Received: 06 October 2017

DOI: 10.1111/zph.12420

ORIGINAL ARTICLE

Survivability of low pathogenic (H9N2) avian influenza virus in

water in the presence of Atyopsis moluccensis (Bamboo shrimp)

A. P. Pathak1  | H. V. Murugkar2 | S. Nagarajan2 | R. Sood2 | C. Tosh2 | 

M. Kumar2 | C. K. Athira3  | A. Praveen4

1
Department of Veterinary Public Health
and Epidemiology, G. B. Pant University Summary
of Agriculture and Technology, Pantnagar, Low pathogenic avian influenza virus (LPAIV) exhibits an ecological climax with the
Uttarakhand, India
2
aquatic ecosystem. The most widely prevalent subtype of LPAIV is H9N2. Wild aquatic
Indian Council of Agricultural Research-
National Institute of High Security Animal birds being the natural reservoirs and ducks, the “Trojan horses” for Avian Influenza
Diseases (ICAR-NIHSAD), Bhopal, Madhya
Virus (AIV), can contaminate the natural water bodies inhabited by them. The virus can
Pradesh, India
3 persist in the contaminated water from days to years depending upon the environmen-
Division of Veterinary Public Health, Indian
Veterinary Research Institute, Bareilly, Uttar tal conditions. Various aquatic species other than ducks can promote the persistence
Pradesh, India
and transmission of AIV; however, studies on the role of aquatic fauna in persistence
4
Veterinary Dispensary, Korukollu, Krishna
District, Andhra Pradesh, India and transmission of avian influenza virus are scarce. This experiment was designed to
evaluate the survivability of H9N2 LPAIV in water with and without Atyopsis moluc-
Correspondence
Anubha P. Pathak, Department of Veterinary censis (bamboo shrimp) for a period of 12 days. The infectivity and amount of virus in
Public Health and Epidemiology, G. B. Pant water were calculated and were found to be significantly higher in water with A. moluc-
University of Agriculture and Technology,
Pantnagar, Uttarakhand, India. censis than in water without A. moluccensis. The study also showed that A. moluccensis
Email: [email protected] can accumulate the virus mechanically which can infect chicken eggs up to 11 days.
The virus transmission potential of A. moluccensis requires further studies.

KEYWORDS
aquatic fauna, avian influenza, bamboo shrimps, persistence, survivability

1 | INTRODUCTION conditions (Markwell & Shortridge, 1982). However, the general im-
portance of water-­borne transmission in natural ecosystems is, as yet,
Low pathogenic avian influenza viruses (LPAIVs) cause huge economic poorly understood. This kind of transmission is of particular interest
losses to the poultry industry due to drop in egg production and loss because the large water bodies fall in the crossroads of many mi-
in body weight. With worldwide prevalence in chickens, H9N2 is the gratory palaeo-­arctic birds (Berthold, 2001), and these water bodies
most common subtype of LPAIV. It has a potential to emerge as an oc- have been identified as potential hot spots for the transmission of
cupational hazard among poultry workers (Trani et al., 2012). In March viral pathogens across the globe (Jourdain, Gauthier-­Clerc, Bicout, &
1999, a new pandemic threat appeared when influenza A H9N2 virus Sabatier, 2007).
infected two children in Hong Kong. These viruses were related to the Large groups of the aquatic fauna are filter feeders and are consid-
H9N2 virus which contributed to internal genes of H5N1 virus that ered as the “ecosystem engineers” due to their feeding habits which
killed six people in Hong Kong in late 1997 (Peiris et al., 1999). If the tend to carry out bioconcentration of the virus present in the water
H9N2 virus adapts to human beings, it can spread rapidly and evolve bodies (Berke, 2010). The varied biotic communities of water including
into a significant public health threat. fishes and shellfishes are often vulnerable to a host of pathogens re-
The water-­borne transmission route spreads the infection with- leased into the aquatic systems. Many types of shellfishes have been
out the need of direct contact between birds and is responsible for shown to accumulate different viruses, viz., hepatitis A virus in mussels
the maintenance of H9N2 LPAIV in domestic ducks under farming and clams, Norwalk virus in oysters and clams and remain infective for a

e124  | © 2017 Blackwell Verlag GmbH

wileyonlinelibrary.com/journal/zph Zoonoses Public Health. 2018;65:e124–e129.
PATHAK et al. |
      e125

substantial duration (Enriquez, Frösner, Hochstein-­Mintzel, Riedemann,

& Reinhardt, 1992; Nenonen, Hernroth, Chauque, Hannoun, & Impacts
Bergström, 2006; Schwab, Neill, Estes, Metcalf, & Atmar, 1998). • The occurrence of avian influenza virus in the aquatic
Freshwater clams can accumulate AIV and have in fact been reported ecosystem is well established. The virus is shed into the
to be an effective tool for the early detection of influenza A viruses in water bodies by wild waterfowl, exposing various species
aquatic environments (Huyvaert et al., 2012). Zebra mussels have estab- of the aquatic fauna to the virus.
lished a potential role as a mechanical vector of influenza A virus (Stumpf • The studies on the role of members of the aquatic com-
et al., 2010). Other than filter feeders, avian influenza viruses have been munity in persistence and survivability of the virus are
detected in haemolymph of the Catfish (Clarias gariepinus), Red Swamp scarce.
Crayfish (Procambarus clarkii), Mediterranean Cone Shell (Conus mediter- • Shrimps (Atyopsis moluccensis) were reared in virus-con-
raneus) and the Puffer fish (Lagocephalus sceleratus) in the vicinity of mi- taminated water for a period of 12 days. It was found that
gratory birds’ natural stop stations (Eissa, Hussein, & Zaki, 2012). Certain shrimps can accumulate the virus. The infectivity and
species of fishes can even serve as a model for avian influenza research. amount of virus are significantly higher in water with
The zebrafish is widely recognized as a valuable model and is amenable A. moluccensis than in water without A. moluccensis.
to visualization of host–pathogen interaction (Gabor et al., 2014).
In this study, shrimp was chosen to be the experimental model as
the farming practices of shrimps require handling and processing by
human beings. The study was carried out with the objective to study the
2.3 | Collection and processing of samples
effect of rearing shrimps, in the persistence of H9N2 virus in water and
the risk posed by shrimps reared in H9N2 virus-­contaminated water. Sampling was carried out for both shrimps as well as water in the
tanks of group 1 and 2. Shrimp samples (1 g) from each tank were
collected using a plastic mesh and sterile forceps at 24-­hr inter-
2 |  MATERIALS AND METHODS
val and transferred to the collection tube using sterile forceps. The
samples were processed immediately after collection. The shrimp
2.1 | Virus
samples were weighed and washed prior to homogenization to re-
A low pathogenic H9N2 avian influenza virus A/chicken/ duce the contamination from the external surface of the body of
India/50438/2007, obtained from the virus repository of ICAR-­ shrimps. The washing was carried out by four serial washes with
NIHSAD, Bhopal, Madhya Pradesh, India, was used in the study. All 100 ml sterile PBS. To ensure that the shrimps were free from de-
the experiments were performed in biosafety level 3 and animal bi- tectable external contamination of the virus, the washings were
osafety level 3 (ABSL3) facility of the institute. The virus was pas- tested for the presence of H9N2 virus by quantitative reverse
saged in 9 to 11-­day-­old specific-­pathogen-­free (SPF) embryonated transcription PCR (RT-­qPCR). For this, one ml of the PBS from the
chicken eggs as per the standard protocol (WHO, 2002). The titre of beakers was collected after proper mixing. RNA was extracted from
the amnio-­allantoic fluid (AAF) from the harvested eggs was deter- these samples and subjected to RT-­qPCR.
mined by haemagglutination assay (HA) (OIE, 2008), and the AAF with One per cent suspension of the shrimp was prepared by homogeniza-
10
HA titre >2 was pooled. Aliquots of 1 ml were prepared and stored tion with 1× PBS. The shrimp was placed in 1.5-­ml Eppendorf tube along
at −80°C until further use. The EID50 of the virus was calculated as per with four ceramic beads. The tube was sealed properly using parafilm to
the method described by Reed and Muench (1938). avoid spillage of the contents. The homogenization was carried out at
30 Hz frequency (Qiagen Mixer Mill 300) for 5 min. The homogenized
shrimp sample was divided into two parts. One part was used for RNA
2.2 | Experimental design
extraction to compare the viral load with water, and the other part was
The tanks (10 L volume), bamboo shrimps and accessories required used for egg inoculation to calculate per cent infectivity.
for survival and maintenance of shrimp (air pump, feed and gravel) Twenty-­five water samples (2 ml each) were randomly collected
were procured from commercial vendors. Atyopsis moluccensis also from each of the tanks at 24-­hr interval and were separately pooled
known as bamboo shrimp was used as the model. One tablet of for group 1 and 2 into 50-­ml tubes. The water samples were processed
algae wafer (as per feed manufacturer’s instructions) was used as using the method of virus concentration reported by Khalenkov, Laver,
feed for shrimps. The experimental design included two groups. and Webster (2008) immediately after collection.
Group 1 was non-­chlorinated demineralized water with the shrimps,
whereas group 2 was non-­chlorinated demineralized water without
2.4 | RT-­qPCR and RNA quantification
shrimps. The water in the tanks of both group 1 and group 2 was
spiked with 106 EID50 of H9N2 virus/100 μl of water. The tempera- One millilitre of TRIzol® reagent (Invitrogen, USA) was added to
ture of the water in the tanks of both group 1 and group 2 was 200 μl of homogenized shrimp, and the RNA was extracted follow-
maintained at 20–22°C (which is the ambient for shrimps) through- ing manufacturer’s protocol. Two hundred and fifty microlitres of
out the period of experiment. processed water sample were mixed in 1 ml TRI Reagent® LS for
 |
e126       PATHAK et al.

fluid samples (Invitrogen), and the RNA was extracted as per the 100
manufacturer’s protocol. Both the RNA samples were stored at 90
80

Per cent infectivity

−80°C after elution in 30 μl of DEPC-­treated nuclease-­free water
70
(Fermentas, USA). 60
One-­step RT-­qPCR was carried out in duplicates for detection 50
of matrix gene of AIV (Payungporn et al., 2006) using SuperScript™ 40 Water samples (group 2)
30 Bamboo shrimps
III Platinum™ One-­Step qRT-­PCR Kit (Invitrogen, Germany) in a total
20
Water samples (group 1)
volume of 25 μl containing 20 pmol each of forward and reverse 10
primers, 10 pmol of probe and 1 μl of RNA. The PCR assay was per- 0
1 2 3 4 5 6 7 8 9 10 11 12
formed in Light Cycler® 480 Real-­Time PCR machine (Roche, USA).
Day
The cycling conditions were 30 min at 50°C for reverse transcrip-
tion, 2 min at 95°C for initial denaturation followed by 40 cycles of F I G U R E   1   Per cent infectivity of water and shrimp samples
a 3-­step PCR including 95°C for 30 s, 50°C for 1 min and 72°C for
30 s). No Template Control (NTC) and No Probe Control (NPC) were
included in each run. day of the experiment, while the water samples of group 1 maintained
For quantification, in vitro transcribed RNA (IVT-­RNA) was syn- it till day 11. The per cent infectivity of the water samples of groups
thesized using linearized (Nco I RE digestion) plasmid clone of matrix 1 and 2 and shrimp samples (of group 1) showed a high negative cor-
gene in pGEMT-­easy vector (Promega, USA) through SP6 promoter relation with number of passing days (r = −.960, .980 and .979, re-
(RiboMAX large-­
scale RNA production system-­
SP6; Promega). spectively). Per cent infectivity of both the water and shrimp samples
Standard curve for the assay was prepared using the 10-­fold serially in the group 1 was significantly higher than water samples of group
diluted IVT-­RNA. Accordingly, the RNA copy numbers in test samples 2. No significant difference was found in the per cent infectivity of
were calculated by extrapolation using the instrument software. the water and shrimp samples present in the tanks of group 1. The
RT-­qPCR of shrimps showed detectable amounts of virus up to the
final day of the experiment (day 12). However, the viral load was sig-
2.5 | Determination of per cent infectivity
nificantly higher (p < .01) in the surrounding water (i.e., water of group
The per cent infectivity of the virus was calculated as per the proce- 1) than shrimps at all points during the experiment (Figure 2).
dure recommended by Lu et al. (2003). Briefly, processed sample of The fourth wash samples of the shrimps were negative for H9N2
water and shrimp was inoculated into five 9 to 11-­day-­old embryo- LPAIV by RT-­qPCR indicating the absence of surface virus contamina-
nated SPF chicken eggs and incubated for 120 hr or until the death of tion. The virus copy number in the water samples containing shrimps
embryo whichever is earlier. The virus isolation was confirmed using (i.e., water samples of group 1) was found to be significantly higher
HA test. The formula used to calculate the per cent infectivity was as (p < .001) than that in the water samples devoid of shrimps (group 2)
follows: across the period of the experiment. The experiment was concluded at
the 12th day as the per cent infectivity of shrimps and water samples
Infected embryos (Number of HA positive)
% Infectivity = × 100 was zero. However, to figure out the end point of quantifiable viral
Total embryos inoculated
RNA, the water samples were collected up to the 20th day. For group

2.6 | Statistical analysis
9 a
a a a
All statistical analysis was conducted using spss 17.0 software (SPSS,
8 a a
Chicago, IL, USA), and p <  .05 was considered to be statistically signif- a
a
7 a
icant. All experiments were performed at least three times. The trends a
b b
of the values of per cent infectivity were analysed using Pearson’s b b b b a
6 b b a
correlation coefficient. The viral RNA copy numbers obtained by RT-­
Log10 value

5
b
qPCR were statistically analysed by Student’s t test. b b
4 b

3
Shrimps
3 |  RESULTS 2
Water
1
The per cent infectivity of the water samples from group 1 and group
0
2 and of the shrimp samples from group 1 was found to be 100% in all 1 2 3 4 5 6 7 8 9 10 11 12

the three samples on the day one post-­spiking. From day 2 onwards, Day
the values followed a decreasing trend and all the samples showed
F I G U R E   2   Viral load in shrimps and surrounding water samples
zero infectivity on day 12, which was the final day of the experiment at various time intervals. Values having different superscripts differ
(Figure 1). Water samples of group 2 lost their infectivity on the ninth significantly
PATHAK et al. |
      e127

9 virus in the shrimps and also to ensure the viability of virus for the
8
entire period of the experiment, initial concentration of the stock was
7
estimated. The EID50 for A/chicken/India/50438/2007 virus was esti-
Log10 value

6
5 mated to be 106/100 μl, and this concentration was used for infecting
4 the water in our study.
3
2
Group 1 To simulate the natural shrimp environment, we studied the sur-
1 Group 2 vivability of bamboo shrimps with natural lake water before conduct-
0 ing the experiment. The shrimps could not survive in the tanks with
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
natural water for more tank 48 hr. The abiotic and biotic components
Day
of water have their individual effect on survivability and persistence
F I G U R E   3   Quantification of viral RNA in the water samples of the virus in the water, and the lake water in the natural environ-
ment has its own mechanism of purification of water like exposure to
1, the log10 values for the day 13, 14 and 15 reduced drastically and sunlight and oxidation which is not possible if the lake water is filled
were zero for the subsequent days up to the 20th day. For group 2, the in the tank (Srivastava, Gupta, & Chandra, 2008). Some workers have
viral RNA was detected only up to 13th day (Figure 3). reported the deleterious effect of chlorinated tap water on virus sur-
vivability (Rice et al., 2007). We, therefore, used non-­chlorinated de-
mineralized water for our study. In order to ensure uniformity in the
4 | DISCUSSION distribution of shrimps in terms of age group and body size, we pre-
ferred to procure the bamboo shrimps from commercial vendor.
Lack of virus-­specific information on the presence and stability of Per cent infectivity and viral load were determined to quantify the
the AIV in the environment warrants the need to study viral per- amount of infectious virus in the water and shrimp samples and mech-
sistence in aquatic ecosystem to understand the ecology of these anism of virus accumulation in the shrimps, respectively. The study
viruses. The dependence of humans on the water bodies for suste- revealed that, both in the case of shrimps and the water containing
nance of life cannot be overemphasized. Interaction of humans with the shrimps, the virus remained infective till the 11th day of the exper-
the aquatic fauna in the form of handling, consumption and trade iment but, in the water without shrimps, the infectivity persisted only
has been well established. In the studies carried out on the aquatic till 9th day of the experiment. The RT-­qPCR was used to compare the
biotic community, it was found that filter-­feeding bivalve shellfish viral load of water samples and shrimps in both the groups. The viral
may accumulate viruses and act as efficient vehicles for their trans- load was found to be higher in the water in which shrimps were reared
mission (Lees, 2000; Skraber, Schijven, Gantzer, & Husman, 2005). during the experiment, suggesting that the virus keeps on entering
Transmission of LPAI viruses among wild water birds is considered and releasing from the body of the shrimps. Both per cent infectivity
to be mainly by the faecal–oral route, with virus particles excreted and quantification studies suggest that the presence of shrimps allows
from infected birds directly from faeces into the water and con- higher copy numbers and higher infectivity of the virus to persist in the
tracted by potential hosts by ingestion of virions in water or on water as compared to the water without bamboo shrimps. However,
food therein (Hinshaw, Webster, & Turner, 1979). LPAI viruses are the viral load in the shrimps was significantly lower (p < .01) compared
reported to be more stable than HPAI in the aquatic environment to surrounding water samples.
(Zarkov, 2006). The survivability of AIV is innately adapted to the In a similar study using zebra mussels, Stumpf et al. (2010) ob-
body temperature of waterfowls that is 40–42°C and to subzero served that virus was detected in the water even after the mussels
temperatures of frozen lakes that is up to −54°C (Shoham, Jahangir, were washed and then transferred into the virus-­free water after
Ruenphet, & Takehara, 2012). In a study carried out in filtered lake 48 hr, indicating that virus from the mussels was released back into
water, infectivity of H9N2 virus could be maintained for 75 days at the water. They were also able to detect the virus in both the mus-
4°C; 25 days at 16°C and 5 days at 28°C (Zhang, Li, Chen, Chen, & sels and the surrounding water on all sampling days that is 16 days.
Chen, 2014). As against these observations, some workers have reported a reduc-
Various species of shrimps are abundantly present in the world and tion in the concentration of virus in the spiked water in the presence
are reared for the purpose of human consumption. Bamboo shrimps of clams (Huyvaert et al., 2012) and daphnids (Meixell, Borchardt, &
are common freshwater filter-­feeding shrimps of South Asia and are Spencer, 2013). Both these studies also reported increased accumu-
may be used as a food source (Chace, 1983). Shrimp rearing is an in- lation of viruses in the clam and Daphnia tissues, respectively. These
tegral part of the integrated farming system in many countries, par- varying trends of accumulation of virus may probably be occurring
ticularly when these are reared in combination with the paddy and due to different feeding behaviours of the aquatic animals used in the
duck rearing. Wild waterfowl, the renowned carriers of LPAIV, are the experiments. A mussel, through its incurrent siphon, draws water in,
constant shedders of AIV in the lake waters. In the marshalling areas, which is then brought into the branchial chamber with the help of the
ducks may shed virus as high as 1010 EID50/g/d infectious virus in the cilia located on the gills. The wastewater exits through the excurrent
faecal matter (Webster, Yakhno, Hinshaw, Bean, & Murti, 1978). In siphon. The digestion begins when the labial palps finally funnel the
order to estimate the infective dose to study the accumulation of the food into the mouth. Water enters the clam via the inhalant siphon
 |
e128       PATHAK et al.

and leaves via the exhalent siphon, driven by ciliary beating on the virus in mussels. Journal of Medical Virology, 37, 174–179. https://doi.
ctenidial filaments. The exhalent water carries away excretory prod- org/10.1002/jmv.1890370305
Foster-Smith, R. L. (1975). The role of mucus in the mechanism of feeding
ucts (Dral, 1967; Foster-­Smith, 1975; Kellogg, 1915). The feeding be-
in three filter-­feeding bivalves. Journal of Molluscan Studies, 41, 571–
haviour of clams helps them to “bioconcentrate” the virus (Huyvaert 588. https://doi.org/10.1093/oxfordjournals.mollus.a065307
et al., 2012). However, shrimps feed on minute particles with the help Gabor, K. A., Goody, M. F., Mowel, W. K., Breitbach, M. E., Gratacap, R. L.,
of claws which are feathery in the bamboo shrimps. A similar phenom- Witten, P. E., & Kim, C. H. (2014). Influenza A virus infection in zebraf-
ish recapitulates mammalian infection and sensitivity to anti-­influenza
enon of accumulation is observed in the shrimps, but the lower viral
drug treatment. Disease Models & Mechanisms, 7, 1227–1237. https://
load in the shrimps compared to the surrounding water samples at all doi.org/10.1242/dmm.014746
the time points of the experiment indicates mechanical accumulation Hinshaw, V. S., Webster, R. G., & Turner, B. (1979). Water-­borne trans-
of the virus in shrimps rather than bioconcentration. Hence, it can be mission of influenza A viruses? Intervirology, 11, 66–68. https://doi.
org/10.1159/000149014
concluded that the mechanism of accumulation of the H9N2 avian in-
Huyvaert, K. P., Carlson, J. S., Bentler, K. T., Cobble, K. R., Nolte, D. L., &
fluenza virus in case of bamboo shrimps is mechanical, unlike mussels Franklin, A. B. (2012). Freshwater clams as bioconcentrators of avian
and clams. influenza virus in water. Vector-­Borne and Zoonotic Diseases, 12, 904–
Shrimps are important members of aquatic fauna as they are 906. https://doi.org/10.1089/vbz.2012.0993
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consumed by large fishes, birds and human beings. Their role in the
migration routes and risk for pathogen dispersion into western
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Mediterranean wetlands. Emerging Infectious Diseases, 13, 365–372.
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are able to accumulate the virus. The virus remains infective in the Kellogg, J. L. (1915). Ciliary mechanisms of lamellibranchs with descrip-
body of the shrimps. The presence of bamboo shrimps in the water tions of anatomy. Journal of Morphology, 26, 625–701. https://doi.
org/10.1002/jmor.1050260403
aids in maintaining higher virus counts as compared to water with-
Khalenkov, A., Laver, W. G., & Webster, R. G. (2008). Detection and iso-
out bamboo shrimps. Further studies will reveal the role of shrimps in lation of H5N1 influenza virus from large volumes of natural water.
transmission of AIV. There are various other members of aquatic fauna Journal of Virological Methods, 149, 180–183. https://doi.org/10.1016/j.
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Lees, D. (2000). Viruses and bivalve shellfish. International Journal
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(2003). Survival of avian influenza virus H7N2 in SPF chickens and
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New Delhi, India, for financial assistance under the Outreach Program sion and maintenance of influenza viruses in domestic ducks. Applied
on Zoonotic Diseases. and Environmental Microbiology, 43, 110–115.
Meixell, B. W., Borchardt, M. A., & Spencer, S. K. (2013). Accumulation and
inactivation of avian influenza virus by the filter-­feeding invertebrate
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A. P. Pathak  http://orcid.org/0000-0003-4441-2456
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