doi:10.1111/jfd.12350
Journal of Fish Diseases 2016, 39, 371–375
Short Communication Isolation and identification of a new reovirus associated with mortalities in farmed oriental river prawn, Macrobrachium nipponense (de Haan, 1849), in China S Zhang1, X Shu2, L Zhou1 and B Fu1 1 Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China 2 Hubei Fisheries Science Research Institute, Wuhan, Hubei, China
Keywords: crustacean virology, freshwater prawn, Macrobrachium nipponense, MnRV, RdRp sequence, reovirus. Since the first report of a crustacean reovirus (Vago 1966), reo-like viruses have been reported in some species of crustaceans. There were several reports about the reovirus infecting marine crabs during the 1970s and early 1980s (Bonami 1973; Bonami & Comps 1976; Bonami 1980; Johnson & Bodammer 1975; Johnson 1977; Mari & Bonami 1988). Recently, another reovirus in marine crabs has been reported in China (Zhang et al. 2007; Weng et al. 2007). In freshwater, two new reoviruses have recently been isolated from the Chinese mitten crab, Eriocheir sinensis, in China (Zhang et al. 2004; Zhang & Bonami 2012). Reoviruses of crabs are known to develop mainly in the connective tissues of the host. In shrimp and prawn, reo-like viruses have been reported in some species, such as white shrimp Penaeus vannamei (Krol, Hawkins & Overstreet 1990), tiger prawn Penaeus monodon (Nash et al. 1988), tiger shrimp Penaeus japonicus (Tsing & Bonami 1987), shrimp Palaemon elegans (Vogt 1992), and freshwater crayfish Cherax quadricarinatus (Edgerton et al. 2000; Hayakijkosol & Owens 2011). Interestingly, unlike crab reoviruses, Correspondence S Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences, 430071 Wuhan, China (e-mail: [email protected]) GenBank Accession number: KM405244 for partial RdRp sequence of MnRV. Ó 2015 John Wiley & Sons Ltd
371
which develop in connective tissues, all the shrimp reoviruses develop in epithelial hepatopancreatic cells. The freshwater prawn (oriental river prawn), Macrobrachium nipponense (de Haan,1849), is widespread in the lakes and rivers of Japan, SouthEast Asia and China. M. nipponense is an economically important species cultured in China with a long history. Prawn culture has increased rapidly in the inland provinces of China, particularly in Hubei and Jiangsu provinces (central and east China). Since 2003, prawn production was estimated at about 20 000 metric tons per year with an average mortality rate of 30–40%. However, with the development of intensive culture, various diseases have emerged and have severely affected production. In the summer of 2008, we investigated mortality problems of M. nipponense in a farm in Hubei province, where nearly half of the farm population was found exhibiting various diseases such as red body disease and black gills. Mortalities reached 15–20% on average. In some other farms, mortality was reported to reach 50–60%. Diseases of freshwater prawns often appear as multifactorial and caused by a combination of pathogenic, nutritional, physiological and environmental factors. During our investigations, we evidenced a new type of virus never reported in crustacean. We report here the general characteristics of this new pathogenic agent closely related with reovirus family group. Diseased prawn samples were collected from June 2008 to September 2012 from a farm in
Journal of Fish Diseases 2016, 39, 371–375
Hubei province, China. They were about 4–10 g in weight and 4–6 cm length, a little smaller when compared to obviously healthy animals of same age, 5–8 cm length. Except the size, no other clinical sign was noted. The whole bodies of naturally infected and healthy prawns were fixed in Davidson’s fixative for 24 h, and tissues were routinely processed and paraffin embedded. Sections (5–7 lm) were stained with haematoxylin and eosin (H&E) and examined with light microscopy. Infection signs were observed only in epithelial cells of hepatopancreas (HP), in which infected cells contained large, round, eosinophilic to pale basophilic inclusion bodies in the cytoplasm. The eosinophilic areas were approximately 10 lm in diameter, nearly half of the size of nuclei. Inclusions were usually surrounded by a large vacuole; however, nuclei looked normal (Fig. 1). Frozen HP tissues were mixed and homogenized in TN buffer (0.02 M Tris–HCl, 0.4 M NaCl, pH 7.4) at 4° C with a Potter tissue blender. After clarification for 30 min at 8000 rpm to pellet cell debris, supernatant was centrifuged for 1 h at 100,000 g. The final pellet was resuspended in TN buffer and extracted twice with Freon before being layered onto a 30% (w/w) sucrose cushion and centrifuged for 1 h at 28 000 rpm. The resuspended pellet was used for
S Zhang et al. Macrobrachium nipponense reovirus
subsequent analysis: electron microscopy, nucleic acid extraction and cloning. A part of the resuspended pellet was used for transmission electron microscopic (TEM) investigations after negative staining with 2% phosphotungstic acid (PTA) pH 7.0. Numerous full and empty, non-enveloped, icosahedral virus-like particles, 60 nm in diameter, were evidenced. An electron-dense, nucleated-like structure was present in the central area of the particles, with a size of 30 nm, surrounded by a bilayered capsid, about 7–10 nm thick (Fig. 2). Nucleic acid of virus-like particles was extracted with TRIzol reagent (Invitrogen) and precipitated with isopropanol. The pellet was resuspended in DEPC (diethyl pyrocarbonate)-treated water. Electrophoresis was performed in 1% agarose gels in 0.59 Tris–borate/EDTA (TBE) buffer at a constant current of 100 V. Gels were stained with ethidium bromide and examined using a UV transilluminator. Treated with proteinase K and sarkosyl, the VLP genome revealed 10 distinctive bands with an electrophoresis type of 5/5 or 5/2/3 (Fig. 3). The RNA extraction performed with the TRIzol reagent obtained the same result. Using DL5000 as marker, the size of the 10 segments was estimated to be 4.2,3.8, 2.8, 2.6, 2.3, 1.9, 1.8, 1.6, 1.4 and 1.3 kbp, respectively. The full length of the genome was about 23.6 kbp. When stained with orange acridine, bands emitted a green fluorescence. All the data reported above, cytoplasmic location, size and icosahedral structure of particles with a double-layered, non-enveloped capsid, multisegmented RNA genome, indicate that this new agent is closely related to the
200 nm
Figure 1 Histology of infected Macrobrachium nipponense. Hepatopancreatic section. White arrow indicates viral cytoplasmic inclusions close to the lumen of the tubule. (Bar is 50 lm.) H&E stain. Ó 2015 John Wiley & Sons Ltd
372
Figure 2 Semi-purified virus particles of MnRV; full and empty particles are present. TEM. PTA2%. (Bar is 100 nm.)
S Zhang et al. Macrobrachium nipponense reovirus
Journal of Fish Diseases 2016, 39, 371–375
5.0 4.2 3.8 2.8 2.6
3.0
2.3 1.9 (2)
2.0
1.6 1.4
1.5
1.3
1.0
Figure 3 Agarose gel (1%) electrophoresis of the MnRV genome. Left: Viral genome. Right: DL5000 DNA marker. Sizes are expressed in kbp.
Reoviridae family. This is confirmed by the distribution and molecular weight of the 10 genomic segments.
Partial cloning and sequencing of the genome was developed for further characterization of the agent. The synthesis of cDNA was performed according to the manufacturer’s instructions for the Universal RiboClone cDNA Synthesis System (Takara). About 20 positive clones were selected for sequencing by the ESGS group (Invitrogen). Sequences were analysed with the DNA Star computer program (DNA Star Inc.). DNA and deduced amino acid sequences were compared with updated databases from GenBank/EMBL and SWISS-PROT. Most of the sequences revealed no significant similarity with other sequences in the GenBank database in NCBI (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi), but one of the deduced amino acid sequences, which contain the putative conserved GDD and ExxK motif regions of RNA-dependent RNA polymerase (Fig. 4), showed some identities (
Journal of Fish Diseases 2016, 39, 371–375
Short Communication Isolation and identification of a new reovirus associated with mortalities in farmed oriental river prawn, Macrobrachium nipponense (de Haan, 1849), in China S Zhang1, X Shu2, L Zhou1 and B Fu1 1 Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China 2 Hubei Fisheries Science Research Institute, Wuhan, Hubei, China
Keywords: crustacean virology, freshwater prawn, Macrobrachium nipponense, MnRV, RdRp sequence, reovirus. Since the first report of a crustacean reovirus (Vago 1966), reo-like viruses have been reported in some species of crustaceans. There were several reports about the reovirus infecting marine crabs during the 1970s and early 1980s (Bonami 1973; Bonami & Comps 1976; Bonami 1980; Johnson & Bodammer 1975; Johnson 1977; Mari & Bonami 1988). Recently, another reovirus in marine crabs has been reported in China (Zhang et al. 2007; Weng et al. 2007). In freshwater, two new reoviruses have recently been isolated from the Chinese mitten crab, Eriocheir sinensis, in China (Zhang et al. 2004; Zhang & Bonami 2012). Reoviruses of crabs are known to develop mainly in the connective tissues of the host. In shrimp and prawn, reo-like viruses have been reported in some species, such as white shrimp Penaeus vannamei (Krol, Hawkins & Overstreet 1990), tiger prawn Penaeus monodon (Nash et al. 1988), tiger shrimp Penaeus japonicus (Tsing & Bonami 1987), shrimp Palaemon elegans (Vogt 1992), and freshwater crayfish Cherax quadricarinatus (Edgerton et al. 2000; Hayakijkosol & Owens 2011). Interestingly, unlike crab reoviruses, Correspondence S Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences, 430071 Wuhan, China (e-mail: [email protected]) GenBank Accession number: KM405244 for partial RdRp sequence of MnRV. Ó 2015 John Wiley & Sons Ltd
371
which develop in connective tissues, all the shrimp reoviruses develop in epithelial hepatopancreatic cells. The freshwater prawn (oriental river prawn), Macrobrachium nipponense (de Haan,1849), is widespread in the lakes and rivers of Japan, SouthEast Asia and China. M. nipponense is an economically important species cultured in China with a long history. Prawn culture has increased rapidly in the inland provinces of China, particularly in Hubei and Jiangsu provinces (central and east China). Since 2003, prawn production was estimated at about 20 000 metric tons per year with an average mortality rate of 30–40%. However, with the development of intensive culture, various diseases have emerged and have severely affected production. In the summer of 2008, we investigated mortality problems of M. nipponense in a farm in Hubei province, where nearly half of the farm population was found exhibiting various diseases such as red body disease and black gills. Mortalities reached 15–20% on average. In some other farms, mortality was reported to reach 50–60%. Diseases of freshwater prawns often appear as multifactorial and caused by a combination of pathogenic, nutritional, physiological and environmental factors. During our investigations, we evidenced a new type of virus never reported in crustacean. We report here the general characteristics of this new pathogenic agent closely related with reovirus family group. Diseased prawn samples were collected from June 2008 to September 2012 from a farm in
Journal of Fish Diseases 2016, 39, 371–375
Hubei province, China. They were about 4–10 g in weight and 4–6 cm length, a little smaller when compared to obviously healthy animals of same age, 5–8 cm length. Except the size, no other clinical sign was noted. The whole bodies of naturally infected and healthy prawns were fixed in Davidson’s fixative for 24 h, and tissues were routinely processed and paraffin embedded. Sections (5–7 lm) were stained with haematoxylin and eosin (H&E) and examined with light microscopy. Infection signs were observed only in epithelial cells of hepatopancreas (HP), in which infected cells contained large, round, eosinophilic to pale basophilic inclusion bodies in the cytoplasm. The eosinophilic areas were approximately 10 lm in diameter, nearly half of the size of nuclei. Inclusions were usually surrounded by a large vacuole; however, nuclei looked normal (Fig. 1). Frozen HP tissues were mixed and homogenized in TN buffer (0.02 M Tris–HCl, 0.4 M NaCl, pH 7.4) at 4° C with a Potter tissue blender. After clarification for 30 min at 8000 rpm to pellet cell debris, supernatant was centrifuged for 1 h at 100,000 g. The final pellet was resuspended in TN buffer and extracted twice with Freon before being layered onto a 30% (w/w) sucrose cushion and centrifuged for 1 h at 28 000 rpm. The resuspended pellet was used for
S Zhang et al. Macrobrachium nipponense reovirus
subsequent analysis: electron microscopy, nucleic acid extraction and cloning. A part of the resuspended pellet was used for transmission electron microscopic (TEM) investigations after negative staining with 2% phosphotungstic acid (PTA) pH 7.0. Numerous full and empty, non-enveloped, icosahedral virus-like particles, 60 nm in diameter, were evidenced. An electron-dense, nucleated-like structure was present in the central area of the particles, with a size of 30 nm, surrounded by a bilayered capsid, about 7–10 nm thick (Fig. 2). Nucleic acid of virus-like particles was extracted with TRIzol reagent (Invitrogen) and precipitated with isopropanol. The pellet was resuspended in DEPC (diethyl pyrocarbonate)-treated water. Electrophoresis was performed in 1% agarose gels in 0.59 Tris–borate/EDTA (TBE) buffer at a constant current of 100 V. Gels were stained with ethidium bromide and examined using a UV transilluminator. Treated with proteinase K and sarkosyl, the VLP genome revealed 10 distinctive bands with an electrophoresis type of 5/5 or 5/2/3 (Fig. 3). The RNA extraction performed with the TRIzol reagent obtained the same result. Using DL5000 as marker, the size of the 10 segments was estimated to be 4.2,3.8, 2.8, 2.6, 2.3, 1.9, 1.8, 1.6, 1.4 and 1.3 kbp, respectively. The full length of the genome was about 23.6 kbp. When stained with orange acridine, bands emitted a green fluorescence. All the data reported above, cytoplasmic location, size and icosahedral structure of particles with a double-layered, non-enveloped capsid, multisegmented RNA genome, indicate that this new agent is closely related to the
200 nm
Figure 1 Histology of infected Macrobrachium nipponense. Hepatopancreatic section. White arrow indicates viral cytoplasmic inclusions close to the lumen of the tubule. (Bar is 50 lm.) H&E stain. Ó 2015 John Wiley & Sons Ltd
372
Figure 2 Semi-purified virus particles of MnRV; full and empty particles are present. TEM. PTA2%. (Bar is 100 nm.)
S Zhang et al. Macrobrachium nipponense reovirus
Journal of Fish Diseases 2016, 39, 371–375
5.0 4.2 3.8 2.8 2.6
3.0
2.3 1.9 (2)
2.0
1.6 1.4
1.5
1.3
1.0
Figure 3 Agarose gel (1%) electrophoresis of the MnRV genome. Left: Viral genome. Right: DL5000 DNA marker. Sizes are expressed in kbp.
Reoviridae family. This is confirmed by the distribution and molecular weight of the 10 genomic segments.
Partial cloning and sequencing of the genome was developed for further characterization of the agent. The synthesis of cDNA was performed according to the manufacturer’s instructions for the Universal RiboClone cDNA Synthesis System (Takara). About 20 positive clones were selected for sequencing by the ESGS group (Invitrogen). Sequences were analysed with the DNA Star computer program (DNA Star Inc.). DNA and deduced amino acid sequences were compared with updated databases from GenBank/EMBL and SWISS-PROT. Most of the sequences revealed no significant similarity with other sequences in the GenBank database in NCBI (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi), but one of the deduced amino acid sequences, which contain the putative conserved GDD and ExxK motif regions of RNA-dependent RNA polymerase (Fig. 4), showed some identities (
