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Occult respiratory viral infections in coronial autopsies: A pilot project

Article  in  Hong Kong medical journal = Xianggang yi xue za zhi / Hong Kong Academy of Medicine · July 2009

Source: PubMed

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Joseph S Malik Peiris

The University of Hong Kong

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koon Ho Chan

The University of Hong Kong

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Leo L. M. Poon

The University of Hong Kong

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Philip S.L. Beh

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Final Report RFCID# 04050132»

RFCID

Research Fund for the Control of Infectious Diseases

«Occult Respiratory Viral Infections in Coronial Autopsies - A pilot

project» Submitted to the Grant Review Board (March 11, 2008)

Investigator(s)

Dr John Nicholls, Department of Pathology, The University of Hong Kong

Professor JS Malik Peiris, Department of Microbiology, The University of Hong Kong

Dr KH Chan, Department of Microbiology, The University of Hong Kong

Dr Leo Poon, Department of Microbiology, The University of Hong Kong

Dr S L Philip Beh, Department of Pathology, The University of Hong Kong

Rev. Aug/06

Contents

Summary

Background : Previous studies have shown the seasonal incidence of respiratory viral infections for

patients presenting to the Hong Kong Hospital Authority but at present there is no data relating to the

number of sudden deaths associated with viral disease in patients who die outside the hospital

system.

Purpose: To analyse the presence of clinically undiagnosed and untreated respiratory viral infections

in autopsies performed under the Hong Kong Coronial system.

Aims and Objectives: To determine and optimize the optimal methods for detecting the presence of

virus in the lungs of patients who have sudden unexplained death, by PCR and/or

immunohisotchemistry

Study Design and Methods: To retrospectively examine the paraffin embedded tissue blocks of

patients who have died of sudden unexplained death outside the hospital setting and who on autopsy

had been found to have pathological evidence of pulmonary infection. Cases perfomed in 2003 were

analyzed. We used archived autopsy specimens from these cases and used consensus or tradiational

PCR protocols for respiratory viruses ( influenzavirus, coronavirus, RSV, etc) followed by

immunohistochemical analysis.

Outcome measures: We found a relatively low incidence 11/130 (8.4%) of PCR positive cases with

RSV and NL63 accounting for 4 and 7 cases respectively. Immunohistochemical did not show any

evidence of viral antigen.

Conclusions: Untreated viral infections constitute or contribute to a minority of sudden respiratory

deaths in Hong Kong but more attention should be focussed on the role of newly discovered agents

such as NL63 to mortality in the general population.

Implications: At this stage occult viral infections appear to play a minor role compared to bacterial

disease.

Rev. Aug/06

Introduction

Respiratory viral infections in Hong Kong have a different seasonality from temperate regions of the

world and in the past few years well controlled studies have investigated the degree to which

influenza and respiratory syncytial virus (RSV) contribute to morbidity and mortality in Hong Kong 1,2. The opportunity for accurate disease modelling in Hong Kong is enhanced because of the central

records of the Hong Kong Census and Statistics Department and the major role of the Hospital

Authority (HA) in hospitalizations – 90% of all admissions of the Hong Kong Special

Administrative Region are managed by the HA. The influenza surveillance program of the

Department of Health has shown that influenza accounted for 4913 cases of influenza in 2004 3 . At

Queen Mary Hospital other viruses such as adenovirus and parainfluenza account for fewer

infections than influenza or RSV 1 . Increasing research effort is being dedicated to patients in whom

no microbiological cause has been detected for respiratory tract infections and this had led to the

discovery of novel infectious agents. Indeed, 3 of the 4 new viral agents discovered over the past 3

years have been coronaviruses 4-7 and one has been a parainfluenza virus, HMPV 8 . One method that

has contributed to this discovery has been PCR amplification of common or consensus regions in

viral genomes that allow for wider detection of viral genes followed by more specific amplification

and sequencing.

In Hong Kong in 2000, there were 33,933 registered deaths and previous reports have investigated

the impact of influenza on the HK death rate 2 . The Forensic Pathology Service (FPS) of HK

provides forensic pathology and clinical forensic medicine services to government departments. It

works closely with the Police and provides professional input on medico-legal aspects of criminal

cases 9 . In 2000, 5,400 post mortems ( that is 16% of all deaths in Hong Kong) were performed by

the FPS to determine a cause of death under the direction of the HK Coroner. Whilst many of these

deaths were violent ( e.g traffic accidents, murder, suicide) under the Coroners Ordinance sudden

unexplained deaths are also required to have a post mortem performed. In these circumstances, if the

lungs show a histological evidence of pneumonia then a cause of death of chest infection or

pneumonia is rendered but under normal circumstances no further investigation is undertaken to

determine whether the agent is viral or bacterial. The newer viral infectious agents for respiratory

tract infection have been identified through the sampling of large numbers of material from the

respiratory tract and this has been done in the past mainly using nasopharyngeal aspirates. The

autopsy material present from the Forensic Pathology Service therefore represents an untapped

source of possible material as the patients have died suddenly so have not been treated with antiviral

agents.

Rev. Aug/06

Aims and Objectives:

1. To retrospectively analyze tissue from the lungs of all autopsies performed under the Government

Forensic System for the calendar year of 2003 where the provisional cause of death was listed as

chest infection or pneumonia to determine if there was a viral aetiology to these deaths.

2. To use either consensus PCR or conventional PCR amplification of tissue blocks from the lungs of

these cases to identify the presence of influenza virus, RSV, adenovirus, herpesvirus, parainfluenza

virus or coronavirus;

3. To determine if newly discovered respiratory infectious agents (e.g. HMPV, CoV-HKU1) are

responsible for mortality due to influenza.

4. To determine the value of whether material from post mortems performed by the Forensic

Pathology Service should routinely be sent for viral study.

Rev. Aug/06

Methods:

Sample selection:

We obtained the causes of death from all autopsies performed in the calendar year 2003 by

pathologists at the three public mortuaries in Hong Kong and all deaths attributable to either “chest

infection” or “pneumonia” was recorded. The calendar year 2003 was chosen to determine if there

may be any undocumented cases of fatal SARS during the months February to June.

In the calendar year 2003 there were a total of 6879 deaths attributable to natural causes and 1293

were due to diseases of the respiratory system. As autopsies were not performed (“waived”) in 810

patients there were 1293 cases in which material was available. The age and sex distribution was as

follows:

0-9 10-19 20-29 30-39 40-49 50-59 60-69 >70 Unk

M 8 4 12 11 42 61 124 608 1 871

F 2 6 15 22 15 30 332 422

1293

We excluded cases of malignancy and in over half of records examined there was insufficient fixed

lung material present for DNA or RNA extraction.

We obtained the paraffin tissue from the cases and haematoxylin and eosin staining was performed to

determine which of the sites in the lung were suitable for further examination. The presence or

absence of viral inclusions was recorded.

PCR amplification:

The first step was to determine if PCR amplification methods used on non-tissue samples in the

clinical laboratory could be applied to tissue sections. Initially, we used consensus coronavirus PCR

for the conserved region of the coronavirus polymerase gene 8 .Two µl of cDNA was added to 48 ul

of reaction mixture containing 1X PCR buffer (Roche Diagnostic Manufacturer, Germany), 2.5μM

of each primer, 100 uM dNTP (Roche Diagnostic Manufacturer, Germany), 3 mM MgCl2 and 2.5

units of AmpliTaq Gold polymerase (Roche Diagnostic Manufacturer, Germany). Samples were

Rev. Aug/06

then subjected to 10 minutes at 94oC followed by 40 cycles of amplification as follows: 1 minute

each of denaturation, annealing and extension at 94oC, 48oC and 72oC, respectively. A final 10

minutes at 72oC will follow to ensure a complete extension of PCR products. Samples were then

subjected to 10 minutes at 94oC followed by 35 cycles of amplification as follows: 1 minute each of

denaturation, annealing and extension at 94oC, 50oC and 72oC, respectively. A final 10 minutes at

72oC followed to ensure a complete extension of PCR products. Two ul of the first PCR product was

then transferred to 48 ul of another tube containing 25 uM of each second primers and other reaction

mixture mentioned as above. The reaction mixture was then subjected to the same cycling condition

as first PCR except 60o C which was used for annealing temperature. Amplicons were be visualized

by ethidium bromide staining following electrophoresis on 2% (w/v) agarose gel.

Subsequent amplifications used the protocols in Appendix 1:

Immunohistochemistry:

Influenza A: The tissue sections were incubated with 0.05% pronase (Roche) in 0.1% CaCl2 pH 7.8

at 37 °C for 2 min, blocked with 3% H2O2 in TBS for 10 min followed by treatment with an

avidin/biotin blocking kit (Vector Lab). After blocking with 10% normal rabbit serum for 10 min at

room temperature, the sections were incubated with 1/100 (15µg/ml) HB65 (EVL anti-influenza NP,

subtype A) antibody for 1 h at room temperature followed by biotinylated rabbit anti-mouse (Dako

Cytomation) diluted 1/100 for 30 min at room temperature. After incubation with strep-ABC

complex (Dako Cytomation) diluted 1/100 for 30 min at room temperature, the sections were

developed with 0.5 mg/ml DAB (Sigma) in 0.02% H2O2 for 20 min

SARS: Antigen retrieval was performed by microwaving sections in 10mM citrate buffer pH 6.0 for

15 min followed by incubation with 1/200 4D11 (SARS N protein) antibody at 4°C overnight.

Secondary labeling was with biotinylated Rabbit Anti-Mouse (Dako, E-0354) at 1/100 for 30 min.

RT followed by incubation with strep-ABC complex (Dako, K-0377) at 1/100 for 30 mins. RT and

colour development by the 3-amino-9-ethylcarbazole (AEC) substrate kit (Vector lab, SK-4200) at

RT (15 to 30 minutes).

RSV: Antigen retrieval was performed by microwaving sections in 10mM citrate buffer pH 6.0 for

15 min and then incubation with 1/200 RSV-3 (NCL-RSV3) antibody at RT for 1 hour. Secondary

labeling was with biotinylated Rabbit Anti-Mouse (Dako, E-0354) at 1/100 for 30 min. RT followed

by incubation with strep-ABC complex (Dako, K-0377) at 1/100 for 30 mins. RT and colour

Rev. Aug/06

development by the 3-amino-9-ethylcarbazole (AEC) substrate kit (Vector lab, SK-4200) at RT (15 to

30 minutes).

NL63: Antigen retrieval was performed by microwaving sections in 10mM citrate buffer pH 6.0 for 5

min and after blocking with 10% normal donkey serum for 10 mins at RT, the sections were

incubated with 1/500 NL63 polyclonal antibody (courtesy of Dr L. van der Hoek, University of

Amsterdam) at 4°C overnight. Secondary labeling was with biotinylated Donkey-Anti-Rabbitt

(Amersham RPN1004) at 1/200 for 30 min. RT followed by incubation with strep-ABC complex

(Dako, K-0377) at 1/200 for 30 mins. RT and colour development by the 3-amino-9-ethylcarbazole

(AEC) substrate kit (Vector lab, SK-4200) at RT (15 to 30 minutes).

Study design:

No randomization was carried out and results were viewed by 2 observers. PCR experiments were

repeated for validity.

Results

Sample selection:

Of the 1293 cases which were referred to the Coroner and had an autopsy performed we focussed on

cases performed by the Forensic Pathology Service as these cases would represent untreated

community aquired cases. From an analysis of the records of the 3 public mortuaries (Tai Wai,

Wanchai and Kwai Chung) we were able to identify 130 cases where a cause of death was recorded

as chest infection and tissue blocks were available for further examination. Histological examination

by H and E confirmed the presence of acute pneumonia, bronchopneumonia, chronic obstructive

lung disease, tuberculosis or acute pulmonary oedema in the sections. Only 1 case showed an

interstitial infiltrate suggestive of viral infection (03-378F). In no cases were intranuclear or

intracytoplasmic viral inclusions seen.

Results of amplification:

Influenza: We used control positive cases of H5N1 proven autopsy material and found that protocols

developed for the clinical virology laboratory did not result in any product (data not shown). When

Rev. Aug/06

we used a protocol developed by the Armed Forces Institute of Pathology 10 using known positive

cases and negative cases spiked with influenza that this required some modification. In particular, we

found that the proteinase K digestion prior to phenol-chloroform-isoamyl alcohol extraction was

more effective than using the commercially available Ambion kit as shown in Figure 1.

M + 1-6 2-6 H1 1-6 2-6 H1 -ve

Proteinase K +

Phonol-Chloroform

Ambion

Figure 1. PCR amplification using known H5N1 positive lung samples (1-6, 2-6) and control

influenza H1 positive cell pellet and 2 methods of RNA extraction.

We then found that 3 x 10µm consecutive sections per sample were just as effective as 10 x 10 µm

consecutive sections for a positive finding and later modified this to 6 x 6 µm sections:

Rev. Aug/06

M + 1-6 2-6 H1 3 H1 10 -ve

Figure 2: Amplification of positive control using 3 and 10 sections, each of 10 µm

In keeping with the findings of Kraaft et al 10 a product size of less than 120 (for influenza) resulted

in a positive band than when a larger amplicon (404bp) was selected:

M + Brain

1

Brain

2

L.

Lung

1

L.

Lung

2

R.

Lung

1

R.

Lung

1

-ve

H5N1 Brain H5N1 lung

Rev. Aug/06

The detection of RSV and NL63 proved less problematic than that for influenza:

Figure 3. Samples from Kwai Ching mortuary showing a single positive result for RSV

When we then analyzed the cases from the 3 public mortuaries using the optimized protocols as per

appendix 1 the following results were obtained:

Table 1:

Mortuary Total number

of cases

RSVA PCR

positive

RSVB PCR

positive

FluA PCR

positive

NL63 PCR

positive

Wan Chai 32 3 0 0 4

Tai Wai 49 0 0 0 0

Kwai Chung 49 1 0 0 3

130 4 0 0 7

Immunohistochemical analysis:

The PCR positive cases of RSV and NL 63 were then analyzed by both immunohistochemistry as

well as sequencing. In the former case there were no positive cells identified in any of the samples

though control samples were positive. Sequencing showed homology with published RSV and NL63

sequences indicating that the results were not artefacts (data not shown). We therefore conclude that

Rev. Aug/06

the positive findings are more indicative of past rather than recent infection as published previously 11 . To confirm that we were not identifying PCR negative cases, we performed

immunohistochemical analysis on all 130 cases using antibodies to RSV and influenza. No staining

was seen indicating that there were no occult cases of active infection identified. There was

insufficient antibody to NL63 present to test the NL63 PCR negative cases..

Discussion

This pilot study aimed to try and optimize the diagnosis of viral infection of archival tissue which had

been fixed in formalin. For over a century formalin has been the fixative of choice for tissue samples 12 . Fixation of tissue is necessary because as soon as death occurs or tissue is removed from the body

in a vital state, it undergoes a process of degeneration called autolysis. The fixation of tissue samples

in formaldehyde leads to extensive cross linking of all tissue components and this preserves tissue,

preventing autolysis. Recently, the safety of formalin has come under scrutiny where in the European

Union it is now classified as a Class I carcinogen 13 . Alternatives have been proposed such as

Glyo-Fixx, STF-Streck, Omnifix, Histochoice, and Histofix 14 , but none of these has met with much

approval by pathologists for morphological diagnosis. The main drawback with formalin fixation

from a research aspect has been the limited ability to extract DNA and RNA from this fixed tissue. A

number of postulated mechanisms for this problem come into play here – firstly, the addition of a

formaldehyde group to a base in the form of N-methylol (N-CH2OH), the second is the attack of

N-methylol on an amino base to form a methylene bridge and the third is the potential deleterious

effects of RNAse which are not inactivated by formalin 15,16 . Indeed, Ding et al demonstrated that

only 0.1% contamination of an extraction reagent by formalin inhibited the ability of the reagent to

extract mRNA 17 .

The ideal solution for molecular diagnosis of a viral infection, therefore, would not to use any form of

fixative as RNA can readily be extracted from fresh tissue using either a combined guanidinium

thiocyanate acid-phenol chloroform procedure 18 or a guanidinium thiocyanate-caesium chloride

gradient. Commercially available RNA extraction methods are available (RNeasy(Qiagen),

Trizol(Invitrogen) and ToTally RNA(Ambion)) but in PCR amplification of fresh samples their

success is varied 19 and this may be due to the steps of the individual techniques and the affinity of

viral RNA to the spin column. It must be stressed that these solutions do not fix the tissues in the

same way as formalin does so will not be useful for morphological analysis or long term storage at

room temperature. For long term storage and transport RNAlater (Ambion) Tissue Protect Tubes

provide pre-measured volumes of RNAlater RNA Stabilization Reagent in re-closable tubes for

Rev. Aug/06

convenient handling and sample storage. The reagent preserves RNA for up to 1 day at 37°C, 7 days

at 18–25°C, or 4 weeks at 2–8°C, allowing processing, transportation, storage, and shipping of

samples without liquid nitrogen or dry ice. Alternatively, the samples can also be placed at –20°C

or –80°C for archival storage. Morphological analysis, however, will be very difficult to interpret

from tissues placed in this reagent.

Fixation in alcohol is superior to formalin for the preservation of RNA but will result in poor

morphology 20 .Whilst these authors found that immunohistochemistry was not altered, it is possible

that in alcohol fixed tissue some antibodies will not react in alcohol as the antibodies are developed

for use in formalin fixed material and small molecules (e.g. peptides) may be solubilized and lost in

alcohol based fixatives 21. In one report Carnoy’s fixative has been found to be superior to formalin

and ethanol for RNA extraction but this fixative is not widely used in routine practice 22 . Bouin’s

fixative is to be avoided as it causes damage to DNA and RNA 23. If ethanol fixation is to be used

70% seems the ideal concentration though this should be used for small samples 24.

If one is going to attempt RNA or DNA extraction from formalin fixed paraffin embedded (FFPE)

tissues a number of factors need to be taken into consideration for successful extraction, especially

for RNA. Firstly, the most successful method of total RNA utilizes a proteinase K digestion prior to

phenol-chloroform-isoamyl alcohol extraction and carrier precipitation 10. Both phenol and

chloroform denature proteins but leave the RNA and DNA in the aqueous phase. The isoamyl

alcohol reduces foaming. Secondly, the RNA that is extracted will be degraded and one should not

aim at amplifying fragments larger than 60-120 bp. 10,25-27 . Thirdly, RNAase from glassware is a

potential source of contamination so all reagents must be RNAse free. Trace amounts of DEPC that

have been used to eradicate RNAse however may be detrimental to RNA by modifying purine

residues 16 . Finally a pre-PCR DNA restoration treatment has been proposed that promises to

increase the length of amplifiable DNA 27 .

Our findings confirm those of others that in an analysis of fatal respiratory lung cases that there is a

problem with the extraction and amplification of RNA which will hamper the ready analysis and

protocols which are used by clinical virology laboratories for non-formalin fixed specimens may not

be suitable for these autopsy specimens. Indeed we found even that using protocols developed for the

extraction of RNA for influenza virus 10 these needed to be modified in order to enhance the

successful amplification of RNA and our results indicate that our protocols were just as effective as

the commercially available kits for RNA extraction.

Rev. Aug/06

We found that from our retrospective analysis of fatal cases dying in the community that had

autopsies performed that there was a low incidence of occult respiratory RNA viral infections as

determined by PCR. As optimization of the previously published techniques required much effort

than anticipated we were unable to fulfil the analysis of more of the novel respiratory agents (such as

HMPV or CoV-HKU1) apart from NL63 within the time frame, but this will be addressed in future

experiments. No cases of undocumented SARS were identified in the 2003 calendar year.

We quite readily accept that there are a number of limitations to this investigation. The first is that the

tissue sampling is dependent on the pathologist’s expertise and given that the conventional practice is

to sample 1 tissue block from each lobe of the lung, in reality only a small area of the lung will be

sampled. The second is that as many of the respiratory viruses are RNA viruses and at this stage the

standard fixative is formalin, the extraction and detection of good RNA is still a valid concern so

whatever positive findings we have most likely represents the lower range of the real situation.

Thirdly, it appears that given the relatively low number of documented viral infections over a

calendar year, at this stage having routine viral culture or analysis of tissue samples of chest infection

is probably not warranted. However, given that we have found 7/130 (5.3%) of these cases (most of

whom are elderly) have coronavirus NL63 PCR positivity, further investigation into the nature and

pathogenesis of this newly discovered coronavirus may be warranted including seasonal range,

existing co-morbidities and cellular tropism, which were beyond the scope of this investigation.

Conclusions:

With respect to the aims and objectives as stated previously the following conclusions are made:

1. This was a pilot project to test the feasibility of a retrospective analysis of formalin fixed tissues

and it demonstrated that this is a less than optimal method for detecting viral infections.

2. Conventional protocols in use by diagnostic laboratories for clinical specimens needs to be

amended – in particular focusing on smaller PCR product sizes

3. A newly discovered CoV (NL-63) is present in 5% of autopsy cases and more investigation into

this novel agent is warranted

4. If prospective studies are to be performed tissue should be fixed in ethanol in addition to formalin

for viral study. At this stage there appears to be little justification for the routine sending of

specimens for viral culture.

Implications:

Rev. Aug/06

We found from this analysis that occult viral infections do not appear to be a major contributor to

sudden unexplained death in the elderly community population. In particular influenza (a treatable

viral infection) did not feature as one of the significant viral infections leading to mortality. The

presence of RSV is not surprising in the elderly but this is a viral disease that is not readily able to be

treated and vaccination is not a viable alternative. We think that further attention should be given to

NL63 and if further funding is awarded the material is stored and available for examination.

Dissemination

A portion of these results detailing the optimization of methods for RNA extraction was presented at

a World Health Organization meeting on autopsy protocols for H5N1 infection in Beijing, PRC in

July 2006.

Publications

Nicholls JM, Peiris JSM. The role of a pathology laboratory in SARS and other emerging infections.

In Lal SK (ed). Emerging viral diseases of Southeast Asia. Issues Infect Dis. Karger, 2007;4:125-135

Rev. Aug/06

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Rev. Aug/06

List of Research Workers: YW Chan (陳韻怡), CHY Wong (黄韾琦), K Fung(馮孝賢) and YS

Chan(陳旭昇)

Financial Statement:

Attached

Rev. Aug/06

Appendix 1:

Protocol for optimized RNA extraction of tissues for influenza PCR:

4 to 8 of 6-micron sections were cut and placed in a 1.5ml

microcentrifuge tube (depending on the type and surface area of

the tissue)

1ml xylene was added RT 10mins

Centrifuge 12000g for 10mins and remove xylene

Repeat steps 2 and 3

1ml of absolute ethanol was added RT 10mins

Centrifuge 12000g for 5 mins and remove ethanol

Repeat steps 4 and 5

Repeat steps 5 and 6 twice by using 90% thenaol and 75%

ethanol instead of absolute ethanol

The tissue pellet was dried briefly in air Until dry

500ul of buffer was added (20mM Tris pH 7.4, 20mM EDTA,

1% sodium dodecyl sulfate, 1mg/ml proteinase K) – 2ml buffer

= 40 ul of 1M Tris + 80ul of 0.5M EDTA + 200 ul of 10% SDS

+ 100ul of 20mg/ml PK + 1580ul of DEPC water)

55°C water bath /

overnight

Day 2

500ul of Phenol:Chloroform:Isoamyl alcohol

(25:24:1) was added

Vertex vigorously for 10 seconds

Centrifuge 12000g for 10 mins

Collect aqueous phase (supernatant) to a new microcentrifuge

tube

Repeat steps 11 to 14

0.l vol of 3M sodium acetate was added and mixed gently by

inverting the tube

0.6vol of pre-cool (-20°C) ispropanol was added too

RNA precipitation On ice / > 10mins

Centrifuge 12000g for 15 mins

Rev. Aug/06

Remove fluid completely and carefully without disturbing the

RNA pellet (RNA pellet may not be visible)

1ml of 75% ethanol was added to wash the pellet by vortex

several times

Centrifuge 12000g for 15mins.

Remove fluid completely and carefully

Allow the pellet air dry

25ul of DEPC H2O was added to resuspend RNA

Store RNA at -70°C

Rev. Aug/06

Appendix 2:

1) Flu A - Forward primer M149F: GAG GYT CTC ATG GAR TGG CT

- Reverse primer M149R: TAC GCT GCA GTC CTC GCT CA

- Product size: 113bp

Optimized Protocol

Reagents Volume (x1)

10uM M149F 1uL (0.5uM)

10uM M149R 1uL (0.5uM)

10x PCR buffer 1uL (0.5x)

2mM dNTPs 1uL (0.1mM)

25mM MgCl2 1uL (1.25mM)

MQ H2O 12.8uL

5U/uL Taq 0.2uL (1U)

cDNA template 2uL

Total volume of reaction

mixture

20uL

94ºC 9mins

94ºC 30s

55ºC 30s 50 cycles

72ºC 30s

72ºC 10mins

15ºC ∞

2) SARS - From World Health Organization

- Forward primer BNIinS: GAA GCT ATT CGT CAC GTT CG

- Reverse primer BNIAS: CTG TAG AAA ATC CTA GCT GGA G

- Product size: 110bp

Optimized Protocol

Rev. Aug/06

Reagents Volume (x1)

10uM BNIinS 1uL (0.5uM)

10uM BNIAS 1uL (0.5uM)

10x PCR buffer 2uL (1x)

2mM dNTPs 2uL (0.2mM)

25mM MgCl2 2uL (2.5mM)

MQ H2O 9.8uL

5U/uL Taq 0.2uL (1U)

cDNA template 2uL

Total volume of reaction

mixture

20uL

94ºC 9mins

94ºC 30s

60ºC 30s 50 cycles

72ºC 30s

72ºC 10mins

15ºC ∞

3) RSV-A - From a paper named Evaluation of a Quantitative Real-time PCR for the Detection

of Respiratory Syncytial Virus in Pulmonary Diseases

- Forward primer RSV-AF: TTG GAT CTG CAA TCG CCA

- Reverse primer RSV-AR: CTT TTG ATC TTG TTC ACT TCT CCT TCT

- Product size: 76bp

Optimized Protocol

Reagents Volume (x1)

10uM RSV-AF 1uL (0.5uM)

10uM RSV-AR 1uL (0.5uM)

10x PCR buffer 2uL (1x)

2mM dNTPs 2uL (0.2mM)

25mM MgCl2 1.2uL (1.5mM)

Rev. Aug/06

MQ H2O 10.6uL

5U/uL Taq 0.2uL (1U)

cDNA template 2uL

Total volume of reaction

mixture

20uL

94ºC 9mins

94ºC 45s

55ºC 45s 50 cycles

72ºC 30s

72ºC 10mins

15ºC ∞

4) RSV-B - Designed by own using Oligo software

- Forward primer RSV-BF: CAT CAC CTC TAT GCA CCA CCA A

- Reverse primer RSV-BR: CAA GTG TCA GCC TGT GGA AAG A

- Product size: 121bp

Optimized Protocol

Reagents Volume (x1)

10uM RSV-BF 1uL (0.5uM)

10uM RSV-BR 1uL (0.5uM)

10x PCR buffer 2uL (1x)

2mM dNTPs 2uL (0.2mM)

25mM MgCl2 1.2uL (1.5mM)

MQ H2O 10.6uL

5U/uL Taq 0.2uL (1U)

cDNA template 2uL

Total volume of reaction

mixture

20uL

Rev. Aug/06

94ºC 9mins

94ºC 45s

55ºC 45s 50 cycles

72ºC 30s

72ºC 10mins

15ºC ∞

5) Beta-Actin - From a paper named Postmortem Diagnosis of Morbillivirus Infection in Bottlenose

Dolphins (Tursiops truncates) in the Atlantic and Gulf of Mexico Epizootics by

Polymerase Chain Reaction-Based Assay (10).

- Forward primer Beta-act-F: ACA GCC TGG ATA GCA ACG TA

- Reverse primer Beta-act-R: CCA CAC CTT CTA CAA TGA GC

- Product size: 156bp

Optimized Protocol

Reagents Volume (x1)

10uM Beta-act-F 1uL (0.5uM)

10uM Beta-act-R 1uL (0.5uM)

10x PCR buffer 2uL (1x)

2mM dNTPs 2uL (0.2mM)

25mM MgCl2 1.2uL (1.5mM)

MQ H2O 10.6uL

5U/uL Taq 0.2uL (1U)

cDNA template 2uL

Total volume of reaction

mixture

20uL

94ºC 9mins

Rev. Aug/06

94ºC 45s

50ºC 45s 50 cycles

72ºC 45s

72ºC 10mins

15ºC ∞

6) NL 63 - Designed by Dr. Leo Poon.

- Forward primer Coro NL-F2: AGG ACC TTA AAT TCA GAC AAC GTT CTG ATG

- Reverse primer Coro NL-R2: GAT TAC GTT TGC GAT TAC CAA GAC TGG TAT

- Product size: 96bp

Optimized Protocol

Reagents Volume (x1)

10uM CoroNL-F2 1uL (0.5uM)

10uM CoroNL-R2 1uL (0.5uM)

10x PCR buffer 2uL (1x)

2mM dNTPs 2uL (0.2mM)

25mM MgCl2 2.8uL (3.5mM)

MQ H2O 9uL

5U/uL Taq 0.2uL (1U)

cDNA template 2uL

Total volume of reaction

mixture

20uL

94ºC 3mins

94ºC 30s

60ºC 45s 40 cycles

72ºC 30s

72ºC 10mins

15ºC ∞

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