Objective To prepare mouse monoclonal antibodies against the ectodomain of E2 (E2ecto) glycoprotein of Western equine encephalitis virus (WEEV). Methods A prokaryotic expression plasmid pET-28a-WEEV E2ecto was constructed and transformed into BL21 (DE3) competent cells. E2ecto protein was expressed by IPTG induction and presented mainly as inclusion bodies. Then the purified E2ecto protein was prepared by denaturation, renaturation and ultrafiltration. BALB/c mice were immunized with the formulated E2ecto protein using QuickAntibody-Mouse5W as an adjuvant via intramuscular route, boosted once at an interval of 21 days. At 35 days post-immunization, mice with antibody titer above 1×10⁴ were inoculated with E2ecto intraperitoneally, and spleen cells were fused with SP2/0 cells three days later. Hybridoma cells secreting specific monoclonal antibodies were screened by the limited dilution method, and ascites were prepared after intraperitoneal inoculation of hybridoma cells. The subtypes and titers of the antibodies in ascites were assayed by ELISA. The biological activity of the mAb was identified by immunofluorescence assay(IFA) on BHK-21 cells which were transfected with eukaryotic expression plasmid pCAGGS-WEEV-CE3E2E1. The specificity of the antibodies were evaluated with E2ecto proteins from EEEV and VEEV. Results Purified WEEV E2ecto protein was successfully expressed and obtained. Four monoclonal antibodies, 3G6G10, 3D7G2, 3B9E8 and 3D5B7, were prepared, and their subtypes were IgG2c(κ), IgM(κ), IgM(κ) and IgG1(κ), respectively. The titers of ascites antibodies 3G6G10, 3B9E8 and 3D7G2 were 10⁵, and 3D5B7 reached 10⁷. None of the four antibody strains cross-reacted with other encephalitis alphavirus such as VEEV and EEEV. Conclusion Four strains of mouse mAb specifically binding WEEV E2ecto are successfully prepared.
Horses ; Animals ; Mice ; Encephalitis Virus, Western Equine ; Ascites ; Immunosuppressive Agents ; Antibodies, Monoclonal ; Immunoglobulin M

Eighteen (18) cases of Chikungunya (or a very close related virus) infection, a newly observed clinical entity in the Philippines, are presented. Diagnosis was established by serological studies consisting of hemagglutination- inhibition (HI) and complement fixation (CF) tests. The clinical picture is characterized by a symptom-complex consisting essentially of fever, severe, incapacitating, recurrent joint pains and a rash. There is no involvement of the respiratory system and no bleeding tendencies were observed. The erythrocyte sedimentation rate was elevated, more so towards the later part of the illness. The platelet counts were normal. No residual joint deformity morbidity or mortality was encountered.
Chikungunya virus

Sparse labeling of neurons contributes to uncovering their morphology, and rapid expression of a fluorescent protein reduces the experiment range. To achieve the goal of rapid and sparse labeling of neurons in vivo, we established a rapid method for depicting the fine structure of neurons at 24 h post-infection based on a mutant virus-like particle of Semliki Forest virus. Approximately 0.014 fluorescent focus-forming units of the mutant virus-like particle transferred enhanced green fluorescent protein into neurons in vivo, and its affinity for neurons in vivo was stronger than for neurons in vitro and BHK21 (baby hamster kidney) cells. Collectively, the mutant virus-like particle provides a robust and convenient way to reveal the fine structure of neurons and is expected to be a helper virus for combining with other tools to determine their connectivity. Our work adds a new tool to the approaches for rapid and sparse labeling of neurons in vivo.
Animals ; Cells, Cultured ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Immunohistochemistry ; methods ; Male ; Mice, Inbred C57BL ; Microscopy, Fluorescence ; methods ; Neurons ; cytology ; metabolism ; Purkinje Cells ; cytology ; metabolism ; Semliki forest virus ; genetics

OBJECTIVES: Dengue epidemic is a dynamic and complex phenomenon that has gained considerable attention due to its injurious effects. The focus of this study is to statically analyze the nature of the dengue epidemic network in terms of whether it follows the features of a scale-free network or a random network. METHODS: A multifarious network of Aedes aegypti is addressed keeping the viewpoint of a complex system and modelled as a network. The dengue network has been transformed into a one-mode network from a two-mode network by utilizing projection methods. Furthermore, three network features have been analyzed, the power-law, clustering coefficient, and network visualization. In addition, five methods have been applied to calculate the global clustering coefficient. RESULTS: It has been observed that dengue epidemic follows a power-law, with the value of its exponent γ = −2.1. The value of the clustering coefficient is high for dengue cases, as weight of links. The minimum method showed the highest value among the methods used to calculate the coefficient. Network visualization showed the main areas. Moreover, the dengue situation did not remain the same throughout the observed period. CONCLUSIONS: The results showed that the network topology exhibits the features of a scale-free network instead of a random network. Focal hubs are highlighted and the critical period is found. Outcomes are important for the researchers, health officials, and policy makers who deal with arbovirus epidemic diseases. Zika virus and Chikungunya virus can also be modelled and analyzed in this manner.
Administrative Personnel ; Aedes ; Arboviruses ; Chikungunya virus ; Critical Period (Psychology) ; Dengue Virus ; Dengue ; Humans ; Methods ; Zika Virus

The recent epidemic of Zika virus in South America caused people around the world to exhibit an increased interest in the impact of arboviral illnesses. In Korea, malaria and Japanese encephalitis are the most important mosquito-borne diseases that occur indigenously. However, with the continuously increasing number of international travelers, the incidence of imported arboviral illnesses is also increasing. Currently, dengue fever is the most common mosquito-borne disease among Korean international travelers. The number of patients with Japanese encephalitis, chikungunya fever, and Zika virus infection is also on the rise. Many countries that have disease-transmitting mosquitoes have already experienced autochthonous arboviral infections due to the introduction of viruses by travelers. Moreover, with global warming and urbanization of the areas in which mosquito-borne diseases occur, the environment is becoming more favorable for mosquito-borne diseases. This concise review describes the current status and outlook of mosquito-borne diseases in Korea.
Chikungunya Fever ; Culicidae ; Dengue ; Encephalitis, Japanese ; Global Warming ; Humans ; Incidence ; Korea* ; Malaria ; South America ; Urbanization ; Zika Virus ; Zika Virus Infection

Getah virus (GETV) infection causes sporadic outbreaks of mild febrile illness in horses and reproductive failure in pigs. In this study, we established a reverse transcription polymerase chain reaction (RT-PCR) method to detect GETV from suspected virus-infected samples. The reaction conditions were optimized and validated by using RNA extracted from GETV propagated in cell culture. A GETV-specific GED4 primer set was designed and used to amplify a 177 bp DNA fragment from a highly conserved region of the E1 glycoprotein gene in the GETV genome. RT-PCR performed with this primer set revealed high sensitivity and specificity. In the sensitivity test, the GED4 primer set detected GETV RNA at the level of 10(2.0) TCID₅₀/mL. In the specificity test, the GED4 primer set amplified only a single band of PCR product on the GETV RNA template, without non-specific amplification, and exhibited no cross-reactivity with other viral RNAs. These results suggest that this newly established RT-PCR method is useful for accurate identification of GETV infection in animals.
Alphavirus* ; Animals ; Cell Culture Techniques ; Diagnosis ; Disease Outbreaks ; DNA ; Genome ; Glycoproteins ; Horses ; Livestock* ; Methods ; Polymerase Chain Reaction* ; Reverse Transcription* ; RNA ; RNA, Viral ; Sensitivity and Specificity ; Swine

Chikungunya infection is caused by an arbovirus transmitted by the Aedes mosquito. A 19-year-old man who had traveled to the Republic of Surinam to perform volunteer work complained of a fever, arthralgia, articular stiffness, and a skin rash on both the arm and trunk. Chikungunya fever was diagnosed using a Chikungunya virus specific IgM antibody in an enzyme-linked immunosorbent assay (ELISA) using blood samples obtained during follow-up visits. In this report, we describe a case of imported Chikungunya fever that presented with arthralgia and a skin rash, with islands of normal skin, that occurred following travel to Surinam, South America.
Aedes ; Arboviruses ; Arm ; Arthralgia ; Chikungunya virus* ; Culicidae ; Enzyme-Linked Immunosorbent Assay ; Exanthema ; Fever ; Follow-Up Studies ; Humans ; Immunoglobulin M ; Islands ; Skin ; South America* ; Suriname* ; Volunteers ; Young Adult

Various new technologies have been applied for developing vaccines against various animal diseases. Virus-like particle (VLP) vaccine technology was used for manufacturing the porcine circovirus type 2 and RNA particle vaccines based on an alphavirus vector for porcine epidemic diarrhea (PED). Although VLP is classified as a killed-virus vaccine, because its structure is similar to the original virus, it can induce long-term and cell-mediated immunity. The RNA particle vaccine used a Venezuela equine encephalitis (VEE) virus gene as a vector. The VEE virus partial gene can be substituted with the PED virus spike gene. Recombinant vaccines can be produced by substitution of the target gene in the VEE vector. Both of these new vaccine technologies made it possible to control the infectious disease efficiently in a relatively short time.
Alphavirus ; Animal Diseases ; Animals* ; Circovirus ; Communicable Diseases ; Diarrhea ; Encephalitis Virus, Venezuelan Equine ; Encephalomyelitis, Equine ; Immunity, Cellular ; Porcine epidemic diarrhea virus ; RNA ; Vaccines ; Vaccines, Synthetic ; Vaccines, Virus-Like Particle ; Venezuela

Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection.
Blotting, Western* ; Chikungunya virus ; Diagnosis ; Disease Outbreaks ; Epitopes ; Humans* ; Sensitivity and Specificity ; Vaccination

Getah virus (GETV) is a member of the genus Alphavirus in the family Togaviridae. GETV infection can occur in a wide range of vertebrate species, and the virus has been known for a pathogen of horses and pigs. To rapidly and accurately diagnose GETV infection of a racehorse, an indirect ELISA (I-ELISA) was developed in the present study for detection of antibodies to GETV in serum samples. To evaluate the developed I-ELISA, a total of 240 serum samples from Thoroughbred racehorses raised in Korea were screened in parallel by a serum neutralization (SN) test. The developed I-ELISA exhibited an efficacy comparable to that of the SN test in terms of a high diagnostic sensitivity (86.3%) and specificity (94.5%) at a cut-off absorbance value of 0.25. In addition, our results showed that the developed I-ELISA had a significant correlation with the SN test (r = 0.91; p < 0.05). Taken together, our findings suggest that the I-ELISA developed in this study is a valuable diagnostic tool for the screening of horses suspected to be infected with GETV.
Alphavirus* ; Antibodies* ; Enzyme-Linked Immunosorbent Assay* ; Horses* ; Humans ; Korea ; Mass Screening ; Sensitivity and Specificity ; Swine ; Togaviridae ; Vertebrates