Citation:
Wang E, He L, Zhang H, Cheng X and Zhang C. Isolation and Molecular Characterization of a Porcine Rotavirus from Henan Province of China.
Austin Virol and Retrovirology
. 2017; 4(1): 1025.
Austin Virol and Retrovirology - Volume 4 Issue 1 - 2017
ISSN: 2472-3517
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Austin Virology and Retro Virology
Open Access
Abstract
A porcine rotavirus, strain HN-001, was isolated from fecal sample of a
diarrhea piglet in Henan province, China. The virus emerged specic CPEs after
6 blind passages on MA-104 cells. The isolate has been successfully adapted on MA-104 cell line and virus titer reached to 105.0 TCID50/ml. Sequencing and phylogenetic analysis showed that the VP4 gene of new isolate had a high homology with that of YM strain (genotype P[7]). The VP7 gene of the isolate belonged to genotype G[5]. An experimental infection was conducted in 3-day old piglets with an oral inoculation of cell culture material of the new porcine rotavirus isolate. The infected piglets showed severe diarrhea at 24h after inoculation. The piglets died or were euthanized 50 hours post inoculation. At necropsy, large amount of watery cheese-like material was found in stomach
and watery uid in small intestines with thinned wall. The jejunum and ileum were partly erated with some yellow contents. The cecum was lled with yellow liquid and inated. The results indicated that the new porcine rotavirus isolate is
pathogenic strain and had caused a severe disease in piglets.
Keywords:
Porcine rotavirus; Isolation; Characterization; Pathogenicity
old in traditional small-scale pig arm in Henan province o China. Te positive ecal samples were collected and mixed with phosphate-buffered saline (PBS, 0.1 M, pH 7.2). Te samples were urther processed by reezing and thawing twice ollowed by a centriugation at 3000 rpm or 12 min at 4°C. Te collected supernatant was filtered with a 0.22 μ filter (Lie science, USA) and aliquoted and stored at -80°C in Laboratory or analysis. All the ecal samples were screened using a Rapid Rota Ag est kit (BioNote.Inc, Korea) beore perorming virus isolation in tissue culture and positive samples were subject to urther virus isolation .Te samples were pretreated with trypsin solution (Hyclone, USA) at a final concentration o 20 μg/mL at 37°C or 1h beore inoculation onto the monolayer o MA-104 cells [9] in 24-well plates (Costar, USA). Te negative control group was treated in the same method instead o maintenance medium (MEM, Hyclone, USA). Each well o the plate was inoculated with 200ul treated samples, 4 wells per sample. Te plates were slightly shaked every 20 minutes or an hour in the incubator. One milliliter o MEM was added to each well o the plates. Te plates were placed into the incubator at 37°C and observed daily or appearance o potential CPEs. Te cell culture supernatant were collected and inoculated into resh cells afer 3 days o incubation. Cell culture supernatant, with or without CPEs, was collected, and stored at -8°C or urther characterization.
Virus Titration and Immunouorescence assay
MA-104 cell monolayers were propagated and prepared in 96-well cell culture plates two days beore virus titration. Te cell monolayers were washed with PBS twice beore virus inoculation. esting virus samples were 10-old serially diluted in MEM in a separate set o test tubes. Samples at dilutions o 10-1, 10-2, 10-3, 10-4, 10-5, and 10-6 were inoculated to the cell monolayers at 100μl per well. Each sample dilution was added to 8 replicated wells on the plates. Cell monolayer
Introduction
Porcine rotaviruses belong to the genus rotavirus in the amily Reoviridae [1]. Porcine rotaviruses have so ar been divided into our serogroups, named as A, B, C, and E [2]. Rotavirus appears as a wheel-like particle under negative staining electronic microscopy (EM) [3]. Te rotavirus genome is composed o 11 double-stranded RNA segments, and the sizes range rom 0.6 to 3.3 kb [4]. VP4, VP6, and VP7 have recently become the ocuses o research due to their unique biological unctionalities. Based on the antigenicity o VP4 and VP7, group A rotavirus is divided into genotypes P and G. Group A porcine rotavirus is consist o 37P genotypes and 27 G genotypes [5]. Te incubation period or acute porcine rotavirus inections in piglets usually ranges rom 16 to 24h, the clinical symptoms or inections include depression, diarrhea, and a large amount o mucosal eces [6]. Porcine rotaviruses mainly exist in the intestines o piglets at beginning o inection excrete into eces and spread throughout to other piglets by a ecal/oral route [7]. Porcine rotavirus is very resistant to surrounding environment and disinectants and viral inectivity in eces can last or 7-9 months at room temperature [8]. In last two years, pig herds with serious diarrhea have been reported in a large number o areas o China. During disease survey and diagnosis, a rotavirus, HN-001, was isolated rom ecal samples o young piglet with acute diarrhea in Henan province o China. Te virus isolation, molecular characterization, and its pathogenic evaluation in piglets will be described.
Materials and Methods
Virus isolation
A total o 50 ecal samples were collected rom piglets o ten day
Research Article
Isolation and Molecular Characterization of a Porcine Rotavirus from Henan Province of China
Erxin Wang
1
, Lei He
1
, Hewei Zhang
2
, Xiangchao Cheng
1
and Chunjie Zhang
1
*
1
Animal Disease and Public Security Academician Workstation of Henan Province/The Key Lab of Animal Disease and Public Health, Henan University of Science and Technology, China
2
State Key Laboratory for Molecular Biology of Special Economic Animals, Institute of Special Economic Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun, P.R. China
*Corresponding author:
Chunjie Zhang, Animal Disease and Public Security Academician Workstation of Henan Province/The Key Lab of Animal Disease and Public Health, Henan University of Science and Technology, China
Received:
July 24, 2017;
Accepted:
September 13, 2017;
Published:
September 22, 2017
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with same amount o medium were served as negative controls. Te CPEs were observed and recorded daily upto 5 days. Te CID50 o each testing samples was determined using the Karber method [10]. Monolayers o MA-104 cells were inoculated with rotavirus at a MOI o 0.01 and incubated or 12 h at 37°C. Te cells were fixed with 80% ice-cold ethanol or 0.5 h at -20°C. 100× diluted monoclonal antibody specially against PoRV was added to the cells as primary antibodies or 40 min at 37°C ollowed by a 2000× diluted FIC-conjugated donkey anti-mouse IgG antibody (Lie echnologies, Carlsbad, CA, USA). Te cells were analyzed by fluorescence microscope.
RT-PCR
otal RNA was extracted rom cell culture supernatant using RIzol reagent (Invitrogen, USA) according to the manuacturer’s instructions. A reverse transcription was perormed using the M-MLV Reverse ran scriptase (araka, Japan) to synthesize cDNA or urther gene amplification. A pair o primers were designed or a 762bp portion o VP4 gene published in GenBank (Accession number: AY523636.1), with a orward primer 5’-GCC C A CA A GA CAC-3’and a reverse primer 5’-G CCA G CA C A GC-3’. PCR reactions were carried out in PCR buffer containing 3 μl o cDNA, 2 μl o each dNPs (2.5mmol), 0.5 μl o each primer, 0. 25 μl o raq in a 25 μL reaction volume. Te PCR program included an initial denaturation at 95°C or 5 min, 35 cycles o 94°C or 45s, 54°C or 45s and 72°C or 1min, ollowed by a 10 min extension at 72°C.A complete VP7 gene o 1041 bp was amplified with a pair o primers, VP7-F 5’-GGC A AGA GAC AA C CG-3’ and VP7-R 5’-GC CAC ACC AA CAC C AA CC AAG-3’.Te same PCR amplification reagents or VP4 were used VP7 gene amplification. Te PCR program included an initial denaturation at 95°C or 5 min, 35 cycles o 94°C or 45s, 57°C or 45s and 72°C or 1min, ollowed by a 10 min extension at 72°C.
Sequencing of the VP4 and VP7 gene
For each amplicons o VP4 and VP7, PCR was run at least twice and purified using the QIA quick Gel Extraction Kit (Qiagen, Germany) according to the instructions. Te purified products o the VP4 and VP7 genes were cloned using the PMD-18 vector (akara, Japan) and transormed into DH5α competent cells. Te plasmid DNAs sequenced using an ABI3730 Prism DNA analyzer.
Pathogenicity evaluation
An experimental inection was carried out in order to determine whether the isolated virus could cause clinical disease in newborn piglets or not. Six 3-day-old piglets, ree o rotavirus antibodies and antigens, were involved in the experimental inection. Piglets were transerred into isolators at Laboratory immediately, with three piglets in each isolator. Te temperature in the isolators was maintained at 32°C or higher using heat lamps. Piglets were ed pasteurized whole milk supplemented with probiotic once a day in the morning and regular milk three times a day and approximately 70mL milk each time.Te 6 piglets, either males or emales, were randomly assigned into two groups o 3 piglets. One group o piglets were orally received 5.0 ml o cell culture material o porcine rotavirus HN-001strain, equal to 105.0 CID50/ml. Te other group was orally received 5.0 ml phosphate buffered saline (PBS) per piglet as control. Afer inoculation, the piglets were closely observed every two hours a day or diarrhea and other gastrointestinal clinical signs. Piglets were humanely euthanized afer the appearance o severe diarrhea or becoming moribund. Te piglets were necropsied and intestinal material rom each piglet was collected and stored at -80°C or urther analysis. At end o the study, all the piglets were euthanized humanely.
Results
Virus isolation
o successully isolate the virus, a blind passage strategy was applied. Structural analysis o the inected cells by electron microscopy confirmed that the rotavirus virion was round with wheel-shape appearance. (Figure 1) Specific CPEs or rotavirus became visible in the MA-104 cells afer continuously eight blind passages. Significant CPEs were detected in cell culture at 24 hours afer 6 passages. Te characteristic CPE or rotavirus, such as cell round up, clustering in grape bunches, seining were observed (Figure 2). As the virus adapting to the MA-104 cell, the virus propagated rapidly and virus titer has reached to 105.0CID50/ml on day 5. Results rom the immunofluorescence assay revealed that the attached fluorophore could be detected in inected MA-104 cells, whereas none were detected in the control group.
Identication of the VP4 and VP7 genes
Te VP4 and VP7 genes were successully amplified by the R-
Figure 1:
Electron microscopy images of rotavirus-like particles of strain HN001 infecting the MA-104 cells in cell culture media.
Figure 2:
Cytopathic effects and Immunouorescence assay of PoRV isolate
A and C were set as control.B: At 24 h postinfection, the cytopathic effects were recorded as clustering, detachment.
B2: Cells were examined by IFA using PoRV-specic monoclonal antibody.
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PCR programs and revealed on agarose gel electrophoresis as the expected ragments, 762bp or VP4 gene and 1041bp or VP7 gene, respectively.
Sequencing and Phylogenetic analysis of the VP4 gene
Te amplified VP4 gene o HN-001 strain was sequenced and its deducted amino acid sequence were compared with known amino acid sequences in the Genebank (able 1) to urther determine the P genotype o the isolate [11]. Te similarity o amino acid sequence o VP4 gene o HN-001 strain to existing rotavirus strains ranges rom 2.7% to 92.1%, with the highest degree o amino acid identity ound in strain YM [12], belonged to genotype P[7]. Te lowest identiy was ound in the strain DEU, o genotype P[35] [13]. Blast analysis revealed that the strain HN-001 had a high homology with porcine rotavirus strain CRW-8 which belonged to P[7].Phylogenetic analysis o the deduced VP4 amino acid sequences indicated that strain HN-001 is closely related to strain YM and the two strains are grouped to orm a distinct cluster (Figure 3A). JS-
StrainGenBank accession No.SpeciesP genotype% Identity of VP4 geneSA11X14204Simian127.60%SA11-N5JQ688676.1Simian224.10%K9EU708926.1Canine324.90%DS-1AB848005.1Human454.80%P343U35851.1Porcine553.50%GottfriedM33516.1Porcine665%YMM63231.1Porcine792.10%DRC88DQ005111.1Human863.80%T152AB077766.1Human956.60%69MEF672556.1Human1072.30%I321L07657.1Bovine1158.40%FI-14D13398.1Equine1273.20%MDR-13L07886.1Porcine1365.60%B4106AY740738.1Human1473.10%Lp14L11599.1Lamb1555.10%EWU08429.1Murine1662.60%PO-13AB009632.2Avian1757.40%GBRJF712558.1Horse1855.30%Mc323D38052.1Human1963.50%EHPU08424.1Murine2065.20%Hg18AF237665.1Bovine2148.80%160-01AF526374.1Rabbit2251.30% A34AY174094.1Porcine2341.40%TUCHFJ816611.1Rhesus2468.40%DhakaGU199520.1Human2557.30%134-04DQ061053.1Porcine2662.30%344-04DQ242615.1Porcine2768%Ecu534EU805773.1Human2852.80% Azuk-1AB454420.1Bovine2937.20%DEU 03NC-021581.1Chicken302.80%03V0567JQ920006Chicken313.60%61-07-ireFJ492835.1Porcine3269.70%Dai-10AB513836.1Bovine3372.30%FGP51AB571047.1Porcine3475.10%DEUNC-021631.1Turkey352.70%SG3AB823215.1Glider3668.60%GERJX204814.1Pheasant3766.30%
Table 1:
Amino acid comparison (% aa identity) of the VP4 of the porcine strain HN-001 with other P genotypes.
Nucleotide Substitutions(x100)0387.450100150200250300350
61-07-ire P[32] FJ492835.1.seq134-04-15 P[26] DQ061053.1.seqHN-001.seqYM P[7] M63231.1.seqK9 P[3 ] EU708926.1.seqSA11-N5 P[2] JQ688676.1.seqEHP P[20] U08424.1.seqP343 P[5] U35851.1.seqFI-14 P[12] D13398.1.seqHg18 P[21] AF237665.1.seqLp14 P[15] L11599.1.seqEcu534 P[28] EU805773.1.seqB4106 P[14] AY740738.1.seqDhaka6 P[25] GU199520.1.seqT152 P[9] AB077766.1.seqEW P[16] U08429.1.seqGERP[37] JX204814.1.seqMDR-13 P[13] L07886.1.seqGottfried P[6] M33516.1.seqMc323 P[19] D38052.1.seqDRC88 P[8] DQ005111.1.seq344-04-1 P[27] DQ242615.1.seqI321 P[11] L07657.1.seq03V0567 P[31] JQ920006.1.seqDEU03 P[30] NC_021581.1.seqPO-13 P[17] AB009632.2.seqTUCH P[24] FJ816611.1.seqSG3 P[36] AB823215.1.seq69M P[10] EF672556.1.seqSA11g4OP[1] X14204.seqFGP51 P[34] AB571047.1.seqDai-10 P[33] AB513836.1.seq160-01 P[22] AF526374.1.seqDS-1 P[4] AB848005.1.seq A34 P[23] AY174094.1.seq Azuk-1 P[29] AB454420.1.seqDEU P[35] NC_021631.1.seq
Figure 3A:
Phylogenetic tree based on the partial VP4 sequence of Porcine rotavirus detected in Henan province.
Figure 3B:
Phylogenetic tree based on the partial VP7 sequence of Porcine rotavirus detected in Henan province.
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