Middle East Respiratory Syndrome in 3 Persons, South Korea, 2015.

In May 2015, Middle East respiratory syndrome coronavirus infection was laboratory confirmed in South Korea. Patients were a man who had visited the Middle East, his wife, and a man who shared a hospital room with the index patient. Rapid laboratory confirmation will facilitate subsequent prevention and control for imported cases.

Differential sensitivity of porcine endogenous retrovirus to APOBEC3-mediated inhibition.

Pigs are considered to be suitable xenotransplantation organ donors. However, the risk of pathogen transmission from pigs to humans is a major concern in the transplantation of porcine tissues. The porcine endogenous retroviruses (PERVs) PERV-A, PERV-A/C, and PERV-B can infect human cells, but PERV-C is an ecotropic virus infecting only pig cells. Thus, several strategies have been proposed to reduce PERV transmission in xenograft recipients. Human APOBEC3G (huA3G) is a single-strand DNA cytosine deaminase, which inactivates the coding capacity of the virus by deamination of cDNA cytosines to uracils. This reaction occurs within the (-) DNA strand during reverse transcription, resulting in a G-to-A mutation in the (+) strand. While recent data have shown that PERV-B is severely inhibited by huA3G and porcine A3Z2-Z3 (poA3F) in a pseudotype assay, little is known about PERV-C. Here, we compare the antiretroviral activities of huA3G, huA3F and poA3Z2-Z3 against PERV-C. Our data show that APOBEC3 was packaged into PERV-C particles and inhibited PERV-C replication in a dose-dependent manner. PERV-C infectivity was strongly inhibited by poA3Z2-Z3, but it did not markedly reduce PERV-B infectivity. This suggests that PERV-C Gag interacts efficiently with poA3Z2-Z3. In addition, we constructed stably huA3G- and poA3Z2-Z3-expressing 293-PERV-PK-CIRCE cells (human 293 cells infected with PK15-derived PERVs) to examine whether PERV is resistant to poA3Z2-Z3 in a virus-spreading assay. The stably expressed huA3G and poA3Z2-Z3 were more packaging-competent than transiently expressed APOBEC3 proteins. These results suggest that poA3Z2-Z3 can inhibit PERV replication in a pseudotype assay as well as in a virus-spreading assay.

Over-expression of PsGPD, a mushroom glyceraldehyde-3-phosphate dehydrogenase gene, enhances salt tolerance in rice plants.

Transgenic potatoes expressing glyceraldehyde-3-phosphate dehydrogenase (GPD), isolated from the oyster mushroom, Pleurotus sajor-caju, had increased tolerance to salt stress (Jeong et al. Biochem Biophys Res Commun 278:192-196, 2000). To examine the physiological mechanisms enhancing salt tolerance in GPD-transgenic rice plants, the salt tolerance of five GPD transgenic rice lines (T1-T5) derived from Dongjin rice cultivar were evaluated in a fixed 150 mM saline environment in comparison to two known wild-type rice cultivars, Dongjin (salt sensitive) and Pokali (salt tolerant). Transgenic lines, T2, T3, and T5, had a substantial increase in biomass and relative water content compared to Dongjin. Stomatal conductance and osmotic potential were higher in the GPD transgenic lines and were similar to those in Pokali. The results are discussed based on the comparative physiological response of GPD transgenic lines with those of the salt-sensitive and salt-tolerant rice cultivars.

Establishment of the Foot-and-Mouth Disease Virus Type Asia1 Expressing the HiBiT Protein: A Useful Tool for a NanoBiT Split Luciferase Assay.

Foot-and-mouth disease virus (FMDV) is a highly contagious virus that affects cloven-hoofed animals and causes severe economic losses in the livestock industry. Given that this high-risk pathogen has to be handled in a biosafety level (BSL)-3 facility for safety reasons and the limited availability of BSL-3 laboratories, experiments on FMDV call for more attention. Therefore, we aimed to develop an FMDV experimental model that can be handled in BSL-2 laboratories. The NanoBiT luciferase (Nano-luc) assay is a well-known assay for studying protein-protein interactions. To apply the NanoBiT split luciferase assay to the diagnosis and evaluation of FMD, we developed an inactivated HiBiT-tagged Asia1 Shamir FMDV (AS-HiBiT), a recombinant Asia1 shamir FMDV with HiBiT attached to the VP1 region of Asia1 shamir FMDV. In addition, we established LgBiT-expressing LF-BK cell lines, termed LgBit-LF-BK cells. It was confirmed that inactivated AS-HiBiT infected LgBiT-LF-BK cells and produced a luminescence signal by binding to the intracellular LgBiT of LgBiT-LF-BK cells. In addition, the luminescence signal became stronger as the number of LgBiT-LF-BK cells increased or the concentration of inactivated AS-HiBiT increased. Moreover, we confirmed that inactivated AS-HiBiT can detect seroconversion in sera positive for FMDV-neutralizing antibodies. This NanoBiT split luciferase assay system can be used for the diagnosis and evaluation of FMD and expanded to FMD-like virus models to facilitate the evaluation of FMDV vaccines and antibodies.

Overexpression of the glutamine synthetase gene modulates oxidative stress response in rice after exposure to cadmium stress.

KEY MESSAGE: Overexpression of OsGS gene modulates oxidative stress response in rice after exposure to cadmium stress. Our results describe the features of transformants with enhanced tolerance to Cd and abiotic stresses. Glutamine synthetase (GS) (EC 6.3.1.2) is an enzyme that plays an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine. Exposure of plants to cadmium (Cd) has been reported to decrease GS activity in maize, pea, bean, and rice. To better understand the function of the GS gene under Cd stress in rice, we constructed a recombinant pART vector carrying the GS gene under the control of the CaMV 35S promoter and OCS terminator and transformed using Agrobacterium tumefaciens. We then investigated GS overexpressing rice lines at the physiological and molecular levels under Cd toxicity and abiotic stress conditions. We observed a decrease in GS enzyme activity and mRNA expression among transgenic and wild-type plants subjected to Cd stress. The decrease, however, was significantly lower in the wild type than in the transgenic plants. This was further validated by the high GS mRNA expression and enzyme activity in most of the transgenic lines. Moreover, after 10 days of exposure to Cd stress, increase in the glutamine reductase activity and low or no malondialdehyde contents were observed. These results showed that overexpression of the GS gene in rice modulated the expression of enzymes responsible for membrane peroxidation that may result in plant death.

Serological Conversion through a Second Exposure to Inactivated Foot-and-Mouth Disease Virus Expressing the JC Epitope on the Viral Surface.

Foot-and-mouth disease (FMD) is a fatal contagious viral disease that affects cloven-hoofed animals and causes severe economic damage at the national level. There are seven serotypes of the causative foot-and-mouth disease virus (FMDV), and type O is responsible for serious outbreaks and shows a high incidence. Recently, the Cathay, Southeast Asia (SEA), and ME-SA (Middle East-South Asia) topotypes of type O have been found to frequently occur in Asia. Thus, it is necessary to develop candidate vaccines that afford protection against these three different topotypes. In this study, an experimental FMD vaccine was produced using a recombinant virus (TWN-JC) with the JC epitope (VP1 140-160 sequence of the O/SKR/Jincheon/2014) between amino acid 152 and 153 of VP1 in TWN-R. Immunization with this novel vaccine candidate was found to effectively protect mice against challenge with the three different topotype viruses. Neutralizing antibody titers were considerably higher after a second vaccination. The serological differences between the topotype strains were identified in guinea pigs and swine. In conclusion, a significant serological difference was observed at 56 days post-vaccination between animals that received the TWN-JC vaccine candidate and those that received the positive control virus (TWN-R). The TWN-JC vaccine candidate induced IFNγ and IL-12B.

Immunogenicity and Protection against Foot-and-Mouth Disease Virus in Swine Intradermally Vaccinated with a Bivalent Vaccine of Foot-and-Mouth Disease Virus Type O and A.

Following the worst outbreak of foot-and-mouth disease (FMD), a highly contagious disease in cloven-hoofed animals caused by the FMD virus, from November 2010-April 2011, the Korean government enforced a mandatory vaccination policy. A bivalent (FMD type O and A; O + A) vaccine has been recently implemented. Although the FMD outbreak was suppressed by vaccination, the intramuscular (IM) injection presents side effects. Therefore, improving FMD vaccine quality is necessary. Here, we investigated the side effects and immune efficacy of the O + A bivalent vaccine using two different routes of administration: intradermal (ID) and IM. To compare the immune efficacy of the two inoculation routes, virus neutralization titers and structural protein (antigen) levels were measured. The protective efficacy of ID vaccines was confirmed using two viruses (FMDV O/AS/SKR/2019 and A/GP/SKR/2018) isolated in the Republic of Korea. Serological analysis revealed that both animals administered by ID and IM injections exhibited equal immune efficacy. A virus challenge test in the target animal (swine) revealed no (or extremely low) clinical symptoms. Swine in the ID injected group exhibited no side effects. In conclusion, we suggest that the ID route of vaccination is an effective alternative to the existing IM route, which is associated with more frequent side effects.

Generation and characterization of a stable full-length ecotropic murine leukemia virus molecular clone that produces novel phenotypes to Fv1 restriction.

Retrovirus tropism can be restricted by host cell factors such as Fv1, TRIM5alpha, and Lv1 that inhibit infection by targeting the incoming viral capsid. The Fv1 gene inhibits murine leukemia virus infection in mice, but the precise mechanism of Fv1-mediated restriction is poorly understood. Our previous studies had demonstrated that Fv1-mediated viral tropism can be determined within the capsid protein at position 114 (Jung and Kozak. 2000. J. Virol. 74: 5385-7). To study the interaction between Fv1 and CA, we introduced amino acid substitution and deletion at this site in the N-tropic AKV capsid gene. The mutated two-LTR proviral DNAs were introduced into SC-1 cells by transfection. After transfection, cell supernatants collected from transfected cells were tested for host range susceptibility. The result indicated that substitution of amino acids did not alter tropism, but the deletion of 114His produced a virus with unusual tropism. The novel phenotype produced here failed to replicate in Fv1-expressing cells. This mutant virus showing such an extreme restriction pattern would be useful for studying the mechanism of Fv1- mediated restriction.

Isolation and characterization of PERV-C env from domestic pig in Korea.

Clone PERV-C (A3) env was isolated from the genomic DNA of domestic pig (Sus scrofa domesticus) in Korea to investigate the molecular properties of PERV-C. The nucleic acid homologies between the PERV-MSL (type C) reference and the PERV-C(A3) clone was 99% for env, but a single base pair deletion was found in the transmembrane (TM) region of the env open reading frame. To examine the functional characteristics of truncated PERV-C env, we constructed a replication-incompetent retroviral vector by replacing the env gene of the pCL-Eco retrovirus vector with PERV-C env. A retroviral vector bearing PERV-C/A chimeric envelopes was also created to complement the TM defect. Our results indicated that truncated PERV-C env was not infectious in human cells as expected. Interestingly, however, the vector with the PERV-C/A envelope was able to infect 293 cells. This observation suggests that recombination within PERV-C TM could render PERV-C infectious in humans. To further characterize PERV-C/A envelopes, we constructed an infectious molecular clone by using a PCR-based technique. This infectious molecular clone will be useful to examine more specific regions that are critical for human cell tropism.

Role of a third extracellular domain of an ecotropic receptor in Moloney murine leukemia virus infection.

The murine ecotropic retroviral receptor has been demonstrated to function as a mouse cationic amino acid transporter 1 (mCAT1), and is comprised of multiple membranespanning domains. Feral mouse (Mus dunni) cells are not susceptible to infection by the ecotropic Moloney murine leukemia virus (MoMLV), although they can be infected by other ecotropic murine leukemia viruses, including Friend MLV and Rauscher MLV. The relative inability of MoMLV to replicate in M. dunni cells has been attributed to two amino acids (V214 and G236) located within the third extracellular loop of the M. dunni CAT1 receptor (dCAT1). Via the exchange of the third extracellular loop of the mCAT1 cDNA encoding receptor from the permissive mouse and the corresponding portion of cDNA encoding for the nonpermissive M. dunni receptor, we have identified the most critical amino acid residue, which is a glycine located at position 236 within the third extracellular loop of dCAT1. We also attempted to determine the role of the third extracellular loop of the M. dunni CAT1 receptor with regard to the formation of the syncytium. The relationship between dCAT1 and virus-induced syncytia was suggested initially by our previous identification of two MLV isolates (S82F in Moloney and S84A in Friend MLV), both of which are uniquely cytopathic in M. dunni cells. In an attempt to determine the relationship existing between dCAT1 and the virally-induced syncytia, we infected 293-dCAT1 or chimeric dCAT1 cells with the S82F pseudotype virus. The S82F pseudotype virus did not induce the formation of syncytia, but did show increased susceptibility to 293 cells expressing dCAT1. The results of our study indicate that S82F-induced syncytium formation may be the result of cell-cell fusion, but not virus-cell fusion.

Complete Genome Sequence of Middle East Respiratory Syndrome Coronavirus KOR/KNIH/002_05_2015, Isolated in South Korea.

The full genome sequence of a Middle East respiratory syndrome coronavirus (MERS-CoV) was identified from cultured and isolated in Vero cells. The viral genome sequence has high similarity to 53 human MERS-CoVs, ranging from 99.5% to 99.8% at the nucleotide level.

Characterization of molecular clones of porcine endogenous retrovirus-A containing different numbers of U3 repeat boxes in the long terminal repeat region.

When full-length molecular clones of porcine endogenous retrovirus (PERV)-A and porcine endogenous retrovirus (PERV)-B were passaged on human cells, an increase in the length of the long terminal repeat (LTR) was reported. A 39-bp repeat box in the LTR U3 region was multimerized dynamically upon replication, acting as a viral enhancer element that contains binding sites for nuclear transcription factor NF-Y. To analyze the optimum number of 39-bp repeats for viral replication, molecular clones of PERV-A with one, two, three, and four 39-bp repeats were constructed. Each full-length PERV-A molecular clone contained a different number of 39-bp repeat boxes and was used to transfect human 293 cells, the relative transcriptional activity of the LTRs 48 h posttransfection was determined. PERV LTRs containing 3 copies of a 39-bp repeat showed the strongest promoter activity by real-time reverse transcription PCR in human 293 cell lines. Virions generated by the transfection of a provirus with 3 enhancer repeats replicated efficiently in human cells and 2.5×10(4)virion copies/µL were released. Although the transcriptional activity of all PERV-A LTRs was lower than 293 cells (293-PERV-PK-CIRCE) infected productively with PERV-A and PERV-B. It was found that 3 was the optimal number of 39-bp repeats for viral replication. This molecular clone with a higher replication capacity could be used to study the biology of porcine endogenous retrovirus by genetic approaches or in vivo infection experiments.

The rapid production of high-titer porcine endogenous retrovirus(PERV)-B env pseudotype and construction of an EGFP-expressing replication competent PERV-A vector.

Porcine endogenous retroviruses (PERVs) present a unique concern associated with xenotransplantation because they have been shown to infect certain human cells in vitro and it is also difficult to generate herds of pigs free of PERVs. A simple system for the production of high-titer MoMLV-PERV pseudotypes is reported; an EGFP-expressing replication-competent molecular clone that allows direct measurement of titer was also constructed. To improve the MLV-based retroviral vector system, a 2.1-kb PERV-B env product was amplified from PK-15 genomic DNA and cloned into the pCL-Eco retroviral vector. The titer of lacZ (PERV-B) from the 293 cells was about 1.0×10(4) CFU/ml. In contrast, the titer of lacZ (PERV-B) from a conventional murine retroviral vector (split genome) was found to be 1.2×10(2) CFU/ml when the PERV-B env expression vector was transfected into TELCeB6 cells, which harbor MFGnlslacZ and the gag-pol-expressing vector. In addition, an infectious PERV-A clone containing enhanced GFP (EGFP) by using a PCR-based method was developed. This EGFP-expressing PERV-A-IRES-EGFP molecular clone was found to be stable genetically on transfection in 293 cells.

An integrated database to enhance the identification of SNP markers for rice.

UNLABELLED: The National Academy of Agricultural Science (NAAS) has developed a web-based marker database to provide information about SNP markers in rice. The database consists of three major functional categories: map viewing, marker searching and gene annotation. It provides 12,829 SNP markers information including gene location information on 12 chromosomes in rice. The annotation of SNP marker provides information such as marker name, EST number, gene definition and general marker information. Users are assisted in tracing any new structures of the chromosomes and gene positional functions using specific SNP markers. AVAILABILITY: The database is available for free at http://nabic.niab.go.kr/SNP/