Pengambilan Spesimen dan

Pemeriksaan Molekuler
2019-nCoV
Wuhan Coronavirus
Juniastuti, Inge Lusida
Dept. of Microbiology Faculty of Medicine
Institute of Tropical Disease
Universitas Airlangga
 Coronaviruses are enveloped non-segmented (+)
sense RNA viruses belonging to the family
Coronaviridae, broadly distributed in humans, other
mammals, birds and that cause respiratory, enteric,
hepatic, neurologic diseases. Both SARS-CoV and
2019-nCoV belong to the b-genus.

(https://media.nature.com/lw800/magazine-
assets/d41586-020-00253-8/d41586-020-
00253-8_17608790.jpg)

(Clinical Virology 4th ed. 2017 ASM)

Previously, alpha-, beta-, gamma- CoVs were designated as group1, group 2,
group 3 CoVs, respectively, with delta CoVs being a recently defined group.

(Clinical Virology 4th ed. 2017 ASM)

Phylogenetic analysis of the receptor-binding domain from
various betacoronaviruses
(Lu et al. Lancet 2020 https://doi.org/10.1016/S0140-6736(20)30251-8)
RESISTANCE
SARS-CoV and MERS-CoV remain viable for much longer than
other HCoV or influenza when dried on surfaces.
SARS-CoV dried on smooth surfaces retains its viability for >5
days at 22–25°C and a relative humidity of 40–50%, whereas
virus viability is rapidly lost at higher temperatures and higher
relative humidity.
MERS-CoV also retains viability on surfaces for many days and
is more stable at low temperature/humidity conditions.
Aerosolized MERSCoV retains viability at low temperature and
low humidity.
Common disinfectants commonly used in hospital and lab
settings are generally effective in inactivating SARS-CoV.
GROWTH IN CULTURE
None of the HCoVs grows easily in cell culture without
adaptation by passage.
SARS-CoV was isolated first in Vero E6 or fetal rhesus
kidney cell lines with production of CPE. Vero E6 cells are
now routinely used for its growth and also for plaque
assays of infectivity. The virus has been adapted for growth
in other cell lines that express the ACE2 receptor.
MERS-CoV was also initially isolated in Vero E6 cells.
Caco-2 cells are more efficient for primary isolation.
(Clinical Virology 4th ed. 2017 ASM)
Healthy monkey kidney cells developed cytopathic effect (CPE)/cell death
after infection with the novel coronavirus (Duke-NUS Medical School)
 A B

Visualization of 2019-nCoV with Transmission Electron Microscopy.

A : Negative-stained 2019-nCoV particles are shown in Panel A
B : 2019-nCoV particles in the human airway epithelial cell ultrathin sections
(Zhu et al. NEJM 2020 DOI: 10.1056/NEJMoa2001017)
Phylogenetic analysis of full-
length genomes of 2019-nCoV
and representative viruses of
the genus Betacoronavirus
(Lu et al. Lancet 2020
https://doi.org/10.1016/S0140-6736(20)30251-8)
Phylogenetic tree of full genome
(Chan et al. Lancet. 2020. doi.org/10.1016/S0140-6736(20)30154-9)
A B

Phylogenetic trees of genetic sequences

(A) RNA-dependent RNA polymerase
(B) Spike gene
(Chan et al. Lancet. 2020. doi.org/10.1016/S0140-6736(20)30154-9)
Spike phylogeny of representative b-genus lineage b coronaviruses
(Wan et al. J. Virol. doi:10.1128/JVI.00127-20)
 RdRP

Wuhan
virus

Wuhan
virus

Wuhan
virus

Partial alignments of oligonucleotide binding regions

(Diagnostic detection of Wuhan coronavirus 2019 by real-time RT-PCR. Corman V, Bleicker T,
Brünink S, Charité CD, Landt O, Tib-Molbiol (Berlin), Koopmans M (Rotterdam), Zambon M
(London) January 2020)
Sequence alignment of 2019-nCoV and SARS-CoV RBDs
RBM : magenta. Five critical residues : blue. ACE2-contacting residues
are shaded. (Wan et al. J. Virol. doi:10.1128/JVI.00127-20)
Sequence identity between the concensus 2019-nCoV & representative
betacoronavirus genomes (SARS-CoV GZ02 & the bat SARS-like CoVs).
(Lu et al. Lancet 2020 https://doi.org/10.1016/S0140-6736(20)30251-8)

S protein is a prototypical class 1 fusion protein and involved in receptor binding

and fusion functions. Following cleavage by cellular proteases into S1 and S2
subunits, the former is involved in interaction with receptors whereas the latter is
involved in fusion of the viral and cellular membranes. CoV cell and tissue
tropism, disease pathogenesis, and host range are initially controlled by
interactions between the S1 subunit and host cell receptors; however, proteolytic
activation of the spike protein by host cell proteases also plays a critical role.
Sequence similarities of SARS-CoV & 2019-nCoV in the spike protein,
RBD and RBM, respectively
(Wan et al. J. Virol. doi:10.1128/JVI.00127-20)
 Genome organization of 2019-nCoV
and the amino acid identities of
different subunits & domains of the
Spike between human 2019-nCoV
strains (HKU-SZ-002a and HKU-SZ-
005b) and bat-SL-CoV-ZC45.
(Chan et al. Lancet. 2020. doi.org/10.1016/S0140-6736(20)30154-9)
 A B

A. Experimentally determined structure of the interface between a designed SARS-CoV

RBD (optimized for human ACE2 recognition) and human ACE2.
B. Modeled structure of the interface between 2019-nCoV RBD and human ACE2.
(Wan et al. J. Virol. doi:10.1128/JVI.00127-20)
DIAGNOSIS LABORATORIUM

CDC has developed a new laboratory test kit for use in testing
patient specimens for 2019 novel coronavirus (2019-nCoV):
“CDC 2019-nCoV Real-Time Reverse Transcriptase (RT)-
PCR Diagnostic Panel”, which is intended for use with the AB
7500 Fast DX Real-Time PCR Instrument with SDS 1.4
software ® for use with upper & lower respiratory specimens
collected from persons who meet CDC criteria for 2019-nCoV
testing (person under investigation: identified as individuals
with a history of travel to China or close contact with a person
confirmed to have the 2019 nCoV illness and symptoms of
respiratory illness such as cough or shortness of breath).
Testing for other respiratory pathogens by the provider should be done as part
of the initial evaluation. If a PUI tests positive for another respiratory
pathogen, after clinical evaluation and consultation with public health
authorities, they may no longer be considered a PUI.

Specimen Type and Priority

For initial diagnostic testing for 2019-nCoV, CDC recommends collecting and
testing upper respiratory (nasopharyngeal AND oropharyngeal swabs), and
lower respiratory (sputum, if possible) for those patients with productive
coughs. Specimens should be collected as soon as possible once a PUI is
identified, regardless of the time of symptom onset. Maintain proper infection
control when collecting specimens.
SPESIMEN
Acceptable Specimens
• Respiratory specimens
- Nasopharyngeal or oropharyngeal aspirates/washes, naso-
pharyngeal or oropharyngeal swabs, bronchoalveolar lavage
(BAL), tracheal aspirates, sputum. Induction of sputum is
not indicated.
- Swab specimens should be collected only on swabs with a
synthetic tip (such as polyester or Dacron®) with aluminum
or plastic shafts. Swabs with calcium alginate or cotton tips
with wooden shafts are not acceptable.
• Serum
PAN AMERICAN HEALTH ORGANIZATION, Regional Office of WHO

https://youtu.be/mfZYAMDpGNk Nasopharyngeal and Oropharyngeal Swabs

https://youtu.be/GJK9FRT4lXM Specimen Packaging

https://youtu.be/TMElw671nJM Personal Protective Equipment

Respiratory Specimens
A. Lower respiratory tract
Bronchoalveolar lavage, tracheal aspirate
Collect 2-3 mL into a sterile, leak-proof, screw-cap sputum collection cup or
sterile dry container. Refrigerate specimen at 2-8°C and send to lab on ice
pack.
Sputum
Have the patient rinse the mouth with water and then expectorate deep
cough sputum directly into a sterile, leak-proof, screw-cap sputum collection
cup or sterile dry container. Refrigerate specimen at 2-8°C and send to lab
on ice pack..
B. Upper respiratory tract
Nasopharyngeal swab AND oropharyngeal swab
(NP/OP swab)
Use only synthetic fiber swabs with plastic shafts.
Place swabs immediately into sterile tubes containing
2-3 ml of viral transport media. NP & OP specimens
should be kept in separate vials. Refrigerate
specimen at 2-8°C and send to lab on ice pack.

Nasopharyngeal swab: Insert a swab into the nostril parallel to the

palate. Leave the swab in place for a few seconds to absorb secretions.
Swab both nasopharyngeal areas with the same swab.
Oropharyngeal swab : Swab the posterior pharynx, avoiding the tongue.
Nasopharyngeal wash/aspirate or nasal aspirate
Collect 2-3 mL into a sterile, leak-proof, screw-cap sputum collection cup or
sterile dry container. Refrigerate specimen at 2-8°C & send to lab on ice pack.
Serum
Minimum volume required:
Children & adults: 1 tube (5-10 mL) of whole blood in a serum separator tube.
Infant: ≥1 mL of whole blood. If possible, 1 mL in a serum separator tube.

Serum separator tubes should be stored upright for ≥30 minutes, and then
centrifuged at 1000–1300 relative centrifugal force (RCF) for 10 minutes before
removing the serum and placing it in a separate sterile tube for shipping (such
as a cryovial). Refrigerate the serum at 2-8°C & send to lab on ice-pack.
Specimen Handling and
Storage
• Specimens can be stored at 4°C
for ≤72 hours after collection.
• If a delay in extraction is
expected, store specimens at -
70°C or lower.
• Extracted nucleic acids should be
stored at -70°C or lower.
Specimen Rejection
criteria
• Specimens not kept at 2-4°C
(≤4 days) or frozen at -70°C or
below.
• Incomplete specimen labeling
or documentation.
• Inappropriate specimen type.
• Insufficient specimen volume
Lab workers should wear appropriate personal
protective equipment (PPE) including disposable
gloves, laboratory coat/gown and eye protection
when handling potentially infectious specimens. Any
procedure with the potential to generate aerosols or
droplets (e.g., vortexing) should be performed in a
certified Class II Biological Safety Cabinet (BSC).
For any procedures outside of a BSC, eye & face
protection (e.g. goggles, mask, face shield) or other
physical barriers (e.g. splash shield) should be used
to minimize the risk of exposure to lab staff.
Virus isolation in cell culture of 2019-nCoV
specimens are NOT recommended at this
time, except in a BSL3 laboratory using
BSL3 work practices.

For 2019-nCoV laboratory waste, follow

standard procedures associated with other
respiratory pathogens, such as seasonal
influenza & other human CoVs.
Assay Controls
• Assay controls should be run concurrently with all test samples.
• PTC - positive template control with an expected Ct value range
• NTC - negative template control added during rt RT-PCR reaction set-up
• HSC – human specimen extraction control extracted concurrently with the
test samples; provides a nucleic acid extraction procedural control and a
secondary negative control that validates the nucleic extraction procedure
and reagent integrity
• RP - all clinical samples should be tested for human RNAse P (RNP)
gene to assess specimen quality
(Diagnostic detection of Wuhan coronavirus 2019 by real-time RT-PCR. Corman V, Bleicker T,
Brünink S, Charité CD, Landt O, Tib-Molbiol (Berlin), Koopmans M (Rotterdam), Zambon M
(London) January 2020)
(Lancet. 2020. doi.org/10.1016/S0140-6736(20)30154-9)

SAMPLES
Nasopharyngeal and throat swabs and stool and urine samples were taken
and put into viral transport media. Plasma was separated from EDTA bottles
and serum were separated from clotted blood bottles

Respiratory samples of the patients were tested for influenza A and B viruses and
RSV using the Xpert Xpress Flu/RSV assay (GeneXpert System).
To detect the presence of 18 respiratory virus targets (i.e. adenovirus, coronaviruses
[HCoV-229E, HCoV-Nl63, HCoV-Oc43, HCoV-HKU1, and MERS-CoV], hMPV, RSV,
human rhinovirus/enterovirus, influenza A viruses [H1, H1-2009 and H3], influenza B
virus, parainfluenza viruses [types 1–4]), and 4 bacteria, samples were tested using
BioFire FilmArray Respiratory Panel 2 plus (bioMérieux, Marcy l’Etoile, France).
Reverse transcription, in-house conventional RT-PCR and sequencing
The 1st set of primers targeting 344 bp of RNA-dependent RNA polymerase (RdRp) gene of
all SARS-CoV.
The 2nd set of primers was designed after our first 2019-nCoV genome sequence by
Nanopore sequencing from the positive clinical samples targeting the 158 bp of Spike (S)
gene of this novel CoV.
During the set up of the assays, we initialy used SARS-CoV cDNA as a positive control for
RdRp assay and gene-synthesized fragment for Spike assay. Thereafter, diluted samples
from positive patients were used as the positive control for both assays.
PCR products were sequenced.

In-house one-step real-time RT-PCR assay

Respiratory, urine, stool, serum, or plasma samples
from each patient was subjected to RNA extraction.
Primers targeting the S gene of this novel
coronavirus were used.