Conceived and designed the experiments: PB. Performed the experiments: VH RG JN. Analyzed the data: VH RG JN PB. Contributed reagents/materials/analysis tools: VH RG JN PB. Wrote the paper: VH RG JN PB.
The authors have declared that no competing interests exist.
Very little is known regarding the persistence of Highly Pathogenic Avian Influenza (HPAI) H5N1 viruses in aquatic environments in tropical countries, although environmental materials have been suggested to play a role as reservoirs and sources of transmission for H5N1 viruses.
The survival of HPAI H5N1 viruses in experimental aquatic biotopes (water, mud, aquatic flora and fauna) relevant to field conditions in Cambodia was 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. They were experimentally contaminated with H5N1 virus. The persistence of HPAI H5N1 virus (local avian and human isolates) was determined by virus isolation in embryonated chicken eggs and by real-time reverse-polymerase chain reaction. Persistence of infectious virus did not exceed 4 days, and was only identified in rain water. No infectious virus particles were detected in pond and lake water or mud even when high inoculum doses were used. However, 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 organisms seemed to be mostly passive carriers of the virus rather than host allowing virus replication.
Although several factors for the survival and persistence of HPAI viruses in the environment are still to be elucidated, and are particularly hard to control in laboratory conditions, our results, along with previous data, support the idea that environmental surveillance is of major relevance for avian influenza control programs.
The highly pathogenic avian influenza (HPAI) H5N1 virus is a major public health concern in Southeast Asia, where it has widely spread since its first detection in 1997
Our objectives in this study were: (1) to describe the survival of H5N1 virus in water and mud in experimental setting reproducing as faithfully as possible natural conditions observed in tropical countries; (2) to determine whether aquatic animals such as fish, tadpoles, clams, snails, mussels and aquatic flora may be contaminated and play a role in the persistence of H5N1 virus in water; (3) to determine whether autochthonous aquatic organisms such as bivalves (fresh water mussels) and labyrinth fish (fighting fish) could transmit the virus to each other.
All experiments using HPAI H5N1 virus and all animal experiments were performed within the Biosafety Level 3 (BSL-3) laboratory of the Institut Pasteur in Cambodia (IPC), complying with the Animal Committee regulations of Institut Pasteur in Paris, France, in accordance with the EC 86/609/CEE directive, and approved by the Animal Ethics Committee of Institut Pasteur in Cambodia (permit number: AEC/IPC/002/2008).
The clade 1, genotype Z, HPAI H5N1 viruses A/Cambodia/408008/2005 (GenBank accession numbers: HQ664938 to HQ664945) and A/chicken/Cambodia/LC1AL/2007 (GenBank accession numbers: HQ200574 to HQ200581) were used to conduct these experiments. The virus stock was obtained after propagation in Specific Pathogen Free (SPF) 9-to-11-day-old embryonated hen eggs, kindly provided by the National Veterinary Research Institute of Cambodia (NaVRI), Ministry of Agriculture, Forestries and Fisheries (MAFF). The amnio-allantoic fluid (AAF) from the second passage on SPF eggs was harvested 48 hours after inoculation and stored at −80°C until further use. Virus titre was determined by calculating the 50% egg infectious dose (EID50) per mL of virus stock. Titration endpoints were calculated using the method of Reed and Muench
Mud and water used in the artificial aquatic settings described below were collected from 2 different ponds (with the landlord's official authorization) and from a lake in areas of Kampong Cham province where an H5N1 virus outbreak had previously occurred. Some of the experiments also involved the use of rain water which was collected and stored in big jars within the IPC external facilities until use. Temperature, pH and conductivity results of all water samples were recorded on site at the time of collection. Additional physico-chemical analyses and all microbiological tests were carried out upon arrival at the laboratory (
Freshwater flora and fauna used for the artificial aquatic settings included guppies (
A total of 4 different series of experiments were carried out. Two series were conducted to investigate the survival and persistence of H5N1 virus in simple biotopes containing only water and mud (A), and in complex biotopes that included aquatic flora and fauna (B) (See
The 3 horizontal rectangles represent different types of water in which mussels were immersed: contaminated vs non-contaminated. Black-filled lines represent mussels immersed in contaminated water from day 0 (D0). White-filled lines represent naïve mussels immersed in non-contaminated water from day 0. The two experiments are discernable and identified as C.1 and C.2 (a, b and c). Water samples were collected daily from day 0 (D0) to day 9 (D9). Numbers not included in boxes correspond to viral loads measured in the water samples. When these numbers are displayed in white color, this indicates that infectious virus was also detected. When the numbers are written in black color, this means that the virus was not recovered after inoculation into eggs. Stars indicate collection of mussels' organs for testing. White stars correspond to detection of infectious virus. Black stars indicate that H5N1 virus was not recovered after inoculation into embryonated eggs. Numbers included in black boxes correspond to average viral loads measured in mussels that were immersed in contaminated water. Numbers in white boxes correspond to average viral loads measured in mussels that were immersed in clean water.
The 3 horizontal rectangles represent different types of water in which animals were immersed: contaminated vs non-contaminated. Tadpoles (T) were immersed along with the male fighting fish (F) in contaminated water. Black-filled lines represent the male fighting fish and the tadpoles immersed in contaminated water from day 0 (D0). At day 5, male contaminated fish were placed over-night in clean water before being exposed to non-contaminated females in clean water. White-filled lines represent naïve female fighting fish immersed in non-contaminated water from day 0. Stars indicate collection of fish and tadpoles (when present) for testing. The two experiments are discernable and identified as D.1 and D.2. White stars correspond to detection of infectious virus in fish. Black stars indicate that H5N1 virus was not recovered from fish's organs after inoculation into embryonated eggs. Numbers included in black boxes correspond to average viral loads measured in fish (F) and tadpoles (T) that were immersed in contaminated water. Numbers in white boxes correspond to average viral loads measured in female fighting fish that were immersed in clean water and exposed to contaminated male fish. Water samples were collected from contaminated water (experiment D.1) from day 0 (D0) up to day 20 (D20). Water samples were collected from non-contaminated water (experiment D.2) from day 0 (D0) to day 12 (D12). The values presented in italic correspond to the viral load measured in water samples. When the numbers in italic are displayed in white color, this indicates that infectious virus was also detected. When the numbers in italic are written in black color, this means that the virus was not recovered after inoculation into eggs.
Series | # | Virus origin |
Virus concentration (EID50/mL water) | T° |
Water origin | Mud origin | Flora/fauna | |
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Avian | 5×104 | 25 | Pond 1 | NA | No | |
Pond 2 | ||||||||
Rain | ||||||||
Human | 5×104 | 25 | Pond 1 | NA | No | |||
Pond 2 | ||||||||
Lake | ||||||||
Rain | ||||||||
|
|
Avian | 5×104 | 25 | Pond 1 | Pond 1 | No | |
Pond 2 | Pond 2 | |||||||
Lake | Lake | |||||||
|
Avian | 5×102 |
|
Pond 2 | Pond 2 | No | ||
25 | Pond 1 | Pond 1 | ||||||
|
Pond 2 | Pond 2 | ||||||
|
Pond 1 | Pond 1 | ||||||
Human | 5×103 | 25 | Lake | Lake | No | |||
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|
Avian | 5×102 | 25 | Pond 1 | Pond 1 | Guppies | |
Snails | ||||||||
Clams | ||||||||
Plants | ||||||||
5×104 | Lake | Lake | Guppies | |||||
Snails | ||||||||
Clams | ||||||||
Mussels | ||||||||
Plants | ||||||||
Human | 5×103 | 25 | Lake | Lake | Guppies | |||
Tadpoles | ||||||||
Plants | ||||||||
5×104 | Guppies | |||||||
Clams | ||||||||
Plants | ||||||||
|
Avian | 5×102 |
|
Pond 2 | Pond 2 | Guppies | ||
Snails | ||||||||
|
Clams | |||||||
Plants | ||||||||
|
Pond 1 | Pond 1 | Guppies | |||||
Snails | ||||||||
Clams | ||||||||
Plants | ||||||||
Human | 5×103 |
|
Lake | Lake | Guppies | |||
Tadpoles | ||||||||
Plants |
Avian origin strain stands for the A/Chicken/Cambodia/LC1AL/2007 strain. Human origin strain stands for the A/Cambodia/408008/2005 strain.
T° = Temperature (°C).
Different virus concentrations were tested in this study. Concentrations of 2×102 and 5×102 EID50/mL of water were chosen based on the quantity of virus found in the natural environment in Cambodia during previous field studies
Experiments of series A and B lasted 14 days each, and were conducted using different H5N1 strains (A/Chicken/Cambodia/LC1AL/2007 and A/Cambodia/408008/2005), different inoculum doses (yielding final estimated concentrations of 5×102, 5×103 or 5×104 EID50/mL water), and different temperatures reflecting the parameters measured in the field during the transmission season in Cambodia (22, 25, 32 or 34°C). Aquariums with a total capacity of 28 litres (38×38×20 cm) were filled with 20 litres water and 5 kilograms mud each, and then were allowed to settle for 24 hours prior to virus inoculation. Water and mud from various origins were also tested: rain, lake, pond 1, pond 2 (as defined above). When flora (about 100 g) and fauna (30 animals of each species) were included in the experiments (B), collection of samples from each of the species used (2 g of plant, 2 animals of each species) was carried out during the first 3 days, and every 3 days from then on. Fifty millilitres of water and 5 grams of mud samples were collected on a daily basis (
Experiments in series C and D lasted from 8 to 20 days depending on the setting chosen. The A/Cambodia/408008/2005 strain was used for experiments C, while experiments D involved the use of the virus A/Chicken/Cambodia/LC1AL/2007. All biotopes created for experiments C and D used rain water only. Aquariums filled with 10 litres of water and 40 mussels each were used for experiments C. In experiments D, series of small aquariums containing 500 mL of water and one male fighting fish in addition to one tadpole were used for experiment D1, while only one fighting fish was included in experiment D2 (
For all aquariums, inoculation was carried out on day 0 (D0). After collection, water and mud samples were stored in sterile tubes at −80°C until testing. All aquatic animals were humanely sacrificed, following the Animal Use Protocol defined by the Animal Ethics Committee of Institut Pasteur in Cambodia (permit number: AEC/IPC/004/2008), and subsequently dissected in order to collect the main organs of interest: gills, intestines, fins, scales, brain, and remaining carcass in fish; gills, digestive gland, intestines, and remaining carcass in clams and mussels; gills, intestines, and remaining carcass in tadpoles; all organs in snails. Before dissection, the animals were washed 2 times with sterile distilled water in order to avoid contamination of the organs by the water contained in the aquarium.
Prior to testing, organs were weighed and placed into vials containing 1 mL of viral transport medium (VTM) (sterile solution at pH 7.2–7.4 containing 26.5 g/l of tryptose phosphate broth, 5 g/l of gelatine, 50 mg/l of fungizon, 1 million units/l of penicillin, 1 g/l of streptomycin and 80 mg/l of gentamycin) and stored at −80°C and in 10% formalin solution (prepared from formaldehyde 37% commercial solution diluted in water) stored at room temperature. Plant samples were also weighed and stored in VTM at −80°C until use.
Each experiment conducted was coupled with a control experiment, using the exactly same conditions, but without virus.
All water samples were concentrated using the method described by Khalenkov et al.
All mud specimens collected went through an elution step with a 10% beef extract solution at pH 7 followed by a polyethylene glycol-precipitation (PEG) step for virus and RNA concentration, as described previously
Precisely, for each mud sample, 5 grams were eluted in 25 mL of elution buffer (10% beef extract solution). One millilitre of the eluted sample was then kept for a first virus detection, while the remaining volume went through the additional concentration step with PEG so as to obtain a final volume of concentrated sample of 1 mL. Mud eluates and concentrates were then used for RNA extraction and qRT-PCR or virus isolation.
All solid samples (animals' organs, plants) were weighed and kept in 1 mL VTM before undergoing a homogenization step using the MagNa Lyser Instrument (ROCHE, Mannheim, Germany) for 3 runs of 50 seconds at 5000× g. The homogenized samples were then used for further RNA extraction and eventually virus isolation.
All samples processed as described above were mixed with a solution containing a mixture of antibiotics and antifungal drugs prior to RNA extraction or virus isolation, in order to reduce the number of contaminating microorganisms in the samples
All samples that tested positive by qRT-PCR were inoculated into 9-to-11-days old SPF embryonated hen eggs. Each specimen was inoculated into 3 eggs. One hundred microlitres were injected into the amniotic cavity and 100 µL into the allantoic cavity. The eggs were then incubated for 48 hours at 37°C and chilled overnight at 4°C. The AAF was then harvested and standard hemagglutination (HA) tests were performed to detect the presence of virus before confirmation by qRT-PCR. HA tests were performed in 96-wells microtiter plates with 0.75% guinea pig red blood cells and serial 2-fold dilutions of AAF. When the HA test was negative, the AAF from each of the three eggs was pooled and inoculated into a second and then a third series of 3 eggs. A maximum of three passages were performed for each sample.
Immunohistochemical staining of the tissues obtained from the mussels and fish of experiment C and D was carried out for the influenza nucleoprotein using HB65 (European Veterinary Laboratories, Netherlands) as described in previously published reports
Contaminated rain water was the only type of water from which infectious particles could be recovered (
A.1: only water of various origins maintained at 25°C and inoculated to a final concentration of 5×104 EID50/mL with H5N1 virus of animal or avian origin. A.2:water and mud containing an estimated final concentration of virus of avian origin of 5×104 EID50/mL water maintained at 25°C (A.2.1) or various concentrations of viruses of avian and human origins and maintained at various temperatures (A.2.2). A: Avian origin strain stands for the A/Chicken/Cambodia/LC1AL/2007 strain. H: Human origin strain stands for the A/Cambodia/408008/2005 strain.*last day of the corresponding experiment at which samples could be collected and tested.
No infectious particle could be isolated from contaminated lake water, although viral RNA was detectable between 1 to 11 days post-inoculation depending on the conditions tested (
No infectious particles could be recovered from pond water. In one instance, viral RNA persisted in water for as long as 14 days (end of the experiment) (experiment A.2.2, Pond 1,
Infectious particles could not be recovered from any mud samples in any of the experiments conducted in this study (
Infectious particles were only isolated from animal organs in experiments C and D, in which mussels, tadpoles and fighting fish were immersed in contaminated rain water in the absence of mud (
C: water inoculated with the virus of human origin, at a final estimated concentration of 5×104 EID50/mL, maintained at 25°C and containing mussels. D: water inoculated with the virus of avian origin, at a final estimated concentration of 2×102 EID50/mL, maintained at 17°C and containing fighting fish and tadpoles.*last day of the corresponding experiment at which samples could be collected and tested.
The clams immersed in contaminated water died very quickly, in contrast with those immersed in non-infected water. The presence of mud in lake or pond water (experiments B.1 and B.2) was associated with the absence of infectious particle isolation in tadpoles and with a shorter persistence of virus RNA at a lower viral load (
B.1: complex biotopes inoculated with virus of avian or human origins at various final concentrations and maintained at 25°C. B.2: complex biotopes inoculated with virus of avian or human origins at various final concentrations and maintained at various temperatures. A: Avian origin strain stands for the A/Chicken/Cambodia/LC1AL/2007 strain. H: Human origin strain stands for the A/Cambodia/408008/2005 strain.*last day of the corresponding experiment at which samples could be collected and tested.
After immersion of mussels in rain water maintained at 25°C and contaminated with the virus A/Cambodia/408008/2005, infectious particles were isolated from water until day 4, from mussels until day 6 and the virus RNA was still detectable in molluscs until the end of the experiment at day 8. Viral loads in mussels varied between 2.40×105 copies on day 1 and 2.30×104 copies per gram of organ on day 8 (experiment C.2,
When transferred into non-contaminated water on day 3 (experiment C.1), RNA was detected in water until day 4 but infectious particles were not found. Infectious virus was not isolated in the infected mussels transferred into clean water on day 6 but the qRT-PCR tested positive until the end of the experiment (experiment C.1.,
When infected mussels were introduced into clean water on day 4 (experiment C.2), no virus could be isolated from the molluscs on day 5, and the water was not contaminated by infectious particles nor by RNA. However, RNA was still detected in infected mussels until day 8 (end of the experiments) and until day 7 in mussels exposed to infected ones (experiment C.2,
During the experiment D, fighting fish and tadpoles were immersed in contaminated rain water. The virus was isolated from animal organs until day 1 in both species and in water until day 2. RNA was detected in tadpoles until day 14 (last tadpole tested) and until day 20 (end of the experiment) in both water and fish. Viral loads varied from 2.8×107 copies on day 1 to 1.55×105 copies per gram of organ on day 14 for tadpoles, and from 4.73×106 copies to 1.48×104 copies per gram of organ on day 20 for fighting fish (experiment D.1,
The viral loads presented here referred to the highest individual values found when analyzing the different organs in each animal group (
Immunohistochemical staining of the tissues did not confirm the presence of the H5N1 virus antigen in any organ of the mussels and fish tested following experiments C and D.
This study aimed to recreate simple as well as complex aquatic environments with parameters (pH, temperature, salinity, microorganisms, flora, fauna, etc.) as close as possible to those observed in Cambodia, where H5N1 outbreaks are regularly reported, and to observe the survival of the HPAI H5N1 virus in all the different compartments of these artificial aquatic biotopes which have been suggested to be at the origin of asymptomatic or sub-clinical human infections
In our experiments, infectious HPAI H5N1 virus could be recovered from water during a maximum of 4 days post-contamination at 25°C but only in rain water. This temperature is commonly observed all year long in Cambodian ponds and lakes, around 20−40 cm beneath the surface, as opposed to the surface where the temperature can easily exceed 30°C. The survival of AIVs in water is known to be shorter when temperature increases
Interestingly, although experiments were conducted under identical conditions, the H5N1 virus obtained from a human case did not survive in rain water in the absence of fauna. Other authors reported that in experiments where only pH, salinity and temperatures varied, H5N1 viruses appeared to persist for shorter periods than other avian influenza viruses tested
The detection of virus RNA by qRT-PCR did not correlate with the recovery of infectious particles. Indeed, in some experiments, infectious virus could not be isolated while RNA could be detected for several days. In the absence of mud, plants or animals, RNA was detected for periods as long as 11 days at 25°C. In complex biotopes, an increase of the temperature from 25°C to 32°C or 34°C reduced the persistence of the RNA (experiments A2.2,
Infectious particles were never isolated in mud specimens although the method used was proven to be efficient
The detection thresholds of the assays could also be questioned but the quantity of virus inoculated at the beginning of the experiments should have ended in theoretical concentrations in water and mud above the limit of detection of these methods. Nevertheless, as we did not perform back-titrations immediately after virus inoculation in water, we cannot dismiss the possibility that the starting concentrations were lower than those calculated by simply applying a dilution factor. The initial virus titers used may appear low but they were comparable to those observed in the field during environmental investigation following outbreak in poultry in Cambodian farms
To our knowledge, data related to the infection of aquatic animals by AIVs in general is very rare and we did not find any study evaluating interactions between H5N1 virus and aquatic plants either.
The plants maintained in H5N1- infected water in conditions meant to simulate natural ones in Cambodia did not show any contamination by the virus regardless of the virus type and of the virus concentration except for one plant specimen in which viral RNA was detected during the first day of the experiment. This finding is in agreement with the investigation conducted on natural environmental samples collected after an H5N1 outbreak in Cambodia in 2006, which assessed the presence of viral RNA in plants from which no live virus particles were recovered
When molluscs, fish or tadpoles were introduced in aquariums containing rain water, infectious particles of the strain of human origin could subsequently be recovered and the RNA persistence of both human and avian strains increased significantly (up to 20 days in one experiment). This suggests a probable impact of these aquatic animals on the biological cycle of H5N1 virus. Faust et al. highlighted in 2009 the role of clams (
Histopathology suggested that the virus was not replicating in mussels and thus that the detection of infectious particles in these molluscs was probably only the result of their natural capacity to filter water. However, this observation may also only be the result of the lower sensitivity of the immunohistochemical method compared to qRT-PCR. Fish (
As often demonstrated through the years, aquatic waterfowl such as ducks, when infected, can shed large amount of virus in their feces, saliva and nasal discharge, all of these potentially resulting in environmental contamination
It should be noted that even though this study was meant to reproduce as faithfully as possible the real field conditions, our experiments differed from those by many elements, including the nature of the inoculum. While most environmental materials are contaminated by faeces, saliva, or other organic secretions, our inoculum was amnio-allantoic fluid. In the field, however, it is noteworthy that influenza viruses are protected by organic materials such as nasal secretions or faeces, which may increase their resistance to physical and chemical inactivation
Although in our experimental study HPAI H5N1 infectious virus could not be detected in environmental water and mud from pond and lake origins, we cannot exclude the possibility that the virus could survive in different areas where physico-chemical and microbiological parameters could differ. Indeed, previous studies suggested that even minor fluctuations in temperature, pH and salinity at levels normally encountered in natural aquatic habitats may enhance or diminish environmental persistence
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