Immunogenicity of a novel inactivated canine adenovirus type 2 variant vaccine for dogs.

Purpose: The immunogenicity of vaccines containing the canine adenovirus (CAdV) type 2 (CAdV-2) variant has not yet been reported. We prepared a novel inactivated CAdV-2 variant vaccine using the CAV2232-41 strain, and evaluated its safety and immunogenicity in raccoon dogs. Materials and Methods: The growth kinetics of CAV2232-41 were determined using Madin-Darby Canine Kidney (MDCK) cells. The nucleotide sequences of CAV2232 and CAV2232-41 were determined by next-generation sequencing. To generate the CAdV-2 variant vaccine, CAV2232-41 propagated in the MDCK cells was inactivated with 0.1% formaldehyde. Two vaccines were prepared by blending inactivated CAV2232-41 with Cabopol and Rehydragel adjuvants. Safety and immunogenicity of the CAV2232C and CAV2232R vaccines were evaluated in guinea pigs. Safety and immunogenicity of the CAV2232C vaccine were also evaluated in raccoon dogs. The virus neutralizing antibody (VNA) titer against CAV2232-41 was measured in sera collected from immunized guinea pigs and raccoon dogs. Results: CAV2232-41 showed the highest viral titer on days 4-6 post-inoculation and had a deletion in the E3 gene, which was confirmed as a CAdV-2 variant. Guinea pigs inoculated with CAV2232C showed slightly higher VNA titers than those inoculated with CAV2232R 2 weeks after booster vaccination. Raccoon dogs immunized with the CAV2232C vaccine developed high mean VNA titers, while non-vaccinated raccoon dogs were antibody-negative. Conclusion: The CAV2232C vaccine is safe and induces a protective VNA titer in raccoon dogs.

A Global Perspective on Oral Vaccination of Wildlife against Rabies.

The long-term mitigation of human-domestic animal-wildlife conflicts is complex and difficult. Over the last 50 yr, the primary biomedical concepts and actualized collaborative global field applications of oral rabies vaccination to wildlife serve as one dramatic example that revolutionized the field of infectious disease management of free-ranging animals. Oral vaccination of wildlife occurred in diverse locales within Africa, Eurasia, the Middle East, and North America. Although rabies is not a candidate for eradication, over a billion doses of vaccine-laden baits distributed strategically by hand, at baiting stations, or via aircraft, resulted in widespread disease prevention, control, or local disease elimination among mesocarnivores. Pure, potent, safe, and efficacious vaccines consisted of either modified-live, highly attenuated, or recombinant viruses contained within attractive, edible baits. Since the late 1970s, major free-ranging target species have included coyotes (Canis latrans), foxes (Urocyon cinereoargenteus; Vulpes vulpes), jackals (Canis aureus; Lupulella mesomelas), raccoons (Procyon lotor), raccoon dogs (Nyctereutes procyonoides), and skunks (Mephitis mephitis). Operational progress has occurred in all but the latter species. Programmatic evaluations of oral rabies vaccination success have included: demonstration of biomarkers incorporated within vaccine-laden baits in target species as representative of bait contact; serological measurement of the induction of specific rabies virus neutralizing antibodies, indicative of an immune response to vaccine; and most importantly, the decreasing detection of rabies virus antigens in the brains of collected animals via enhanced laboratory-based surveillance, as evidence of management impact. Although often conceived mistakenly as a panacea, such cost-effective technology applied to free-ranging wildlife represents a real-world, One Health application benefiting agriculture, conservation biology, and public health. Based upon lessons learned with oral rabies vaccination of mesocarnivores, opportunities for future extension to other taxa and additional diseases will have far-reaching, transdisciplinary benefits.

Immunomodulatory effects of canine mesenchymal stem cells in an experimental atopic dermatitis model.

Mesenchymal stem cells (MSCs) have the potential to differentiate into multi-lineage cells, suggesting their future applicability in regenerative medicine and biotechnology. The immunomodulatory properties of MSCs make them a promising replacement therapy in various fields of animal research including in canine atopic dermatitis (AD), a skin disease with 10-15% prevalence. We investigated the immunomodulatory effects of MSCs in an experimental canine AD model induced by Dermatophagoides farinae extract ointment. Canine adipose tissue-derived MSCs (cAT-MSCs) were differentiated into mesodermal cell lineages at the third passage. Alterations in immunomodulatory factors in control, AD, and MSC-treated AD groups were evaluated using flow cytometric analysis, enzyme-linked immunosorbent assay, and quantitative reverse transcription PCR. In the MSC-treated AD group, the number of eosinophils decreased, and the number of regulatory T cells (Tregs) increased compared to those in the AD group. In addition, the immunoglobulin E (IgE) and prostaglandin E2 levels were reduced in the MSC-treated AD group compared to those in the AD group. Furthermore, the filaggrin, vascular endothelial growth factor, and interleukin-5 gene expression levels were relatively higher in the MSC-treated AD group than in the AD group, however, not significantly. cAT-MSCs exerted immunomodulatory effects in an AD canine model via a rebalancing of type-1 and -2 T helper cells that correlated with increased levels of Tregs, IgE, and various cytokines.

Evaluation of porcine intestinal organoids as an in vitro model for mammalian orthoreovirus 3 infection.

BACKGROUND: Mammalian orthoreovirus type 3 (MRV3), which is responsible for gastroenteritis in many mammalian species including pigs, has been isolated from piglets with severe diarrhea. However, the use of pig-derived cells as an infection model for swine-MRV3 has rarely been studied. OBJECTIVES: This study aims to establish porcine intestinal organoids (PIOs) and examine their susceptibility as an in vitro model for intestinal MRV3 infection. METHODS: PIOs were isolated and established from the jejunum of a miniature pig. Established PIOs were characterized using polymerase chain reaction (PCR) and immunofluorescence assays (IFAs) to confirm the expression of small intestine-specific genes and proteins, such as Lgr5, LYZI, Mucin-2, ChgA, and Villin. The monolayered PIOs and three-dimensional (3D) PIOs, obtained through their distribution to expose the apical surface, were infected with MRV3 for 2 h, washed with Dulbecco's phosphate-buffered saline, and observed. Viral infection was confirmed using PCR and IFA. We performed quantitative real-time reverse transcription-PCR to assess changes in viral copy numbers and gene expressions linked to intestinal epithelial genes and antiviral activity. RESULTS: The established PIOs have molecular characteristics of intestinal organoids. Infected PIOs showed delayed proliferation with disruption of structures. In addition, infection with MRV3 altered the gene expression linked to intestinal epithelial cells and antiviral activity, and these effects were observed in both 2D and 3D models. Furthermore, viral copy numbers in the supernatant of both models increased in a time-dependent manner. CONCLUSIONS: We suggest that PIOs can be an in vitro model to study the infection mechanism of MRV3 in detail, facilitating pharmaceutical development.

Immunogenicity of an inactivated rabies vaccine for animals derived from the recombinant ERAGS strain.

PURPOSE: The aims of the present study were to evaluate the immunogenicity of an inactivated rabies vaccine based on the ERAGS strain. MATERIALS AND METHODS: The ERAGS virus propagated in Vero cells was inactivated with 3 mM binary ethylenimine for 8 hours. Three types of inactivated rabies vaccines were prepared to determine the minimum vaccine virus titers. Four further types of inactivated rabies vaccines were prepared by blending inactivated ERAGS with four different adjuvants; each vaccine was injected into mice, guinea pigs, and dogs to identify the optimal adjuvant. The immunogenicity of a Montanide (IMS) gel-adjuvanted vaccine was evaluated in cats, dogs, and cattle. Humoral immune responses were measured via a fluorescent antibody virus neutralization method and a blocking enzyme-linked immunosorbent assay. RESULTS: The minimum virus titer of the inactivated rabies vaccine was over 107.0 50% tissue culture infectious doses (TCID50 values)/mL. Of the four kinds of adjuvants, the IMS gel-adjuvanted vaccine induced the highest mean viral neutralizing antibody (VNA) titers of 6.24 and 2.36 IU/mL in guinea pigs and dogs, respectively, and was thus selected as the vaccine for the target animals. Cats, dogs, and cattle inoculated with the IMS gel-adjuvanted vaccine developed protective VNA titers ranging from 3.5 to 1.2 IU/mL at 4 weeks post-inoculation (WPI). CONCLUSION: Our data indicate that cats, dogs, and cattle inoculated with an inactivated rabies vaccine derived from the ERAGS strain developed protective immune responses that were maintained to 12 WPI.

Generation of a recombinant rabies virus expressing green fluorescent protein for a virus neutralization antibody assay.

BACKGROUND: Fluorescent antibody virus neutralization (FAVN) test is a standard assay for quantifying rabies virus-neutralizing antibody (VNA) in serum. However, a safer rabies virus (RABV) should be used in the FAVN assay. There is a need for a new method that is economical and time-saving by eliminating the immunostaining step. OBJECTIVES: We aimed to improve the traditional FAVN method by rescuing and characterizing a new recombinant RABV expressing green fluorescent protein (GFP). METHODS: A new recombinant RABV expressing GFP designated as ERAGS-GFP was rescued using a reverse genetic system. Immuno-fluorescence assay, peroxidase-linked assay, electron microscopy and reverse transcription polymerase chain reaction were performed to confirm the recombinant ERAGS-GFP virus as a RABV expressing the GFP gene. The safety of ERAGS-GFP was evaluated in 4-week-old mice. The rabies VNA titers were measured and compared with conventional FAVN and FAVN-GFP tests using VERO cells. RESULTS: The virus propagated in VERO cells was confirmed as RABV expressing GFP. The ERAGS-GFP showed the highest titer (108.0 TCID50/mL) in VERO cells at 5 days post-inoculation, and GFP expression persisted until passage 30. The body weight of 4-week-old mice inoculated intracranially with ERAGS-GFP continued to increase and the survival rate was 100%. In 62 dog sera, the FAVN-GFP result was significantly correlated with that of conventional FAVN (r = 0.95). CONCLUSIONS: We constructed ERAGS-GFP, which could replace the challenge virus standard-11 strain used in FAVN test.

THE PRESENCE OF ANTIBODIES AGAINST PSEUDORABIES VIRUS IN WILD BOARS (SUS SCROFA) IN KOREA.

The control and elimination of pseudorabies (PR) is one of the most important goals in the pig industry. After the first PR outbreak in Korea in 1986, all pigs infected with PR virus (PRV) were removed, and a vaccination program for pigs was implemented. No PR has occurred in Korea since 2010, and vaccination was discontinued after 2013. Information on the seroprevalence of PRV in pigs, including wild boars (Sus scrofa), is important for evaluating the PR status in a country. In this study, 2.65% (28/1057) of the wild boars tested had antibodies against PRV in 2018, indicating that PRV has been circulating continuously in the wild boar population in Korea. Effective means should be implemented to prevent the transmission of PRV between wild and domestic pigs, because the wild boar is a potential reservoir host for PRV.

Role of Oral Rabies Vaccines in the Elimination of Dog-Mediated Human Rabies Deaths.

Domestic dogs are responsible for nearly all the ¼59,000 global human rabies deaths that occur annually. Numerous control measures have been successful at eliminating dog-mediated human rabies deaths in upper-income countries, including dog population management, parenteral dog vaccination programs, access to human rabies vaccines, and education programs for bite prevention and wound treatment. Implementing these techniques in resource-poor settings can be challenging; perhaps the greatest challenge is maintaining adequate herd immunity in free-roaming dog populations. Oral rabies vaccines have been a cornerstone in rabies virus elimination from wildlife populations; however, oral vaccines have never been effectively used to control dog-mediated rabies. Here, we convey the perspectives of the World Organisation for Animal Health Rabies Reference Laboratory Directors, the World Organisation for Animal Health expert committee on dog rabies control, and World Health Organization regarding the role of oral vaccines for dogs. We also issue recommendations for overcoming hesitations to expedited field use of appropriate oral vaccines.

Isolation and molecular characterizations of canine distemper virus from a naturally infected Korean dog using Vero cells expressing dog signaling lymphocyte activation molecule.

BACKGROUND: Canine distemper virus (CDV) infection results in high morbidity and mortality in dogs. There has been no report about isolation of Korean CDV since 1980 in Korea. OBJECTIVES: To investigate the biological properties and the genetic characterization of Korean CDV. METHODS: Vero cells expressing dog signaling lymphocyte activation molecule (dSLAM) gene named as Vero/dSLAM were used to isolate CDV using 17 samples. Diagnostic methods such as cytopathic effects, immunofluorescence assay, peroxidase linked assay, electron microscopy, rapid immunodiagnostic assay, and reverse transcription polymerase chain reaction were used to confirm the Korean CDV isolate as a CDV. The genetic analysis was performed through cloning and sequencing of hemagglutinin gene of CDV isolate. RESULTS: A virus propagated in Vero/dSLAM cell was confirmed as CDV (CD1901 strain) based on the above methods. The CD1901 strain showed the highest viral titer (105.5 50% tissue culture infectious dose [TCID50]/mL) in the Vero/dSLAM cells at 4 days post inoculation, but did not form a fork on chorioallantoic membrane of 7-day-old egg. Ribavirin, a nucleotide analogue anti-viral agent, inhibits moderately the Korean CDV propagation in the Vero/dSLAM cells. The nucleotide and amino acid sequences of the H gene of CD1901 strain were compared with those of other CDV strains. The CD1901 strain belonged to Asia 1 group and had the highest similarity (99.9%) with the BA134 strain, which was isolated in China in 2008. CONCLUSIONS: We constructed successfully Vero/dSLAM and isolated one Korean CDV isolate (CD1901 strain) from a naturally infected dog. The CD1901 strain belonged to Asia 1 genotype.

Genotype-specific neutralizing antibody titers against Japanese encephalitis virus genotypes 1 and 3 in horses immunized with a genotype 3 vaccine.

PURPOSE: Japanese encephalitis is one of the most important mosquito-borne and zoonotic diseases in Asia and the Pacific region. Although the dominant Japanese encephalitis virus (JEV) genotype has shifted from G3 to G1 in Korea since 1990, a G3 strain (Anyang 300) has been used in vaccines for horses for almost 40 years. This study aimed to investigate the seroconversion rates and geometric mean titers (GMTs) of virus-neutralizing antibodies (VNAs) against JEV G1 and G3 in horses immunized with the G3 vaccine. MATERIALS AND METHODS: Serum samples of 1,231 horses immunized with the Anyang 300 vaccine were collected in 2018. VNA titers against JEV KV1899 (G1) and Anyang 300 (G3) were measured in all serum samples using the virus neutralization test. Titers were analyzed according to blood sampling time (prior to and following annual revaccination), age, and region. RESULTS: Rates of VNA titer >10 were 45.1% and 77.8% for G1, and 49.1% and 82.9% for G3 in samples taken before and after revaccination, respectively. GMTs of genotype-specific VNAs against JEV G1 and G3 were 8.3 and 11.6 before revaccination and rose to 27.2 and 65.4 following revaccination. Overall sero-positivity did not significantly differ between genotypes, but GMTs significantly differed among genotypes and sampling times. No significant difference was found in GMTs among age groups or regions. CONCLUSION: Genotype-specific neutralizing antibody titers against JEV G1 and G3 differed significantly in horses immunized with the G3 vaccine. Antigenic differences between genotypes could reduce the vaccine's efficacy, requiring the development of a new vaccine.

Development of indirect ELISA for the detection of canine adenovirus type 2 antibodies in dog sera.

BACKGROUND: Canine adenovirus type 2 (CAV-2) induces infectious laryngotracheitis in members of the family Canidae, including dogs. To date, no ELISA kits specific for CAV-2 antibody have been commercialized for dogs in Korea. OBJECTIVES: We aimed to develop new indirect enzyme-linked immunosorbent assay (I-ELISA) to perform rapid, accurate serological surveys of CAV-2 in dog serum samples. METHODS: In total, 165 serum samples were collected from dogs residing in Chungbuk and Gyeongbuk provinces between 2016 and 2018. The Korean CAV-2, named the APQA1701-40P strain, was propagated in Madin-Darby canine kidney cells and purified in an anion-exchange chromatography column for use as an antigen for I-ELISA. The virus-neutralizing antibody titers of CAV-2 in the dog sera were measured by virus neutralization (VN) test. RESULTS: We compared the results obtained between the VN and new I-ELISA tests. The sensitivity, specificity, and accuracy of new I-ELISA were 98.6%, 86.4% and 97.0% compared with VN test, respectively. New I-ELISA was significantly correlated with VN (r = 0.91). CONCLUSIONS: These results indicate that new I-ELISA is useful for sero-surveillance of CAV-2 in dog serum.

The Presence of Rabies Virus-Neutralizing Antibody in Wild Boars (Sus scrofa), a Non-Target Bait Vaccine Animal in Korea.

Oral vaccination with bait is an effective method to prevent rabies in wildlife, but non-target wild animals may also ingest the bait vaccine. In Korea, the target animal of the rabies bait vaccine is the raccoon dog (Nyctereutes procyonoides). Bait vaccines have been distributed in Korea for 20 years; although wild raccoon dogs have been tested for antibodies, rabies antibodies have never been investigated in non-target wild animals. Therefore, this study investigated rabies antibody formation in wild boars (Sus scrofa), which is likely the main competitor for the bait vaccine in Korea. In bait areas, 20 of 109 wild boars (18.3%) were seropositive, and 39 of 470 wild boars (8.3%) in non-bait areas were also seropositive. These results provide insights regarding bait uptake or vaccination in non-target wild boars.

Establishment of multiplex RT-PCR for differentiation between rabies virus with and that without mutation at position 333 of glycoprotein.

Rabid raccoon dogs (Nyctereutes procyonoides koreensis) have been responsible for animal rabies in South Korea since the 1990s. A recombinant rabies vaccine strain, designated as ERAGS, was constructed for use as a bait vaccine. Therefore, new means of differentiating ERAGS from other rabies virus (RABV) strains will be required in biological manufacturing and diagnostic service centers. In this study, we designed two specific primer sets for differentiation between ERAGS and other RABVs based on mutation in the RABV glycoprotein gene. Polymerase chain reaction analysis of the glycoprotein gene revealed two DNA bands of 383 bp and 583 bp in the ERAGS strain but a single DNA band of 383 bp in the field strains. The detection limits of multiplex reverse transcription polymerase chain reaction (RT-PCR) were 80 and 8 FAID50/reaction for the ERAGS and Evelyn-Rokitnicki-Abelseth strains, respectively. No cross-reactions were detected in the non-RABV reference viruses, including canine distemper virus, parvovirus, canine adenovirus type 1 and 2, and parainfluenza virus. The results of multiplex RT-PCR were 100% consistent with those of the fluorescent antibody test. Therefore, one-step multiplex RT-PCR is likely useful for differentiation between RABVs with and those without mutation at position 333 of the RABV glycoprotein gene.

Immunogenicity of a new, inactivated canine adenovirus type 2 vaccine for dogs.

PURPOSE: We constructed a new canine adenovirus type 2 (CAV-2) vaccine candidate using the recently isolated Korean CAV-2 strain; we termed the vaccine APQA1701-40P and evaluated its safety and immunogenicity in dogs. MATERIALS AND METHODS: To generate the anti-CAV-2 vaccine, APQA1701 was passaged 40 times in MDCK cells growing in medium containing 5 mM urea and the virus was inactivated using 0.05% (volume per volume) formaldehyde. Two vaccines were prepared by blending inactivated APQA1701-40P with two different adjuvants; both were intramuscularly injected (twice) into guinea pigs. The safety and immunogenicity of the Cabopol-adjuvanted vaccine were evaluated in seronegative dogs. The humoral responses elicited were measured using an indirect enzyme-linked immunosorbent assay (I-ELISA), and via a virus neutralization assay (VNA). RESULTS: The new, inactivated CAV-2 vaccine strain, APQA1701-40P, lacked six amino acids of the E1b-19K protein. In guinea pigs, the Cabopol-adjuvanted vaccine afforded a slightly higher VNA titer and I-ELISA absorbance than an IMS gel-adjuvanted vaccine 4 weeks post-vaccination (p>0.05). Dogs inoculated with the former vaccine developed a significantly higher immune titer than non-vaccinated dogs. CONCLUSION: The Cabopol-adjuvanted, inactivated CAV-2 vaccine was safe and induced a high VNA titer in dogs.

Rabies immune status of raccoon dogs residing in areas where rabies bait vaccine has been distributed.

Since 2000, large amounts of rabies bait vaccine have been distributed in two provinces where raccoon dog-mediated rabies has occurred. A total of 146 raccoon dogs were caught in Gangwon and Gyeonggi Provinces from January 2017 to June 2018, and raccoon dog blood samples were collected. Of the 146 raccoon dogs, 13.7% (20/146) had rabies antibodies. In Gyeonggi and Gangwon provinces, the rate of rabies antibody was 8.5% (5/59) and 17.2% (15/87), respectively. Considering these results, it would be desirable to improve the distribution method or use a new bait vaccine to prevent animal rabies in South Korea.

Strategies to maintain Korea's animal rabies non-occurrence status.

Endemic animal rabies causes >99% of all human rabies cases; elimination of animal rabies reduces the rate of human infections. The most recent animal rabies cases in the Gangwon and Gyeonggi provinces of Korea occurred in November 2012 and February 2013, respectively. Here we explore ways to ensure that Korea remains animal rabies non-occurrence. The government must prevent rabies recurrence by vaccinating dogs, distributing bait vaccine in regions with a high rabies risk, performing laboratory-based surveillance, preventing the flow of rabies-suspect animals from neighboring countries, and enhancing border quarantine. As has already been shown in several developed countries, careful and ongoing rabies control will allow Korea to remain animal rabies-free.

Serosurvey of rabies virus, canine distemper virus, parvovirus, and influenza virus in military working dogs in Korea.

Rabies virus (RABV), canine distemper virus (CDV), canine parvovirus type-2 (CPV-2), and canine influenza A virus (CIV) are important contagious pathogens in canine populations. To assess post-vaccination immunity against RABV, CDV and CPV-2, and serological evidence of exposure to influenza A virus in military working dogs (MWDs) in Korea, we tested blood samples of 78 MWDs by fluorescent antibody virus neutralization (FAVN) for RABV, and by commercially available enzyme-linked immunosorbent assay (ELISA) for CDV, CPV-2, and CIV. Korean MWDs had high antibody-positive rates against RABV (97.4%, ≥0.5 IU/ml), CDV (94.8%), and CPV (100%). All dogs tested seronegative (0/78; 0%) for influenza A virus. Two 1-year-old dogs stationed in known rabies outbreak areas (Gangwon and Gyeonggi) exhibited VNA titers below the protective level (0.06 and 0.29 IU/ml, respectively). The breed and sex of MWDs were not significantly associated with antibody titers for RABV, CDV, or CPV; however, age was significantly associated with CPV antibody titers, while region of residence was associated with CDV antibody titer. Taken together, the data presented here provide important insights necessary for post-vaccination management and control of infectious diseases in MWDs.

Efficacy of inactivated variant porcine epidemic diarrhea virus vaccines in growing pigs.

PURPOSE: The first aim of this study was to develop a novel inactivated porcine epidemic diarrhea virus (PEDV) vaccine using the recently isolated Korean PEDV QIAP1401 strain and to evaluate its protective efficacy in growing pigs. The second was to determine the optimum adjuvant formulation of the inactivated PEDV vaccine that induces protection against viral challenge. MATERIALS AND METHODS: To generate high titers of infectious PEDV, the QIAP1401 isolate was passaged in Vero cells. The experimental vaccines were prepared from a binary ethyleneimine-inactivated QIAP1401 strain passaged sequentially 70 times (QIAP1401-p70), formulated with four commercial adjuvants, and administered twice intramuscularly to growing pigs. Challenge studies using a virulent homologous strain of PEDV QIAP1401-p11, which was passaged 11 times after isolation, were performed to assess protection against disease progression and viral shedding during the 15-day observation period. The vaccine-induced antibody responses were measured in serum samples collected at predetermined time points by indirect enzyme-linked immunosorbent assay and virus neutralization test. RESULTS: The QIAP1401-p70 strain had 42 amino acid (aa) mutations, including a 25 aa deletion, and was selected as the inactivated PEDV vaccine candidate. Although none of the pigs that received the experimental vaccines were completely protected against subsequent viral challenge, they exhibited a significantly higher immune response than did non-vaccinated control pigs. Among the vaccine groups, the highest antibody responses were observed in the pigs that received an oil-based multiphasic water/oil/water (W/O/W) emulsion adjuvanted vaccine, which delayed the onset of clinical symptoms and viral shedding. CONCLUSION: A novel inactivated PEDV vaccine formulated with a W/O/W emulsion adjuvant was both immunogenic and protective against viral challenge.

Isolation and characterization of a new porcine epidemic diarrhea virus variant that occurred in Korea in 2014.

Outbreaks of porcine epidemic diarrhea (PED) have resulted in significant economic losses in the swine industry, and another PED outbreak occurred in 2014 in Korea. Isolating and culturing PED virus (PEDV) allow investigations into its pathogenesis and the development of vaccines and diagnostic assays. In this study, we successfully isolated two PEDV isolates (QIAP1401 and QIAP1402) from naturally infected piglets at Jeju-do, Korea. Viral propagation was confirmed in Vero cells based on cytopathic effect, immunofluorescence assay, reverse transcription-polymerase chain reaction, and electron microscopic analyses. The QIAP401 isolate propagated well in Vero cells for 70 passages, with titers of 106.5 to 107.0 50% tissue culture infectious dose/mL, which increased gradually with passaging. The nucleotide and amino acid sequences of the QIAP1401 isolate were determined and compared with those of other PEDV isolates. The QIAP1401 isolate was determined to be closely related to the USA/Minnesota271/2014 strain (> 99.9% nucleotide similarity) that was isolated in the USA in 2014. Phylogenetic analysis based on several PEDV genes suggested that a new PEDV variant is circulating in the Korean swine industry, with 93.08% similarity to the SM98 strain isolated in 1998. In addition, the QIAP1401 strain showed strong virulence in 3-day-old piglets and 11-week-old growing pigs.

Development and evaluation of an anti-rabies virus phosphoprotein-specific monoclonal antibody for detection of rabies neutralizing antibodies using RFFIT.

BACKGROUND: Rabies is a major public health problem with a fatality rate close to 100%; however, complete prevention can be achieved through pre- or post-exposure prophylaxis. The rapid fluorescent focus inhibition test (RFFIT) is one of the recommended testing methods to determine the production of neutralizing antibodies after vaccination. Here, we report the development of a new monoclonal antibody (mAb) designed to react specifically with Rabies virus (RABV) phosphoprotein (P protein), and the evaluation of its applicability to the RFFIT and its effectiveness as a diagnostic reagent for human rabies. METHODOLOGY/PRINCIPAL FINDINGS: The mAb KGH P 16B8 was produced to target the P protein of the Korean KGH RABV strain. An indirect immunofluorescence assay (IFA) was conducted to detect various strains of RABV in various cell lines. Alexa-conjugated KGH P 16B8 (16B8-Alexa) was developed for the RFFIT. The IFA test could detect RABV up to a 1:2,500 dilution, with a detection limit comparable to that of a commercial diagnostic reagent. The sensitivity, specificity, positive predictive value, and negative predictive value of the RFFIT using 16B8-Alexa in 414 clinical specimens were 98.67%, 99.47%, 99.55%, and 98.42%, respectively. The results of the RFFIT with 16B8-Alexa were strongly correlated with those obtained using an existing commercial diagnostic reagent (r = 0.995, p<0.001). CONCLUSIONS/SIGNIFICANCE: The mAb developed in this study shows high sensitivity and specificity, confirming its clinical utility with the RFFIT to measure the rabies neutralizing antibody titer and establish a diagnosis in human. Thus, 16B8-Alexa is expected to serve as an alternative diagnostic reagent that is widely accessible, with potentially broad applications beyond those of the RFFIT in Korea. Further studies with 16B8-Alexa should provide insight into the immunological mechanism of the P protein of Korean RABV.