This WPG deals with the survival of avian influenza viruses in all kinds of waters as well as in aquatic biological systems such as fresh water aquatic snail and mollusc bivalves.

Workpackage 0: Molecular basis of virus survival and virus strain ‘bio-equivalence’

(WP leader: India LECLERCQ, Université Denis Diderot/ Institut Pasteur de Paris)
Participants : IPP
This WP is uptsream all the others in the RIVERS project. It is included in WPG I because this is probably the WPG that will start first and very quickly. The data produced in WP0 are also relevant to WPG II.
Apart from the presence or absence of an envelope, little is known about viral features involved in AIV survival in the environment. As the envelope is a critical feature, we propose to study the influence of the origin of the virus lipid bilayer on virus stability. Indeed, most data about virus survival in waters were obtained with AIVs whereas the rare publication of virus stability on surfaces dealt with human strains. Using parallel avian (chicken fibroblasts) and mammalian (SK93/2) cell culture systems, we will compare viruses with the same genome but differing in the origin of their lipid bilayer and glycosylation: avian-like or mammalian-like. To confirm the impact of the nature of lipids, we will load each cell system with various glycolipids. To evaluate the involvement of carbohydrate moieties on virus stability, we will digest virus HA and NA in viral preparation with a number of glycosidases.
The comparison on virus survival between avian cell derived viruses and mammalian cell derived viruses will be made after two simple assays are set up: one with virus in suspension submitted to 2 or 3 different temperatures and one with virus suspensions spotted on a glass slide and dried at room temperature.
Avian and mammalian grown viruses will be prepared and compared in relation to survival in the above mentioned assays at least for the following strains: a HPAIV H5N1 genotype Z strain, A/Scotland/59(H5N1), a LPAIV strain and a human strain such as A/Sydney/5/97(H3N2). The idea would be to select a non HPAI strain, possibly not H5 with the same survival profile as the tested HPAIV H5N1 genotype Z strain as a model virus. This would allow not working in BSL3 conditions and would make the assay portable outside the EU if needed (H5 IVs being listed as double usage goods, they are quasi-impossible to export outside the EU).

Workpackage 1: Concentration, identification and quantification of avian influenza viruses from waters, muds and aquatic animals

WP leader: Jean-Marie DELATTRE, Institut Pasteur de Lille, France
Participants: IPL, CIRAD, IPC, IPP
Given the needs :
- to understand the perpetuation of the virus in nature, and hence its survival in lakes (water, sediment, biota) and maybe in wastewaters (in case faecal excretion would occur);
- to evaluate the risks for poultry from contact with surface waters, or with contaminated drinking water inside premises;
- and the risks for humans from use of surface water in case of disinfection failure, or in absence of treatment (open shallow wells, roof-collected water,…), or from  recreational waters (bathing, …),
there is clearly a need for quantification techniques, adapted to various matrices (clean and polluted waters, mud, biota, food). Detection techniques may be modified to become quantitative with sufficient sensitivity for the analysis of heavily contaminated samples (such as duck ponds,…) as did  Halvorson et al .(1983). But for most lakes and especially for drinking water, a concentration step will be necessary. Principles used for enteroviruses (adsorption/elution) can be kept but filters will have to be adapted, for the electric properties of the viruses are not the same. This has been attempted by several authors, but only one has measured and reported the yield (Roepke et al., 1989) after spiking tap water. A different yield may be obtained with naturally contaminated surface water, so other trials will be necessary. Alternatively, concentration with chicken erythrocytes, or with poly-ethylene glycol, have been used, sometimes in combination with  filter adsorption (Ito et al., 1995, Sinavandan et al., 1991).
Another question is the analysis of solid matrices (sediments, biota, food) : there the concentration is not necessarily the limiting factor, but strong matrix effects occur. For example, molluscs concentrate a lot of pollutants, some of which act as polymerase inhibitors, lowering PCR performances. Such effects must be studied and overcome.
The development of quantitative methods for H5N1 virus is a prerequisite for workpackages 2 (environmental contamination in tropical areas and eastern Europe) and workpackages 3 & 4 (experimental) . A minimum of quality control will also be necessary : characterisation of the detection and quantification thresholds, repeatability/reproducibility. Thus interlaboratory trials will have to be organised with other participants, with pertinent statistical treatment .

Workpackage 2: Observation of influenza viruses in natural environments

WP leader: Ze CHEN, Wuhan Institute of Virology, China
Participants: WIV, IPS, IPC, CIRAD, MICB, IC, IPP
Lakes most probably play a central role in virus transmission between birds and possibly constitute efficient relays for virus transmission from one year to the other one. If this role of virus conservatory is plausible in the North according to some data, no data are available in the south such as Africa. Moreover, available data deal with the contamination of lakes by AIVs but no data are specific of rivers and of the effect of water flow on virus dilution/transport. The role of biotic components of fresh water systems (ponds, lakes, rivers) will also be investigated in this WP. Traditional and molecular techniques will be ‘imported’ from WP0 and WP1 in order to evaluate the viability of detected viruses in theses circumstances.
The following settings will be investigated:
1/ Natural waters: rivers, lakes and ponds
2/ Running waters, wells and drinking water
3/ Biological systems: in particular molluscs (gastropods and bivalves)
Samples of water are to be collected from water collections where migratory birds are known to concentrate if possible throughout the year but at least before migration. Sampling on wild birds in Asia and in Africa has already started in humid zones and will be continued within the frame of other projects to study the reservoir and disseminating role of AIVs by avifauna. It is proposed in the present project to take advantage of bird sampling to investigate the role of waters in AIV perpetuation in Nature and in particular to study whether fresh water molluscs are involved in this perpetuation. Basically, sampling will be focused on zones with high bird concentrations and water analysis for the presence of virus will thus be done without concentration to start with. If relevant, water concentration through protocols developed in WP1 will be implemented. If relevant and possible, water sampling will be envisaged in additional areas of bird concentration that will be followed in bird ecology studies in liaison with other tasks of this call (tasks 3 and 4 mainly).
The build up of the collaboration took into account the geographic complementarity of the participants to this WP. Three regions of the world will be concerned by this WP: Africa, where CIRAD has been operating; Asia in Cambodia in particular where IPC is already involved in such issues, in China as well as in Europe (in Bulgaria and Romania). The priority areas for sampling will be determined for Asia, Africa and Europe. In Africa, these areas are the delta of the Senegal river (Senegal and/or Mauritania side), the central delta of Niger river in Mali, the lake Chad region. In Asia: areas are to be chosen in Cambodia and China. In Cambodia: the Mekong river in its Cambodian part, in China: On the Yellow River (Huang He): Qinghai Lake, On the Yangtze (Chang Jiang) River upstream of Wuhan (Hubei Province) Lake Dongting, downstream of Wuhan Lake Poyang and downstream in the delta of the river in the Shanghai Province. In Europe, at least two countries are concerned, Romania with the delta of the Danube and France with the Bay of the Somme

Workpackage 3: Observation of influenza virus survival and concentration in experimental settings

WP leader: Philippe BUCHY, Institut Pasteur du Cambodge, Phnom Penh, Cambodia)
Participants: IPC
Basically, WP3 is the experimental counterpart of WP2. Data will be generated by the observation of strategic water collections details in WP2 and will probably lead to hypothesis about the survival of AIVs in waters concerning the effect of protein content, temperature, sunshine. These effects will be simulated in water tanks such as aquarium and jars. The progressive addition of biological systems will mimic natural environments. Water plants will be added into the system as a first step. For only one parameter can be changed at a time, the interaction between virus survival and water features can be investigated and possibly attributed. The addition of beings belonging to the animal kingdom such as aquatic molluscs gastropod (breathing and not breathing water) or bivalves will help to determine whether they can be virus concentrators and conservatories and this at various temperatures. It is also envisaged that fish will be added to the tank with contaminated water to assess their capacity to store AIVs in their gills or to rule out their ability to be infected by AIVs, and this at tropical temperatures.

Workpackage 4: Impact of water treatments on virus survival

WP leader: Emilia LUPULESCU, Institutul Cantacizino, Romania)
Participants: IC, IPC, IPP
In birds, especially in Anatidae species, AIVs replicate preferentially in the cells lining the intestinal tract of aquatic birds and can be excreted in high concentrations in the faeces. Indeed, a Muscovy Duck (Cairina moschata) can produce 6.4G of faeces per hour with a virus titre of 107.8  EID50, which means that a single of these infected ducks can shed 1010 EID50 par 24 hours! (Stallknecht et al., 1990b). Waters can be heavily contaminated and although, viruses can be degraded and become non infectious, the titre might decrease dramatically without becoming null. IV infectivity and resistance in water is dependent on the salinity, pH and temperature (Stallknecht et al., 1990a).
Chemical conditions in various combinations will be applied to various concentration of virus in water: pH, salinity, heavy metals etc ….. Physical treatments in combination between them will also be experimented to evaluate their impact on virus viability and concentration: temperature, ultra-violet light at various depths, water filtration systems etc ….
As part of control efforts, water treatment including the use of disinfectants will probably be needed at some stage. Regarding the disinfectants, every compound has its limitations, which are often linked to the content of the waters: amount of proteins, pH etc…. A multichemical approach will be considered and compared with single chemical strategy. A number of disinfectants will first be selected and include environment friendly compounds. Virus survival will be assessed using the outcome from WP0 and WP1 and experiments will be designed according to the existing National, European ou International standards.
This WP will primarily be done at IC, which has the leadership. To guaranty coherence throughout the project concerning standardized assays and compliance with existing standards, IPP (Ana Maria Burguière) will be involved in study design with the leader team.