glycoproteins
 HA – Hemagglutinin
 NA – Neuramidase activity

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 F – mediates membrane fusion and hemolysin activity

 2 Sub Families
 Paramyxovirinae
 Paramyxovirus – PIV 1,3
 Rubulavirus – PIV 2,4, Mumps
 Morbilivirus - Measles
 Nonclassified – Nipah and Hendra Virus
 Pneumovirinae
 Pneumovirus - RSV
 Metapneumovirus

 Virulence Factors
Virus HA NA F
Measles + - +
Mumps + + +
Respiratory Synctial - - +
Virus
Parainfluenza + + +

 MOT: Respiratory Droplets (Droplet Infections), direct contact, respiratory secretions, Zoonotic Transmission
(Hendra and Nipah Viruses)
 Affected: Most infections are seen in children
 Target Area: Initial Infection in the Respiratory Tract; some may become generalized in cases of measles

PARAINFLUENZA VIRUS
 Clinical Manifestations:
 Common Cold Syndrome (Cold Like Symptoms),
 Bronchitis
 Croup (laryngotracheobronchitis)
 bronchiolitis,
 pneumonia
 Parainfluenza Serotypes:
 PIV 1 – Croup
 PIV3 – bronchiolitis and pneumonia
 PIV 4 – mild upper respiratory disease
 Diagnosis of Parainfluenza
 Specimens: Aspirated and Nasopharyngeal Washes
 Cell Culture (PMK or LLC-MK2 cells)
 Serology (Hemadsorption, IF or EIA)
 Direct Methods: IF
MUMPS VIRUS
 Disease: Mumps
 acute illness characterized by fever, malaise, anorexia and followed by unilateral or bilateral
swelling of the parotid gland and other organs.
 Highly Affected Children aged 5-9 years
 1 serotype; infection or vaccination provides lifelong immunity
 Diagnosis:
 Direct Methods, Culture, Shell Vial,
 Serology (Hemagglutination Inhibition test, CF detecting the Ab against the Soluble Antigen)
 Molecular Methods
 Mumps Skin Test
 Specimens: Saliva, Swabs from Stensen’s Duct, Pharynx, Urine, CSF
MEASLES VIRUS
 Disease: MEASLES (RUBEOLA) - highly contagious disease of children with signs and symptoms of:
 high fever
 Coryza
 characteristic KOPLIK’s spots – lesions on oral mucosa which consists of irregular red spots with a
bluish white speck in the center
 maculopapular rash - due to T-cell response to virus infected endothelium lining the capillaries
 Common Complications of Measles
 otitis media; diarrhea
 Severe Complications:
 Bronchopneumonia, Encephalitis
 Long Term Complications
 Subacute sclerosing panencephalitis (SSPE)
 Defective Measles Virus that persists in the brain and acts as a slow virus
 Occurs after 7-10 years after acquiring the infection
 Central nervous system manifestations (e.g. personality, behavior and memory changes,
myoclonic jerks, spasticity and blindness)
 Death within 6-12 months
 Measles among pregnant women – risk for stillbirth
 Diagnosis
 Specimen: Nasopharynx and Urine
 Tests: Culture (PMK) – look for characteristic CPE (War thin Finkeldey Giant Cells), Serology,
 Prevention: Vaccination (Live, attenuated Vaccine)
 2 doses: Given at 15months of age, then at 4-6yrs subcutaneously
 Booster is recommended
RESPIRATORY SYNCYTIAL VIRUS - most common virus isolated from infants
 Two Serotypes: A and B

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Common cause of viral pneumonia and bronchiolitis among children 5 years below
 Also causes cold, bronchitis, lower RT infections among adults as well and elderly
 Infections does not confer complete immunity; infections can occur throughout life
 MOT: Respiratory Droplet; direct contact; entry through nose and eyes
 Laboratory Diagnosis:
 DFA or EIA using nasopharyngeal swabs
 Cultures (Quite Difficult): Culture in HEp2 (Syncytia formation)
 Rapid Ag Detection Kits
METAPNEUMOVIRUS
 First described in 2001
 Occurs most often in children
 Causes a wide range of respiratory illness from mild upper respiratory symptoms to severe lower
respiratory tract disease
 In general, symptoms are similar to those caused by RSV
 Detected by polymerase chain reaction using clinical samples from children with respiratory illness
 Nipah and Hendra Viruses
 Zoonotic Viruses endemic to SouthEast Asia and Australia
 Nipah (South East Asia)
 Hendra (Australia)
 Causes encephalitis and severe interstitial pneumonia
 High Mortality (40%)

֍ Family Orthomyxoviridae

 ssRNA viruses, segmented virus

 Enveloped, Icosahedral Capsid
 Ortho – “true” (to differentiate from Paramyxoviruses)
 Myxo – “mucus” (ability to attach to mucoproteins on the cell surface)
 Genus: Influenza Viruses
 Causes the “FLU”
 Hemagglutins
 Ability to agglutinate RBCs
 Plays a role in the attachment and entry of the virus to the host’s cells
 determines virulence
 major antigen against which neutralizing antibodies are directed
 Currently there are 16 types of HA of Influenza A
 Neurmaminidase
 mushroom-like spikes
 Destroys neuraminic (sialic) acid, a component of the specific cell receptor for these viruses
 main function is the release of the new virus from cells
 determines the subtype of influenza A virus isolates
 currently there are 9 NA types of Influenza A
 Matrix Proteins – anchors the HA and NA glycoproteins; found in the envelope
o Influenza A Virus possess 2 types of M proteins
 M1 protein
 M2 protein – forms an ion channel
 MOT: Inhalation of small aerosol droplets released by talking, breathing, coughing
 Virus “likes cool,” less humid atmosphere (e.g. winter heating season)
 Genera
 Influenza A
 Influenza B
 Influenza C

 Basis of difference:
 Group Specific Antigen: Viral Nucleoproteins and Matrix Proteins
 Type Specific Antigen: HA and NA
GENETIC VARIATION OF INFLUENZA VIRUSES
 Antigenic Shift
 occurs in Influenza A
 abrupt or sudden change in the genetic material
 gene reassortment between two influenza viruses
 reassortment of HA and NA antigens to create a novel flu virus
 can lead to FLU PANDEMICS
 3 Possible Mechanisms Of Antigenic Shift
 Genetic Reassortment of non-human and human influenza viruses to produce a virus with
new HA and NA
 Direct infection of humans by Flu virus of other animals (pigs or birds) without genetic
reassortment
 Non-human flu virus is passed from one type of animal (ex. Birds) through an
intermediate host (pigs) to humans
 Antigenic Drift
 occurs in Influenza A, B and C
 minor antigenic change; seen as point mutations in the genetic material during replication
 changes in HA and NA
 slower occurrence leading to new viral strains
WHO are RISK for Flu illness
o Seronegative Individuals
o Adults
o School aged children

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

WHO are at RISK for hospitalization due to FLU

 Children less than 2 years old
 Persons with certain medical conditions (diabetes, heart disease, lung disease or immunocompromising
condition)
 Pregnant women
 Persons 65 years old and above
3 Major Flu Pandemics That Occurred In The 20th Century
 Spanish Flu (1918-1919) – caused by A(H1N1)
 Asian Flu (1957-1958) – caused by A(H2N2)
 More than 1 million dead worldwide
 Hong Kong Flu (1968-1969) – caused by A(H3N2)
 More than 1 million dead worldwide
 Recent Flu Pandemics
 A(H5N1) – Bird Flu (1998 – HongKong)
 Sporadic human infections are far more severe with high mortality; Longer incubation
period
 A(H1N1) – Swine Flu;
 2009; Similar to seasonal flu
 Dominant Strains of Influenza A since 1977 are H1N1 and H3N2
 Virus attacks the upper respiratory tract
 Severity of the disease depends on the immune status, presence or absence of underlying disease of
the infected patient
 Disease is more common in the winter months
Clinical Syndrome of Influenza
 Incubation period of 1-4 days
 Prodrome of malaise, headache
 Followed by abrupt onset of fever, myalgia and usually nonproductive cough, coryza (rhinitis)
 Acute illness lasts for 1 week but malaise and cough may persist for 2 weeks
 Diarrhea may also be seen in influenza caused by pandemic strains (AH5N1)
Complications
 Secondary bacterial complications
 Exacerbation of pre-existing conditions
 Otitis media especially in children
 Other complications: Reye’s syndrome, myositis, myocarditis
Diagnosis
 Specimens: Nasopharyngeal swabs, washes, or aspirates
 DFA, EIA, Optical Immunoassays, Cell Cultures, Commercially Available Rapid Kits
 Specific ID of Flu Strain is done through immunofluorescence or inhibition of hemadsorption or
hemagglutination with specific antibodies
Prevention:
 Vaccination (Trivalent Vaccine)
 Annual Vaccination for high risk groups
 Vaccination (Live Attenuated or Inactivated Forms)
Benefits of Vaccination
 Reduction of influenza related respiratory illnesses
 Reduction of hospitalizations and deaths
 Reduction of otitis media
 Reduction of work absenteeism

֍ Family Rhabdoviridae

 Rhabdo – “bullet shaped”

 ssRNA; negative stranded
 Helical Structure
 Genus: Lyssavirus
 Causes: Rabies (“Madness”)
 Genus: Lyssavirus has 7 Genotypes
 Genotypes 1-7 are Human Pathogens
 Broad Host Range
 TYPE 1: Classic, Worldwide Type
 Street Virus – isolated from humans and animals
 Virus Fixe (Pasteur)
RABIES
 major public health problem; Zoonotic disease with many animal reservoirs
 Acute infection of CNS (Very FATAL)
 MOT: Transmitted by a bite, scratch of a rabid animal Virus is widely distributed in the nervous system,
saliva, urine, lymph, milk and blood of infected animals
 MOT: can also be through organ transplantation, inhalation of aerosolized virus; inoculation through
intact mucus membrane
Pathogenesis
 Receptor: acetylcholine receptors
 Virus multiplies in the muscle or CT of the site of bite
 Virus slowly travels through the peripheral nerves
 Reaches the CNS for further replication; Encephalitis occurs and neuronal degeneration
 Dissemination to other organs
 RABIES IS FATAL ONCE CLINICAL DISEASE IS SEEN
Clinical Manifestation Of Rabies In Dogs
 Prodrome Stage – increased alertness or apathy, fever, papillary dilatation, increased muscular tone
 Excitement Stage – unusual restlessness, biting at inanimate objects, aimless running, difficulty in
swallowing, change in bark or growl

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FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Paralytic Stage – dog unable to take food or swallow water, paralysis of the jaw and tongue, drooling
saliva, paralysis of hindquarters
 Death occurs within 3-7 days after the initial symptoms
Forms Of Rabies In Dogs
1. Furious Rabies – AKA Excitatory Rabies
2. Dumb Type – opposite of furious type
Three Stages Of Human Rabies
1. Prodromal Phase – 2-3 days. Symptoms of Fever, Vomiting, Loss of Appetite, Headache and pain at
the site of bite. Autonomic Nervous System is Affected. Salivation and sadness is noted
2. Anger Stage – Restlessness and Irritable. Patient becomes aggressive. Seizures may develop. This
Stage lasts for 2-4 days
3. Neurological Stage or Paralytic Stage
 Paralysis beginning in the bitten area
 Difficulty in swallowing; Uncontrolled movement, Confusion, Delirium
 Hydrophobia, Aerophobia
 Anxious and Hyperactive;
 COMA and DEATH then occurs
Pathogenesis
 CPE: Presence of Eosinophilic Cytoplasmic Inclusion: NEGRI BODIES
 Incubation Period: 2-16 weeks or longer
 Majority of Cases (90-95%) – 1 year or less
 Few Cases (5-10%) – 1-5 years
 Length of Incubation Period depends on
 Severity of Bite
 Distance of bite to the CNS
 Viral Load
 Immune Status
Prevention: Post-Exposure Prophylaxis by Vaccination
 Vaccines available in the Philippines (Active Immunization)
 Purified Verocell Rabies Vaccine (PVRV)
 Purified Duck Embryo Vaccine (PDEV)
 Purified Chick Embryo Cell Vaccine (PCECV)
 Passive Immunization
 Rabies Immune Globulin (either HRIG or ERIG)
 Regimen of Vaccination (WHO Standard Intramuscular Regimen)
 5 doses at Day 0,3,7,14 and 28
 Administer at deltoid area or anterolateral aspect of the thigh of infants
 Contraindication of Vaccination: Allergy to Eggs
Diagnosis
 DOGS: Specimen: Brain Tissue
 Fluorescent Antibody Test – Gold Standard
 Direct Microscopic Examination/Seller’s Test
 Mouse Inoculation Test
 RT-PCR
 Diagnosis of Rabies in Humans
 Fluorescent Antibody Test on brain or skin
 PCR
 Serology – Serum Rapid Fluorescent Focus Inhibition Test
 Histology

֍ ARBOVIRUSES

 Arthropod Borne Viruses

 Viral Infections that require arthropods (i.e. mosquitoes, ticks and sandflies) to transmit the
disease to susceptible humans
 Arthropods act as Vectors
 Hosts: Man and other vertebrates
 Man can either be a dead end host or a reservoir host
 3 Clinical Syndromes Of Arthropod Borne Viruses
 Fever, with myalgias, arthralgias and nonhemorrhagic rash
 Encephalitis
 Hemorrhagic Fever

 Major Arboviruses

 Bunyaviridae |Bunyavirus
 Flaviviridae | Flavivirus
 Togaviridae | Alphavrius
 Reoviridae | Orbivirus

Medically Important Arboviruses

 Yellow fever
 Dengue
 Chikungunya
 Japanese B encephalitis
 St Louis encephalitis
 Western equine encephalitis
 Eastern Equine encephalitis
 Russian Spring-Summer encephalitis
 West Nile fever

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Sandfly fever

1. Arbovirus - Bunyaviridae
 Segmented (-) sense RNA Viruses that are helical and enveloped
 RNA Viruses that are Majority Vector Borne but some members are rodent borne (robovirus)
 Causes Viral Hemorrhagic Fevers and damage to vital organs
 Disease Manifestation of Bunyaviruses
 Patients experience febrile illness, hemorrrhagic fever or encephalitis
 Infection starts as fever, muscle pain, joint pain and photophobia
 Mental Status Changes occur
 Rashes, Ecchymoses are also seen
 Bleeding in the gums, nose
 Vector Borne Viruses
 Genus Orthobunyavirus
 California Encephalitis Virus and La Crosse Virus
 Transmitted by Mosquitoes
 Causes Encephalitis
 Other viruses include
 Genus Phlebovirus – Rift Valley Fever Virus
 Disease: Rift Valley Fever – encephalitis and hepatitis
 Transmitted by ticks; aerosols of the virus
 Phlebovirus
 Sandfly Fever (Phlebotomus Fever)/ Pappataci Fever/3day fever
 Transmitted by Sand flies
 Endemic in Europe, North Africa, Asia and South Africa
 Nairovirus – Crimean Congo Hemorrhagic Fever Virus
 Transmitted by ticks
 Target Organs of Bunyaviruses
 Rift Valley Fever – Brain and Liver
 La Crosse and California Encephalitis and Crimean Congo Fever – vascular endothelium and liver

2. Arbovirus - Flaviviridae
 (+) ssRNA enveloped icosahedral virus
 Vector borne Zoonotic Viruses
 Primarily transmitted by mosquitoes and ticks
 Genus: Flavivirus
 Japanese Encephalitis Virus
 Dengue Virus,
 West Nile Virus,
 Yellow Fever Virus,
 St. Louis Encephalitis Fever
 Disease manifestation include fever, headache, muscle pain and exanthems, encephalitis, sometimes
hemorrhagic fever can occur depending on the virus
 Yellow Fever – Caused by Yellow Fever Virus
 Transmitted by Aedes aegypti or Haemagogus
 Cases seen in Africa and South America
 Manifestations
 Acute infection of fever, myalgia, backache, headache, anorexia, vomiting (black
vomit/hematemesis), nausea
 Toxic Phase: Development of Jaundice
 Distinct Cycles: Jungle Yellow Fever & Urban Yellow Fever
 Live attenuated Vaccine is available
 St. Louis Encephalitis – most common flavivirus infection in the US (Eastern and Central States)
 most have inapparent illness;
 initial symptoms: fever, headache, nausea, vomiting and tiredness
 Neuroinvasive disease – encephalitis (Adults)
 infection is milder in children
 transmitted by Culex mosquitoes
 West Nile Virus – causes febrile illness, encephalitis or meningitis
 Transmitted by Culex mosquitoes
 Documented in Europe, Middle East, Africa, India, Australia, US
 Risk factor: age (50 years old)
 Classic WNV infection: fever, headache, fatigue, skin rashes, swollen lymph glands, and/or eye
pain
o Japanese Encephalitis – leading vaccine preventable cause of encephalitis in the Asia and Western Pacific
region
 Vector: Culex tritaeniorhynchus
 Incubation period 5-15 days after the bite of a mosquito
 Occurs in rural and agricultural areas (rice farming); More common during the rainy season
 Patients experience Fever, chills, headache, fatigue, nausea and vomiting. The disease can
progress to encephalitis.
 Vaccine is available
DENGUE VIRUS
 5 serotypes (DENV1, DENV2, DENV3, DENV4, DENV5)
 Most rapid spreading mosquito borne disease in the world (WHO)
 Vectors: Aedes aegypti and Aedes albopictus are the two most important vectors of dengue
 Classic Dengue – “Breakbone Fever”
 Flu-like illness (high fever of 39-40C after 3-14 days from infection, myalgia, malaise,
headache)

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Maculopapular Rash, Leukopenia, Lymphadenopathy, thrombocytopenia (less than

150,000/cu mm), increasing Hct (5-10%)
 Rarely fatal and has few sequelae
 Dengue Hemorrhagic Fever
 More severe and sometimes fatal
 Similar to Classic Dengue but with shock and hemorrhage
 Occurs when the fever subsides
 WHO Criteria
 Fever or recent history of fever lasting for 2-7 days
 Hemorrhagic Manifestation
 Thrombocytopenia (<100,000/cu mm)
 Evidence of increased vascular permeability
 Evidences of Plasma Leakage include
 Elevated Hematocrit (>/= 20% above the mean Hct)
 Hypoproteinemia or Hypoalbuminemia
 Decline in Hct (after volume replacement)
 Dengue Shock Syndrome
 Occurs if the criteria for DHF is present and
 Evidence of circulatory failure
 rapid, weak pulse and narrow pulse pressure (≤ 20 mmHg [2.7 kPa])
 or (2) hypotension for age, restlessness, and cold, clammy skin
 Lab Findings
 Dengue Endemic Areas
 Positive Tourniquet Test
 Leukopenia (WBC ≤ 5000 cells/mm3)
 Helps in making an early diagnosis of dengue infection
 Total WBC is usually normal at the onset of fever; then leucopenia
develops with decreasing neutrophils and lasts throughout the febrile
period.
 Mild thrombocytopenia (100 000 to 150 000 cells/mm3) is common
 Below 1000,000 cells/mm3 are seen in about half of DF patients
 A Mild Rise of Hct (around 10%) –consequence of dehydration (associated
with high fever, vomiting, anorexia and poor oral intake)
 Liver Enzymes such as AST may be elevated
 Lab Methods
 Cell Culture – Confirmatory
 Acute Serum Sample is needed (0-5 days)
 Nucleic Acid Amplification Tests (NATs)
 RT-PCR
 Serology (IgM/IgG ELISA)
 Detection of Antigens (NS1 Antigen)
 Rapid Methods (ICT methods)
 Infection due to one serotype confers lifelong protection
 Re-infection with a different serotype leads to a more severe form of Dengue Fever
 Especially if DENV1 is followed by DENV2 or 3
 Or if DENV3 is followed by DENV2

 Why are secondary infections more severe??

 This is due to ANTIBODY DEPENDENT ENHANCEMENT
 Circulating Strains of Dengue in the Country
 DENV2 and DENV3

Flavivirus - Zika Virus

 1st identified in Uganda and Tanzania in 1952
 MOT: Mosquito bite (Aedes); sexual contact; vertical transmission
 Signs and Symptoms are similar to dengue fever;
 include fever, skin rashes, conjunctivitis, muscle and joint pain, malaise, and headache. These
symptoms are usually mild and last for 2-7 days.
 Possible Complication: Microcephaly and mental retardation; autoimmune disease, Guillian Barre
Syndrome

3. Arbovirus - Togaviridae
 (+) sense ssRNA Enveloped Icosahedral Virus
 Genera:
 Alphavirus (Arbovirus)
 Rubivirus (causes German Measles/Rubella)*
Alphavirus
 Cause a variety of mosquito borne diseases that usually cause encephalitis
 Eastern Equine Encephalitis
 Occurs in Eastern US
 High Fatality Rate; Half of those who survive suffer permanent CNS damage
 Vector: Culex, Aedes
 Natural Hosts: Birds
 Dead-end Hosts: Humans and Horses
 Western Equine Encephalitis - Milder than EEE
 Venezuelan Equine Encephalitis
 Mortality is less common
 Encephalitis is seen more in children
 Chikungunya Virus

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 CHIKUNGUNYA
 Kimakonde language, meaning "to become contorted”
 Transmitted by Aedes aegypti and Aedesalbopictus
 Symptoms appear between average 4 and 7 days (2-12 days) after the patient has been
bitten by the infected mosquito and these include:
 High fever (40°C/ 104°F) & Joint pain (lower back, ankle, knees, wrists or phalanges),
Joint swelling
 Predominant Symptoms are fever and arthritis
 Other signs and symptoms include rash, Headache, Muscle pain, Nausea, Fatigue
 Joint pain can last for weeks and even years
 Usually affects adults
 Rarely fatal and is self limiting
 Infection confers immunity
 Lab Diagnosis
 Serology (IgM/IgG)
 Culture
 PCR
 Laboratory Findings of Chikungunya
 Lymphopenia (Low Lymphocyte Count)
 Thrombocytopenia
 Elevated Creatinine
 Elevated Liver Enzymes
Robovirus - Bunyaviridae
 Rodent Borne*
 Hantavirus
 Hemorrhagic Fever with Renal Syndrome
 Hantaan Virus, Seoul Virus, Puumala Virus and Dobrava
 Old World Hantaviruses
 Puumala Hantavirus –causes nephropathia epidemica
 Reservoir Host of Hantaviruses: Deer Mouse
 MOT: of Hantavirus: Inhalation of aerosols of urine, feces and saliva of infected
rodents
 Sin Nombre Virus – newly identified virus in the American South west
 Causes Hantavirus Pulmonary Syndrome
 MOT: Inhalation of aerosolized mouse urine, saliva, saliva and feces
 Fever, chills, myalgia. Hypotensive shock may also occur and development of DIC
 Diagnosis: EIA, Immunohistochemistry
Togaviridae
 Genus Rubivirus
 Rubella Virus
 Causes: Rubella/German Measles/3 Day Measles
 highly contagious viral infection of children and young adults; milder than measles
 MOT: Coughing and Sneezing (Respiratory Droplets)
 Incubation period: 2-3 weeks
 Signs and Symptoms: low grade fever, maculopapular rashes, mild conjunctivitis, nausea, posterior
auricular lymphadenopathy, arthritis (in adults and women)
 Forscheimer spots, an exanthem consisting of petechial lesions on the soft palate can also be seen
 Congenital Rubella Syndrome
o Infection of pregnant woman in the 1st trimester particularly 1st month
o Can cause congenital malformations on the unborn child
 Patent ductus arteriosus, cataracts, deafness and mental retardation, premature
birth, microcephaly
 Teratogen
 Prevention: MMR Vaccine (Attenuated Vaccine)
 Given 2 doses
 1st dose (12-15 months)
 2nd dose (4 or 6 years old)
 Persons who are working in hospitals, soon to be pregnant women or women who are in their
child bearing age should be vaccinated (1 or 2 doses)
 Diagnosis: Culture, Serology (ELISA, hemagglutination inhibition), PCR

4. Arbovirus - Reoviridae
 “Respiratory Enteric Orphan Virus”
 Segmented dsRNA naked icosahedral virus
 Colorado Tick Fever – transmitted by ticks (Dermacentor andersoni).
 Dengue-like infection in the Western US
 Fever, myalgia, nausea, vomiting and rarely encephalitis
 dsRNA virus, naked
 Double capsid structure
 Medically Important Genera:
 Rotavirus
 Coltivirus –tick borne causing Colorado Tick Fever Virus
Rotaviruses
 Most important cause of infant gastroenteritis in the world
 5 groups (A,B,C,D,E);
 A is the most frequent
 Causes death and failure to thrive among infants
 Leading cause of death for children under 5 years old
 MOT: oral-fecal route; more common in developing countries

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Incubation period: 1-3 days

 S/S: fever, abdominal pain, vomiting, watery diarrhea, dehydration
 Pathology
 Affects villi of small intestine,
 Impairs transport mechanism of nutrients
 Diarrhea is due to impaired sodium and glucose absorption
 Disease lasts for 3-8 days
 Complications: Dehydration and metabolic acidosis
 Diagnosis
 Direct Methods (EM)
 Serology (IgM/IgG)
 ELISA
 PCR
 Prevention: Wash Hands; Basic Sanitation and Hygiene
 Vaccination: Given Orally (3 doses or 2 doses) starting at 2 months

֍ RETROVIRIDAE

 RNA Viruses that replicate in the nucleus

 Requires REVERSE POLYMERASE (RNA-Dependent DNA Polymerase)

 Some members are members possess Oncogenes and Proto-oncogenes

 Some members are Cytocidal (Cytotoxic)

 Subfamilies

 Oncovirinae – oncogenic viruses; associated with leukemias, sarcomas and lymphomas

 HTLV-1, HTLV-2 (seen in Hairy Cell Leukemia) and HTLV-5

 Lentivirinae

 HIV-1 and HIV-2

 Subfamily Oncovirinae

 Human T-cell Lymphotropic Virus (HTLV)

 HTLV1 – first Human retrovirus

 Causes adult T-cell leukemia, lymphoma and tropical spastic paraparesis

 HTLV2- isolated from a patient with Hairy Cell Leukemia

 Subfamily Lentivirinae

 Genus Lentivirus – ”slow”

 Slow viral diseases

 HIV-1: Worldwide Distribution

 Causes AIDS

 HIV-2: Seen in West Africa

 less severe as compared to HIV-1

Viral Characteristics (HIV)

 Structural Components

 Capsid

 2 identical ssRNA pieces

 3 essential retroviral enzymes: protease, reverse transcriptase and integrase

 Capsid proteins – p24 (major)

 Matrix Protein (p17)

 Surface glycoproteins (gp) – gp160 (gp120 and gp41)

 Structural genes present

 gag – “group specific antigen”

 Codes for p24 and matrix proteins

 pol – “polymerase” proteins

 Codes for reverse transcriptase, endonuclease

 env – “envelope glycoprotein”

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Code for glycoprotein that adhere to host cells and produce Cytopathic effect (fusion)

 Regulatory Genes

 tat gene – “transactive transcription”; enhances transcription and expression of viral

proteins

 rev gene – “posttranscriptional activator”; production of structural proteins

HIV

 Causes AIDS (Acquired Immunodeficiency Syndrome)

 First recognized among homosexual males in the US in 1981

 Discovered in 1981 by Dr. Robert Gallo and Dr. Luc Montagnier

Transmission of HIV Infection

Routes Specific Transmission

Known routes of Transfusion of blood and blood

transmission products

1. Inoculation of Needle sharing among intravenous drug

blood users

Needlestick open wounds, and mucous

membrane exposure in health care
workers

Tattoo needles

2. Sexual Anal and vaginal intercourse

3. Perinatal Anal and vaginal intercourse

4. Perinatal Intrauterine, Peripartum, Breastmilk

Characteristics of Lentivirus Infection

 Transmitted thru body fluids

 Virus persists indefinitely in infected hosts

 Viruses have high mutation rates

 Infection progresses slowly through stages

 It takes years for the disease to develop

 NB. Host factors like age, stress, genetics and concurrent infections are important in the pathogenesis

HIV: Disinfection & Inactivation

 10% hypochlorites

 50% ethanol, 35% isopropanol

 0.5% lysol, 0.5% paraformaldehyde

 0.3% H2O2

 pH 1.0, pH 13.0

 Readily inactivated in liquids

 Dried proteinatious materials need increased temperature & extended heating time

HIV Receptors

 Main Receptor is CD4 Molecule (particular its gp120 receptor)

 Found in T-helper cells and other CD4+ cells (monocytes, phagocytes)

Disease Mechanisms of HIV / AIDS

 HIV infects CD4+ T cells and cells of the macrophage lineage

 monocytes, macrophages , alveolar macrophage of the lung, dendritic cells of the skin & microglial
cells of the brain

 Causes lytic and subsequently latent infection of

 CD4 T cells and persistent low-level productive infection of macrophage lineage cells

 Stages of HIV Infection

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Acute Infection: Acute Retroviral Syndrome/ Primary HIV infection

 2-4 weeks after infection (may be 6-8 weeks)

 Flu-like illness; some are asymptomatic

 Large amounts of HIV in the blood

 Replication of HIV in CD4 cells

 Clinical Latency – Asymptomatic or Chronic HIV infection

 No symptoms or illness

 Low viral replication

 Lasts for a decade or less for untreated patients

 In the middle and end of the phase: Increased Viral Replication and Decreased levels of CD4 cells

 AIDS – immunocompromised state of the infected person

 Patients are vulnerable for opportunistic infections and certain cancers

 Definition: CD4 count is LESS THAN 200/ul

 NV: 500-1600/ul

 AIDS is also considered if patient suffers at least one opportunistic infection

regardless of the CD4 count

 Death: 3 years after (if untreated)

Pediatric AIDS

 Usually presents with clinical symptoms by age 2, death follows in 2 years "

 Particularly susceptible since the immune system has not developed at the time of primary infection

Indicator Diseases of AIDS

Opportunistic Bacterial Tuberculosis

Infections
Listeriosis
Protozoal Toxoplasmosis
Nocardiosis
Cryptosporidiosis
Salmonellosis
Isosporiasis
Streptococcal infection
Fungal Candidiasis

Pneumocystis
pneumonia

Cryptococcosis Opportunistic
Neoplasms
Histoplasmosis
Kaposi’s sarcoma
Coccidiomycosis
Primary lymphoma of
Viral CMV, EBC Infection the brain

Herpes simplex virus Other non-Hodgkin’s

infection lymphomas

Hepatitis B and C Others

Varicella-Zoster HIV wasting syndrome

Adenovirus infection HIV encephalopathy

Polyomavirus Lymphoid insterstitial

pneumonia
JC Virus

Laboratory Diagnosis

 Screening Tests:

 ELISA (detection of anti-HIV antibodies)

 IF

 1st Generation Tests: Purified Viral Lysates as Antigens

 2nd Generation: Recombinant Viral Proteins

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 3rd Generation: Double Antigen Sandwich Assay

 4th Generation: Detection of Ag and Ab

 Screening Test

 Rapid Diagnosis (using serum, plasma, blood, saliva)

 For ELISA:

 If Non-reactive: Report as Negative

 If Reactive: the test should be done in duplicate

 Confirmatory Testing Using Western Blot

 For Confirmation, send to STD/AIDS Central Cooperative Laboratory or RITM

 In SACCL and RITM

 Serum Samples are screened twice

 Rapid testing (Serodia HIV1/2)

 4th generation EIA (HIV1/2 and p24)

 If Positive to either, WB is then done

o Western Blot - confirmatory

 Detects Antibodies specific to p24, p31, gp41 and gp120/gp160

 Positive: 2 out of the 3 major antigens (p24, gp41 and gp120/gp160)

 Negative

 Indeterminate: (re-test after 6months)

o Viral proteins are separated first and immobilized. In subsequent steps, the binding of serum
antibodies to specific HIV proteins is visualized

o Result: Positive or negative

Plasma Viral Load

 Prognostic value

 Predictor of long term clinical outcome:

 CD4 lymphocyte count – best predictor of short term risk of developing an opportunistic disease

 AIDS: CD4 is less than 200/ul

 Normal Ratio of CD4:CD8 CELLS: 2:1

 Ratio of CD4: CD8 Cells in HIV: 0.5:1

 Immunophenotyping (CD4 count)

 Principle: Flow cytometry

 Measures amount of CD4 left in a host.

 Immunophenotyping or CD4 count is one of the monitoring tests for HIV

 Specimen of choice: EDTA anticoagulated whole blood

PRIONS

 Proteinaceous infectious particles

 Causes Transmissible Spongiform Encephalopathies

 Causes Neurodegenerative Diseases affecting humans and animals

 Characteristics

 Long Incubation Time

 Gradual Increase in Severity

 No-host immune response

 Non-inflammatory Process

 Presence of Spongiform changes, vacuoles, plaques on brain tissue

PRION Diseases

 Among Humans

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Creutzfeldt Jakob Disease

 Kuru

 Gertsmann-Straussler Scheunker Disease

 Fata Familial Insomnia

 Animals

 Scrapie

 Bovine Spongiform Encephalopathy

 MOT: ingestion

 Diagnosis: Clinical Findings; Histopathology; PrPScimmunostaining, Serology, Nucleic Acid Testing

 HIGHLY RESISTANT to INACTIVATION

HEPATITIS
Viral Hepatitis
 Systemic disease primarily involving the liver.
 Most cases of acute viral hepatitis in children and adults are caused by one of the following agents:
 Hepatitis A virus (HAV) – infectious
 Hepatitis B virus (HBV) – serum
 Hepatitis C virus (HCV) – post transfusion non A non B
 Hepatitis D Virus (Delta Virus) – delta
 Hepatitis E virus (HEV) – enteric non A non B
 Additional well-characterized viruses that can cause sporadic hepatitis, such as:
 Yellow fever virus
 Cytomegalovirus
 Epstein-Barr virus
 Herpes simplex virus
 Rubella virus
 Enteroviruses
 Hepatitis viruses produce acute inflammation of the liver, resulting in a clinical illness
 Early signs and symptoms include:
 Flu-like illness (such as fever, nausea, vomiting, myalgia, diarrhea)
 Mild to moderate pain on the right upper quadrant of abdomen
 Progression can lead to:
 Hepatomegaly, liver tenderness, jaundice, dark colored urine, light colored feces
 Elevated bilirubin and liver enzymes
 Regardless of the virus type, identical histopathologic lesions are observed in the liver during acute
disease

Properties of Viral Hepatitis

Virus Hepatitis A Hepatitis B Hepatitis C Hepatitis D Hepatitis E
Family Picornaviridae Hepadnaviridae Flaviviridae Unclassified Unclassified
Genus Hepatovirus Orthohepadnavirus Hepacivirus Deltavirus Hepevirus
Virion 27 nm, 42 nm, spherical 60 nm, 35 nm, 30 – 32 nm,
icosahedral spherical spherical icosahedral
Envelope No Yes (HBsAg) Yes Yes (HBsAg) No
Genome ssRNA dsDNA ssRNA ssRNA ssRNA
Genome size 7.5 kb 3.2 kb 9.4 kb 1.7 kb 7.6 kb
Stability Heat and acid Acid-sensitive Ether- Acid-sensitive Heat stable
stable sensitive, acid-
sensitive
Transmission Fecal-oral Parenteral Parenteral Parenteral Fecal-oral
Prevalence High High Moderate Low, regional Regional
Fulminant Rare Rare Rare Frequent In pregnancy
disease
Chronic disease Never Often Often Often Never

Oncogenic No Yes Yes ? No

Hepatitis A
 Picornaviridae Family
 Formerly classified as Enterovirus 72, now Genus Hepatovirus
 Only one serotype is known.
 There is no antigenic cross-reactivity with HBV or with the other hepatitis viruses.
 ssRNA enveloped icosahedral virus
 MOT: Almost Always Transmitted by the Fecal Oral Route
 Ingestion of Contaminated Shellfish or other food and water
 Outbreaks can occur
 Most frequently infected group: CHILDREN
 Risk Factors
 Sexual Contact with Infected Person
 Household contact with Infected Person
 Daycare contacts
 Food borne or Waterborne outbreaks

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 IV drug use
 International Travel
 Incubation Period: 28 days (1month)
 Clinical Features
 Children (<5yrs): are usually ASYMPTOMATIC
 Adults usually manifest the disease (mild to severe prolonged)
 No Chronic Infection
 Symptoms usually last for 2 months
 Shedding of the virus in the feces
 Usually self limiting and non fatal
 Infection Provides lifelong immunity
 Laboratory Diagnosis
 Serology
 Detection of Total Anti-HAV
 IgM anti-HAV: recent infection (acute Hepatitis A)
 IgG anti-HAV: past infection/indicates immunity to the infection
 Hepatitis A

 Administration of Immune Globulin to Exposed Individuals

 80% effective if given before or early in the incubation period
 Given 2 weeks after exposure
 Short Term (3months)
 Prevention:
 Hepatitis A Vaccine (2 Doses)
 Indicated for:
 Infants aged 12-23 months
 High-risk individuals
 Individuals travelling to high risk areas
 Sanitation
 Chlorination

Hepatitis B
 Family Hepadnaviridae
 DNA virus; enveloped
 8 Genotypes
 Virus can survive in the environment for 7 days!! And still viable
 MOT: Blood-borne, Sexual, Perinatal
 High Risk Groups
 IV Drug Users
 MSMs
 Individuals from endemic areas
 Sexual Contact with infected persons
 Health Care Personnel
 People with tattoos and piercings
 Infants born to HBV(+) mothers
 Incubation period: 10-12 weeks
 Clinical Features
 Duration of Symptoms can last for days to up to 6 months
 Manifestations are similar to Hepatitis A but more severe and can be life-threatening
 Acute Hepatitis – usually asymptomatic;
 some may experience flu-like symptoms
 <1% can develop fulminant hepatitis
 Occurrence of Chronic Carrier State
 Persistent infection of hepatocytes
 Dependent on the levels of T-cytotoxic cells
 HIGH RISK for DEVELOPMENT of HEPATOCELLULAR CARCINOMA
 HIGH RISK for CHRONIC Hepatitis B
 80-90% of Infected infants will eventually develop Chronic hepatitis B infection
 30-50% of children infected before 6 years old
 ADULTS: <5%
 From the 5%, 20-30% of these will develop Liver cirrhosis or Liver cancer
 RECOVERY: Majority of Adults (95%) recover completely
 Diagnosis
 Clinical Presentation
 Demonstration of Serologic Markers
 HBsAg – detected in high levels of acute or chronic HBV

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 HBeAg – product of nucleocapsid; viral replication;

 infected person has high levels of HBV; high likelihood of transmissibility
 Anti-HBe – seroconversion; indicates lower levels of HBeAg
 Total anti - HBc – lifelong marker
 IgM anti-HBc – recent infection (<=6mos); acute infection
 Anti-HBs –recovery and immunity; develops in vaccinated persons
 HBcAg – not detected in serum; forms the nucleocapsid core

 Chronic HBV Infection

 Chronic Infection Low Infectivity
 HBsAg (+)
 Anti-HBc (+)
 Anti-Hbe (+)
 Chronic Infection High Infectivity
 HBsAg (+)
 HBeAg (+)
 Anti-HBc (+)
 Tx, Prev’n, Control
 Treatment
 Acute Hepatitis: No antiviral therapy is typically used
 Chronic HBV: Interferons such as peginterferon alfa-2a or peginterferon alfa-2b;
nucleoside analogues such as lamivudine, adefovir
 Prevention:
 Blood screening
 Vaccination (Recombinant Vaccine)
 3 Doses
 Hepatitis B Immune-globulin – post-exposure prophylaxis

Hepatitis C
 Non-A, non-B (NANB) hepatitis agent
 HCV is a positive-stranded RNA virus, classified as family Flaviviridae, genus Hepacivirus
 6 Genotypes
 Genotypes 1a and 1b – most common in US
 Usually related to blood transfusions
 Most cases of post-transfusion NANB hepatitis were caused by HCV.
 MOT
 Injection Drug Use (Common MOT in US)
 Transfusion of Infected Blood
 Organ Transplants
 Needlesticks
 Birth to an HCV (+) mother
 Sexual Contact (not an efficient MOT)
 Symptoms appear 4-12 weeks after infection
 Most patients are asymptomatic
 Acute Form- similar to acute Hepatitis A & B but
 inflammatory response is less and symptoms milder
 Chronic Form- more prevalent than HBV, often leads to cirrhosis
 Most patients (70-90%) develop chronic hepatitis
 20-30% of Chronic Hepatitis Patients develop cirrhosis which could further lead to hepatocellular
carcinoma
 Infects liver cells primarily
 Mediated by T-cytotoxic Cells
 Viral replication is enhanced by a liver specific micro-RNA
 Alcoholism – enhances rate liver carcinoma
 Laboratory Diagnosis
 Serology: Anti-HCV is not protective;
 HCV infection dose not provide persistent and life long levels of the antibody
 Appear 6 weeks or 12 weeks after infection
 Elevation of ALT can also be seen
 Methods:
 Gene Amplification Tests to detect HCV RNA
 EIA – screening test
 2nd Generation Immunoblot tets - RIBA (Recombinant Immnunoblot Assay) – used to
confirm

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Tx, Prvn and Control

 Interferon alpha and ribavirin: the only effective treatment (50% response rate)
 liver transplant
 supportive
 blood screening
 No Vaccine Available

Hepatitis D
 Delta agent: HDAg surrounded by HBsAg envelope
 HDV: ssRNA, smallest of known human pathogens
 responsible for about 40% of fulminant hepatitis
 Highly dependent on HBV for replication
 In blood, HDV (delta agent) contains delta-Ag (HDAg) surrounded by an HBsAg envelope.
 HDV is a defective virus that acquires an HBsAg coat for transmission.
 It is often associated with the most severe forms of hepatitis in HBsAg-positive patients.
 MOT:
 Parenteral or percutaneous or mucosal contact with infected blood
 Co-infection can occur alongside HBV
 Superinfection can also occur
 High Risk:
 IV drug users and hemophiliacs
 Agent increases severity of Hepatitis B infection
 Delta agent carriers: more likely to develop fulminant hepatitis
Anti-HBc IgM HBsAg Anti-HDV Anti-HDV IgM
Co-infection + + + +
Super-infection - + + N/A

 Laboratory Diagnosis
 ELISA & RIA (Ag /Ab detection)
 HDV-RNA by PCR
 Markers:
1. HDV- the agent
2. HD Ag -delta antigen (detectable in early acute infection)
3. Anti-HDV indicates past or present infection
 Treatment
 No known specific treatment
 HBV Vaccination is protective

Hepatitis E
 MOT: Similar to Hepatitis A
 Transmitted enterically and can cause epidemics in developing countries, where water or food
supplies are sometimes fecally contaminated
 HEV causes an acute self-limiting disease similar to HAV
 Incubation Period: 2-9 weeks
 Mortality rate is higher as compared to HAV
 Pregnant women may have a high (20%) mortality rate if fulminant hepatitis develops.
 Diagnosis: ELISA (IgM anti-HEV, IgG anti-HEV)

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

Other Hepatitis Viruses

 Hepatitis F – virus like particles (dsDNA)
 Thought to be a mutant strain of HBV
 Hepatitis G – Flaviviridae family
 Now called as GBV-C virus
 Transfusion transmitted Hepatitis
 Related to HCV
 No associated disease yet

-end-

REFERENCES

Carroll, K. C., Hobden, J. A., Miller, S., Morse, S. A., Mietzner, T. A., Detrick, B., Mitchell, T. G., ... Sakanari,

J. A. (2016). Jawetz, Melnick, & Adelberg's medical microbiology.

Carter, J. B., Saunders, V. A., & John Wiley & Sons. (2015). Virology: Principles and applications. Chichester:

John Wiley & Sons.

Jorgensen, J. H., In Jorgensen, J. H., Pfaller, M. A., Carroll, K. C., In Carroll, K. C., In Pfaller, M. A., &

American Society for Microbiology,. (2015). Manual of clinical microbiology.

Mahon, C. R., Lehman, D. C., & Manuselis, G. (2015). Textbook of diagnostic microbiology.

Ryu, W.-S. (2017). Molecular virology of human pathogenic viruses.

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