Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 4 de 4
Filter
1.
Journal of South China Agricultural University ; 41(5):27-35, 2020.
Article in Chinese | CAB Abstracts | ID: covidwho-2040361

ABSTRACT

Objective: To prepare monoclonal antibodies against porcine epidemic diarrhea virus (PEDV) N protein, and develop an indirect immuno-fluorescence assay method used for detecting PEDV. Method: The expressed recombinantly PEDV N protein was used as an immunogen and 8-week-old female BALB/c mice were immunized. Then their spleen cells with high antibody titer were isolated and fused with SP2/0 cells. The hybridoma cell lines secreting monoclonal antibodies against PEDV N protein were screened. In Vero cells infected with PEDV, monoclonal antibody of anti-PEDV N protein was used as the primary antibody and FITC-goat-anti-mouse IgG was used as the secondary antibody to develop indirect immuno-fluorescence assay method used for detecting PEDV. Result: The prepared hybridoma cell lines could stably secrete anti-PEDV N protein antibodies, ELISA antibody titer in cell supernatant was above 1:3 200, and in mouse ascites above 1:1 000 000. While monoclonal antibodies were applied in established indirect immuno-fluorescence assay, the optimal conditions were that cells were fixed with 80% () acetone at -20 degrees C for 30 min;The primary antibody was diluted 1 000 times by PBS buffer solution and incubated at 4 degrees C overnight;The secondary antibody was diluted 100 times by PBS buffer solution and incubated at 37 degrees C for 1 h. Transmissible gastroenteritis virus (TGEV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine reproductive virus (PRV), porcine enteric a corone virus (PEAV), porcine rotavirus (PoRV) and PEDV were detected by established indirect immuno-fluorescence assay method, only PEDV showed positive, all the else viruses showed negative.

2.
Acta Veterinaria et Zootechnica Sinica ; 53(6):2024-2028, 2022.
Article in Chinese | CAB Abstracts | ID: covidwho-2025545

ABSTRACT

This study aimed to analyze the proliferation characteristics of porcine deltacoronavirus (PDCoV) in suspension cultured porcine kidney cells LLC-PK1, so as to provide Candidate cell for large-scale production of PDCoV inactivated vaccine. LLC-PK1 cells were suspended by gradually decreasing serum method. PDCoV adaptive monoclonal cell lines were screened by limited dilution method. Indirect immunofluorescence method was used to identify the infectivity of PDCoV. The initial cell density, MOI, time of receiving virus collection and TPCK pancreatin concentration were screened to determine the best suspension culture conditions. The suspension cell strain LLC-PK1Sa which can proliferate PDCoV efficiently was screened out;PDCoV can specifically infect LLC-PK1 cells;PDCoV inoculated LLC-PK1Sa cells with a density of 2 x 106 cells.mL-1 according to the MOI of 10-3, When the final concentration of TPCK pancreatin reached 7.5 g.mL-1, the titer of virus solution harvested 48 h after inoculation was the highest. In this study, the efficient proliferation of PDCoV in LLC-PK1Sa suspension cells was realized for the first time, and the suspension culture conditions were preliminarily optimized, which could provide theoretical reference for large-scale production of PDCoV inactivated vaccine.

3.
Wiener Tierarztliche Monatsschrift ; 109(Artikel 11), 2022.
Article in English | CAB Abstracts | ID: covidwho-2025202

ABSTRACT

We have evaluated the diagnostic performance of immunochromatographic point-of-care tests (POCT) for the detection of rotavirus, coronavirus, Escherichia (E.) coli F5, Cryptosporidium (C.) parvum, Clostridium (Cl.) perfringens and Giardia (G.) intestinalis in fresh and thawed faecal samples from calves aged up to six months with diarrhoea. We performed POCTs to detect rotavirus, coronavirus, E. coli F5, C. parvum, Cl. perfringens and G. intestinalis on fresh samples in a field study and re-evaluated the performance for C. parvum, Cl. perfringens and G. intestinalis using thawed samples. We calculated the performance based on the results of the reference methods, which were RT-qPCR for the detection of rota- and coronavirus and bacteriological culturing and PCR to detect E. coli F5 and Cl. perfringens a and ss2 toxins. C. parvum was detected by phase-contrast microscopy and G. intestinalis by immunofluorescence microscopy. We collected 177 faecal samples from diarrhoeic calves. We found good performance for the POCT targeting rotavirus (sensitivity (SE)=92.9%;specificity (SP)=95.6%) and C. parvum (SE=63.3%;SP=96.2%). For E. coli F5, the number of true positive samples (n=1) was too low to evaluate the performance. The POCT to detect coronavirus gave a poor performance (SE=3.3%;SP=96.6%) and the POCT to detect Cl. perfringens a moderate performance (SE=52.8%;SP=78.2%). G. intestinalis POCT showed a higher sensitivity to immunofluorescence microscopy in thawed than in fresh faecal samples (SE=43.9% versus SE=29.2%). There are substantial differences in diagnostic performance between the commercially available immunochromatographic POCTs. Still, POCT can make a valuable contribution to the diagnosis and prevention of calf diarrhoea.

4.
Acta Microbiologica Sinica ; 2:672-685, 2022.
Article in Chinese | CAB Abstracts | ID: covidwho-1841702

ABSTRACT

[Objective] To explore whether porcine deltacoronavirus (PDCoV) can infect and proliferate in different animal species-derived cell lines. [Methods] The Sichuan isolate CHN-SC2015of PDCoV was inoculated in twelve cell lines derived from hamster,poultry,monkey, human and swine. After at least five blindly passages in each cell line, the virus was identified by RT-PCR,RT-q PCR, indirect immunofluorescence assay (IFA), and sequencing. [Results] PDCoV caused distinct cytopathic effect (CPE) in Vero,PAM,PK15,ST, and LLC-PK1 cells at the 1st passage (P1) and proliferated to various degrees in PAM,PK15,ST, and LLC-PK1 cells, while the CPE gradually disappeared during subsequent passages in Vero and PAM cells. Except that in the three susceptible cell lines (PK15,LLC-PK1, and ST), the viral copies of the infected cell lines gradually decreased with the increase in passages, and PDCoV could not be detected at P4 or P5 of DEF,Marc-145,HEK-293,ZYM-SIEC02, and PAM cells. PCR results showed that PDCoV could be detected only in CEF and Vero cells at P5. The IFA results showed that PDCoV could infect other cell lines except BHK-21 and ZYM-SIEC02, and specific immunofluorescence was observed in PK15,LLC-PK1, and ST cells at P1,P3, and P9. Therefore, only three cell lines (PK15,LLC-PK1, and ST) were suitable for serial passage, with the virus titers up to 107.11,107.00, and 107.37 TCID50/mL at P9,respectively. After passage in different cell lines,CHN-SC2015 accumulated 14 nucleotide mutations corresponding to 12 amino acid mutations. [Conclusion] This study indicates that PDCoV can infect a variety of cells in vitro, suggesting that it may have the potential of cross-species transmission.

SELECTION OF CITATIONS
SEARCH DETAIL