Fig 1.
Outbreaks, clinical symptoms and pathological analysis.
(A) Healthy tilapia; (B) Tilapia disease outbreak in cage-cultured results in massive mortality (August 2015; Jinmen, Hubei province, China); (C, D) Gross pathological signs of infected tilapia, including hemorrhages on the lower jaw, anterior abdominal and the fin bases, accompanying with exophthalmos eyes and pronounced ocular lesions. (E) Healthy spleen; (F) Moderated diseased spleen infected at 3rd days after post TiPV-infection; (G, H) Severe diseased spleen infected at 5th days after post TiPV-infection. lymphocytes (white arrow) and macrophages (white triangular arrowheads) in diseased spleen sinusoids, necrotic splenocytes (red arrow), virus inclusion body (black arrow) and melano-macrophage centers (asterisk) in affected spleen; (I) Healthy brain; (J) Moderated diseased brain infected at 3rd days after post TiPV-infection, vacuolated neurons with marginated nuclear (red arrow); (K, L) Severe diseased brain infected at 5th days after post TiPV-infection, vacuolated neurons with marginated nuclear (red arrow), lymphocytes (white arrow) in the blood vessel, edema of cerebral cortex (black arrow). HE staining. Bar = 20um (A, B, E, F), 50um (D), 5 um(C).
Fig 2.
Morphology of the Tilapia brain cells (TiB) and cytopathic effect (CPE) induction on TiB induced by TiPV and transmission electron micrographs of the TiPV-infected TiB cells.
(A) The TiB cells at passage 1, 10 days; (B) The TiB cells at passage 2, 3 days; (C) TiB cells infected with TiPV at passage 3 at 5 days post infection; (D) TiB cells infected with TiPV at passage 6 at 3 days post infection (Bar = 100 μm). (E) Virus particles existed in the cytoplasm and nuclei (white arrow), Nu: nucleus.(Bar, 1 μm); (F) High magnification of the region in the white rectangular box indicated in Panel A, virus particles aggregated in the cytoplasm (Bar, 200 nm); (G) The virus releasing at the plasma membrane of the TiB cell. (Bar, 200 nm); (H) Purified TiPV particles negatively stained with 2% phosphotungstic acid (Bar, 200nm).
Fig 3.
Transmission electron micrographs of the TiPV from the tissues of infected tilapia.
(A) Brain. Large aggregates of parvovirus particles were present in the brain. Virus particles were located in the cytoplasm and nuclei (white arrow), Nu: nucleus (Bar, 1 μm); (B) High magnification of the region in the white rectangular box indicated in Panel A (Bar, 200 nm); (C) Heart. Large numbers of virus particles occupy the interfibrillar spaces, Mf: Myofibril (Bar, 500nm); (D) Eye. (Bar, 500nm); (E) Spleen. The mature virus clustered in the cytoplasm near the cell nucleus. (Bar, 500nm); (F) Spleen. Small amounts of bacterial pathogens (Streptococcus agalactiae) and virus co-infected the same spleen cell (bar, 1μm).
Fig 4.
TiPV replication and viral protein expression in naturally infected tilapia.
(A, B) in situ hybridization of the TiPV-infected kidney and spleen cells, respectively; (Ab, Bb) positive signals presented in kidney and spleen cells, respectively; (Aa, Ba) No signals presented in healthy kidney and spleen cells. Arrows indicate positive signals (bar = 20 mm); (C, D) Immunofluorescence assay of the TiPV infected kidney and spleen. The red color indicates the presence of viral protein. The nuclei are stained blue (bar = 50 mm); (Cd, Dd), (Ce, De) and (Cf, Df) Mock infected kidney and spleen cells, respectively; (Ca, Da), (Cb, Db), and (Cc, Dc) No fluorescence signals were observed in kidney and spleen cells of heathy fish.
Fig 5.
Characterization of the TiPV genome, PCR detection of TiPV in different samples and cumulative mortality of artificial infected tilapia.
(A) The genome organization of TiPV. The NS1, NP, VP1 proteins, the alternative ORF1 and ORF2 are showed in different colors; (B) Alignment of conservative domains of NS1 proteins from different parvoviruses by Muscle package. The HuH (u indicates hydrophobic residues) and the walker loop motif, including A, B, B′ and C Walker box of helicase domains are marked; (C) Lane M: DL1000 bp DNA ladder; Lane 1: positive control; Lane 2: negative control; Lanes 3: the kidney tissues sample from natural infected tilapia; Lanes 4: TiB cell culture 3rd-passage viral supernatant; Lane 5: The artificial infected tilapia peritoneal injection with 0.5 ml of 0.22μm filtrate of diseased fish tissue homogenates; Lanes 6: the mock-infected tilapia; (D) Fish in the test group 1 (▲) were challenged by intraperitoneal injection with the 2nd-passage of viral supernatant (104.0TCID50/ml) from cell culture; fish in test group 2 (■) were challenged by intraperitoneal injection with 0.5 ml filtrated supernatant homogenate from diseased fish tissues; and fish in the control group (●) were injected intraperitoneally with Dulbecco’s PBS.
Fig 6.
TiPV genome and transcription show its unique characterizations.
(A) Assembled genome of TiPV. The major hypothesized ORFs, NS1 and VP1, including start and stop sites, and their orientation of them are both from 5′ end to 3′ end. The primers and probe below show their indicated locations in TiPV genome; (B, C) 3′ RACE analysis of NS1 and VP1. Total RNA was isolated from diseased kidney tissue and subjected to reverse transcription. The cDNA was then subjected to 3′ RACE analysis. Amplified DNA fragments were electrophoresed on 2% agarose gel and visualized using ethidium bromide staining; (D) The polyadenylation sites of TiPV. Sequencing of the PCR products from lanes 2 and 4 (B and C) show TiPV has two different polyadenylation sites; (E) Northern blot analysis. Total RNA was isolated from 293 cells transfected with pTiPV and empty vector, and used for Northern blot analysis. The blots were hybridized to probes NS1.
Fig 7.
TiPV genome copies and distribution in affected tilapia and the prevalence of TiPV infection in different locations of China.
(A) qPCR assay of TiPV genome copies in different tissues of the naturally infected tilapia. Each column represents the mean ± standard error of mean, with three independent replicates; (B) PCR detection of TiPV in naturally infected tilapia tissues samples. Lane M: DL1000 bp DNA ladder; Lane 1: positive control; Lane 2: negative control; Lanes 3: spleen; Lanes 4: kidney; Lanes 5: intestine; Lanes 6: heart; Lanes 7: brain; Lanes 8: liver; Lanes 9: gill; Lanes 10: eye; Lanes 11: muscle; (C) TiPV genome copies in different tissue samples of experimentally infected tilapia at different time point (1, 2, 3, 4, 5 dpi). Each column represents the mean ± standard error of mean, with three independent replicates; (D) Details of tilapia collection in Hubei province, Henan province, Guangdong province, Guangxi autonomous region and Hainan province. Circles: tilapia-sampling locations. Samples positive for parvovirus are identified in red, with negative in black.
Fig 8.
Phylogenetic analysis based on amino acid sequences of the non-structural 1 (NS1) protein from the family Parvoviridae.
The phylogenetic tree is constructed based on the NS proteins of 55 representative parvoviruses using the Maximum Likelihood method with 500 bootstrap replicates. TiPV are indicated with shading and the number under the branch indicates the bootstrap values (values lower than 50 were hided). The scale bar means the genetic distance, number of substitutions per site.
Table 1.
Primers used in present study.
Table 2.
Origins of the TiPV isolates from different provinces used in this study.