Chinese Mitten Crabs (Eriocheir Sinensis) Could Act As Vehicles of Spreading Avian In uenza Virus


 Avian influenza virus (AIV) possessed significant risk to various animals and human health. Wild birds, especially waterfowls are considered to be the natural reservoir of AIVs. The ecology of AIV is still far from being fully understood. Chinese mitten crabs are nonnegligible biotic factor in AIV ecosystem. We analyzed the ability of Chinese mitten crabs accumulate and spread AIV. We found that AIV remain infectious in water only for 36 hours but persist in crabs for 48 hours. Crabs grills and gastrointestinal tracts accumulated AIV with higher titers than viral water. Crabs could accumulate AIV from contaminated water, carry the virus and spread to naïve crabs via surrounding water. Our study identified Chinese mitten crab as a novel transmission vehicle in AIV ecosystem.


Introduction
Avian In uenza virus (AIV) is important pathogen for both human and animals. AIV is a RNA virus composed of 8 genome segments. Hemagglutinin (HA) and neuraminidase (NA) are two major glycoproteins on the surface of AIV. AIV can be classi ed into 18 HA (H1-H18) and 11NA (N1-N11) subtypes based on the different antigenicity of HA and NA. All of these subtypes have been found in wild birds excepting for H17N10 and H18N11 subtypes [1,2]. So it has been widely accepted that wild birds, especially waterfowls are the natural reservoir of AIVs. Occasionally, AIV in waterfowls could spill over to domestic poultry, livestock, marine mammals and even humans [3,4].
AIVs mainly replicate in the intestine tract cells of waterfowls [5], so the infected bird faeces may contain high concentration of AIVs. The faecal-oral route is considered to be the primary AIV transmission mode in waterfowls. It has been reported that AIVs can remain infectious in aquatic environment for more than seven months [6]. As a result, it makes the aquatic environment become an epidemic focus where AIV could transmit among waterfowl and other animal living in the same area [7].
Chinese mitten crab (Eriocheir sinensis) belongs to Malacostraca, Decapoda, Grapsidae. It is an omnivorous animal which mainly feed on plant and animal detritus [8]. In the wild, the crabs could also be the prey of waterfowl and poultry. The farming industry of Chinese mitten crabs involves 30 provinces in China, which has become the largest industry of single species of freshwater shery [9]. Chinese mitten crabs are widely existing in freshwater lakes, rivers and brackish waters in China [10]. Sharing same aquatic habitats and being in predation relation with waterfowls, Chinese mitten crabs might become a transmission biotic factor in AIV ecosystem. In this study we evaluated the function of Chinese mitten crab in AIV ecosystem.

Chinese mitten crabs
The Chinese mitten crabs which weighed 10.0 ± 1.0 g were giving by Panjin Guanghe Crab industry Co Ltd. The crabs were kept in aerated water for 2 weeks at 18 ℃ ahead. Aerated water was made by pumping air into 20 Liter tap water with an air pump (20L/min) overnight. Finally, the dissolved oxygen and pH level of the aerated water was 6.4mg/ml and 7.2 respectively.

Virus and cell
H9N2 avian in uenza virus A/chicken/Liaoning/07/2016 was isolated from chicken during routine surveillance. Virus stock was prepared by inoculation of Madin-Darby canine kidney (MDCK) cells. The viral titer was determined by 50 % tissue culture infectious dose (TCID 50 ).

Persistence of Avian in uenza virus in the aerated water
The viral water was made by adding 10 7.5 TCID 50 AIV into 1 Liter aerated water, mixing thoroughly. 3 tanks with 1L viral water were put into a biosafety cabinet at 18 ℃. 1 ml water sample was taken from each tank after 0, 1, 3, 8, 12, 24, 36, 48 and 60 hours(h) respectively to test the viral titer in MDCK cells.
The limitation of virus detection was set as 0.5 TCID 50 /ml. 2.5 AIV accumulating limitation of crabs 6 groups of 3 crabs were incubated in viral water. Every 12h, the tank water was changed with fresh viral water until 60 hours later. Since 0h after incubation, 1 group of crabs were rinsed and euthanized for gills collection before water changing. The viral water sample was collected at the same time.

AIV spreading activity of Chinese mitten crabs
Groups of 3 crabs were incubated in viral water for 8h as inoculated groups. After rinsing thoroughly, the inoculated crabs were transferred into fresh water. Groups of 3 naïve crabs, as sentinel groups, were put into each tank. During the rst co-tanking 4h, every 0.5h 1 group of inoculated and sentinel crabs were rinsed and euthanized. Their grills and water sample were collected at the same time for viral titration.

AIV persisted for a shorter time in lab condition
As shown in gure 1, AIV could maintain similar infectivity for 3h. Viral titer began to drop at 8hpi. At 24hpi, viral titer dropped by half. There's no detectable virus in water after 36h.
3.2 Chinese mitten crabs accumulated and preserved AIV longer than in water Groups of 3 crabs were inoculated by incubating in viral water. As shown in Table 1, AIV could be detected in crabs' grills and intestinal tracts since 1hpi. The crabs' grills accumulated AIV more e ciently than their gastrointestinal tracts. The grills viral titers were higher than viral water since 8hpi. At 36hpi, the crab viral titers began to drop. Whereas, water viral titer kept on dropping from the beginning of the experiment. AIV could still be detected in 1 crab's grill at 36hpi and in 1crab's grill and gastrointestinal tract at 48hpi. No virus was detected in other organs of the crabs. It's indicated that AIV could "infect" crabs through their respiratory and digestive systems. There's no detectable AIV after 48hpi in neither crabs nor in water.

Chinese mitten crabs accumulated AIV with a titer but not time limitation
The crabs surrounding viral water was refreshed every 12h. The crab viral titers were consistent but higher than viral water. So the Chinese mitten crabs could continue accumulating AIV but might be con ned by their size or water viral titer.

Chinese mitten crabs spread AIV to surrounding water and naïve crabs
We inoculated crabs by incubating them in viral water for 8h and used them as the inoculated groups. Groups of 3 naïve crabs were treated as sentinel groups. As shown in Table2, AIV could be detected from inoculated crabs until 8hpi. Since 1hpi, AIV was detected from 2 of the 3 sentinel crabs. At 2.5hpi, AIV was only detected from 1 sentinel crab. There's no detectable AIV in sentinel groups after 3hpi. AIV was detected from water at 1 and 1.5hpi. No alive virus in water after 2h. The inoculated groups contained higher viral titer than sentinel groups and water. The result indicated that, AIV contaminated Chinese mitten crab could spread virus into water and "infect" the naïve crabs.  Researchers found AIV RNA in small aquatic vertebrates and invertebrates, such as water eas [11], bamboo shrimp (Atyopsis moluccensis), clams (Corbicula uminea), freshwater snails (Physa spp.), zebra mussels (Dreissena ploymorpha), cray sh and Mediterranean cone shell (Conus sp.). Experiments indicated these aquatic animals can accumulate AIVs through water ltering but the infectivity of these accumulated AIV had not been evaluated yet [12][13][14][15][16][17]. Our study for the rst time evaluated the infectivity of accumulated AIV in crabs. Furtherly, we determined the AIV spreading activity of Chinese mitten crabs. Further study should be conducted to evaluate the AIV transmission between Chinese mitten crabs and their predators.

Conclusion
In the present study, we evaluated the Chinese mitten crab AIV accumulating and spreading activity. AIV could be accumulated in multiple organs of the crabs and stay infectious longer than in water. Most importantly, AIV could be carried by the crabs into freshwater and transmitted to the naïve crabs. Our study indicates that Chinese mitten crabs is an important factor in the AIV ecosystem. All data generated or analysed during this study are included in this published article Figure 1 Persistence of AIV in aerated water. 107.5TCID50 AIV virus was added to 1L aerated water at 18°C. Water samples were collected at designated time points and titrated on MDCK cells. The data shown are the means of three replicates; the error bars indicate standard deviations. The dashed lines indicate the lower limit of virus detection. Data were analyzed using analysis of variance (ANOVA) in GraphPad Prism version 9.0 (GraphPad Software Inc., CA, USA). Signi cance was analyzed by using a one-way ANOVA with post-hoc tests. a, P<0.001; b, P<0.05.