• Journal of Microbiology and Biotechnology
• /
• v.34 no.4
• /
• pp.804-811
• /
• 2024

Foamy viruses (FVs) are generally recognized as non-pathogenic, often causing asymptomatic or mild symptoms in infections. Leveraging these unique characteristics, FV vectors hold significant promise for applications in gene therapy. This study introduces a novel platform technology using a pseudo-virus with single-round infectivity. In contrast to previous vector approaches, we developed a technique employing only two vectors, pcHFV lacking Env and pCMV-Env, to introduce the desired genes into target cells. Our investigation demonstrated the efficacy of the prototype foamy virus (PFV) dual-vector system in producing viruses and delivering transgenes into host cells. To optimize viral production, we incorporated the codon-optimized Env (optEnv) gene in pCMV-Env and the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) at the 3' end of the transgene in the transfer vector. Consequently, the use of optEnv led to a significant enhancement in transgene expression in host cells. Additionally, the WPRE exhibited an enhancing effect. Furthermore, the introduced EGFP transgene was present in host cells for a month. In an effort to expand transgene capacity, we further streamlined the viral vector, anticipating the delivery of approximately 4.3 kbp of genes through our PFV dual-vector system. This study underscores the potential of PFVs as an alternative to lentiviruses or other retroviruses in the realm of gene therapy.

• The Plant Pathology Journal
• /
• v.40 no.4
• /
• pp.390-398
• /
• 2024

The Chinese artichoke (Stachys affinis syn. S. sieboldii) is a widely cultivated crop, and its rhizome is used as a medicinal vegetable. To investigate the causes of viral diseases in Chinese artichokes, the infection rates of four virus species infecting Chinese artichoke were investigated. Since the Chinese artichoke propagates through its tuber, this study aimed to determine whether viral transmission to the progeny is possible through the tuber, by identifying the virus present in the tuber and investigating its accumulation. First, reverse transcription polymerase chain reaction analysis was performed to detect viruses using total RNA extracted from the flowers, leaves, and tubers of Chinese artichoke plants. Alfalfa mosaic virus (AMV) and Chinese artichoke mosaic virus (ChAMV) had high infectivity in Chinese artichoke and most plants were simultaneously infected with AMV and ChAMV. These viruses were present in all tissues, but their detection frequency and accumulation rates varied across different tissues of the Chinese artichoke. Also, we sequenced the coat protein (CP) genes of AMV and ChAMV to investigate genetic variations of virus between the leaf and tuber. It provides information on CP gene sequences and genetic diversity of isolates identified from new hosts of AMV and ChAMV. This study offers valuable insights into the distribution and spread of the ChAMV and AMV within Chinese artichoke plants, which have implications for the management and control of viral infections in crops.

• Parasites, Hosts and Diseases
• /
• v.24 no.2
• /
• pp.109-114
• /
• 1986

In order to observe the growth and development of Fibricola seoulensis metacercariae, the tadpoles of Rana nigromaculata were experimentally infected with the cercariae. The meta cercariae of various developmental stages were recovered from the tadpoles after 2 to 65 days of infection. They were prepared for morphological observation, and were given orally to mice to observe their infectivity. The following results were obtained. 1. All of the tadpoles exposed to the cercariae were observed to harbour the larvae in their abdominal cavity. 2. The young metacercariae of 2 days after infection were $121.1{\mu}m$ long and $63.3{\mu}m$ wide. They grew linearly for the first 14 days to be $262.0{\mu}m$ long and $166.4{\mu}m$ wide. Thereafter, no more growth recognized until 65 days. 3. The larvae of 2 days old were similar with cercarial body and had 2 suckers, a pharynx, 2 ceca and a primordium of germ cells but no tribocytic organ. On the 8th day, they had tribocytic organ, and their morphology resembled that of mature metacercariae. 4. The metacercariae younger than 10 days could not infect the mice. Only the metacercariae older than 14 days had infectivity. The recovery rates increased by the age of metacercariae from 19.0% in 14 days old to 70.0% in 40 days old. Above findings indicate that the tadpole is indispensable for metacercarial development and it needs at least 2 weeks for maturation. The tadpole is a pivotal host in the life cycle of F. seoulensis for connection between the snail and the frog.

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

• Parasites, Hosts and Diseases
• /
• v.33 no.4
• /
• pp.297-304
• /
• 1995

To evaluate the feasibility of irradiation as a control measure for metagonimiasis, the metacercariae of Metagonimus yokogcwni were irradiated with gamma ray, either after isolation from the sweetfish (Plecoglossus cltivelis) or in situ of the fish, and their survival and development in rats were observed at 7 days post-infection. The radiation dose varied from 5 to 100 Gy for the metacercaria-irradiation group and from 5 to 500 Gy for fish-irradiation group. The results showed that the worm recovery rate from the irradiation groups decreased as the radiation dose was increased. Higher doses of radiation were required for the fish-irradiation group to obtain the same results as the metacercaria-irradiation group. The LD50 of the metacercaria-irradiation group was 4.5 Gy, whereas that of the fish-irradiation group 6.2 Gy A few number of worms which survived until 7 days in rats were severely retarded especially in the growth of their reproductive organs, j.e., complete or partial failure in the development of testes and formation of uterine eggs . The present study revealed that irradiation of sweetfish by 200 Gy is effective to control infectivity as well as development of M. vokogawai metacercariae in rats.

• ALGAE
• /
• v.22 no.4
• /
• pp.287-295
• /
• 2007

Infection of free-living dinoflagellates by endoparasitic dinoflagellates of the genus Amoebophrya are thought to have significant impacts on host population dynamics and have long been proposed to be a potential biological agent for controlling harmful algal bloom (HAB). To understand the impact of Amoebophrya on particular host species, however, it is necessary to quantify aspects the parasites life cycle. Here we used cultures of Amoebophryahost systems from Jinhae Bay, Korea to determine, parasite generation time, and dinospore survival and infectivity. The proportion of host cells infected by Amoebophrya sp. changed sharply from 5% to 87% with increasing dinospore:host inoculation ratios. In the absence of H. triquetra, most free-living dinospores died within 72 hours and their ability to infect host cells decreased remarkably in a day. The relatively short free-living phase of Amoebophrya suggests that the spread of infections is most likely to occur during seasons of high host abundance, as that is when dinospores have the greatest chance of encountering host cells. Infection of host cells inoculated with dinospores during the day was higher than when inoculated during the night, suggesting that infection rates might be related to environmental light conditions and/or diurnal biological rhythm of host species. Total generation times of parasite strains from a thecate dinoflagellate Heterocapsa triquetra were nearly the same regardless of dinospore:host inoculation ratios, representing 54 ± 0.5 h in a 1:1 ratio and 55 ± 1.2 h in a 20:1 ratio. Dinospore production of Amoebophrya sp. infecting Heterocapsa triquetra was estimated to be 125 dinospores per a strain of Amoebophrya sp. There is a growing need to maintain a variety of host-parasite systems in culture and to examine their autecology under various environmental conditions. Such studies would be very helpful in understanding ecological role of these parasites, their overlooked importance in the flow of material and energy in marine ecosystem, and their practical use as biological control agents applied directly to areas affected by HAB.

• Research in Plant Disease
• /
• v.16 no.3
• /
• pp.247-253
• /
• 2010

Apple scar skin viroid (ASSVd) is one of the smallest viral pathogens infecting fruits, especially apple, and causes a significant damage to fruit trees. ASSVd usually induced the skin-dapple ring symptoms, but in 'Fuji' varieties, corked spot were occurred on the fruit skin in 2009. This new symptom will be of great helpful to diagnosis ASSVd in sight. ASSVd was surveyed in apple and pear from 2009 to 2010 in Korea, and ASSVd was identified in 20 out of 1,193 trees. The infection rate was 1.7%. To screen the infectivity of ASSVd among apple cultivars, real-time RT-PCR was applied followed by designing of ASSVd specific primers based on highly conserved regions of several ASSVd isolates including Korean isolate. NADH dehydrogenase subunit 5 (nad 5) gene, which is mRNA of the mitochondrial gene, was used for internal control. In this study, ASSVd infected apples were classified into 12 groups depending on different symptoms and symptom severity (scaring, rusting or malformation). Taken together, this study suggested that real-time PCR analysis was more sensitive to detect the low copy of ASSVd on early viroid infected apple skins than regular RT-PCR method.

• Parasites, Hosts and Diseases
• /
• v.30 no.4
• /
• pp.237-244
• /
• 1992

In order to observe the infectivity, growth and development and adult morphology of Metorchis orientalis, a total of 40 chicks were experimentally infected with 100 metacercariae respectively, collected from Pseuderasbora larva. The worms of various developmental stages were recovered from chicks at 1.5, 3, 5, 7, 9, 11, 1:k and 21 days after infection, and they were prepared for morphological observations and measurements. All of the worms were found in the gallbladders of chicks, and their recovery rate was 32% in average. The growth of the body was rapid from 9 to 11 days after infection. The genital primordia appeared in 1.5 and 3-day old worms, and ovary and testes were first observed in 5-day old worms. Thereafter, genital organs gradually matured and completed up to 11 days after infection. The adult worm was leaf-like, and possessed a con- voluted tubular seminal vesicle, an ovoid ovary, a sac-like seminal receptacle, 2 lobed-testes and follicular vitellaria. Eggs were $31.9{\times}15.3{\;}\mu\textrm{m}$ in average size, ellipsoid to elliptical in shape and possessed abopercular thickenings. From the above results, it is concluded that M. orientalis grows in sigmoid pattern in chicks, and their genital organs fully matul.e between days 9 and 11. It is also confirmed that a chick is a new definitive host of M. orientalis.

• Korean Journal of Veterinary Research
• /
• v.34 no.3
• /
• pp.517-527
• /
• 1994

An attempt was made to isolate a causative viral agents from the fecal specimens of the diseased dogs with the gastroenteritis symptoms. Two coronavirus-like agents were isolated by serial dilution end point method and plaque assay. The isolates were characterized in terms of cytopathology, antigenicity, replication, physicochemical and morphological properties. The results obtained through the experiment were as follows; 1. Among 7 fecal specimens collected from the dogs with enteric disease, 2(28.6%)coronavirus-like agents showing typical cytopathic effects of canine coronavirus were isolated, and designated as CCV D1 and CCV D2, respectively. 2. By the cross-neutralization test and indirect immunofluoresence antibody test, the isolates were antigenically indentified as the standard CCV. The viruses were replicated only in the cytoplasm of A-72 cells. 3. The isolates showed no haemagglutinating activity against the erythrocytes from 11 kinds of animals. 4. The electron microscopic observation for the isolates showed spherical and pleomorphic features, covered with club-shaped projections on the surface. The size of particles was ranged from 70 to 150nm. 5. In one-step growth curve for the isolates in A-72 cells, maximum titers of intracellular vius was $10^{4.6}$ $TCID_{50}/0.1ml$ at 46 hrs postinoculation(pi) of CCV Dl and $10^{4.4}$ $TCID_{50}/0.1ml$ at 34 hrs pi of CCV D2. The maximum titers of extracellular virus was $10^{5.5}$ $TCID_{50}/0.1ml$ at 58 hrs pi of CCV D1 and $10^{5.8}$ $TCID_{50}/0.1ml$ at 46 hrs pi of CCV D2. 6. In physicochemical property test, the isolates were very sensitive to choroform and were found to be RNA virus. The viruses was stable at pH 3.0 for 1 hr and at $22{^{\circ}C}$ for 5 hrs. However, infectivity titers reduced remarkably by treatment with $56{^{\circ}C}$ for 10min.

• Journal of Sericultural and Entomological Science
• /
• v.34 no.2
• /
• pp.26-31
• /
• 1992

Flacherie virus (FV) and Densonucleosis virus (DNV) of the silkworm, Bombyx mori, which give the most severest damage to the silk production in korea, were fed on the mulberry wild silkworm, Bombyx mori mandarina, the mulberry pyralid, Gryphodes phyloalis, and the American fall webworm, Hypantria cunea, to investigate cross infectivity by serological and histopathological at observation. By the Ouchterlony's double difusion test the mulberry wild silkworm was infected with both FV and DNV type 1 (DNV-1) and the mulberry pyralid with DNV-1, so those were confirmed the cross infection. But the American fall webworm was not recognized the cross infection by the same method. The infection and multiplication of the FV in the mulberry wild silkworm was observed in the cytoplasm of the goblet cell with the appearance of the virus-specific vesicle. In DNV-1 infection to the mulberry wild silkworm and the mulberry pyralid, the nuclei of columnar cell in the midgut of both insects was hypertrophied and the nuclei of midgut cell of the mulberry pyralid positively stained with the feulgen stain. Multiplication of DNV-1 in the midgut cell of the mulberry wild silkworm was replicated in two different patterens as linear arrays and large masses, while that of DNV-1 in the muberry pyralid was multiplied as virus masses in several portion of the nuclei of the midgut cell.