A serological study of severe fever with thrombocytopenia syndrome using a virus neutralization test and competitive enzyme-linked immunosorbent assay

Severe fever with thrombocytopenia syndrome (SFTS) is caused by the SFTS virus (SFTSV). The SFTSV appears to have a wide host range, as SFTSV-positive ticks have been isolated from both farm animals and wild rodents. Therefore, it is important to monitor SFTSV-positive animals to prevent the transmission of SFTSV from animals to humans. Previously, we developed a competitive enzyme-linked immunosorbent assay (cELISA) to detect SFTSV-specific antibodies from field animals and compared the cELISA results to those from an indirect immunofluorescence assay (IFA). In this study, cELISA results were compared to and evaluated against the results from both an IFA and a virus neutralization (VN) test of 193 bovine serum samples (including two bovine positive control sera) and 70 horse serum samples. The consistency (98.9%) between cELISA and VN results was higher than that (97.4%) between cELISA and IFA for the bovine serum samples. Similarly, for the horse serum samples, the consistency (88.6%) between cELISA and VN results was higher than that (84.3%) between the cELISA and IFA. These findings indicate that our newly developed cELISA can be used for surveillance or epidemiological studies of SFTSV in animals.

Development and evaluation of a competitive enzyme-linked immunosorbent assay using a monoclonal antibody for diagnosis of severe fever with thrombocytopenia syndrome virus in bovine sera

Severe fever with thrombocytopenia syndrome (SFTS) caused by the SFTS virus (SFTSV), a phlebovirus in the family Bunyaviridae, is an emerging tick-borne infectious disease that impacts humans. This disease manifests as a decreased blood cell count and multi-organ failure, with a case-fatality rate of more than 12% in China. Because vaccines or antiviral drugs for the treatment of this disease are not available, monitoring the SFTS circulation in animals and controlling the tick-mammal cycle are important for preventing SFTS. Monoclonal antibodies against the recombinant nucleoprotein of SFTSV were generated to develop a competitive enzyme-linked immunosorbent assay (cELISA) for the detection of antibodies against SFTSV infection in cattle. The specificity and sensitivity of cELISA was assessed by comparing the results of this assay to those of an immunofluorescence assay (IFA). The results of the cELISA using 416 field bovine serum samples and laboratory-immunized positive sera showed 98.1% consistency with those of the IFA. The cELISA used in this study did not show cross-reactivity with antisera against other viral cattle diseases. The cELISA presented in this study can be applied to detect antibodies against SFTSV in cattle.

Uracil-DNA glycosylase-treated reverse transcription loop-mediated isothermal amplification for rapid detection of avian influenza virus preventing carry-over contamination

Here, we describe a uracil-DNA glycosylase (UNG)-treated reverse transcription loop-mediated isothermal amplification (uRT-LAMP) for the visual detection of all subtypes of avian influenza A virus (AIV). The uRT-LAMP assay can prevent unwanted amplification by carryover contamination of the previously amplified DNA, although the detection limit of the uRT-LAMP assay is 10-fold lower than that of the RT-LAMP without a UNG treatment. To the best of our knowledge, this is the first successful application of deoxyuridine triphosphate/UNG strategy in RT-LAMP for AIV detection, and the assay can be applied for the rapid, and reliable diagnosis of AIVs, even in contaminated samples.

Development and evaluation of an immunochromatographic assay using a gp51 monoclonal antibody for the detection of antibodies against the bovine leukemia virus

Infection of cattle with bovine leukemia virus (BLV) has been observed and reported worldwide, including in Korea. The onsite identification of infected cattle would help decreasing and eradicating BLV infections on farms. Here, we present a new immunochromatographic assay that employs monoclonal antibodies (MAbs) for the detection of antibodies against BLV in the field. BLV envelope glycoprotein (gp)51 was expressed in E. coli, and MAbs against recombinant BLV gp51 were generated for the development of an immunochromatographic assay to detect BLV antibodies in cattle. The sensitivity and specificity of the assay were determined by comparing these results with those obtained from a standard enzyme linked immunosorbent assay (ELISA). A total of 160 bovine sera were used to evaluate the new immunochromatographic assay. Using ELISA as a reference standard, the relative specificity and sensitivity of this assay were determined to be 94.7% and 98%, respectively. Because of its high sensitivity and specificity, this BLV antibody detection assay would be suitable for the onsite identification of BLV infection in the field.

Genetic diversity of porcine reproductive and respiratory syndrome virus in Korea

The high genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) has been an obstacle to developing an effective vaccine for porcine reproductive and respiratory syndrome (PRRS). This study was performed to assess the degree of genetic diversity among PRRSVs from Korean pig farms where wasting and respiratory syndrome was observed from 2005 to 2009. Samples from 786 farms were tested for the presence of PRRSV using reverse transcription PCR protocol. A total of 117 farms were positive for type 1 PRRSV while 198 farms were positive for type 2. Nucleotide sequences encoding the open reading frame (ORF) 5 were analyzed and compared to those of various published PRRSV isolates obtained worldwide. Sequence identity of the ORF 5 in the isolates was 81.6~100% for type 1 viruses and 81.4~100% for type 2 viruses. Phylogenetic analysis of the ORF 5 sequences showed that types 1 and 2 PRRSVs from Korea were mainly classified into three and four clusters, respectively. The analyzed isolates were distributed throughout the clusters independent of the isolation year or geographical origin. In conclusion, our results indicated that the genetic diversity of PRRSVs from Korean pig farms is high and has been increasing over time.

Temporospatial clustering analysis of foot-and-mouth disease transmission in South Korea, 2010~2011 / 대한수의학회지

To investigate the transmission pattern of geographical area and temporal trends of the 2010~2011 foot-and-mouth disease (FMD) outbreaks in Korea, and to explore temporal intervals at which spatial clustering of FMD cases space-time analysis based on georeferenced database of 3,575 burial sites, from 30 November 2010 to 23 February 2011, was performed. The cases represent approximately 98.1% of all infected farms (n = 3,644) during the same period. Descriptive maps of spatial patterns of the outbreaks were generated by ArcGIS. Spatial Scan Statistics, using SaTScan software, was applied to investigate geographical clusters of FMD cases across the country. Overall, spatial heterogeneity was identified, and the transmission pattern was different by province. Cattle have more clusters in number but smaller in size, as compared to the swine population. In addition, spatiotemporal analysis and the comparison of clustering patterns between the first 7 days and days 8 to 14 of the outbreak revealed that the strongest spatial clustering was identified at the 7-day interval, although clustering over longer intervals (8~14 days) was also observed. We further discussed the importance of time period elapsed between FMD-suspected notice and the date of confirmation, and emphasized the necessity of region-specific and species-specific control measures.

A survey of porcine reproductive and respiratory syndrome among wild boar populations in Korea

No information is currently available on porcine reproductive and respiratory syndrome virus (PRRSV) infection in wild boars (Sus scrofa) in Korea. In this study, the status of PRRS in wild boars was investigated. Blood samples were collected from 267 wild boars from eight provinces in Korea. Four of the samples tested (1.5%) were positive for PRRSV antibodies and eight (3.0%) were positive for antigens. Of the virus-positive samples, three and five samples were typed as containing European (EU, type 1) or North American (NA, type 2) viruses, respectively. Two amplicons (one from type 1 and one from type 2) were used to analyze the PRRSV open reading frame 7 (ORF7) sequence. The nucleotide sequences of type 1 PRRSV ORF7 had identities between 96.1% and 98.4% with PRRSVs from domestic pigs in Korea. The sequences of type 2 PRRSV ORF7 had identities of 100% with the PRRSV strain VR-2332, which was prototypic North American strain. These results show that PRRSVs are present in wild boars in Korea, and effective PRRSV surveillance of the wild boar population might therefore be useful for disease control.

Seroepidemiological Studies of Aino Virus Infection in Korean Cattle

Aino virus infection is characterized by abortion, stillbirth, and congenital abnormalities such as arthropgryposis-hydranencephaly syndrome in calves. In Korea, Aino virus infection was first reported in 1997 by researchers who were investigating the cause of newborn calf deformities. Given the incidence of Aino-related deformities, the need for a study of the Aino virus infection status in Korea was recognized. In this study, we investigated the nationwide seroepidemiological status of Aino virus infection. A total of 9,921 serum samples collected between 1993 and 2001, and 23,760 serum samples between 2002 and 2007 were tested using a virus neutralization assay. The seroprevalence of Aino virus was 73.1, 63.8, 44.9, 56.0, 38.5, 28.4 18.3, 19.6, and 23.2%, respectively, between 1993 and 2001, and 43.8, 42.9, 50.7, 55.3, 31.4, and 25.4%, respectively, between 2002 and 2007. Aino virus infection does not pose a major threat to the bovine industry in Korea till now. The future prospects for Aino virus infection in cattle, however, may change with the global warming phenomena. The results of this study may serve as a basis for future epidemiological studies on Aino virus infection.

Monitoring of Five Bovine Arboviral Diseases Transmitted by Arthropod Vectors in Korea

A survey was performed in Korea to monitor the prevalence of five bovine arboviruses [Akabane virus, Aino virus, Chuzan virus, bovine ephemeral fever (BEF) virus, and Ibaraki virus] in arthropod vectors, such as Culicoides species. To determine the possible applications of survey data in annual monitoring and warning systems in Korea, we examined the prevalence of bovine arboviruses in arthropod vectors using RT-PCR. To compare the sensitivity and specificity of virus detection, nested PCR was also performed in parallel for all five viruses. Using the RT-PCR, the detection limits were at least up to 10(1.5), 10(2.8), 10(2.0), 10(1.8), and 10(4.0) TCID50/ml for Akabane virus, Aino virus, Chuzan virus, BEF virus, and Ibaraki virus, respectively. When nested PCR was performed using 1 micronl of PCR product, the detection limits were increased, to 10(0.05), 10(1.8), 10(1.0), 10(0.008), and 10(2.0) TCID50/ml for Akabane virus, Aino virus, Chuzan virus, BEF virus, and Ibaraki virus, respectively. Thus, nested PCR increased the sensitivity of the virus detection limit by 1~2 log. We pooled 30~40 mosquitoes in one sample. We collected 113 samples in 2006, 135 samples in 2007, and 100 samples in 2008. Among these samples, Chuzan virus and BEF virus genes were detected at a range between 0.82% and 1.19%, and Akabane virus, Aino virus, and Ibaraki virus genes were detected at less than 0.20%. These data may provide some insight into future epidemiological studies of bovine arboviral diseases in Korea.