Diagnosis Laboratorium dan

Aspek Virologi Influenza A H1N1

Budiman Bela

Fakultas Kedokteran Universitas Indonesia

Centers for Disease Control and
Prevention
Interim Guidance on Specimen
Collection, Processing, and Testing for
Patients with Suspected Swine-Origin
Influenza A (H1N1) (S-OIV) Virus
Infection
CDC Guidance on Specimen
Collection, Processing, and Testing
Objective: To provide interim guidance on
appropriate specimen collection, storage,
processing, and testing for patients with
suspected swine-origin influenza A (H1N1)
virus infection.
CDC Guidance on Specimen
Collection, Processing, and Testing
Case Definitions
A confirmed case of S-OIV infection is
defined as a person with an acute febrile
respiratory illness with laboratory confirmed
S-OIV infection at CDC by one or more of the
following tests:
real-time RT-PCR
viral culture
CDC Guidance on Specimen
Collection, Processing, and Testing
Case Definitions
A probable case of S-OIV infection is defined as a person with an
acute febrile respiratory illness who is positive for influenza A, but
negative for H1 and H3 by influenza RT-PCR
INDONESIA: perlu mempertimbangkan RT-PCR H5 negatif dalam
algoritme diagnosis laboratorium berdasarkan RT-PCR, sebelum
pasangan primer spesifik galur H1N1 baru telah diperoleh

A suspected case of S-OIV infection is defined as a person with acute

febrile respiratory illness with onset
within 7 days of close contact with a person who is a confirmed case of S-
OIV infection, or
within 7 days of travel to community either within the United States or
internationally where there are one or more confirmed cases of S-OIV
infection, or
resides in a community where there are one or more confirmed cases of S-
OIV infection.
CDC Guidance on Specimen
Collection, Processing, and Testing
The duration of shedding with swine-origin
influenza A (H1N1) virus is unknown.
until data are available, the estimated
duration of viral shedding is based upon
seasonal influenza virus infection.
CDC Guidance on Specimen
Collection, Processing, and Testing
At the current time, CDC believes that this
virus has the same properties in terms of
spread as seasonal flu viruses.
Seasonal flu:
studies have shown that people may be
contagious from one day before they develop
symptoms to up to 7 days after they get sick.
Children, especially younger children, might
potentially be contagious for longer periods.
Testing for swine-origin Influenza A (H1N1)
virus (CDC)
Clinicians should consider testing suspected cases of
swine-origin influenza A (H1N1), especially those with
severe illness, by obtaining:
an upper respiratory specimen to
test for swine-origin influenza A
(H1N1) virus.
Preferred respiratory specimens
(CDC)
Specimen that should be collected as
soon as possible after illness onset:
nasopharyngeal swab/aspirate
or nasal wash/aspirate.
Preferred respiratory specimens
(CDC)
If nasopharyngeal swab/aspirate or nasal
wash/aspirate cannot be collected:
a combined nasal swab with an
oropharyngeal swab is acceptable.
Preferred respiratory specimens
(CDC)
For patients who are intubated:
an endotracheal aspirate should also be
collected.
BIOSAFETY MEASURES (CDC)
Recommended infection control guidance
is available for persons collecting clinical
specimens in clinics and other clinical
settings and laboratory personnel.
Specimen transportation (CDC)
Specimens should be placed into sterile
viral transport media (VTM) and
immediately placed on ice or cold packs or
at 4C (refrigerator) for transport to the
laboratory.
CDC Guidance on Specimen
Collection, Processing, and Testing
Swabs
Ideally, swab specimens should be collected using
swabs with a synthetic tip (e.g. polyester or Dacron)
and an aluminum or plastic shaft.
Swabs with cotton tips and wooden shafts are not
recommended.
Specimens collected with swabs made of calcium
alginate are not acceptable.
The swab specimen collection vials should contain 1-
3ml of viral transport medium (e.g. containing, protein
stabilizer, antibiotics to discourage bacterial and
fungal growth, and buffer solution), such M4RT or the
BD Universal Viral Transport System .
CDC Guidance on Specimen
Collection, Processing, and Testing
Storing Clinical Specimens:
All respiratory specimens should be kept at 4C until they can be
placed at -70C.
If a -70C freezer is not available, specimens should be kept at
4C, preferably no longer than 1 week.
Shipping clinical specimens:
Clinical specimens should be shipped on dry ice in appropriate
packaging.
All specimens should be labeled clearly and include information
requested by your state public health laboratory. Suspected
case specimens shipped from the state public health laboratory
to CDC should include all information required for seasonal
influenza surveillance isolate or specimen submission.
Recommended Tests (CDC)

Real-time RT-PCR for influenza A, B,

H1, H3 :
Departemen Mikrobiologi FKUI:
RT-PCR Influenza A: Sudah tersedia (IHVCB-UI)
RT PCR Influenza B: Primer sudah tersedia
RT PCR H1 : Sudah tersedia (IHVCB-UI)
RT PCR H3 : Primer perlu dirancang
Recommended Tests (CDC)
Real-time RT-PCR for influenza A, B,
H1, H3 :
Currently, swine-origin influenza A (H1N1)
virus will test positive for influenza A and
negative for H1 and H3 by real-time RT-PCR:
Recommended Tests (CDC)
Real-time RT-PCR for influenza A, B, H1, H3 :
Departemen Mikrobiologi FKUI:
RT-PCR Influenza A:
daerah target pasangan-pasangan primer: M dan N telah
dianalisis dengan software bioedit pada berbagai jenis
influenza A dan terlihat sebagai daerah yang terkonservasi
RT PCR Influenza B: Primer sudah tersedia:
belum dianalisis
RT PCR H1 :
Daerah target sudah dianalisis dan terlihat tidak bereaksi
dengan H1N1 galur baru
Analisis bioinformatik untuk perancangan pasangan primer
pendeteksi H1 dan N1 galur baru sedang dilakukan
RT PCR H3 : Primer perlu dirancang
Recommended Tests (CDC)
If reactivity of real-time RT-PCR for
influenza A is strong (e.g. Ct <30) it is
more suggestive of a novel influenza A
virus.
Confirmation as swine-origin influenza A
(H1N1) virus is performed at CDC
currently, but may be available in state
public health laboratories soon
Other influenza tests (CDC)
Rapid influenza antigen tests:
unknown sensitivity and specificity to
detect human infection with swine-origin
influenza A (H1N1) virus in clinical specimens
suboptimal sensitivity to detect seasonal
influenza viruses.
A negative rapid test could be a false negative
and should not be assumed a final diagnostic
test for swine-origin influenza infection.
Other influenza tests (CDC)
Immunofluorescence (DFA or IFA):
These tests can distinguish between influenza
A and B viruses.
A patient with a positive for influenza A by
immunofluorescence may meet criteria for a
suspected case.
it is not possible to differentiate from seasonal
influenza A viruses.
Other influenza tests (CDC)
Immunofluorescence (DFA or IFA):
Immunofluorescence depends upon the
quality of a clinical specimen, operator skills
unknown sensitivity and specificity to detect
human infection with swine-origin influenza A
(H1N1) virus in clinical specimens.
a negative immunofluorescence could be a
false negative and should not be assumed a
final diagnostic test for swine-origin influenza
infection.
Other influenza tests (CDC)
Viral culture:
Isolation of swine-origin influenza A (H1N1) virus is
diagnostic of infection, but may not yield timely results
for clinical management.
A negative viral culture does not exclude infection
with swine-origin influenza A (H1N1) virus.
Dapat dilakukan di Universitas Indonesia
Penting untuk analisis evolusi virus dan penentuan
sequence asam amino yang akan digunakan untuk
merancang vaksin dan sistem diagnostik
 VIROLOGY
FAMILI : ORTHOMYXOVIRIDAE
GENUS : INFLUENZAVIRUS
TYPE ( protein NP & M) :
A (Human and Animal)
B (Human)
C (human)
Influenza A Virus (highly variable) and Influenza B Virus
outbreak in human
Influenza C Virus spreads periodically, mild, not causing
outbreaks
GENOM ssRNA (SINGLE STRANDED, NEGATIVE
SENSE), SEGMENTED
Influenza Virion
Influenza Virus Proteins
RNA PROTEIN Function
Segment
1 PB2 RNA transcription component (Polymerase)
2 PB1 RNA transcription component (Polymerase)
3 PA RNA transcription component (Polymerase)
4 HA Hemaglutinin, envelope glycoprotein, viral attachment, fusion, subtype
specific, target of neutralizing antibody
5. NP Nucleocapsid., associate with RNA and polymerase, type specific
6 NA Neuraminidase, envelope glycoprotein, enzyme (cleaves sialic acid and
promotes virus release), subtype specific
7 M1 Matrix protein, major component of virion, promote viral asembly, structural
protein, type specific, interacts with nucleocapsid and envelope
M2 Integral membrane protein, ion channel, from spliced mRNA, type specific,
target for amantadine, facilitates uncoating and HA production
8 NS1 Nonstructural, inhibits nuclear export of mRNA (inhibits cellular messenger
RNA translation)
NS2 Minor component of virion, function unknown, from spliced mRNA
Influenza A Virus
Influenza A Virus has 2 Surface
Glycoprotein antigens :
hemaglutinin(H)
neuraminidase (N)
 NEURAMINIDASE ACTIVITY

Antigenic drift can also occur in the NA. The NA carries several important amino acid residues which, if they mutate,
can lead to resistance against neuraminidase inhibitors. Mutations that have been observed include:
R292K, H274Y, R152K, E119V . The letters represent amino acids (R, arginine; K, lysine; H, histidine; Y, tyrosine; E,
glutamic acid; V, valine): the former letter is the original amino acid, and the latter the amino acid after mutation occurred.
Influenza A Virus
Variations of H dan N are the basis for
subtyping H1 - H16 dan N1 - N9
Influenza viruses in human 4 H (H1,
H2, H3, H5) and 2 N (N1, N2)
H1N1,H2N2 and H3N2, Avian Virus H5N1
Designation :
human A/Hongkong/03/68(H3N2), other:
A/swine/Iowa/15/30(H1N1)
Hosts of Influenza A Virus

Human
Pig
Horse
Bird
Other Mammals
Properties of Influenza Virus
The virus can survive in water from 4 days at
22oC and more than 30 days at 0oC

AI (Avian Influenza) Virus in poultry meat will die

in 1 minute at 80oC or in 30 minutes at 60oC

The virus is very labile and can easily change

from virulent to avirulent strain and vice versa
Inactivation of Influenza Virus
LIPID SOLVENT, PROTEIN DENATURANT,
FORMALDEHYDE AND IRRADIATION
INFLUENZA VIRUS WILL BE INACTIVATED BY:
DETERGENT
70 % ALCOHOL PREPARATION,
QUARTERNERY AMMONIUM
CHLORIN
FORMALIN 2-5%
IODOFORM COMPLEX (IODINE)
PHENOL
SODIUM/POTASSIUM HYPOCHLORIDE (bleach/pemutih)
THE EMERGENCE OF NEW STRAIN OF
INFLUENZA VIRUS
Key molecular basis that drives the
virulence change of a virus is: amino acid
mutation.
Dynamic gene mutant has been shown to
play an important role in the virulence
change of AIV (Hatta et al., 2001;Hulse-
Post et al., 2007)
THE EMERGENCE OF NEW STRAIN OF INFLUENZA VIRUS

- ANTIGENIC SHIFT
- ANTIGENIC DRIFT
- RECOMBINATION
new
reassortants
with altered
host range

Pathogenic for
ducks
wild birds
humans

Peiris M, 2004
GENETIC EVOLUTION OF
H5N1 FROM 1996 TO2002 DI CINA
Pandemic influenza virus
PIG AS MIXING VESSELS
OF INFLUENZA VIRUS STRAINS
How do animal influenza viruses adapt to efficient human human
transmission ?

Pigs: A mixing vessel

Many avian and human viruses replicate in pigs

Kida et al J Gen Virol. 1994; 75: 2188-8
 H5N1: What is risk of a pandemic ?

In SARS, with repeated

challenge, the animal-
human species barrier
was breached
Swine Virus
Swine flu infects people every year and is found
typically in people who have been in contact
with pigs, although there have been cases of
person-to-person transmission.
Swine influenza is known to be caused by
influenza A subtypes H1N1, H1N2, H3N1,
H3N2, and H2N3.
Symptoms include fever, disorientation,
stiffness of the joints, vomiting, and loss of
consciousness ending in death.
THE EMERGENCE OF NEW STRAIN OF
OTHER RNA VIRUSES
homologous recombination plays an
important role in the evolution of some
RNA viruses (Kirkegaard and Baltimore,
1986; Lai, 1992; Nagy and Simon, 1997).
Virulent variants of some other viruses
have been generated by homologous
recombination (Anderson et al., 2000;
Kewet al., 2002; Pita et al., 2001;Worobey
et al., 1999).
THE EMERGENCE OF NEW STRAIN OF
INFLUENZA VIRUS
Evidence that influenza viruses undergo various
forms of non-homologous recombination.
Recombination can occur between HA and
nucleoprotein gene (Orlich et al., 1994).
Increased viral pathogenicity after insertion of a 28S
ribosomal RNA sequence into the haemagglutinin
gene of an influenza virus was found (Khatchikian et
al., 1989).
The occurrence of homologous recombination
within segments has recently been proven (He
et al., Virology 380 (2008) 1220)
The evidences for recombination in PA gene
of the strain A/swine/Ontario/57561/03(H1N1).
He et al., Virology 380 (2008) 1220
Tantangan Menghadapi Virus Influenza A
Baru di bidang Virologi
Indonesia memerlukan peningkatan
kapasitas sumber daya manusia untuk
melakukan Penelitian dan Pengembangan
di bidang virologi, khususnya mengenai
Virus Influenza A
Perlu dilakukan peningkatan kapasitas
secara terarah untuk mengantisipasi
munculnya galur-galur Influenza A Baru
Penerapan Ilmu Virologi di FKUI sebagai
persiapan menghadapi Pandemi Influenza

Pelaksanaan Tri Dharma Perguruan Tinggi

Pendidikan sumber daya manusia untuk

meningkatkan pengetahuan dan
keterampilan melalui penelitian virologi
Penerapan ilmu virologi di FKUI sebagai
persiapan menghadapi Pandemi Influenza
Pendidikan sumber daya manusia untuk
meningkatkan pengetahuan dan keterampilan
melalui penelitian virologi
Peningkatan pengetahuan mengenai aspek virologi dan
biologi molekular
Peningkatan keterampilan:
Kultur Virus
Teknik-teknik biologi molekular
Aplikasi Bioinformatika dalam analisis molekular virus
influensa
Teknik simulasi komputasional untuk analisis resistensi obat
anti virus influenza dan analisis epitop netralisasi
(bekerjasama dengan FMIPA-fisika, FMIPA-kimia dan
FMIPA-farmasi)
Penerapan ilmu virologi di FKUI sebagai
persiapan menghadapi Pandemi Influenza

Penelitian Virologi:
Pengembangan vaksin DNA dan VLP (viral
like particle):
Sintesis DNA tanpa meggunakan RNA virus
sebagai asam nukleat pola cetak (memakai
informasi sekuen dari GenBank)
Telah diterapkan pada H5N1
Akan dilakukan pada H1N1
Bekerjasama dengan FTUI, FMIPA-UI dan Biofarma
 Why DNA and VLP Vaccine?
USA experience with seasonal influenza vaccine,
produced using traditional vaccine production
technologies :
during the 2004 flu season Chiron could not supply vaccine to
the U.S. market
Recent years also have seen surges in demand and delays in
the delivery of influenza vaccine mismatches between
vaccine availability and supply, and enhanced demand at other
times shortages of vaccine (Industry Analysis of Influenza
Vaccine by Rodman and Renshaw, 2006)

How possible will it be that the demand for vaccine supply be

fullfiled by the national capacity of Indonesia to produce
pandemic strain vaccine, during the next strike of pandemic
influenza?
 Industry Analysis by Rodman and
Renshaw Investment Bank, 2006
We however favor investing in companies with
technologies that are capable of quickly producing
vaccines against novel strains and have surge capacity.
Of the companies listed in Figure 1, we believe:
Novavax Inc. (NVAX, Market Outperform) with its virus-like
particle (VLP) vaccine technology,
and Vical Inc. (VICL, Market Outperform) with its DNA vaccine
technology,
have the most promising technologies that could meet
the desirable traits for coping with pandemic flu
outbreaks.
 DNA vaccine
Simple and powerful concept:
the coding sequence of an antigenic pathogen gene
is incorporated into plasmid DNA, which will allow its
expression in host cells
DNA vaccines circumvent the need for:
Preparation
Purification
Delivery
of a pathogen or antigenic protein.
Utilize the intrinsic machinery of host cells
 AI DNA Vaccine
Immune response to DNA Vaccine:
Phase I Clinical Trial:
Epidermal DNA vaccine for influenza is
immunogenic in humans (Drape et al, 2006)
Delivery system:
Gold Particle
Biolistic
DNA Vaccine
 DNA Vaccine
DNA Delivery by biolistic approach delivers the
DNA directly to the intracellular compartment
1,000-fold less DNA than needle and injection
administration
the cost per dose would be commercially attractive
(PMED platform)
 Viral Like Particle Vaccine

Expression can be performed in

Mammalian Cells
Baculovirus expression system
Relatively easy to adjust with the
sequence of a new H5N1 AIV
Administration: injection or nasal spray
Penerapan ilmu virologi di FKUI sebagai
persiapan menghadapi Pandemi Influenza

Penelitian Virologi:
Pengembangan RT-PCR multiplex:
Deteksi simultan Influenza A dan H5N1
Akan dikembangkan sehingga dalam satu reaksi
juga dapat mendeteksi H1N1 dan H3N2 seasonal
meningkatkan kemungkinan penyebab infeksi
sebagai H1N1 galur baru
Penerapan ilmu virologi di FKUI sebagai
persiapan menghadapi Pandemi Influenza

Penelitian Virologi:
Pengembangan rapid diagnostic:
Deteksi antigen
Deteksi IgM
Simulasi Komputasional:
Docking molekul antivirus pada protein target
(oseltamivir dan Neuraminidase)
Docking antibodi monoklonal pada epitop netralisasi
Pengembangan molekul terapetik:
Humanized neutralizing antibody IgA dan IgG
Penerapan ilmu virologi di FKUI sebagai
persiapan menghadapi Pandemi Influenza

Penelitian Virologi lain yang dapat dilakukan:

Uji sensitivitas anti influenza
Pengembangan bahan alam sebagai anti
influenza
Evolusi virus influenza di Indonesia
Identifikasi pola transmisi virus influenza
(epidemiologi molekular)
Drug design
dll.
Penerapan ilmu virologi di FKUI sebagai
persiapan menghadapi Pandemi Influenza

Pelayanan kepada Masyarakat:

Lab Regional jejaring pemeriksaan flu burung
Uji efektivitas sistem inaktivasi virus:
Uji inaktivasi H1N1 oleh Plasmacluster ion (dalam
laboratorium BSL3)
 KESIMPULAN
Virus Influenza H1N1 galur baru merupakan
ancaman yang harus diwaspadai dengan
meningkatkan kapasitas di bidang virologi

Sumber daya manusia FKUI di bidang

virologi telah diberdayakan untuk
kepentingan nasional dalam menghadapi
ancaman pandemi influenza