Virology Unit 2012 - pasteur-kh.org€¦ · Epi Predict (USAID) WCS/UC Davis WCS/UC Davis Etiology...

Prepared by: Philippe Buchy, Head, Virology Unit Institut Pasteur du Cambodge, 5, Bvd. Monivong BP 983 - Phnom Penh, Royaume du Cambodge

Virology Unit

2012

The scientific activity report of the Virology Unit of the Institut Pasteur du Cambodge for the period January 1st to December 31st, 2012.

Scientific

activity

report

Virology Unit, Scientific Activity Report 2012

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1. Organigram of the Virology Unit


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2. General presentation of the main activities

Unit Themes Studies / Projects Main collaborations

Seasonnal fluWHO National Reference Centre - Virus evolution -

Drug resistanceMoH/WHO

IMMI Epi - Bacterio

Avian influenzaWHO National Reference Centre - Virus evolution -

Drug resistance MoH/MAFF/WHO/FAO

Human/animal/Environment interface (markets, ..,) Epi/MoH/MAFF/WHO/FAO

Pathogen

discoveryVirus discovery in severe pneumonia (Pathoquest)

IP Paris

National Reference Centre MoH

Dengue DENFREE (genomic, immuno,

entomo.)

Epi/IP Paris

Bio-markers (BioMerieux and PTR) IP Paris/BioMerieux

National Reference Centre Epi/MoH/WHO

Molecular Epidemiology§/entomology Epi/MoH/WHO

Epi

Predict (USAID) WCS/UC Davis

WCS/UC Davis

Etiology of meningo-encephalitis in

CambodiaBacterio/OUCRU

AVIESAN Regional Project Epi/Bacterio/IP Paris/RIIP

Enteroviruses EV71 epidemic Epi/MoH/IP/RIIP

IP ParisPathogen

Discovery

Virus discovery in acute encephalitis

(Pathoquest/AVIESAN)

Zoonoses

Vir

olo

gy

National Reference Center - Molecular Epidemiology

Neurotropic

infections

Eme

rgin

g &

Ep

ide

mic

vir

use

s

Arboviral

diseases

Rabies

Chikungunya

Respiratory

viruses

Zoonotic viruses transmitted by bats, rodents,

primates

Pathogen Discovery

Predict (USAID)

Meningo-

encephalitis


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3. Respiratory viruses

a. Seasonnal influenza & other respiratory viruses: virus evolution, drug resistance, co-infections

Coordination:

Buchy P. (NIC Director)

Partners:

MoH & WHO Cambodia (NIC), Tarantola A. and coll. (IMMI & SISEA), Vong S. and coll.

(SISEA), M. Von Itzstein (Australia), WHOCC Melbourne, WHO Geneva

Objectives:

Influenza and some other viruses are responsible for respiratory infections that represent an

important disease burden in children and adults. The etiology of respiratory infections in

tropical countries is not well-known and influenza viruses require a constant monitoring to

follow their genetic evolution, antigenic shift, and drug resistance patterns. The role of

bacterial and viral co-infections in the respiratory disease severity has rarely been assessed

and described, especially in developing tropical countries.

Core of the project:

The Influenza-like illness (ILI) surveillance established in 2006 in collaboration with MoH and

WHO allows the collection of influenza strains and data on seasonality. Based on this

information, the National Influenza Centre (NIC) based in the IPC Virology Unit makes every

year recommendations to MoH regarding the influenza vaccine strategy and the use of

antiviral drugs for the treatment of severe cases. The characteristics of influenza virus

circulation and evolution have been described in the past and a new publication reporting the

data since 2008 has been recently submitted. The NIC is part of the Global Influenza

Response and Surveillance system.

Since the global burden of disease attributable to seasonal influenza virus in children was not

known, we aimed to estimate the global incidence of and mortality from influenza-associated

lower respiratory infections in children younger than 5 years. In collaboration with many

groups, we participated at a study that estimated that 28000-112000 deaths in children


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younger than 5 years could be attributed to influenza-associated ALRI, with 99% of these

deaths occurring in developing countries.

The SISEA project aimed to study the etiology of respiratory infections in Cambodia, Vietnam,

Laos, and China, and generated an important amount of data (almost 4000 patients

hospitalized for ALRI were tested in the Virology Unit for 18 respiratory viruses). After

describing the epidemiological, clinical and virological characteristics of several respiratory

viruses of interest (RSV, HMPV, bocavirus, etc.) in Cambodia, it was decided to extend these

studies at a regional level and several manuscript are in preparation.

The influenza pandemic in 2009 gave an opportunity to better understand the role of bacterial

and viral co-infection in the disease severity. This is the goal of the IMMI project that involves

several countries (International Network of Pasteur Institutes).

Prospective:

In order to decrease the burden of public health surveillance activities for the Virology Unit

and in the frame of the reinforcement of national capacities, the Virology Unit will stop to test

all the specimens collected by the ILI surveillance. From January 2013, the Virology Unit will

keeps its role of NIC but will only perform the 2nd line tests (quality control and confirmation of

samples analysed by National Institute of Public Health/US CDC, NAMRU, AFRIMS;

antigenic characterization, susceptibility to drugs, sequencing and phylogenetic analysis).

This will allow to develop the research activities, in particular on new drugs using in vitro and

in vivo models (collaboration with the Institute of Glycomics, Griffith University, Australia and

with the WHOCC for influenza in Melbourne)

Recent publications (2011-2012):

1. BUCHY P. Clinical experience of influenza virus sialidase inhibitors. In: Influenza virus sialidase - A drug discovery target. Ed: M. von Itzstein. Springer Basel Switzerland 2012. DOI 10.1007/978-3-7643-8927-7

2. MARDY S, RITH S., LY S, HENG S., HORM SV, VONG S, HUCH C, NORA C, ASGARI N, VEASNA D, TOUCH S, BUCHY P. Seasonal influenza and 2009 H1N1pandemic characterization in Cambodia. (submitted)

3. Members of the Western Pacific Region Global Influenza Surveillance and Response System. Epidemiological and virological characteristics of influenza in the Western Pacific Region of the World Health Organization, 2006-2010. PLoS ONE 2012, 7:5; e37568.


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4. GUERRIER G, GOYET S, CHHENG ET, RAMMAERT B, BORAND L, TE V, TRY PL, SARETH R, CAVAILLER P, MAYAUD C, GUILLARD B, VONG S, BUCHY P, TARANTOLA A. Acute viral lower respiratory tract infections in Cambodian children: clinical and epidemiological characteristics. Pediatr Infec Dis J 2012; Aug. 24. [Epub ahead of print]

5. RITH S, NETRABUKKANA P, SORN S, MUMFORD E, MEY C, HOLL D, GOUTARD F, Y B,

FENWICK S, ROBERTSON I, ROGER F, BUCHY P. Serologic evidence of human influenza virus infections in swine populations, Cambodia. Influenza Other Respi Viruses 2012; May 30. doi: 10.1111/j.1750-2659.2012.00382.x.

6. DAWOOD FS, LULIANO AD, REED C, MELTZER MI, SHAY DK, CHENG PY, BANDARANAYAKE D, BREIMAN RF, BROOKS WA, BUCHY P, FEIKIN DR, FOWLER KB, GORDON A, HIEN NT, HORBY P, HUANG QS, KATZ MA, KRISHNAN A, LAL R, MONTGOMERY JM, MOLBACK K, PEBODY R, PRESANIS AM, RAZURI H, STEENS A, TINOCO YO, WALLINGA J, YU H, VONG S, BREESE J, WIDDOWSON MA. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modeling study. Lancet Infect Dis., 2012 Jun 25 [Epuib ahead of print]

7. ARNOTT A, VONG S, RITH S, NAUGHTIN M, LY S, GUILLARD B, DEUBEL V, BUCHY P. Human bocavirus (HBoV) amongst an all-ages population hospitalized with acute lower respiratory infections in Cambodia. Influenza and Other Respiratory Viruses 2012, doi: 10.1111/j.1750-2659.2012.00369.x

8. RAMMAERT B, GOYET S, BEAUTÉ J, HEM S, TE V, LORN P, MAYAUD C, BORAND L, BUCHY P, GUILLARD B, VONG S. Klebsiella pneumoniae related community-acquired acute lower respiratory infections in Cambodia: clinical characteristics and treatment. BMC Infect Dis 2012; 12:3

9. ARNOTT A, VONG S, MARDY S, CHU S, NAUGHTIN M, LY S, BUECHER C, BEAUTE J, RITH S, BORAND L, ASGARI N, FRUTOS R, GUILLARD B, SOK T, DEUBEL V, BUCHY P. A study of the genetic diversity of human respiratory syncytial virus (HRSV) reveals the existence of a new HRSV group B genotype. J Clin Microbiol 2011, 49:3504-3013.

10. H NAIR, WA BROOKS, M KATZ, A ROCA, JA BERKLEY, SA MADHI, JM SIMMERMAN, A GORDON, M SATO, S HOWIE, A KRISHNAN, M OPE, KA LINDBLADE, P CAROSONE-LINK, M LUCERO, W OCHIENG, L KAMIMOTO, E DUEGER, N BHAT, S VONG, E THEODORATOU, M CHITTAGANPITCH, O CHIMAH, A BALMASEDA, P BUCHY, E HARRIS, V EVANS, M KATAYOSE, B GAUR, C O’CALLAGHAN-GORDO, D GOSWAMI, W ARVELO, M VENTER, T BRIESE, R TOKARZ, MA WIDDOWSON, AW MOUNTS, RF BREIMAN, DR FEIKIN, KP KLUGMAN, SJ OLSEN, BD GESSNER, PF WRIGHT, I RUDAN, S BROOR, EAF SIMÕES, H CAMPBELL. The global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis. Lancet 2011, 378:1917-1930.

11. RAMMAERT B, BEAUTE J, BORAND L HEM S, BUCHY P, SOPHIE GOYET S, OVERTOOM R, ANGEBAULT C, TE V, LORN TRY P, MAYAUD C, VONG S, GUILLARD B. Pulmonary Melioidosis in Cambodia: a Prospective Study. BMC Infect Dis 2011; 11:126.

12. ARNOTT A, VONG S, SEK M, NAUGHTIN M, BEAUTE J, RITH S, GUILLARD B, DEUBEL V, BUCHY P. Genetic variability of human metapneumovirus amongst an all ages population in Cambodia between 2007 and 2009. Infect Genet Evol 2011 Feb 1 [Epub ahead of print]


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Financial support:

WHO – French Agency for Development (SISEA project) - US CDC

b. Avian influenza: virus evolution, transmission, mechanisms of drug resistance and new therapeutic approaches

Coordination:

Buchy P., Horm SV

Partners:

Rith S. (NIC), Tarantola A., MoH, Ministry of Agriculture (Sorn S.), WHO, FAO

Objectives:

To study the evolution and the dynamic of circulation of H5N1 virus clade 1 in Cambodia. In

addition, as the treatment of H5N1 poses a great challenge (60% of mortality) we decided to

study some mechanisms associated with a resistance to antiviral drugs, to evaluate the

neutralizing activity of polyclonal antibodies raised against H5N1 virus and, in collaboration for

IP Shanghai, to evaluate the protection obtained with antibodies against conserved epitopes

of H5N1 virus. The transmission of H5N1 to human being sometimes associated with an

exposure to a contaminated environment, we evaluated the virus persistence and the role of

the environment contamination in life bird markets or in farms after outbreaks in poultry. This

integrative study of the virus cycle at the human/animal/environment interface allows to better

understand why the avian influenza virus is still actively circulating since 2003 and thus still

posing a pandemic threat.


From 2004 to 2012, there have been 32 outbreaks in poultry and 21 human cases reported in

the country but the origin of these epizootics remains unclear. The phylogenetic relationships

among the H5N1 strains revealed that 6 different lineages were circulating in the country

since 2004. Lineage 6 is endemic to Cambodia since 2010 and could be classified as a new

clade according to WHO criteria for H5N1 virus nomenclature.Currently two lineages with

totally different dynamics are cocirculating in Cambodia.


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We aimed at analyzing the mutational events occurring during the infection of chickens by the

Highly Pathogenic Avian Influenza (HPAI) H5N1 virus and to demonstrate for the 1st time that

the quasi-species concept could apply to an avian influenza virus. We also observed a strong

mutational bias, resembling host-driven ADAR1 adenosine deamination, which is responsible

for 81% of all mutations.

All H5N1 viruses detected in Cambodia are sensitive to neuraminidase inhibitor (NAI) drugs.

However, we identified a virus with a mildly decreased sensitivity to zanamivir, and have

predicted that a V149A mutation was responsible or this phenomenon by affecting the

catalytic site of the neuraminidase. We also identified a virus with a hemagglutinin A134V

mutation, present in a subpopulation amplified directly from a fatal human patient's sample.

Using reverse genetics, we verified that this mutation was adaptative for human α2,6-linked

sialidase receptors. The importance of ongoing surveillance of H5N1 antigenic variance and

genetic drift which may alter receptor binding and sensitivities of H5N1 viruses towards NAIs,

cannot be underestimated whilst avian influenza remains a pandemic threat. The cross-

neutralization activity of anti-H5N1 polyclonal Fab2’ produced in horse was demonstrated in

vitro. Antibodies could be used, in a near future (phase 1 trial ongoing) to treat the infected

patients or to prevent the disease in exposed individuals.

Very little is known regarding the persistence of Highly Pathogenic Avian Influenza H5N1

viruses in natural settings during outbreaks in tropical countries, although environmental

factors may well play a role in the persistence and in the transmission of H5N1 virus. We

therefore used an integrated approach and investigated various environmental compartments

surrounding outbreak areas as potential sources for H5N1 virus transmission.

For instance, we demonstrated that during H5N1 virus outbreaks, number of environmental

samples surrounding outbreak areas were contaminated by the virus and may act as potential

sources for human and/or animal contamination. The survival of HPAI H5N1 viruses in

experimental aquatic biotopes (water, mud, aquatic flora and fauna) relevant to field

conditions in Cambodia was also investigated. Artificial aquatic biotopes, including simple

ones containing only mud and water, and complex biotopes involving the presence of aquatic

flora and fauna, were set up. Viral RNA persisted up to 20 days in rain water and 7 days in

pond or lake water. Viral RNA was also detected in mud samples, up to 14 days post-

contamination in several cases. Infectious virus and viral RNA was detected in few cases in

the aquatic fauna and flora, especially in bivalves and labyrinth fish, although these


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organisms seemed to be mostly passive carriers of the virus rather than host allowing virus

replication. Our results, along with previous data, support the idea that environmental

surveillance is of major relevance for avian influenza control programs.

Merit Release Birds, such as the Eurasian Tree Sparrow, are believed to increase one’s

positive karma when kissed and released during Buddhist rituals. Since these birds are often

in close contact with both poultry and humans, we investigated their potential role in the

spread of H5N1 virus. This study indicated that under experimental conditions, Eurasian Tree

Sparrows were susceptible to H5N1 infection, either by direct inoculation or by contact with

infected poultry. Their ability to transmit H5N1 infection to other birds was also demonstrated,

suggesting that the sparrows may play a role in the dissemination of the virus. Finally, the

presence of significant quantities of H5N1 virus on sparrows’ feathers, including infectious

particles, would suggest that Merit Release Birds represent a risk for human contamination in

countries where avian influenza virus is circulating and where this religious ritual is practiced.

The role of the soil in the indirect transmission of the highly pathogenic H5N1 virus through

the environment was also addressed by experimentally reproducing the conditions met within

an actual epizootic context in Mekong Region and using soils of various origins. For the first

time, our results provided evidence that even when abundantly contaminated, some soils did

not pose a significant risk of infection to poultry and consequently also probably not to other

animals or humans. These results suggest that some of the often limited resources could be

spared and put at better use in high-risk areas where the nature of the soils would be more

likely to lead to poultry infection after natural contamination.

A H5N1 virus surveillance was conducted in 4 wet markets of 3 Cambodian provinces during

6 weeks. Out of the 502 environmental samples collected, 10 (2%) contained infectious virus

particles and 90 (18%) tested positive by qRT-PCR for H5N1 virus. All the strains detected

belonged to a lineage endemic in Cambodia. The impact for human health of such high

contamination of the market’s environment in Cambodia is not known and should be

evaluated through clinical and serological surveillance of vendors and poultry workers.

Prospective:

We demonstrated the importance of the environment in the natural cycle of the H5N1 virus

through field and experimental studies. This role has been poorly studied in the past and the


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Virology Unit is now internationally recognized for its expertise in this field. The impact on

human health will now be addressed, in 2013, through a combined surveillance in markets by

following the virus circulation in the environment and by monitoring the individuals exposed

(vendors, butchers, middle-men, ...) to the contaminated environment (serological

surveillance). This should make the proof of the environmental risk associated with H5N1

virus and lead to public heath measures.

The monitoring of the H5N1 virus evolution, the study of mechanism of resistance, of

adaptation to human host will still be research topics as they are directly linked to the testing

activity of the WHO National Influenza Centre. New molecules, antibodies will be evaluated in

the coming months to try to improve the clinical management of this deadly infection.


1. HORM SV, SORN S, ALLAL L, BUCHY P. Environmental analysis for avian influenza surveillance in Cambodian wet markets. Emerg. Infect. Dis. (in revision)

2. S SORN, T SOK, S LY, S RITH, N TUNG , AL VIARI, L GAVOTTE, D HOLL, H SENG, N ASGARI, B RICHNER, D LAURENT, N CHEA, V DUONG, T TOYODA, CY YASUDA, P KITSUTANI, P ZHOU, S BING, V DEUBEL, R DONIS, R FRUTOS, P BUCHY. Dynamic of H5N1 virus in Cambodia and emergence of a novel endemic sub-clade. Infect Genetics Evol. 2012 Jun 7. [Epub ahead from print]

3. GUTIERREZ RA, BUCHY P. Contaminated soil and transmission of influenza virus (H5N1). Emerg Infect Dis 2012; 18:9. http://dx.doi.org/10.3201/eid1809.120402

4. HORM SV, GUTIERREZ RA, NICHOLLS JM, BUCHY P. Highly Pathogenic Influenza A (H5N1) virus survival in complex artificial aquatic biotopes. PLOS One 2012;7(4):e34160

5. HORM SV, GUTIERREZ RA, SORN S, BUCHY P. Environment: a potential source of animal and human infection with influenza A (HN) virus. Influenza and Other Respiratory Viruses 2012; doi: 10.1111/j.1750-2659.2012.00338.x

6. HU H, VOSS J, ZHANG G, BUCHY P, ZUO T, WANG L, WANG F, ZHOU F, WANG G, TSAI C, CALDER L, GAMBLIN SJ, ZHANG L, DEUBEL V, ZHOU B, SKEHEL JJ, ZHOU P. A human antibody recognizing a conserved epitope of H5 hemagglutinin broadly neutralizes highly pathogenic avian influenza H5N1 viruses. J Virol 2012; Jan 11 [Epub ahead of print]

7. WANG Q, ZHANG S, JIANG H, WANG J, WENG L, MAO Y, SEKIGUCHI S, YASUI F, KOHARA M, BUCHY P, DEUBEL V, XU K, SUN B, TOYODA T. PA from an H5N1 highly pathogenic avian influenza virus activates viral transcription and replication and induces apoptosis and interferon expression at an early stage of infection. Virol J. 2012; 9:106.


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8. ZHOU F, WANG G, BUCHY P, CAI Z, CHEN H, CHEN Z, CHENG G, WAN XF, DEUBEL V, ZHOU P. A tri-clade DNA vaccine designed on the basis of a comprehensive serologic study elicits neutralizing antibody responses against all clades and subclades of HPAI H5N1 viruses. J Virol 2012, April 11 [Epub ahead of print]

9. N DEBOOSERE, SV HORM, A DELOBEL, J GACHET, P BUCHY, VIALETTE. Viral Elution and Concentration Method for Detection of Influenza A Viruses in Mud by real-time RT-PCR. J Virol Methods 2012, 179:148-153.

10. GUTIERREZ RA, SORN S, NICHOLLS JM, BUCHY P. Eurasian tree sparrows, risks for H5N1 virus spread

and human contamination through Buddhist ritual: an experimental approach. PLoS ONE 2011; e28609

11. XU C, HU WB, XU K, HE YX, WANG TY, CHEN Z, LI TX, LIU JH, BUCHY P, SUN B. Amino acids 473V and

598P of PB1 from avian origin influenza A virus contribute to polymerase activity especially in mammalian cells. J Gen Virol 2011, Nov 16 [Epub ahead of print]

12. GUTIERREZ RA, VIARI A, BODELLE B, FRUTOS R, BUCHY P. Biased mutational pattern and quasi-species hypothesis in H5N1 virus. Infect Genet Evol, Oct 29 2011 [Epub ahead of print]

13. SV HORM, N DEBOOSERE, RA GUTIERREZ, M VIALETTE, P BUCHY. Direct detection of H5N1 virus from mud specimens. J Virol Methods 2011; 176:69-73.

14. DING H, TSAI C, ZHOU F, BUCHY P, DEUBEL V, ZHOU P. Heterosubtypic antibody response elicited with seasonal influenza vaccine correlates partial protection against Highly Pathogenic H5N1 virus. PLoS One 2011; 6:e17821

15. NAUGHTIN M, DYASON J. C, MARDY S, SORN S, VON ITZSTEIN M, BUCHY P. Neuraminidase inhibitor sensitivity and receptor-binding specificity of Cambodian clade 1 highly pathogenic H5N1 virus. Antimicrobial Agent Chemotherapy 2011; 55:2004-10.

16. N DEBOOSERE, SV HORM, A PINON, J GACHET, C COLDEFY, P BUCHY, M VIALETTE. Development and Validation of a Concentration Method for the Detection of Influenza A Viruses from Large Volumes of Surface Water. Applied Environmental Microbiology 2011; 77:3802-8.

17. DING H, TSAI C, GUTIERREZ RA, ZHOU F, BUCHY P, DEUBEL V, ZHOU P. Superior neutralizing antibody response and protection in mice vaccinated with heterologous DNA prime and virus like particle boost against HAPI H5N1 virus. PLoS ONE 2011;6(1):e16563

Financial support:

DHHS - WHO – European Union – Fab’entech (start-up)

c. Pathogen Discovery

Coordination:


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Buchy P

Partners:

Marc Eloit (IP), Valérie Caro (IP)

Objectives:

To identify unusual or new pathogens in severe acute lower respiratory infections in

Cambodia


Out of the 4000 patients screened for 18 viruses during the SISEA project, we selected

patients who tested negative but presented with severe respiratory symptoms. After nucleic

acid extraction, the samples were screened by high throughput sequence (HTS) using

Illumina technology. On the 1st series of specimens, we identified virus species that has

mutations in the region amplified by the primers and rare respiratory viruses recently

identified.

Prospective:

Patients hospitalized for suspicion of H5N1 infection (severe pneumonia) will be prospectively

included in a new study which aims to identify all the unusual/rare/new pathogens at the origin

of severe/lethal respiratory infections in Cambodia. The samples will be tested on the HTS

Roche 454 platform of the Virology Unit and on a new Ion Torrent HTS platform in France.

Recent publications:

In preparation

Financial support:

Institut Pasteur Paris – Pathoquest (start-up)

4. Arboviral diseases

a. Dengue: role of asymptomatic infections in dengue epidemiology


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Coordination:

Buchy P. (PTR) and Anavaj Sakuntabhai (DENFREE)

Partners:

Tarantola A. and coll (Epi), Anavaj Sakuntabhai, Rick Paul and coll. (IP), members of the

DENFREE consortium (Mahidol U., Imperial College, Institute Pedro Kuri, ...), Marie Flamant

(IP), J-Y Coppée (IP), Ivan Moszer (IP), Marco Vignuzzi (IP),Philippe Dussart (IP Guyane),

etc.

Objectives:

Individuals infected asymptomatically by the dengue virus are believed to represent to

majority of the infections although their role as potential reservoir is not known. In this project,

we aim to compare the immune response (DC, NK, etc.), the viral genetic characteristics (full

genome, quasi-species, virus population dynamics), the human genetic background, the

epidemiological characteristics, the transmissibility by vectors (direct and indirect feedings)

that distinguish the asymptomatic carriers from the patients who develop mild to severe

disease.


DENFRAME European project generated preliminary data regarding the clinical and

virological characteristics of asymptomatic dengue infections. Strains isolated during the last

decade by our National Reference Centre for arboviruses were analyzed to better understand

the virus population dynamics in the country and in the region while our cohort study in

Kampong Cham province provided very useful and novel epidemiological data of dengue

disease in Cambodia.

The PTR that stared in 2010 aimed to study both viral and host factors and their interaction

and its effect on the outcome of infection by studying viral quasi-species and host whole

genome expression in the individuals from the same household who had dengue virus

(DENV) infection with or without clinical symptoms using samples that were collected in 2006-

2007 during DENFRAME project (EU). This project should be finished by June 2013 and

preliminary data are currently being analyzed.

The DENFREE European project started in June 2012. Over 150 patients were included and

sequential samples collected. Over 1500 family members of these patients were followed for


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1 week and sequential samples collected to detect asymptomatic infections. Comparison

between direct and indirect mosquito feeding was conducted in patients and asymptomatic

individuals. Mosquito were collect in the household of the patients and each mosquito tested

individually before nucleic acid extraction for full genome sequencing and quasi-species

studies followed by comparison with DENV isolated from patients by using HTS.

Prospective:

DENFREE is a 4-years project that should lead to number of discoveries in the field of

dengue immunology, genomic evolution (following recent work on DENV population

dynamics), human genetic predisposition, entomology (a new thematic in the Unit that is

being strengthen), diagnostic development, etc. This project is complementary from the PTR

project that should deliver interesting data in 2013. These data will be eventually used to

adjust some of the initial research questions of the DENFREE program.


1. P DUSSART, L BARIL, L PETIT, L BENIGUEL, LC QUANG, S LY, R DO SOCORRO

AZEVEDO, J-B MEYNARD, S VONG , L CHARTIER, A DIOP, O SIVUTH, V DUONG, CM

THANG, M JACOBS, A SAKUNTABHAI, MR TEXEIRA NUNES, VTQ HUONG, P BUCHY,

PFC VASCONCELOS. Clinical and virological study of dengue cases and the members of their

households: the multinational DENFRAME project. PLoS Neglected Tropical Diseases 2012;

e1482

2. VONG, S GOYET, S LY, C NGAN, R HUY, S ONG, O WICHMANN, G.W LETSON, HS.

MARGOLIS, P BUCHY. Under-recognition and reporting of dengue in Cambodia: a capture-

recapture analysis of the National Dengue Surveillance System. Epidemiol Infect 2012;

140:491-9.

3. V DUONG, C SIMMONS, L GAVOTTE, A VIARI, S ONG, N CHANTHA, NJ LENNON, BW

BIRREN, S VONG, JJ FARRAR, MR HENN, V DEUBEL, R FRUTOS, P BUCHY. Genetic

diversity and population dynamic of dengue virus serotype 1 (DENV-1) in Cambodia. Infect

Genet Evol 2011; doi:10-1016/j.meegid.2011.06.019


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4. WICHMANN O, YOON IK, VONG S, LIMKITTIKUL K, GIBBONS R, MAMMEN MP, LY S,

BUCHY P, SIRIVICHAYAKUL C, BUATHONG R, HUY R, LETSON W, SABCHAREON A.

Dengue in Thailand and Cambodia: an assessment of the degree of underrecognized disease

burden based on reported cases. PLoS Negl Trop Dis 2011; 29:e996

5. V DUONG, C SIMMONS, L GAVOTTE, A VIARI, S ONG, N CHANTHA, NJ LENNON, BW

BIRREN, S VONG, JJ FARRAR, MR HENN, V DEUBEL, R FRUTOS, P BUCHY. Genetic

diversity and population dynamic of dengue virus serotype 1 (DENV-1) in Cambodia. Infect

Genet Evol 2011; doi:10-1016/j.meegid.2011.06.019

Financial support: European Union – Institut Pasteur Paris – Paediatric Vaccine Initiative

b. Dengue: markers of disease severity

Coordination:

Anavaj Sakuntabhai (BioMérieux)

Partners:

Anavaj Sakuntabhai and coll. (IP), Marie Flamant (IP), BioMérieux

Objectives:

This project is part a larger collaborative program on dengue that is being established

between the Biomérieux (BM) and the Institut Pasteur (IP). BM is pursuing two major goals:

become involved in the dengue diagnostic field and provide new prognostic markers for

predicting an evolution towards the severe dengue hemorrhagic fever and dengue shock

syndrome.


Using the samples collected during the DENFRAME project, we demonstrated that the level

of NS1, a protein secreted by the virus, as well as several other factors were associated with

disease severity. In a mouse model as well as in vitro, we observed that some strains isolated

for severe human cases were more virulent or were replicating differently.


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Many other factors, including soluble molecules could eventually serve as prognostic

markers. To address this question, over 1000 sequential serum samples obtained from 120

patients hospitalized with mild (DF) to severe (DHF, DSS) dengue infections extensively

documented (standard biology, virology, ultrasound, etc.) were collected in 2011-2012 in

Cambodia. The proteomic study is currently being performed on the plateform of BioMerieux

France.

Prospective:

An additional 100 patients will be recruited in 2013 to obtain a significant sample size of each

group of disease severity. In this project, we expect to identify biomolecules which are

differentially expressed in serum from patients with Dengue Hemorrhagic fever (DHF)/

Dengue Shock syndrome (DSS) compared to patients with Dengue Fever (DF). The

identification of potent markers of disease severity would allow a reliable prognosis within the

first days of hospitalization and significantly improve the clinical management, reduce the

mortality and limit unnecessary hospitalization in resource-limited countries.


1. ANDRIES AC, DUONG V, NGAN C, ONG S, HUY R, SROIN KK, TE V, Y B, LORN TRY P,

BUCHY P. Field evaluation and impact on clinical management of a rapid diagnostic kit that

detects dengue NS1, IgM and IgG. PLoS Neglected Tropical Diseases (in revision)

2. DUONG V, LY S, PATRICH LT, TUISKUNEN A, ONG S, NORITH C, LUNKVIST A, LEPARC-

GOFFART I, DEUBEL V, VONG S, BUCHY P. Clinical and virological factors influencing the

performance of a NS1 Antigen capture assay and potential use as a marker of dengue disease

severity. PLoS Neglected Tropical Diseases 2011; 5(7):e1244

3. TUISKUNEN A, WAHLSTROM M, BERGSTROM J, BUCHY P, LEPARC-GOFFART I,

LUNDKVIST A. Phenotypic characterization of patient dengue virus isolates in BALB/c mice

differentiates dengue fever and dengue hemorrhagic fever from dengue shock syndrome.

Virology J. 2011; 8:398


17 | P a g e

4. TUISKUNEN A, MONTEIL V, PLUMET S, BOUBIS L, WAHLSTROM M, DUONG V, BUCHY

P, LUNDKVIST A, TOLOU H, LEPARC-GOFFART I. Phenotypic and genotypic

characterization of dengue virus isolates differentiates dengue fever and dengue hemorrhagic

fever from dengue shock syndrome. Arch Virol, 2011; 156:2023-2032.

Financial support: BioMerieux

c. Chikungunya

Coordination:

Buchy P

Partners:

Tarantola A. and coll. (Epi), MoH, WHO

Objectives:

Characterize the Chikungunya virus strains detected by our national arbovirus surveillance

and encephalitis project for the first time in 2011


The study documented the re-emergence, after 50 years, of Chikungunya virus (CHIKV)

belonging to the East /Central African genotype which most probably originated from

Thailand.

Prospective:

The vector of the ECSA genotype of the virus has not being yet identified in Cambodia.

Following the detection of cases, entomological investigations are conducted to collect Aedes

mosquitoes and to try identifying the virus in those vectors. This re-emerging disease is

considered as a public health concern by national authorities.



18 | P a g e

1. V. DUONG, A-C ANDRIES, C. NGAN, T. SOK, B. RICHNER, N. ASGARI-JIRHANDEH, S.

BJORGE, R. HUY, S. LY, D. LAURENT, B. HOK, MC ROCES, S. ONG, M C CHAR, V.

DEUBEL, A. TARANTOLA, P BUCHY. Emergence of Central/East African genotype of

Chikungunya virus in Cambodia. Emerg. Infect. Dis., Dec. 2012 (online)

2. Chikungunya outbreak - Cambodia, February-March 2012. MMWR Morb Mortal Wkly Rep. 2012 Sep 21; 61:737-40.

Financial support: WHO – Li Ka Shing Foundation

5. Zoonoses

a. Zoonotic viruses transmitted by bats, rodents and primates/Pathogen discovery

Coordination:

Buchy P

Partners:

Wildlife Conservation Society, University California Davis, CNRS, Ministry of Agriculture Laos,

Objectives:

The objective of this project is to identify, in high-risk areas, the spillover of well-known as well

as novel infectious diseases in high-risk animal species (bats, rodents, primates) which may

constitute a risk to the human population. This project is following a National Agency for

Research project on pathogen/rodent co-evolution in Southeast Asia (CEROPATH).


CEROPATH gave the opportunity to study hantaviruses (with the identification of a new

variant of Seoul virus in Laos), leptospirosis, srub typhus and to discover a new virus that

belongs to a viral family never described in Asia (manuscript in preparation).

Few thousands of bat and rodent samples (swabs, organs) were collected by WCS in Laos

and Cambodia other the last 2 years. These samples were tested in our laboratory by generic


19 | P a g e

PCRs for the detection of the following genus/virus families: flaviviruses, henipaviruses,

coronaviruses, astroviruses, lyssaviruses, filoviruses, paeamyxoviruses, hantaviruses,

arenaviruses, reoviruses. Over 150 viruses were detected and their identification confirmed

by amplicon sequencing. Most of them demonstrated a very significant phylogenetic distance

with known viruses suggesting that most of them may represent new species.

Prospective:

New specific PCR as well as serological tests have been developed for the detection of our

“new” virus in rodent and human samples. Preliminary data suggest that a significant number

of patients could contract the virus infection. This should be further studied on a larger scale.

Primary bat and rodent cell lines are being generated to better amplify these viruses before

full/partial genome sequencing on our Roche 454 HTS platform in order to genetically

characterize these 150 potentially new viruses.


1. K. BLASDELL, H. HENTTONEN, P. BUCHY. Hantavirus genetic diversity. In: New frontiers of

molecular epidemiology of infectious diseases. Springer edition Basel 2012. DOI 10.1007/978-

94-007-2114-2

2. SVILENA I, HERBRETEAU V, BLASDELL K, CHAVAL Y, BUCHY P, GUILLARD B, MORAND S. Leptospira

and rodents in Cambodia. Am J Trop Med Hyg 2012; 86:1032-38

3. BLASDELL K, COSSON JF, CHAVAL Y, HERBRETEAU V, DOUANGBOUPHA B,

JITTAPALONG S, LUNDQVIST A, HUGOT JP, MORAND S, BUCHY P. Rodent-borne

hantaviruses in Cambodia, Lao PDR and Thailand. Ecohealth 2011; 8:432-43.

4. P WONGPROMPITAK, V DUONG, W ANUKOOL, L GAVOTTTE, C MOULIA, K BLASDELL, P

BUCHY, P EKPO, R FRUTOS, Y SUPUTTAMONGKOL. Broad-coverage molecular

epidemiology of Orientia tsutsugamushi in Thailand. Infect Genet Evol 2011; June 25 [Epub

ahead of print]

5. DUONG V, MAI TT, BLASDELL K, LO LV, MORVAN S, LAY S, ANUKOOL W,

WONGPROMPITAK P, SUPUTTAMONGKOL Y, LAURENT D, RICHNER B, RA C, CHIEN


20 | P a g e

BT, FRUTOS R, BUCHY P. Molecular epidemiology of Orientia tsutsugamushi in Cambodia

and Central Vietnam reveals a broad region-wide diversity. Infect Genet Evol 2011 Jan 15

[Epub ahead of print]

Financial support: ANR (National Research Agency) – USAID - Institut Pasteur

6. Neurotropic infections

a. Etiologies of meningo-encephalitis in Cambodia and Southeast Asia

Coordination:

Buchy P

Partners:

Kantha Bopha Hospitals, Oxford University Clinical Research Unit Vietnam (J. Farrar), Epi.

Unit (A. Tarantola), AVIESAN, Institut Pasteur (M. Lecuit, O. Lortholary, M. Eloit), NIHE

Hanoi, IRD (X. de Lamballerie), Oxford University Clinical Research Unit Laos (P. Newton).

Objectives:

To describe the etiologies of meningo-encephalitis in Cambodia (project that started in 2010)

and to extend the study to Laos, Vietnam, and potentially Thailand, Indonesia, etc.


Although Cambodia is suspected to be highly endemic for Japanese encephalitis virus (JEV),

there were no nationally representative data on JEV transmission. Most of the existing data

on human disease came from few sentinel hospitals, and there have been no previous

studies or surveillance for JEV transmission among pigs - the amplifying hosts in the natural

cycle of JEV transmission. Analysis of JEV transmission among pigs provided additional data

on geographical scope and intensity of JEV transmission in Cambodia.


21 | P a g e

This has been confirmed by our study on meningo-encephalitis in children. During a 2 year-

period, over 450 patients were recruited, clinically characterized and tested for the most

common known etiologies of meningo-encephalitis (viruses and bacteria). JEV and DENV are

the most common etiologies of encephalitis in Cambodia but we also detected a significant

number of treatable infections like scrub typhus, Streptococcus suis, etc. This study also

participated to the detection of the re-ermergence of Chikungunya in Cambodia.

Prospective:

The results of the 1st study are currently being analyzed while the regional study is being

developed. The initial step consists in developing and standardizing PCR methods for the

detection of approximately 50 viruses, virus families, bacteria and parasites. The standardized

methods will be then transferred to the study sites. A pathogen discovery work-package

(similar to the one described below) will be added in 2013-2014.


1. V DUONG, S SAN, D HOLL, M RANI, V DEUBEL, P BUCHY. Evidence of Japanese

encephalitis virus infections in swine populations in 8 provinces of Cambodia: implications for

national Japanese encephalitis vaccination policy. Acta Tropica 2011; 120:146-150.

2. YZ LI, D COUNOR, P LU, GD LIANG, VT HUONG, PT NGA, HT LOAN, G SUN, M

GRANDADAM, S BUTRAPET, JP LAVERGNE, M FLAMAND, YX YU, T SOLOMON, P

BUCHY, V DEUBEL. A specific and sensitive antigen capture assay for NS1 protein

quantitation in Japanese encephalitis virus infection. J Virol Methods 2012; 179: 8-16.

Financial support: Li Ka Shing Foundation – AVIESAN

b. Enteroviruses

Coordination:

Buchy P


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Partners:

IP Shanghai (Ralf Altmeyer and coll.) – IP Paris (JC Manuguerra, Francis Delpeyroux, Marc

Lecuit, Thérèse Couderc, Marc Eloit, etc.) – Key State Laboratory Hong Kong University

Objectives:

To characterize the epidemic of EV71 and other enteroviruses that started in April 2012 and

was responsible for the death of over 150 children in 5 months.


Following the detection in our lab of EV71 as the main etiologic agent of the epidemic of

encephalitis associated with severe pulmonary oedema that recently started in Cambodia, we

started several studies aiming to describe the circulating genotype (currently C4), to generate

full genome sequences from mild to deadly form of the disease in order to identify eventually

some molecular markers of virulence. Our partners in IP started to study the pathogenicity in

mice, to develop new serological tools (Luminex).

Prospective:

Viral and host factors responsible for severe EV71 infections in children should be explored:

innate, T cell, and B cell immune response in severe disease, identification of viral and host

biomarkers, case fatality rate (seroprevalence study in 1000 children), pathogenicity in other

animal models.


None

Financial support: Institut Pasteur – Asian Development Bank – WHO – Other

c. Pathogen discovery

Coordination:

Buchy P

Partners:


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Marc Eloit (IP/Pathoquest), Valérie Caro (IP)

Objectives:

Detect unusual or new pathogens associated with meningo-encephalitis in Cambodia


On a limited number of human specimens (CSF) selected, we detected in some cases some

viruses rarely found in encephalitis. A larger number of samples are currently being

processed in a HTS facilities in France.

Prospective:

Detection of new pathogens, development of new tests, of new treatments, etc.


None

Financial support: Institut Pasteur – Pathoquest (Start-up)

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