DHF,

HIV, Poliomyeli/s, Herpes

simpleks 1 & 2, Varicella Zoster,
Morbilli, Mumps Rubulavirus, RSV,
Molluscum contagiosum, HPV
By Rizalinda Sjahril
Microbiology Department
SISTEM TROPIS
FK UNIVERSITAS HASANUDDIN
2019
 Virus and Diseases Caused
Family Gen Genus (--Virus) Route of Disease
(-idae) transmission
Retrovir- HIV Blood/body AIDS
fluid/sexual
Flavivir- RNA Dengue V. Vectorborne DF/DHF/DSS
PicoRNAvir- RNA Polio V. Feco-Oral Poliomyeli/s
*Pneumovir- RNA Orthopneumovirus Respiratory Cold-like /pniae
Paramyxovir- RNA Morbillivirus Respiratory Measles
Mumps Rubulavirus Paro//s
Herpesvir- DNA Herpes simpleks 1 Respiratory/ Herpes labialis
Herpes simpleks 2 Contact Herpes genitalis
Varicella Zoster V. Ch. pox /shingles
Poxvir- DNA Molluscum Contact/ Molluscum
contagiosum virus sexual contagiosum
Papovavir- DNA HPV Sexual/ Warts/Ca
Contact/
fomites
* New taxonomy of the Mononegavirales as of 2016
Incidence/Epidemiology
Clinical symptoms
Virology/Taxonomy/Genomic
Virulence/Host Immune response
Laboratory Diagnosis
PrevenLon/Control

DENGUE HEMORRHAGIC FEVER

VIRUS
 Dengue Virus (DENV)
•  Arbovirus = arthropod borne virus
•  4 serologically different Dengue viruses
•  Clinical cases:
–  Dengue fever (DF) presents an acute self-limi(ng fever
–  Dengue hemorrhagic fever (DHF) Trombositopenia and increased
–  Dengue shock syndrome (DSS). vascular permeability

•  No effec/ve DENV vaccine and an/virus.

 Clinical Symptoms of Dengue
•  Varies from asymptoma/c to severe with
bleeding and shock (DHF/DSS).
–  DHF is marked by plasma leakage and
hemorrhagic diathesis observed before
defervescence, usually day 5 of fever.
–  Death is due to hypotension and shock, o`en with
coagula/on disorders and bleeding.
 Taxonomy/Classifica/on
•  Family: Flaviviridae
•  Genera: Flavivirus
•  Type virus: Dengue Virus
 Life Cycle of DENV
An/viral Res. 2009 January; 81(1): 6–15.
In the low pH of the trans-Golgi network (TGN),
prM is cleaved by furin.
acachment

endocytosis

A`er replica/on,
assembly occurs
in ER membrane.
Encapsida/on( produces capsid and RNA)
Result in immature virion
Virology Journal 2005: 2; 26

Electron
Microscopy of
Virus DEN2
An/viral Res. 2009 January; 81(1): 6–15.
3 factors determining severeness of
dengue infec/on
 Host Factor
•  Example: During the epidemic of DHF/DSS in
Cuba infected Europeans were more severely
ill than African
•  Different alel class I of HLA shows different
suscep/bility to DHF.
 Other Disease and the role of Age
•  Other disease can be correlated with
severeness DHF/DSS: asthma bronchial,
diabetes mellitus, pep/c ulcer, and sickle cell
anemia
•  Age: di South EastAsia > children than adults,
probably due to more permeable vascular
endothelial among children.
•  Other countries found > more in children,
some found more in adults.
 Dengue Incuba/on Period
•  Extrinsic Incuba/on period
ü  Virus replicates within female mosquito a`er bi/ng an infected human
un/l the mosquito becomes infec/ous (8-12 days)
•  Intrinsic Incuba/on period
ü  A`er a mosquito bites a suscep/bel human (one who has never been
infected before) un/l symptoms appear (4-7 days; may range 3-14
days)
•  Period of communicability (human transmit virus to
bi/ng mosquito)
–  Shortly before un/l the end of symptoma/c viremia (4-5
days)
–  Human never transmit to human; but transfusion related
case is possible
 DHF/DSS: Autoimmune Response
•  Antibodi terhadap serotype-specific bersifat protektif seumur
hidup terhadap serotype yang sama(homolog), dan protektif
silang terhadap serotipe yang beda (heterolog) selama 34 bulan.
•  Antibodi terhadap protein E pada permukaan virus bereaksi
silang dengan plasminogen dan menyebabkan perdarahan, dan
Ab anti-DENV NS1 bereaksi silang sehingga merusak
platelet (trombosit) dan sel endotel inang
•  Respon imun tjd terhadap berbagai komponen DENV pada
DHF/DSS dengan bukti adanya immune activation markers
(IL-6, IL-8, TNFa, IFNg, dan komponen 3A dan 5A
complement) dengan perubahan fungsi platelet, DC, monosit,
dan sel T.

Viral Immunol. 2006 Summer;19(2):127-32.

An/body Dependent Enhancement
•  ADE terjadi pada masa protektif silang (infeksi kali ke2):
–  Antibodi yang sudah ada thd satu tipe Dengue karena infeksi
sebelumnya namun dlm konsentrasi non/subnetralisasi terhadap virus
heterolog (virus serotipe beda yang menginfeksi sekarang) akan
melekat pada DENV (membentuk imunkompleks Ab-DENV) dan akan
meningkatkan terikatnya DENV dengan sel yang mempunyai FcR ,
kemudian virus bereplikasi di dalam sel tersebut, mengaktivasi
komplemen sehingga dilepaskan banyak sitokin dengan target sel
endotel pembuluh darah.
•  ADE terjadi pada beberapa jenis sel yang mempunyai FcR yi
human plasmacytoid DC, mature DC dan monosit.
•  Selain ADE ada faktor lain (respons imun innate) yang ikut
berperan menentukan beratnya gejala klinis dan banyaknya
VL .

Virology. 2011 Dec 20;421(2):245-52.

Dengue in Infants

FEMS Immunology & Medical Microbiology Volume 59, Issue 2, pages 119–130, July 2010
 The Role of cytokine, chemokine and
leukocyte in Dengue pathogenesis
Virus àha/ dan limpa à bereplikasi dan
menyebabkan inflamasi à banyak
kemokin CC di dalam darah, ha/ dan
limpa à mengak/vasi sel (melalui
reseptor kemokin) àkerusakan jaringan
ha//limpa.

Lekosit yang terak/vasi (terutama
limfositTh17/Th22dan trombosit) ini
bersama kemokin CC masuk ke ha/.

Sel iNKT yang direkrut karena memiliki
reseptor CCR2 dan CCR4 akan
menyebabkan kerusakan ha/.

Sel NK menghasilkan IL22 dalam ha/. Sel
T gammadelta menghasilkan IL-17
bersama kemokin CC menyebabkan
proses inflamasi. Immunology. 2013 Oct 12.
 Laboratory Tests for Defini/ve
Diagnosis
Either one:
•  Nucleic Acid Tes/ng (PCR)
•  NS1 (nonstructural protein 1) in serum
•  VIRAL ISOLATION (/ssue culture)
•  IgG seroconversion or significant increase of an/body (by a
qualita/ve assay)
•  4 fold increase of an/body (quan/ta/ve assay) in paired
sera which is proven by neutraliza/on test or another
specific test
•  Dengue specific IgM in cerebrospinal fluid (in the abscence
of IgM to Murray Valley Encephali/s/Kunjin/Japanese
encephali/s virus)
•  Dengue specific IgM which is confirmed by a reference lab.
Time Sequence of Dengue Infec/on
Time of Laboratory Tes/ng

www.health.qld.gov.au
 Available tests
Five serological tests:
1.  hemaggluLnaLon-inhibiLon (HI)
2.  complement fixaLon (CF)
3.  neutralizaLon test (NT)
4.  immunoglobulin M (IgM) capture enzyme linked immunosorbent assay (MAC-ELISA)
5.  Indirect immunoglobulin G ELISA.
The limita*ons of these techniques are the high cross-reac*vity observed with these tests.

Four methods of viral isolaLon:
1.  intracerebral inoculaLon of newborn mice
2.  inoculaLon on mammalian cell cultures
3.  intrathoracic inoculaLon of adult mosquitoes
4.  inoculaLon on mosquito cell cultures.

Molecular diagnosLc techniques:
1.  nucleic acid hybridizaLon
2.  RT-PCR.

The Brazilian Journal of InfecLous Diseases 2004;8(6):390-398

Incidence/Epidemiology
Virology/Taxonomy/Genomic
Virulence/Host Immune response
Laboratory Diagnosis
PrevenLon/Control

HUMAN IMMUNODEFICIENCY VIRUS

 HIV
•  Simian Immunodeficiency Virus (SIV) terdapat
pada 26 spesies primata non-human di Afrika
•  Dua dari SIV diketahui dapat menyebabkan
HIV pada manusia:
–  HIV-1 – SIVcpz dari chimpanzee
–  HIV-2 – SIVsm dari sooty mangabey
–  Len/virus ditransmisikan ke manusia 8 kali

Emerging Infec/ous Diseases 2002; 8: 451-7

Epidemiology

hcp://aids.gov/hiv-aids-basics/hiv-aids-101/global-sta/s/cs/
 Taxonomy/Classifica/on of HIV
•  Family: Retroviridae (transcribed to DNA by RT’ase)
•  Genera: LenLvirus (slow onset of disease)
•  Type virus: HIV-1 and HIV-2

•  HIV-1 is most common world wide.

•  “HIV” refers to HIV-1.
•  HIV-2 reported only in Africa; clinical symptoms
are the same but HIV-2 progresses slower.
 Subtypes of HIV
Group P
Alfa, beta,
gamma
HIV-1 Group O
ORTHO
RETRO Len/virinae
Group N
VIRIDAE HIV-2
RETRO Group M Subtype A,B,C,D,F,G,H,J,K,CRFs
Delta,
VIRIDAE epsilon SIV
SPUMA
RETRO SPUMAVIRUS
VIRIDAE

HIV subtypes in Indonesia:

CRF_AE and B
 RNA
HIV structure Enzyme
Capsid
Capsomer
Lipid layer
glycoprotein
 Important enzymes of HIV
Enzymes for viral replicaLon:
•  Reverse transcriptase – tRNA serves as primer
•  Integrase –
•  Protease –
•  Ribonuclease --
 Gene and gene products of HIV
Protein Gene Product FuncLon
Structural Gag P17 (matrix) Core Protein
P24 (capsid)
P7(nucleocapsid)
Env Gp120 envelope
Gp41
Cataly/c Pol Protease Enzyme
Reverse transcriptase
Integrase
Regulator Tat Tat increase mRNA produc/on
Rev Rev Transport mRNA
Addi/onal vpu Vpu Degrade CD4, viral release
proteins vif Vif Inhibit cell ac/on that inhibits
vpr Vpr virus
nef Nef Import to nucleus
HIV replica/on within Ly T and
monocyte
 Lab Diagnosis
1.  An/body (rapid test) – prick blood test or
oral test
•  Not for a very recent contact
•  True nega/ve if repeated result remains nega/ve
a`er 12 weeks since contact
2.  An/gen (detect p24)
•  Can detect the presence of an/gen at day 22-28
a`er contact
3.  Western Bloqng (detect protein pol, gag,
env)
–  Posi/ve à if pacern similar to “HIV posi/ve”
Control à report as HIV posi/ve
–  Nega/ve à no pacern
–  Indeterminate à lacking enough detected
protein as compared to “HIV posi/ve Control”
•  Test again within the next 6 months
•  Should convert to ‘determinate” at one month
a`er contact
•  Indeterminate is assumed as nega/ve if no change
of detected protein a`er 6 months
4.  NAAT/PCR
•  Few days - 1 week a`er contact

Ref: Alexander TS. 2016. Human immunodeficiency virus diagnos/c

tes/ng: 30 years of evolu/on. Clin Vaccine Immunol 23: 249-253.
Ref: Alexander TS. 2016. Human immunodeficiency virus diagnos/c
tes/ng: 30 years of evolu/on. Clin Vaccine Immunol 23: 249-253.
 Cell Receptor for HIV
For entry to the cell HIV must ahach to CD4 receptor and a co receptor:
Co receptor CCR5 is found on macrofage and T lymfocyte;
CXCR4 is found on T lymphocyte.

Virus entry No Virus entry

enabled due to is due to the
availability of chemokine co-
CD4 receptor receptor (CCR5
and a chemokine or CXCR4) is
co-receptor engaged in the
binding to its
ligand

Ref: Sultan Qaboos Univ Med J. 2007 August; 7(2): 82–96.

 HIV Replica/on
2. RNA, enzim RT,
1. Fusi virus pada
integrase, protein
permukaan sel
lainnnya
Masuk ke dalam sel

3.Pembentukan DNA
virus

4. Masuk ke nukleus
dan
alami integrasi
7. Maturasi terjadi;
virion siap keluar sel
5. RNA baru dari
6. RNA virus baru
virus berperan
beserta protein yang
dalam pembentukan
diperlukan
protein
dibawa ke permukaan
sel (bentuk imatur)
 HIV-1-infected MT-4 cells
A. infected cells with budding structures and immature virus.
B .mature extracellular HIV-1 par/cles, and
C.a higher-magnifica/on view of an immature par/cle s/ll connected to the cellular
membrane
 HIV Entry into Cell

HIV entry starts by Gp120 binding to CD4 receptor. à

binding changes the conforma/on and causes Gp120 binds
to co receptor CCR5 or CXCR4 à binding to co receptor
triggers membrane fusion and forms six-helix bundle.
From:Ref: Chukwuka et al, in Viruses. 2012 February; 4(2): 309–324.
How ARV works
HIV Resistance to ARV drugs

Ref: Daniel R. Kuritzkes, MD

HIV Drug Resistance: New Insight and Updated PracLces
 HIV Gene/c Varia/ons
hcp://www.ncbi.nlm.nih.gov/books/NBK2245/

•  Gene/c varia/ons among subtypes of HIV:

–  Allow virus evade the immune system
–  Persistence
–  An/viral resistance

In subtype B, only one nucleo/de change is required to switch from the wild-type (WT) valine to
the mutant adenine, whereas in subtype G two nucleo/de changes are required (a).

Adenine will therefore be the preferen/al drug-resistant subs/tu/on in subtype B.
In subtype G, resistance is preferen/ally acquired by changing the WT isoleucine for the
mutants threonine or methionine, requiring a single nucleo/de change (b).
 In lymphnodes

HIV
during
acute In Bloodstream
phase

Disseminate to brain, spleen, gut

mesenteric lymphoid /ssue and
lymphnodes
Ref: Kahn, 1998
Time frame, site of infec/on, major
events at vaginal transmission

Haase, AT,
Nature: 464, March 2010
HIV Transmission via Mucous Membrane

Viruses enter
through minor
lesions on the
mucous layer;
bind to CCR5 (on
macrophage and
Lymphocytes)
and CXCR4 (on
Lymphocytes)

Ref: Davis and Doms, J Exp Med. 2004 April 19; 199(8): 1037–1040
 Preven/on of HIV Transmission
• 

Ref: Lancet. 2013 Nov 2;382(9903):1515-24.

 HIV Reservoir in the body
•  The main obstacles to viral eradica/on are:
–  HIV-1 remains latent in resLng CD4+ T Lymphocytes
(In Vivo)
–  Blood-/ssue barrier inhibits ARV drugs entering the
CNS, the reLna, and the testes.
–  Cryp/c viral replica/on in infected CD4 lymphocytes
–  Virions bound to dendri/c cells in the lymphnode
persist for a long /me
 HIV Crisis: world’s Largest Challenge
•  33.4 millions of people died with HIV/AIDS
•  >25 millions died due to AIDS since 1981
•  2008: 2 million died but 2.7 million new cases
iden/fied.
•  97% pa/ents reside in low-moderate especially
sub-Sahara Africa: a country which cannot
prevent the spread.
•  No cure (ARV only supresses viral replica/on)
 HIV Cure
•  FuncLonal cure: When
a`er the pa/ent stop ART
their HIV remains in
remission and does not
damage their immune
system enough to cause
any adverse health
consequences. Examples of HIV functl’ cure:
•  Sterilizing cure: the The Berlin Pa/ent (2008)
complete eradica/on of all The Mississippi baby (2013)
HIV from a person’s body. The VISCONTI Cohort (2014)
Viscon/=virological and Immunological Study on Controllers a`er Treatment Interrrup/on
 Elite Controller of HIV
•  Are a unique subset of HIV-infected
individuals who spontaneously
control HIV (=normal CD4+ and low or
undetectable virus) without given ARV à
'func/onal cure' (long term control of viral
replica/on and remission from symptoms
of HIV infec/on in the absence of
an/retroviral therapy)
 Man Func/onally Cured from HIV
•  The Berlin PaLent – Timothy Brown was an American
who was diagnosed as HIVposi/ve in Berlin in 1995, received
ART for 10 years and then was diagnosed with AML (acute
myeloid leukemia). He then was transplanted stem cells from
a Caucasian donor who gene/cally lack the CCR5 gene
encoding the host cell’s HIV co receptor) and had claimed HIV
cured in 2008.
The ‘Berlin pa/ent’ has shown that the re-engra`ment
of bone marrow stem cells with certain gene/c
traits can cure AIDS and at least inhibit HIV infec/on in
the long term to below detectable levels.
 The Mississippi Baby
•  The baby girl born in 2010 was given ART at 31
days a`er birth from a HIV pos mother, given
/ll 18 months, ART stopped due to lost from
care, a`er 28 month without ART she
remained with no detectable HIV RNA and
was reported as func/onally cured (March
2013).
•  Unfortunately Rebound was detected in this
baby at the age of 4 years old (July 2014!!!)
The HIV Cured Baby Rebound
 VISCONTI Cohort
•  14 persons in France (diagnosed HIV pos from
between 1996-2002) who were soon started
ART (during acute phase), given for at least 1
year and then stopped, they showed
undetectable virus RNA for 4 years now.
Incidence/Epidemiology
Clinical Appearance
Virology/Taxonomy/Genomic
Virulence/Host Immune response
Laboratory Diagnosis
PrevenLon/Control

POLIOMYELITIS VIRUS
‘I was diagnosed with paraly(c poliomyeli(s,
which is experienced in less than 1 percent of
poliovirus infec(ons. Not only did it immobilize
me completely from the neck down, it also
aLacked my lungs. It was August (1949), a
popular month for polio, and I was six years
old.”

hcps://www.huffpost.com/entry/my-polio-story-is-an-inconvenient-truth-
to-those-who-refuse-vaccines_b_57b22672e4b07184041270f6
 Poliomyeli/s Akut
•  WHO 1988: targetkan eradikasi global poliomielitis paralitik
sblm 2000
•  Strategi:
–  1) vaksinasi massal dgn vaksin live attenuated oral pd anak <5 th
–  2) surveillans
–  3) imunisasi pada daerah/populasi yang kemungkinan masih
terdapat penyebaran polio
•  Kenyataan: Virus wild-type polio masih terus ada pada daerah
endemik, dan kadang2 pada daerah yg dianggap sudah bebas
polio.
–  PV type 2 terakhir diidentifikasi 1999 –declared eliminated 2015
–  PV type 3 terakhir diidentifikasi 2012 –declared eliminated 2019
–  Sisa PV type 1 belum eliminasi
Deteriorasi
Fungsional
Akibat kelemahan otot
dan
gangguan bentuk
rangka tubuh

Sindrom Deteriorasi Fungsional

yangTerlambat deteriorasi tjd
selama bbrp tahun setelah
poliomyelitis paralitik akut atau
'post-polio syndrome’ àgejala
motor neuron terjadi setelah
bertahun2 infeksi dimasa kecil –
salah satu contoh: Franklin D.
Roosevelt
 Perjalanan penyakit
•  Reservoir : GIT
•  Rute infeksi: oral - oral dan faecal - oral.
•  Viruses multiplikasi dalam farings dan usus masa
inkubasi 1-3 minggu – diseminasi darah – seluruh
tubuh
•  Eksresi saliva 2-3 hari, selanjutnya pada faeces 2-3
minggu kemudian.
•  Sifat infeksius: 7-10 sebelum dan setelah gejala
muncul.
•  95% infeksi virus ini asimptomatik atau seperti flu-like'
illnesses yangsembuh sendiri
 POLIOMIELITIS NON-PARALITIK atau
PREPARALITIK

•  Setelah gejala prodromal, pasien mengalami
demam tinggi, faringitis, myalgia, anorexia,
nausea dan muntah-muntah, dan sakit kepala,
kaku leher (meningitis).
•  Gejala ini menghilang dalam 1-2 minggu.
 POLIOMIELITIS PARALITIK

•  Setelah fase meningitis à poliomyelitis tipe spinal (nyeri
otot hebat, kadang dengan kejang otot, melemah asimetris,
sensorik baik hanya paraestesi, sering pada tungkai saja dan
fasikulasi).
•  Bentuk bifasik: ketika kelemahan otot baru terjadi setelah
melewati periode singkat dimana kondisi pasien membaik.
•  Infeksi tanpa kelemahan otot bisa terjadi terutama pada
anak-anak yang sudah diangkat tonsil/adenoid
•  Orang dewasa bisa bergejala spinal. Infeksi ke medulla bisa
menyebabkan disfagi, disfonia dan gagal nafas
•  Gangguan Vasomotor (hipertensi, hipotensi dan kolaps
sirkulasi menyebabkan kematian).
•  Gangguan kencing dan gangguan BAB.
•  Tipe encefalitik: agitasi, confusion, stupor, coma.
Rancho Los Amigos Rehab Cntr 1953

J Neurosci. 2013 January 16; 33(3): 855–862.

 Taxonomy/Classifica/on
•  Family: PICORNAVIRIDAE
•  Genus: Enterovirus
•  Type name: Poliovirus

There are 3 Wild Type Polioviruses :

1.  Type 1
2.  Type 2
3.  Type 3

TEM Photograph of 30 nm virus par/cle

. J Neurosci. 2013 January 16; 33(3): 855–862
 Structure of
Poliovirus
•  Pos strand ss RNA

J Neurosci. 2013 January 16; 33(3): 855–862.

Pathogenesis infeksi Poliovirus
Proc. Jpn. Acad., Ser. B 83 (2007)
 Strategi Virus Bertahan di dalam sel
neuron
•  Sel neuron dapat membatasi replikasi dan
penyebaran virus ini à virus hilang tanpa
banyak neuron rusak atau terjadi infeksi
persisten non sitoliLk.
–  Dugaan mekanisme terjadinya post polio
syndrome
•  Reaksi IgM thd virus yang baru muncul lagi belakangan
hari (terbuk/ dengan pemeriksaan PCR posi/f)
 Acute Flaccid Paralysis
•  Jika ada kejadian AFP di faskes, dilakukan
s u r v e i l a n s d e n g a n k u e s i o n e r ; d i s e r t a i
pengumpulan sampel faeces 2 kali dalam interval
>24 jam, dalam waktu 14 hari sejak onset
paralisis.
•  Konsultasikan cara pengiriman sampel dengan
Dinas Kesehatan Propinsi Sul-Sel à kirim sampel
ke pusat pemeriksaan Poliomyeli/s Virus di
Indonesia:
•  Litbangkes, Jakarta
•  Biofarma, Bandung
•  Balai Besar Lab Kesehatan, Surabaya
 Surveillance
Acute Flaccid Paralysis (AFP)
–  Isolasi virus
•  Faeces + medium kultur sel 10% Chloroform v/v
–  sel kultur (L20B) atau RD-A à ama/ adanya CPE à sel hancur
•  Konfirmasi dengan netralisasi an/sera dan/atau
–  RT (reverse transcriptase) PCR dan sequencing atau
Nucleic acid probe hybridiza/on
–  ELISA (typing) strain Sabin dan WT à tentukan:
•  strain Sabin (=strain vaksin)
•  Wild Type strain
•  Double reac/ve terhadap Sabin dan WT (akibat mutasi
dari strain Sabin)
Ref: Kelly et al, Journal of Paediatrics and Child Health 42 (2006) 370–376
 Specimen collec/on
Stool Serum
•  > 1 gram •  0.2 mL
•  Sterile container, do not •  Sterile container
add transport medium
•  Highest likely to detect •  Less likely to detect virus
virus

Nasopharyngeal/ CSF
Oropharyngeal swab
ü  0.2 mL
ü  Store in 1 ml sterile viral
transport medium (VTM) ü  Sterile container, no
media, do not dilute


Storage and shipping condiLon:

Freeze at -20oC
Ship on dry ice
 CELL CULTURE
Penggan/an medium (nutrisi) sel: sedot sel dan medium
lama, isi 1/10 jumlah sel lama lalu tambahkan medium baru

Plate berisi
kultur sel

Masukkan volume spesimen yang diperiksa (

 Cytopathic effect (CPE) pada kultur sel
Sel L20b
cytopathic effect (CPE)

Sel RD
cytopathic effect (CPE)

Thorley and Roberts, Chapter 4. Isola/on and

L20B = a mouse fibroblast line with a stable
Characteriza/on of Poliovirus in Javier Mar/n (ed);
gene/c integra/on of the human poliovirus receptor, CD-155 Poliovirus: Methods and Protocols, Methods in
RDA = human rahbdomyosarcoma cell line Molecular Biology, vol 1387
 Kultur Sel Virus Polio
•  Menggunakan rhabdomyosarcoma cell line (RD
cells) dan mouse L cell line yang mempunyai PV
receptor (L20B cells)
–  Keuntungan:
•  1) /dak perlu alat molekuler
•  2) sangat sensi/f (detec(on limit pada konsentrasi virus 50
– 1.000 virion
–  Kelemahan:
•  Perlu keahlian dalam mengevaluasi CPE*.
•  Perlu menjaga sterilitas kultur
•  (me-consuming: 10 hari

*CPE = cytopathic effect

 Deteksi RNA Virus
BMC Infect Dis. 2009 Dec 16;9:208

•  Reverse transcrip/on-loop-mediated isothermal

amplifica/on (RT-LAMP) system.
–  Keuntungan:
•  1) Alat minimal – cukup isothermal heat bath (suhu 60-65oC)
•  2) sangat sensi/f karena amplikon lebih banyak
–  detec(on limit 0.01 PFU* utk respiratory syndrome coronavirus
–  0.1 PFU utk mumps virus
–  0.4 focus forming units utk hepa//s A virus
–  50 copies utk swine vesicular disease virus
•  3) cepat -- hanya 1 jam
•  4) kurang kemungkinan kontaminasi silang antar sampel.
•  5) pembacaan amplikon dengan fotometer atau fuorometer
(jika ditambahkan dye fluorescense)

*PFU = plaque forming unit

 Deteksi Poliomyeli/s virus
Arita, J Clin Microbiol. 2010 August; 48(8): 2698–2702.

•  Par/cle agglu/na/on test

(A) Schema/c view of a sensi/zed

gela/n par/cle with a soluble PVR
(PVR-IgG2a).
(B) The order of sample addi/on to
the reac/on plate is as follows: 1,
an/-PV an/bodies; 2, PV solu/on;
and 3, sensi/zed gela/n par/cle
solu/on. r.t., room temperature.
 Pencegahan
•  Salk trivalent inactivated polio vaccine: tahun 1956: PER
INJEKSI à dapat mengstimulasi IgM, IgG and IgA tidak
mengstimulasi secretory IgA.
•  Sabin trivalent oral live attenuated polio vaccine (OPV):
tahun 1962, mengandung poliovirus I, II* and III* yang
tumbuh dalam sel kultur. PER ORAL à infeksi aktif
orofarings dan endotel usus shg IgA dihasilkan juga.
•  Timbulkan herd immunity karena disekresikan lewat feses.
•  Dewasa yg lahir sblm 1958 dan belum imunisasi harus
diimunisasi
•  Tiga dosis: 2, 3, 4 bulan
•  Eradikasi global tercapai bila semua orang berhasil
divaksinasi

* Komponen vaksin OPV tyoe 2 modifikasi

 OPV
•  tOPV = Sabin vaccine (1952) = acenuated type
1, 2, 3
•  Type 2 di eradikasi (2015) à sejak April 2016
bOPV (hanya acenuated type 1 dan 3) untuk
menghindari kejadian circula/ng vaccine
derived Poliovirus (cVDPV) type 2
•  nOPV2 = dikembangkan gene/cally stable
type 2 vaccine acenuated OPV
•  mOPV 1 dan 3 licenced kembali thn 2005

 Family Herpesviridae à subfamily
Alphaherpesvirinae
Type virus Nama lain Sel target Tempat patofi Transmisi
name laten
Human HSV-1 Oral dan
Herpesvirus 1 atau genital
(HHV1) (utama oro- Kontak
fasial) langsung
Human HSV-2 Oral atau (oral atau
Herpesvirus 2 Mukoepitel Sel saraf genital seksual)
(HHV2) (utama
genital)
Human VZV Cacar air(Ch Respirasi
herpesvirus 3 pox) atau atau kontak
(HHV3) Cacar api langsung
(Shingles)
Incidence/Epidemiology
Clinical Appearance
Virology/Taxonomy/Genomic
Virulence/Host Immune response
Laboratory Diagnosis
PrevenLon/Control

HUMAN HERPES VIRUS 1 & 2

 HSV infec/ons
Foto: FatahzadehM, 2007

Primary herpe/c ginggivostoma//s

in a young child

Herpes labialis Recurrent herpeslabialis in

Immunocmprs pa/ent

Recurrent intraoral herpes

Primary herpe/c whitlow in

Genital herpes in
2 yrs old with primary oral
Immunocompetent person
erpes
Recurrent intraoral herpes in immncmprs pa/ent
 Taxonomy/Classifica/on
•  Famili: Herpesviridae
•  Subfamili: Alphaherpesvirus
•  Genus: Herpes Virus Simpleks1
 HSV Structure

•  Icosahedral
•  Out to inside:
–  Envelope + glycoprotein knobs
–  Tegumen
–  CAPSID
–  DNA
Genom HSV: 152000 bp

Daerah unik panjang dan pendek, diapit dengan sekuens berulang

Virus Replica/on of HSV-1
Garpu replikasi virus HSV-1
A. Molekul DNA untai ganda terlepas oleh H/P.
Polymerase HSV (oval hitam didalamnya ada
UL30 oval dan UL42 bentuk bulan sabit)
menyebabkan
sintesis DNA leading strand. Dibawah H/P ada
RNA primer .
ssDNA keluar dari balik helicase terbungkus
ssDNA-binding protein ICP8.

Terbentuk replica/on loop pada lagging strand

agar dapat bersesuaian
dengan leading strand.
DNA polimrs pada Strand lagging memulai
okazaki fragment dengan menggunakan primer
RNA.
 Ac/va/on of alphaviruses

Neurotropic and neuroinvasive virus

Produc/ve vs latent

•  Jika ada IE ICPO

protein, enzim repair
DNA terhambat,
genom tetap linier =
virus produk/f.
•  Jika ada enzim repair
DNA maka genom
menjadi sirkuler=
laten.
 Jalur infeksi alfaherpesvirus pada
sistem saraf mamalia

Alfaherpesvirus:
• HSV-1
• HSV-2
• VZV

•  Infeksi biasa berawal pada perifer yakni epitel mukosa à virus masuk pada bagian terminal
neuron sensorik pada sistem saraf perifer (PNS) ; secara retrograd sepanjang akson mencapai
badan sel, dan disimpan (seumur hidup) dalam in/ sel.
•  Ke/ka ada reak/vasi, par/kel virus baru dibentuk dan terletak dekat lokasi akan dikeluarkan
dari sel. Infeksi menyebar ke arah anterograd ke arah perifer.
•  Infeksi juga bisa berlangsung secara trans-neuronal, dari PNS ke CNS à fatal encephali/s .
Sequen/al of Herpes simplex virus
Infec/on

Legoff et al, 2014, Virology Journal, 11:83

Genom Herpes simpleks 2 (HSV-2)
Gambar ds-DNA HSV-2
Daerah Unik: UL dan US
Bagian Repeat Mayor: TRL, IRL

Ref: Dolan etal. 1998

 Ref: Anderson, 2012

HSV 1 and HSV 2

•  Acute – chronic disease
•  Mild – severe disease
•  No cura/ve therapy
•  Lifelong carriage
•  Recurrent outbreaks
 Diagnosis of HSV
•  Cell culture – microscopy
•  ELISA – only for past exposure (not acute)
•  PCR
 Direct Laboratory Methods for HSV
Diagnosis
Method Principle Sample
VIRAL ANTIGEN Immunoperoxidase staining Swab, smears from lesion, smear or
DETECTION vesicular fluid or exudate fr. base of
vesicle
Capture ELISA Swab, or vesicular fluid or exudate fr.
base of vesicle
Rapid Test Device Swab, or vesicular fluid or exudate fr.
base of vesicle
VIRUS CULTURE HSV Isola/on suscep/bel test Swab, Skin Lesions, vesicular fluid or
exudate fr. base of vesicle, Mucosal
sample w.o. Lesions biopsies, conj./
corneal Smears, neonates
MOLECULAR HSV DNA Detec/on and or Swab, Skin Lesions, vesicular fluid or
BIOLOGY qua/ta/on by NAAT/inhouse exudate fr. base of vesicle, Mucosal
classical PCR/Real/me PCR/ sample w.o. Lesions biopsies,
commercial PCRs Aqueous/Vitreous humor, CSF, blood
CYTOLOGICAL Tzank smears Skin/mucosal lesions.
EXAMINATION Papanicolau or Romanovski stain Biopsies, Conjunc/val/mucosal smears.
Direct Immunofluorescence Smears, /ssue sec/on smear fr base of
vesicle.
 Indirect Laboratory detec/on of HSV
(serological tests)
Method Principle Sample
Western Blot Western Blot HSV 1 Serum
Western Blot HSV2
EIA (enzyme immunoassay) Monoclonal An/body Serum
Blocking EIA
ELISA
POC (Point of Care tests) Immunofiltra/on Serum, capillary blood
Herpe/c Whitlow
Incidence/Epidemiology
Clinical Appearance
Virology/Taxonomy/Genomic
Virulence/Host Immune response
Laboratory Diagnosis
PrevenLon/Control

HUMAN HERPES VIRUS 3 (VARICELLA

ZOSTER VIRUS)

 Structure of Virus Varicella Zoster
•  Virion size 80-120 nm
(Rash without pain)
VZV (VIRUS VARICELLA ZOSTER)
 Herpes zooster= shingles

Zoster a`er given Mycophenolate Mofe/l (an/metabolit) in Sarcoidosis pa/ent

Ref: Hegde,2012
Herpes zoster = shingles

Ref: Sampath Kumar, 2009

Rarely occurring: Herpes zoster duplex
bilateralis
•  Case report (in) A 15-year-old Chinese boy presented with a bilateral and
symmetrical painful erup/on on the upper abdomen of 7 days' dura/on.
The erup/on was preceded by a 2-day history of malaise and low grade
fever. He did not have the varicella vaccine but had chickenpox at 3 years
of age. His past health was otherwise unremarkable. In par/cular, he did
not have recurrent or chronic infec/ons. The pa/ent did not have recent
weight loss and was not on any medica/ons. There was no history of
recent travel. He did not have exposure to venereal or other infec/ous
diseases. The family history was noncontributory. (Ref: Leung et al, 2015)

Previously the same case had been reported: in 1947

(Thomas), in 2003 (Arfan ul-bari ), and in 2006
(Brandon)
The role of Varicella laboratory test in
pregnant women:
1.  Determining immune status (a`er a VZV exposure) à
does she require VZIG? or is she safe?
2.  Diagnosing varicella in pregnant women whose
clinical symptoms are not clear (e.g
immunocompromised, post vaccina/on, orpost VZIG)
and to DD/ with other vesicle forming infec/ons.
3.  Prenatally diagnosing intrauterine VZV infec/on and/
or CVS à to detect congenital
4.  Diagnosing neonatal infec/on (test a`er the baby has
been born)

Ref: De paschale and Clerici, World J Virol 2016 August 12; 5(3): 97-124
 Lab tests for pregnant women
suspected with Varicella
•  If no history of previous •  Complement Fixing
infec/on, and test is •  ACIIF, IAHA, PHA, RIA
nega/ve à give VZIG within •  Neutraliza/on test
96 hour
•  LA
•  If no history of previous
infec/on, but test is posi/ve •  IFA
during the first 7-10 days of •  FAMA = gold standard
contact à a prior infec/on •  TRFIA
•  IB
•  Elisa/EIA
•  Gp ELISA
•  Quan/ta/ve CLIA
•  Microarray

Ref: De paschale and Clerici, World J Virol 2016 August 12; 5(3): 97-124
 Lab Test of Varicella in Pregnant
women
1.  Tzanck test (Cytology) à HE/Giemsa stain à
observe CPE as mul/nucleated giant cells,
syncy/a and ballooning cell degenera/on)
2.  Direct fluorescent an/body assay
3.  Molecular test
4.  Electron microscopy
5.  Virus isola/on à Gold standard

Ref: De paschale and Clerici, World J Virol 2016 August 12; 5(3): 97-124
 Preven/on
•  Vaccina/on: live acenuated OKA virus
–  Should not be given to pregnant women
–  can be given to postpartum or 1 month before
pregnancy or women during breast feeding
•  VZIG:
–  can be given to pregnant women à Ig provides
protec/on for 3 weeks à wanes off
–  For neonates and preterm babies whose mother are
infected 7 days before and 7 days a`er birth
–  Should not be given if symptoms of varicella has
already appeared

Ref: De paschale and Clerici, World J Virol 2016 August 12; 5(3): 97-124
Incidence/Epidemiology
Clinical Appearance
Virology/Taxonomy/Genomic
Virulence/Host Immune response
Laboratory Diagnosis
PrevenLon/Control

MUMPS VIRUS

 Taxonomy
•  Family: Paramyxoviridae
•  Genus: Mumps Rubulavirus
•  Type: Mumps rubulavirus
 Incidence/Epidemiology
•  1st described by Hippocrates (5th Century) à 1930
Johnson and Goodpasture inoculated paro/d /ssue
from Macaca mulaLa into some children.
•  World wide distribu/on
•  Virus causes encephali/s, meningi/s, orchi/s,
myocardi/s, pancrea//s and nephri/s
•  Global resurgence of cases were reported in highly
vaccinated popula/ons
•  Neurotropic à 50% cases are CNS associated
•  Cases of asep/c meningi/s are associated with some
vaccine strains
 Mumps virus structure
•  Nega/ve sense, single stranded (non
segmented) RNA virus
•  15.384 nucleo/des long
•  Helical/pleomorphic par/cle 120-450 nm
(average 200 nm)
•  Enveloped
•  Serotypes: A-N (A, B, C, D, F, G, H, I, J, K, L, N)
excluding (E and M).
–  Vaccine strains: A, B, or N
 Mumps Clinical symptoms
•  Fever
•  Headache
•  Muscle aches
•  Tiredness
•  Loss of appe/te
•  Swollen and tender salivary glands under the
ears or jaw on one or both sides of the face
(paro//s)
www.CDC.gov
Mumps virus by EM
EM of mumps virus by dr. F.A.Murphy (1976, CDC)

Hemagglu/nin and Neuraminidase

Protrude from the envelope
 Paro//s
•  Self limi/ng disease; recover within few weeks
•  Long-term sequelae: paralysis, seizures,
cranial nerve palsies, hydrocephalus and
deafness
Mumps clinical presenta/on

Rubin S, et al, J Pathol 2015; 235: 242–252

 Mumps Pathogenesis
and the Unresolved Ques/ons

Rubin S, et al, J Pathol 2015; 235: 242–252

 Laboratory Methods for Mumps
Diagnosis

1.  Virus Isola/on (Gold standard)

2.  Nucleic acid detec/on (now gold standard)
3.  Serological confirma/on (IgM detec/on)

Incidence/Epidemiology
Clinical Appearance
Virology/Taxonomy/Genomic
Virulence/Host Immune response
Laboratory Diagnosis
PrevenLon/Control

MORBILLI VIRUS

 Virus Classifica/on
•  Family: Paramyxoviridae
•  Genus: Paramyxovirus
•  Type name: Morbillivirus
Distribu/on of Measles Genotypes
Measles Virus Structure
 Griffin et al 2012

Morbillivirus Life Cycle

Morbillivirus infects
myeloid cells
(CD150+ lymphocytes and
dendri/c cells)
Measles Mode of Transmission

Ref: Laksono, 2016

 Measles pathogenesis
Bone marrow, Thymus,
Spleen, tonsil,
Lymphnodes, Bronchus
Associated Lymphoid Lssue
Or ConjuncLval CD150+
lymphocytes/dendri/c cells

MV infects myeloid cells à Infected cells enters circula/on Reduced immune cells à
enters lymphnodes to infect (Viremia) à enters organ and ResulLng transient immunosuppression
lymphocytes (B cells, CD4 /ssues àinfects nec/n4+ and skin rash occurs
and CD8 T cells) (epithelial) cells

Ref: Laksono, 2016

 Morbilli Rash
•  Skin rash is due to leukocytes, CD4+ and CD8+
T lymphocytes infiltra/on in the skin to clear
infected epithelial cells

Griffin, et al, 2012

 Lab diagnosis for Measles
•  IgM an/body : from day 3- 4 weeks a`er rash
•  RNA detec/on
Incidence/Epidemiology
Clinical Appearance
Virology/Taxonomy/Genomic
Virulence/Host Immune response
Laboratory Diagnosis
PrevenLon/Control

RESPIRATORY SYNCYTIAL VIRUS

 Virus Classifica/on
•  Family: Paramyxoviridae
•  Genus: Pneumovirus
•  Type name: Respiratory Syncy/al Virus
 Viral Characteris/c of RSV
•  Enveloped
•  Spherical or long filaments
•  Nega/ve sense single stranded RNA
•  15.222 nucleo/des
•  10 genes encoding 11 proteins
 Structure and Genome Organiza/on

SPHERICAL FILAMENT
RSV SHAPED RSV

10 genes of RSV
encodes 11 proteins

Lambert L, et al, 2014 Front Immunol. 2014; 5: 466.

 Incidence and Epidemiology
•  A novel virus was isolated from chimpanzee with
respiratory symptoms in 1955, was named chimpanzee
coryza agent.
•  Soon was understood as human virus, renamed as RSV
to reflect the giant syncy/a formed in /ssue culture
•  RSV is the most common cause of severe respiratory
infec/on in infants and young children.
•  RSV is also important pathogens among:
–  Elderly
–  Those with underlying lung disease
–  Those with impaired immunity
 Reinfec/on of RSV
•  Highest incidence at 2-3 months old babies
•  World wide 33.000.000 RSV cases; 3.400.000
death; Mortality rate 66.000-199.000/year
(Lanari et al, 2005)
•  Able to reinfect repeatedly although no
change of viral an/genicity, but previous
infec/ons cause reduced severity.
•  Such reinfec/on is due to the ability of RSV to
inhibit or subvert host immune system
RSV Life Cycle
RSV
spread
from cell to
cell by
causing
cell fusion
And
syncy/a
forma/on

Bawage et al. 2013, Advances in Virology vol 2013

 Lab methods for Diagnosis
1.  Tissue Culture
2.  Direct Immunofluorescence or IFA or EIA
3.  Chromatographic Rapid An/gen Detec/on by
Rev Transf PCR
4.  An/genic Capture ELISA, serology ELISA (IgM
and IgG), HA, HAI, Neutraliza/on test.
Infected Cells Fuse and Form Syncy/a

Hep2 cells = laryngeal carcinoma cell line

Day 1 no change in cells; day 3 some cells fused and form syncy/a;
day 5 fused cells are dead and detaches from the base of plate
Domachowske et al, Clini Microbiol Reviews Vol 12 no 2, 1999
Cell Culture for Diagnosis of RSV

Uninfected Hep-2 cells RSV Infected Hep-2 cells

Leland and Ginocchio, Clinical Microb Rev, Vol 20 no 1, 2007, p 49-78

Incidence/Epidemiology
Clinical Appearance
Virology/Taxonomy/Genomic
Virulence/Host Immune response
Laboratory Diagnosis
PrevenLon/Control

MOLLUSCUM CONTAGIUM VIRUS

 Taxonomy
•  Family: Poxviridae
•  Genus: Molluscipoxvirus
•  Type name: Molluscum contagiosum virus
 Moluscum contagiosum
•  double-stranded DNA genome of
190.289 base pair
•  Preadolescent children à
sanita/on, crowds, swimming pool
•  Adolescent and adults à sexual
transmission
•  Self limi/ng disease 6-9 months to 3
-4 years – unless HIV/
immunocompromise
 HyperplasLc, acanthoLc squamous epithelium forming a central
crater filled with keraLn fragments and molluscum bodies

Molluscum
contagiosum virion
with envelope
measuring
340×265 nm

Inclusion bodies
in cytoplasma
 BOTE Phenomenon =
Beginning of the End

BOTE : swelling and erythema at

the beginning of the regression phase

Ref: Meza Romero, 2019

 Papules of Molluscum contagium
•  Single -- mul/ple, 2-5mm some/mes ,
rounded, dome-shaped, pink, waxy,
umbilicated with caseous plug Axilla of HIV infected man
Chest
Leg of HIV infected child

Cheek
Ref: Chen 2013
Incidence/Epidemiology
Clinical Appearance
Virology/Taxonomy/Genomic
Virulence/Host Immune response
Laboratory Diagnosis
PrevenLon/Control

HUMAN PAPILLOMA VIRUS

 Taxonomy
•  Family: Papillomaviridae
•  Genus:
•  Type name: Human papilloma virus
Cancer e.c HPV in man and women

Ref: Brian/ et al, 2017, New Microbiologica, 40, 2, 80-85

Natural History of HPV infec/on in

women

* Red box and green boxes are

based on molecular tests

Ref: Gravit and Winer, 2017, Viruses, 9, 267

 HPV types and disease

Ref: Brian/ et al, 2017, New Microbiologica, 40, 2, 80-85 Bosn J Basic Med Sci. 2014 Aug; 14(3): 136–138
 Virology
•  Small
•  Non-enveloped
•  Icosahedral viruses
•  50–60 nm in diameter
•  Circular, doublestranded
DNA genome (~7000–
8000 bp).

Ref: Brian/ et al, 2017, New Microbiologica, 40, 2, 80-85

Associa/on of HPV and Children
HPV on skin layer

Virus shedding
Molecular tests for diagnosis of HPV

Ref: Chan, 2016