• The Plant Pathology Journal
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• v.37 no.2
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• pp.162-172
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• 2021

Soybean mosaic virus (SMV) is the predominant viral pathogen that affects the yield and quality of soybean. The natural host range for SMV is very narrow, and generally limited to Leguminosae. However, we found that SMV can naturally infect Pinellia ternata and Atractylodes macrocephala. In order to clarify the molecular mechanisms underlying the cross-family infection of SMV, we used double-stranded RNA extraction, rapid amplification of cDNA ends polymerase chain reaction and Gibson assembly techniques to carry out SMV full-length genome amplification from susceptible soybeans and constructed an infectious cDNA clone for SMV. The genome of the SMV Shanxi isolate (SMV-SX) consists of 9,587 nt and encodes a polyprotein consisting of 3,067 aa. SMV-SX and SMV-XFQ008 had the highest nucleotide and amino acid sequence identities of 97.03% and 98.50%, respectively. A phylogenetic tree indicated that SMV-SX and SMV-XFQ018 were clustered together, sharing the closest relationship. We then constructed a pSMV-SX infectious cDNA clone by Gibson assembly technology and used this clone to inoculate soybean and Ailanthus altissima; the symptoms of these hosts were similar to those caused by the virus isolated from natural infected plant tissue. This method of construction not only makes up for the time-consuming and laborious defect of traditional methods used to construct infectious cDNA clones, but also avoids the toxicity of the Potyvirus special sequence to Escherichia coli, thus providing a useful cloning strategy for the construction of infectious cDNA clones for other viruses and laying down a foundation for the further investigation of SMV cross-family infection mechanisms.

• Journal of Animal Science and Technology
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• v.64 no.1
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• pp.123-134
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• 2022

Toll-like receptors (TLRs), as a part of innate immunity, plays an important role in detecting pathogenic molecular patterns (PAMPs) which are structural components or product of pathogens and initiate host defense systems or innate immunity. Precise negative feedback regulations of TLR signaling are important in maintaining homeostasis to prevent tissue damage by uncontrolled inflammation during innate immune responses. In this study, we identified and characterized the function of the pancreatic progenitor cell differentiation and proliferation factor (PPDPF) as a negative regulator for TLR signal-mediated inflammation in chicken. Bioinformatics analysis showed that the structure of chicken PPDPF evolutionarily conserved amino acid sequences with domains, i.e., SH3 binding sites and CDC-like kinase 2 (CLK2) binding sites, suggesting that relevant signaling pathways might contribute to suppression of inflammation. Our results showed that stimulation with polyinosinic:polycytidylic acids (Poly [I:C]), a synthetic agonist for TLR3 signaling, increased the mRNA expression of PPDPF in chicken fibroblasts DF-1 but not in chicken macrophage-like cells HD11. In addition, the expression of pro-inflammatory genes stimulated by Poly(I:C) were reduced in DF-1 cells which overexpress PPDPF. Future studies warrant to reveal the molecular mechanisms responsible for the anti-inflammatory capacity of PPDPF in chicken as well as a potential target for controlling viral resistance.

• The Plant Pathology Journal
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• v.38 no.5
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• pp.533-540
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• 2022

Thunberg fritillary (Fritillaria thunbergii), a perennial used in traditional Chinese herbal medicine, is a members of the family Liliaceae. The degeneration of germplasm is a severe problem in the production of Fritillaria thunbergii var. chekiangensis. However, no information about viral infections of F. thunbergii var. chekiangensis has been reported. In this study, we sequenced the small RNAs of F. thunbergii var. chekiangensis from leaves and bulbs, and viruses were identified using a phylogenetic analysis and BLAST search for sequence. In addition, multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) was used to rapidly detect viruses in this variety. Our study first reported that five viruses infected F. thunbergii var. chekiangensis. Among them, fritillary virus Y (FVY), lily mottle virus (LMoV), Thunberg fritillary mosaic virus (TFMV), and hop yellow virus (HYV) had been reported in F. thunbergii, while apple stem grooving virus was first reported in the genus Fritillaria. A multiplex RT-PCR method was developed to rapidly test the four viruses FVY, LMoV, TFMV, and HYV in F. thunbergii var. chekiangensis. Our results provide a better understanding of the infection of F. thunbergii var. chekiangensis by viruses and a basic reference for the better design of suitable control measures.

• IMMUNE NETWORK
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• v.22 no.6
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• pp.48.1-48.13
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• 2022

With the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, which are randomly mutated, the dominant strains in regions are changing globally. The development of preclinical animal models is imperative to validate vaccines and therapeutics against SARS-CoV-2 variants. The objective of this study was to develop a non-human primate (NHP) model for SARS-CoV-2 Delta variant infection. Cynomolgus macaques infected with Delta variants showed infectious viruses and viral RNA in the upper (nasal and throat) and lower respiratory (lung) tracts during the acute phase of infection. After 3 days of infection, lesions consistent with diffuse alveolar damage were observed in the lungs. For cellular immune responses, all macaques displayed transient lymphopenia and neutrophilia in the early stages of infection. SARS-CoV-2 Delta variant spike protein-specific IgM, IgG, and IgA levels were significantly increased in the plasma of these animals 14 days after infection. This new NHP Delta variant infection model can be used for comparative analysis of the difference in severity between SARS-CoV-2 variants of concern and may be useful in the efficacy evaluation of vaccines and universal therapeutic drugs for mutations.

• The Korean Journal of Pesticide Science
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• v.15 no.4
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• pp.545-572
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• 2011

The genetic diagnosis methods by RT-PCR and Virion capture (VC)/RT-PCR against Rice stripe virus (RSV) were developed. Three diagnosis methods of seedling test, ELISA and RT-PCR were compared in virus detection sensitivity (VDS) for RSV. The VDS of ELISA for RSV viruliferous small brown plant hopper (SBPH) was higher with 40.5% than that of seedling test. The VDS of RT-PCR was higher with 21% than that of ELISA. The VDS of ELISA and VC/RT-PCR was same with 9.2% in average on the SBPH collected from fields at the areas of Gimpo, Pyungtaeg and Sihueng, Gyeonggi province in 2009. The specific primers of RSV for SBPH and rice plant were developed for the diagnosis by Real time PCR. The RQ value of Real time PCR for the viruliferous and non viruliferous SBPH was 1 for 50 heads of non viruliferous SBPH, 96.5 for 50 heads of viruliferous SBPH, 23.1 for 10 heads of viruliferous SBPH + 40 heads of non viruliferous SBPH, and 75.6 for 30 heads of viruliferous SBPH + 20 heads of non viruliferous SBPH. The RQ value was increased positively by the ratio of viruliferous SBPH. Full sequences of 4 genomes of RSV RNA1, RNA2, RNA3 and RNA4 were analysed for the 13 RSV isolates from rice plants collected from different areas. Genetic relationships among the RSV isolates of Korea, Japan and China were classified as China + Korea, and China + Korea + Japan by phylogenetic analysis for RSV RNA1 and RNA2. In case of RNA3 involved in pathogenicity, genetic relationship of RSV among the three countries was grouped into 3 as China, China + Korea, and Korea + Japan. According to the genetic relationships in RSV RNA4, RSV isolates were grouped into 4 as China, Korea, China + Korea + Japan, and Korea + Japan. Viruliferous insect rate (VIR) of RSV in average increased in each year from 2008 to 2010, and the rates were 4.3%, 6.1%, and 7.2%, respectively, at the 28 major rice production areas in 7 provinces including Gyeonggido. The highest VIR in each year was 11.3% of Gyeonggido in 2008, 20.1% of Jellanamdo in 2009 and 14.2% of Chungcheongbukdo in 2010. The highest VIR depending upon the investigated areas was 22.1% at Buan of Jellabukdo in 2008, 36% at Wando and Jindo of Jellanamdo in 2009, and 30.0% at Boeun of Chungcheongbukdo in 2010. Average population density (APD) of overwintered SBPH was 13.1 heads in 2008, 13.9 heads in 2009 and 5.6 heads in 2010. The highest APD was 39.1 and 60.4 heads at Buan of Jellabukdo in 2008 and 2009, respectively, and 14.0 heads at Pyungtaeg of Gyeonggido. The acreage of RSV occurred fields was 869 ha in the western and southern parts, mainly at Jindo and Wando areas, of Jellanamdo in 2008. In 2009, RSV occurred in the acreage of 21,541 ha covered whole country, especially, partial and whole plant death were occurred with infection rate of 55.2% at 3,025 plots in 53 Li, 39 Eup/Myun, 19 Si/Gun of Gyeonggido, Incheonsi, Chungcheongnamdo, Jeollabukdo and Jeollanamdo. Seasonal development of overwintered SBPH was investigated at Buan, Jeollabukdo, and Jindo, Jeollanamdo for 3 years from 2008. Most SBPH developed to the 3rd and 4th instar on the periods of May 20 to June 10, and they developed to the adult stage for the 1st generation on Mid and Late June. In 2009, all SBPH trapped by sky net trap were adult on May 31 to June 1 at Mid-western aeas of Taean, Seosan and Buan, and South-western areas of Sinan and Jindo. The population density of adult SBPH was 963 heads at Taean, 919 at Seocheon and 819 at Sinan area. The origin of these higher population of adult SBPH were verified from the population of non-overwintered SBPH but immigrant SBPH. From Mid May to Mid June in 2010, adult SBPH could not be counted as immigrant insects by sky net trap. The variation of RSV VIR was high with 2.1% to 9.5% for immigrant adult SBPH trapped by sky net trap at Hongsung of Chungcheongbukdo, Buan of Jeollabukdo and so forth in 2009. The highest VIR for the immigrant adult SBPH was 9.5% at Boryung of Chungcheongnamdo, followed by 7.9% at Hongsung of Chungcheongnamdo, 6.5% at Younggwang of Jeollanamdo, and 6.4% at Taean of Cheongcheongnamdo. The infection rate of RSV on rice plants induced by the immigrant adult SBPH cultivated near sky net trap after about 10 days from immigration on June 12 in 2009 was 84.6% at Taean, 65.4% at Buan and 92.9% at Jindo, and 81% in average through genetic diagnosis of RT-PCR. Barley known as a overwintering host plant of RSV had very low infection rate of 0.2% from 530 specimens collected at 10 areas covering whole country including Pyungtaeg of Gyeonggido. Twenty nine plant species were newly recorded as natural hosts of RSV. In winter annual plant species, 11 plants including Vulpia myuros showed RSV infection rate of 24.9%. The plant species in summer annual ecotype were 13 including Digitaria ciliaris with 44.9%, Echinochloa crusgalli var. echinata with 95.2% and Setaria faberi with 65.5% in infection rate of RSV. Five perennial plants including Miscanths sacchariflorus with infection rate of 33.3% were recorded as hosts of RSV. Rice cultivars, 8 susceptible cultivars including Donggin1 and 17 resistant ones including Samgwang, were screened in field conditions at 3 different areas of Buan, Iksan and Ginje in 2009. All the susceptible cultivars were showed typical symptom of mosaic and wilt. In 17 genetic resistant cultivar, 12 cultivars were susceptible, however, 5 cultivars were field-resistant plus genetic resistant to RSV as non symptom expression. When RSV was artificially inoculated at seedling stage to 4 cultivars known as genetic resistant and 3 cultivars known as genetic susceptible, the symptom expression in resistant cultivars was lower as 19.3% in average than that of 53.3% in susceptible ones. In comparison of symptom expression rate and viral infection rate using resistant Nampyung and susceptible Heugnam cultivars by artificial inoculation of RSV at seedling stage, the symptom expression of Heugnam was higher as 28% than 12% of Nampyung. However, virion infection of resistant Nampyung cultivar was higher as 12% reversely than 85% of susceptible Heugnam. Yield loss of rice was investigated by the artificial inoculation of RSV at the seedling stage of resistant cultivars of Nampyung and Onnuri, and susceptible cultivars of Donggin1 and Ungwang for 3 years from 2008. The average yield per plant was 7.8 g, 8.5 g and 13.8 g on rice plants inoculated at seedling stage, tillering stage and maximum tillering stage, respectively. The yield loss rate was increased by earlier infection of RSV with 51% at seedling stage, 46% at tillering stage and 13% at maximum tillering stage. In resistant rice cultivars, there was no statistically significant relation between infection time and yield loss. In natural fields on susceptible rice cultivar of Ungwang at Taean and Jindo areas in 2009, the yield loss rate was increased with same tendency to the infection hill rate having the corelation coefficient of 0.94 when the viral infection was over 23.4%.

• Korean Journal of Microbiology
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• v.44 no.3
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• pp.228-236
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• 2008

Validation of viral safety is essential in ensuring the safety of mammalian cell culture-derived biopharmaceuticals, because numerous adventitious viruses have been contaminated during the manufacture of the products. Mammalian cells are highly susceptible to Reovirus type 3 (Reo-3), and there are several reports of Reo-3 contamination during the manufacture of biopharmaceuticals. In order to establish the validation system for the Reo-3 safety, a real-time RT-PCR method was developed for quantitative detection of Reo-3 in cell lines, raw materials, manufacturing processes, and final products as well as Reo-3 clearance validation. Specific primers for amplification of Reo-3 RNA was selected, and Reo-3 RNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be $3.2{\times}10^0\;TCID_{50}/ml$. The real-time RT-PCR method was proven to be reproducible and very specific to Reo-3. The established real-time RT-PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with Reo-3. Reo-3 RNA could be quantified in CHO cell as well as culture supernatant. When the real-time RT-PCR assay was applied to the validation of virus removal during a virus filtration process, the result was similar to that of virus infectivity assay. Therefore, it was concluded that this rapid, specific, sensitive, and robust assay could replace infectivity assay for detection and clearance validation of Reo-3.

• Research in Plant Disease
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• v.12 no.3
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• pp.298-302
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• 2006

An isolate of Cucumber mosaic virus(CMV), called as Can-CMV, was originally isolated from Canna generalis showing typical streak mosaic foliar symptoms, and its properties were investigated in this study. Whereas all known isolates of CMV could induce symptoms on their systemic hosts(four kinds of Nicotiana spp and a zucchini squash), Can-CMV induced no symptoms on its systemic hosts tested. Replication and movement of the virus on upper leaves as well as inoculated leaves-were confirmed by RT-PCR suggesting that Can-CMV could only infect systemically on N. benthamiana and N. glutinosa. Size of local lesions on the Can-CMV-inoculated leaves of Chenopodium amaranticolor was much smaller than that of Fny-CMV. Whereas Fny-CMV and LS-CMV could induce distinct necrotic local lesions on Vigna unguiculata 2 to 3 days postinoculation(dpi), chlorotic spots symptom was expressed by Can-CMV 4 to 5 dpi. Virus-specific 4 kinds of dsRNAs were isolated from leaves of N. benthamiana infected with Can-CMV, and these dsRNAs corresponded to the viral genomic RNAs and subgenomic RNAs and their patterns were indistinguishable to those of Fny-CMV and LS-CMV. By restriction mapping analysis of 950 bp of RT-PCR amplified products of coat protein gene of the virus as well as by serological analysis of gel diffusion test, Can-CMV belongs to a typical member of CMV subgroup IA. These results suggest that the Can-CMV isolated from C. generalis possesses unique pathological properties to understand further insight into the various interactions between virus and host.

• Journal of Life Science
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• v.33 no.6
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• pp.498-505
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• 2023

Solanum tuberosum Linnaeus cv Hongyoung, which represents red potato, was developed in Korea. Hongyoung is known to have anti-oxidant, anti-inflammatory, anti-viral, and anti-tumor properties, but no research has been conducted on the growth inhibition and apoptosis effects of hongyoung in YD-10B oral cancer cells. In this study, the combined treatment of hongyoung ethanol extract (HEE) and cisplatin were examined to determine its ability to inhibit cancer cell growth, induce apoptosis, and inhibit matrix metalloproteinases (MMP)-2 and MMP-9 cancer metastasis. The cell viability was investigated using a 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H- tetrazolium monosodium salt (MTS) assay, and the ability to induce apoptosis was analyzed using an FACS analyzer. The mRNA expression and protein activity of MMP-2 and MMP-9 were measured via RT-PCR and zymography. The YD-10B oral cancer cells showed an increase in growth inhibition as the concentration of HEE increased. The combination of 200 µM cisplatin and 500 ㎍/ml HEE reduced the growth of the YD-10B oral cancer cells by more than 50% compared to cisplatin alone. When phorbol 12-myristate 13-acetate (PMA)-treated YD-10B oral cancer cells were co-treated with 200 µM cisplatin and 500 ㎍/ml HEE, both the mRNA expression and protein activity of the MMP-2 and MMP-9 decreased. In addition, the percentage of the sub-G1 phase, which indicates apoptosis ability, more than doubled when treated in combination with 200 µM cisplatin and 500 ㎍/ml HEE than when cisplatin alone was used. The results of this study therefore suggest the possibility of using a combination of HEE and cisplatin in the development of effective drugs to treat oral cancer.

• Research in Plant Disease
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• v.12 no.2
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• pp.139-143
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• 2006

Virion captured reverse transcription polymerase chain reaction (VC/RT-PCR) could detect plant virus quickly and accurately. In the VC/RT-PCR, no antibody is needed unlike immuno-captured RT-PCR (IC/RT-PCR) which had been improved method of RT-PCR for plant viruses, and virus nucleic acids can be obtained easily within 30minutes by property of polypropylene PCR tube which is hold and immobilized viral particles on its surface. For the virion capture of Tomato spotted wilt virus (TSWV), the extraction buffer was tested. The optimum macerating buffer for TSWV was 0.01M potassium phosphate buffer, pH 7.0, containing 0.5% sodium sulfite. The viral crude sap was incubated for 30 min at $4^{\circ}C$. The virions in the PCR tubes were washed two times with 0.01M PBS containing 0.05% Tween-20. The washed virions were treated at $95^{\circ}C$ immediately for 1 min containing RNase free water and chilled quickly in the ice. Disclosed virions' RNAs by heat treatment were used for RT-PCR. Dilution end point of $10^{-5}$ from plant's crude sap infected with TSWV showed relatively higher detection sensitivity for VC/RT-PCR. During multiple detection using two or more primers, interference was arisen by interactions between primer-primer and plant species. The result of multiplex RT-PCR was influenced by combinations of primers and the kind of plant, and the optimum extraction buffer for the multiplex detection by VC/RT-PCR should be developed.

• IMMUNE NETWORK
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• v.1 no.1
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• pp.77-86
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• 2001

Background: Hepatitis C virus(HCV), a family of Flaviviridae, has a host cell-derived envelope containing a positive-stranded RNA genome, and has been known as the maj or etiological agent for chronic hepatitis, hepatic cirrhosis, and hepatocellular carcinoma. There remains a need to dissect a molecular mechanism of pathogenesis for the development of therapeutic and effective preventive measure for HCV. Identification of cellular receptor is of central importance not only to understand the viral pathogenesis, but also to exploit strategies for prevention of HCV. This study was aimed at identifying peptide mimotopes inhibiting the binding of E2 protein of HCV to MOLT-4 cell. Methods: In this study, phage peptide library displaying a random peptides consisting of 7 or 12 random peptides was employed in order to pan against E2 protein. Free HCV particles were separated from the immune complex forms by immunoprecipitation using anti-human IgG antibody, and used for HCV-capture ELISA. To identify the peptides inhibiting E2-binding to MOLT-4 cells, E2 protein was subj ect to bind to MOLT-4 cells under the competition with phage peptides. Results: Several phage peptides were selected for their specific binding to E2 protein, which showed the conserved sequence of SHFWRAP from 3 different peptide sequences. They were also able to recognize the HCV particles in the sera of HCV patients captured by monoclonal antibody against E2 protein. Two of them, showing peptide sequence of HLGPWMSHWFQR and WAPPLERSSLFY respectively, were revealed to inhibit the binding of E2 protein to MOLT-4 cell efficiently in dose dependent mode. However, few membrane-associated receptor candidates were seen using Fasta3 programe for homology search with these peptides. Conclusion: Phage peptides containing HLGPWMSHWFQR and WAPPLERSSLFY respectively, showed the inhibition of E2-binding to MOLT-4 cells. However, they did not reveal any homologues to cellular receptors from GenBank database. In further study, cellular receptor could be identified through the screening of cDNA library from MOLT-4 or hepatocytes using antibodies against these peptide mimotopes.