Differential recognition of the ORF2 region in a complete genome sequence of porcine circovirus type 2 (PCV2) isolated from boar bone marrow in Korea.

Porcine circovirus type 2 (PCV2) is the causative agent of post-weaning multisystemic wasting syndrome (PMWS) in swine. Here, a phylogenetic tree was constructed using PCV2 nucleotide sequences derived from the bone marrow of Korean boar and previously reported PCV2 sequences isolated from various countries. PCV2 from Korean boar bone marrow (KC188796) was classified into the group containing PCV2a-Canada and other PCV2 strain from Korea. While the ORF1 region of the PCV2 genome was highly conserved, ORF2 (the capsid protein coding region) was relatively variable. The nucleotide sequences for bone marrow-derived PCV2 were 93.4-99.0% homologous to the other reference sequences. The deduced amino acid sequences for the ORF1 and ORF2 coding regions were 97.4-99.3% and 84.5-97.4% homologous with the other reference strains, respectively, indicating that KC188796 did not differ markedly from the other PCV2 strains. Phylogenetic analysis demonstrated that bone marrow-derived PCV2 was highly similar to PCV2a from Canada and may be related to persistent PCV2 infections in swine.

Derivation of cell-adapted Sacbrood virus (SBV) from the native Korean honeybee.

Sacbrood virus (SBV), a causative agent of larval death in honeybees, is one of the most devastating diseases in bee industry throughout the world. Lately the Korean Sacbrood virus (KSBV) induced great losses in Korean honeybee (Apis cerana) colonies. However, there is no culture system available for honeybee viruses, including SBV, therefore, the research on honeybee viruses is practically limited until present. In this study, we investigated the growth and replication of SBV in cell cultures. The replication signs of KSBV after passages from mammalian cells was identified and confirmed by using combined approaches with nested, quantitative, negative-strand PCR and electron microscopy along with in vivo experiment. The results revealed that mammalian cell lines, including Vero cells could support the replication KSBV. Although there were no signs of cytopathic effect (CPE) in cells, it was for the first time demonstrated that SBV could be replicated in cells through the sequential passages linked with cell adaptation. KSBV from the present study would be a valuable source to understand the mechanism of pathogenicity of sacbrood virus in the future.

Seroprevalence of Toxoplasma gondii and Trichinella spiralis infections in wild boars (Sus scrofa) in Korea.

Toxoplasma gondii and Trichinella spiralis are important zoonotic pathogens with worldwide distributions. In Korea, several outbreaks of human toxoplasmosis and trichinellosis due to the consumption of infected wild animals have been reported. The purpose of this study was to determine the seroprevalence of T. gondii and T. spiralis infections in wild boars killed in Korea from December 2009 to October 2011. A total of 521 wild boars hunted in eight provinces were examined for antibodies to T. gondii and T. spiralis by using commercial ELISA kits. Overall, 25.1% of serum samples from individual boars were seropositive for T. gondii and 1.7% were seropositive for T. spiralis. Seropositive for T. gondii was found in the boars in all the eight provinces investigated and for T. spiralis in four provinces. This is the first report on the seroprevalence of T. gondii and T. spiralis infections in wild boars in Korea. The consumption of undercooked wild boar meat may expose humans to a high risk of infection.

Vero cells expressing porcine circovirus type 2-capsid protein and their diagnostic application.

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS) in swine. Although the incidences of PCV2-related diseases are ubiquitous throughout the world, the serological tools are rather limited, mainly because the virus does not induce any cytopathic effects in cells. The purpose of this study was to develop a rapid, sensitive and easy quantitative immunofluorescence assay (QIFA) using the recombinant PCV2 nucleocapsid protein (NCP) for the detection of PCV2-specific antibodies in pig sera. The recombinant PCV2 NCP was expressed in Vero cells by a lentivirus system. The performance of QIFA using these Vero cells as a diagnostic antigen was compared with currently available C-ELISA and I-ELISA; the relative sensitivity turned out to range from 92.5% up to 99.3%. The relative specificity was 93.3% when compared to C-ELISA as the gold standard. The serological experiment also indicated the inverse relationship between QIFA and the viral load in serum, semen, feces samples from 7 PCV2-positive boars. In addition, the PCV2 sequence detected from bone marrow cells shows 99% of sequence identity with PCV2 genome, confirming the infectivity of PCV2.

Molecular detection and phylogenetic analysis of Anaplasma bovis from Haemaphysalis longicornis feeding on grazing cattle in Korea.

Ticks are vectors of various pathogens that affect humans and animals throughout the world. Anaplasma bovis is one of the most important tick-borne pathogens that cause cattle diseases but there is still very little information available about this agent in Korea. In the present study, 535 Haemaphysalis longicornis tick pools were analyzed from grazing cattle in five Korean provinces. A. bovis was detected in 50 (9.3%) of 535 tick pools using 16S rRNA-based PCR. A. bovis infections were detected for the first time in ticks feeding on cattle in Chungbuk, Geongbuk, and Jeonbuk provinces in Korea. The 50 positive PCR products were sequenced successfully and compared with sequences in GenBank. Phylogenetic analysis of the Korean isolates classified them into four genotypes with nucleotide sequence identities of 99.4-100%. Two of the four genotypes had high similarity (99.8-100%) with known sequences. The other two genotypes have never been identified.

Analysis of the complete genome sequence and capsid region of black queen cell viruses from infected honeybees (Apis mellifera) in Korea.

Black queen cell virus (BQCV) infection is one of the most common viral infections in honeybees (Apis mellifera). A phylogenetic tree was constructed for 19 partial nucleotide sequences for the capsid region of South Korean BQCV, which were also compared with 10 previously reported BQCV sequences derived from different countries. The Korean BQCV genomes were highly conserved and showed 97-100% identity. They also showed 92-99% similarity with other country genotypes and showed no significant clustering in the phylogenetic tree. In order to investigate this phenomenon in more detail, the complete genome sequence of the Korean BQCV strain was determined and aligned with those from a South African reference strain and European genotypes, Poland4-6 and Hungary10. A phylogenetic tree was then constructed. The Korean BQCV strain showed a high level of similarity (92%) with Hungary10, but low similarity (86%) with the South African reference genotype. Comparison of the Korean and other sequences across different genome regions revealed that the 5'-UTR, the intergenic region, and the capsid regions of the BQCV genome were highly conserved. ORF1 (a non-structural protein coding region) was more variable than ORF2 (a structural protein coding region). The 5'-proximal third of ORF1 was particularly variable and contained several insertions/deletions. This phenomenon may be explained by intra-molecular recombination between the Korean and other BQCV genotypes; this appeared to have happened more with the South African reference strain than with the European genotypes.

Molecular investigation of tick-borne pathogens in ticks from grazing cattle in Korea.

This study was carried out to identify the tick species that infest grazing cattle and to determine the presence of tick-borne pathogens transmitted by these ticks in Korea. A total of 903 ticks (categorized into 566 tick pools) were collected from five provinces during 2010-2011. The most prevalent tick species was Haemaphysalis longicornis, followed by three Ixodes spp. ticks. The collected ticks were infected with both rickettsial and protozoan pathogens. In all, 469 (82.9%) tick pools tested positive for the Anaplasma/Ehrlichia 16S rRNA gene, whereas 67 (11.8%) were positive for the Babesia/Theileria 18S rRNA gene. Among the rickettsial pathogens, E. canis was detected with the highest rate (22.3%), followed by A. platys (20%), E. chaffeensis (19.4%), E. ewingii (19.3%), Rickettsia sp. (12.4%), A. phagocytophilum (5.5%) and E. muris (0.5%). Among the protozoan pathogens, T. equi was detected with the highest rate (7.2%), followed by T. sergenti/T. buffeli (3.7%) and B. caballi (0.35%). Simultaneous infections with up to seven pathogens were also identified. In particular, ticks infected with rickettsial pathogens were also infected with protozoan pathogens (22 samples). All five provinces investigated infected with tick-borne pathogens.

Phylogenetic analysis of black queen cell virus genotypes in South Korea.

The black queen cell virus (BQCV), a picorna-like honeybee virus, was first isolated from queen larvae and pupae of honeybees found dead in their cells. BQCV is the most common cause of death in queen larvae. Phylogenetic analysis of two Apis cerana and three Apis mellifera BQCV genotypes collected from honeybee colonies in different regions of South Korea, central European BQCV genotypes, and a South African BQCV reference genotype was performed on a partial helicase enzyme coding region (ORF1) and a partial structural polypeptide coding region (ORF2). The phylogeny based on the ORF2 region showed clustering of all the Korean genotypes corresponding to their geographic origin, with the exception of Korean Am str3 which showed more similarity to the central European and the South African reference genotype. However, the ORF1-based tree exhibited a different distribution of the Korean strains, in which A. cerana isolates formed one cluster and all A. mellifera isolates formed a separate cluster. The RT-PCR assay described in this study is a sensitive and reliable method for the detection and classification of BQCV strains from various regions of Korea. BQCV infection is present in both A. cerana and A. mellifera colonies. With this in mind, the present study examined the transmission of honeybee BQCV infections between A. cerana and A. mellifera.

Reverse transcription loop-mediated isothermal amplification for sensitive and rapid detection of Korean sacbrood virus.

Sacbrood virus (SBV) is one of the most serious honeybee viruses. The virus causes failure to pupate and death in both larvae and adult bees. Recently, the Korean sacbrood virus (KSBV) caused great losses in Korean honeybee (Apis cerana) colonies. Although KSBV shows high homology with SBV strains, it has unique motifs and causes different symptoms. Therefore, a simple, sensitive and specific method for detecting KSBV is needed urgently. In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting KSBV using total RNA extracted from honeybees (A. cerana) infected with SBV. The LAMP and the polymerase chain reaction (PCR) methods were then compared for their ability to detect KSBV in clinical samples. The virus was detected in RT-LAMP reactions containing 10(3) copies of pBX-KSBV within 30min, which was comparable to RT-PCR. In addition, the LAMP was able to distinguish between KSBV and other closely-related SBV strains, indicating a high degree of specificity. This simple and sensitive RT-LAMP assay is a useful method for the rapid diagnosis of KSBV infection in honeybees.

Phylogenetic analysis of benign Theileria species based on major piroplasm surface protein (MPSP) genes from ticks of grazing cattle in Korea.

Complete major piroplasm surface protein (MPSP) gene sequences of benign Theileria parasites were isolated from ticks of grazing cattle in Korea. A total of 556 tick samples were collected in five provinces: Chungbuk, Jeonbuk, Jeonnam, Gyeongbuk, and Jeju during 2010-2011. Fifteen samples from Chungbuk and Jeonnam were positive for the Theileria MPSP gene by PCR amplification using a specific primer set. A phylogenetic tree was constructed with the amplified gene sequences and 26 additional sequences published in GenBank. The benign Theileria parasites were classified into eight types, those isolated from Korean cattle ticks belonged to Types 1 (Ikeda), 2 (Chitose), 4, and 8. Types 2 and 4 were the most common types, with the rate of 40%, followed by Types 1 and 8 (with the rate of 13% and 7%, respectively). Nucleotide sequence identities of 23 theilerial MPSP sequences (15 MPSP gene sequences amplified and 8 sequences published) ranged from 67.3 to 99.8%. Multiple alignments of the deduced amino acid sequences also showed that each type was characterized by specific amino acids: 7 for Type 1, 9 for Type 2, 4 for Type 4, and 3 for Type 8.

Prevalence and distribution of six bee viruses in Korean Apis cerana populations.

The prevalence and distribution of six bee viruses was investigated in 527 Apis cerana samples which were collected from five provinces in South Korea. The most prevalent virus, black queen cell virus (BQCV), was present in 75.11% of 446 adult bee samples, followed by sacbrood virus (SBV) in 30.71%. Deformed wing virus (DWV), Kashmir bee virus (KBV), and chronic bee paralysis virus (CBPV) were present at lower levels of 8.07%, 1.56%, and 0.44%, respectively. The most prevalent virus in 81 larvae samples was SBV, with an incidence of 60.49%, followed by BQCV in 48.14%, DWV in 6.17%, and KBV in 1.23% of samples. CBPV infection was not detected in larvae samples, and acute bee paralysis virus (ABPV) was not present in both larvae and adult bee. Simultaneous infections with up to four viruses were also identified. Of these, infections with SBV and BQCV were most frequent in 25.61% of samples. The distribution of these viruses varied considerably throughout the geographic regions investigated. The three provinces of Gyeongbuk, Jeonnam, and Chungbuk had the highest frequency of bee viruses.

Competitive ELISA for the detection of antibodies to Rift Valley fever virus in goats and cattle.

Rift Valley fever virus (RVFV) is one of the important emerging viral diseases of serious impact in public health and animal hygiene both in human and animal industries. In this study, we developed a monoclonal antibody-based competitive ELISA for the detection of antibodies to RVFV in goats and cattle. The recombinant N protein of RVFV was expressed in E. coli with a six-histidine tag, and the purified N protein was used for detecting antigen with a competitive monoclonal antibody against RVFV antibodies. The competitive ELISA (C-ELISA) could detect antibodies at 9-11 days after inoculation in goats and cattle with a sensitivity of 94.7% (virus neutralization titer >32) and specificity of 99.7%, respectively. In addition, the C-ELISA did not show any cross-reactivity with positive sera against arboviruses such as Akabane, Aino, Chuzan, Ibaraki and bovine ephemeral fever virus, which are prevalent viral agents in ruminant animals throughout Southeast Asia. The results of the present study indicate that the C-ELISA is a simple, rapid and convenient serodiagnostic method for RVFV in goats and cattle.

Genetic analysis of Fasciola isolates from cattle in Korea based on second internal transcribed spacer (ITS-2) sequence of nuclear ribosomal DNA.

Nuclear ribosomal DNA sequence of the second internal transcribed spacer (ITS-2) has been used efficiently to identify the liver fluke species collected from different hosts and various geographic regions. ITS-2 sequences of 19 Fasciola samples collected from Korean native cattle were determined and compared. Sequence comparison including ITS-2 sequences of isolates from this study and reference sequences from Fasciola hepatica and Fasciola gigantica and intermediate Fasciola in Genbank revealed seven identical variable sites of investigated isolates. Among 19 samples, 12 individuals had ITS-2 sequences completely identical to that of pure F. hepatica, five possessed the sequences identical to F. gigantica type, whereas two shared the sequence of both F. hepatica and F. gigantica. No variations in length and nucleotide composition of ITS-2 sequence were observed within isolates that belonged to F. hepatica or F. gigantica. At the position of 218, five Fasciola containing a single-base substitution (C>T) formed a distinct branch inside the F. gigantica-type group which was similar to those of Asian-origin isolates. The phylogenetic tree of the Fasciola spp. based on complete ITS-2 sequences from this study and other representative isolates in different locations clearly showed that pure F. hepatica, F. gigantica type and intermediate Fasciola were observed. The result also provided additional genetic evidence for the existence of three forms of Fasciola isolated from native cattle in Korea by genetic approach using ITS-2 sequence.

Development of inactivated trivalent vaccine for the teratogenic Aino, Akabane and Chuzan viruses.

Aino, Akabane and Chuzan viruses are arthropod-borne (arbo) viruses transmitted by blood-sucking insects like mosquitoes and Culicoides biting midges. These arbovirus infections are mainly associated with abortion, stillbirth and congenital defects in pregnant cattle, sheep and goats, which induces a considerable economic loss in livestock industry. The viruses seem to be widely distributed in Southeast Asia and Australia. As a control strategy, an inactivated trivalent vaccine against Aino, Akabane and Chuzan virus was developed by using binary ethylenimine or formalin as an inactivating agent. The newly developed trivalent vaccine is evaluated for its safety and immunogenicity in animals such as mice, guinea pigs and cattle. The immune responses were significantly detected within 2-weeks after second vaccination without any side effects. Since the field application of experimental vaccine also revealed increased antibodies in inoculated cattle, we demonstrated that these trivalent vaccines could be used as a vaccine to control the arboviral infections in ruminants.

Direct typing and molecular evolutionary analysis of field samples of foot-and-mouth disease virus collected in Viet Nam between 2006 and 2007.

In this study, we used universal or duplex serotype-specific (O and Asia 1) RT-PCR to analyze clinical field samples of foot-and-mouth disease virus (FMDV) or virus isolates collected in Viet Nam between 2006 and 2007. We found viral serotypes O and Asia 1 circulating concurrently during this period. Direct sequencing of type-specific RT-PCR products revealed the existence of three different topotypes of serotype O: Southeast Asia (SEA), Middle East-South Asia (ME-SA), and Cathay. Of these, SEA was most prevalent during the period. All samples of serotype Asia 1 belonged to genetic group V. Based on the rooted maximum likelihood phylogenetic trees inferred from the VP1 region, new lineages in topotype SEA were originating from Viet Nam, and group V strains of Asia 1 have undergone fewer passages from the common ancestor, compared with other genetic groups. The co-circulation of different types of FMDV may complicate the individual or population genomic structures of FMDV and make conventional multiplex diagnostic methods and phylogenetic analyses with relevant evolutionary models essential in Viet Nam.

Multiple shRNAs driven by U6 and CMV promoter enhances efficiency of antiviral effects against foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is an economically significant animal disease because of the speed of its transmission. The current vaccine for FMD provides no protection until 7 days post-vaccination, thus reducing its effectiveness in the case of an outbreak. Small interfering RNA (siRNA) is a promising antiviral approach because it can induce a protective response much more rapidly. This study is the first report to apply multiple short hairpin RNA (shRNA) expression systems to inhibit foot-and-mouth disease virus (FMDV) replication. Three different shRNAs, one targeting 2B region and two targeting 3C region, were driven by three RNA Polymerase III (Pol III) promoters, U6 or a combination of two U6 promoters and one RNA Polymerase II (Pol II) promoter, CMV. The adenoviruses simultaneously expressing three different shRNAs in a single construct had significantly enhanced antiviral effects compared with those expressing only a single shRNA, those expressing double shRNAs or a mixture of adenoviruses expressing a single shRNA and the adenovirus expressing double shRNAs, both in vitro and in vivo. The adenoviruses had broad antiviral effects against seven serotypes of FMDV, including O, A, Asia1, C, SAT1, SAT2, and SAT3 in vitro, but differed in their efficacy. The adenovirus expressing multiple shRNAs driven by three U6 promoters had strong antiviral effects in suckling mice challenged with O, A, and Asia1 serotype of FMDV.

Use of a baculovirus-expressed structural protein for the detection of antibodies to foot-and-mouth disease virus type A by a blocking enzyme-linked immunosorbent assay.

A blocking enzyme-linked immunosorbent assay (ELISA) with a baculovirus-expressed structural protein was developed for the detection of antibodies to foot-and-mouth disease virus type A. It exhibited 99% specificity with a cutoff of 53% inhibition. Its sensitivity was comparable to the sensitivities of the virus neutralization test and the liquid-phase blocking ELISA, indicating its potential as an alternative assay.

Development of a blocking ELISA using a recombinant glycoprotein for the detection of antibodies to vesicular stomatitis New Jersey virus.

A recombinant glycoprotein (R-GP) of vesicular stomatitis New Jersey virus (VSV-NJ) was expressed in insect cells by a baculovirus system. Its utility as a diagnostic antigen in a blocking ELISA was investigated as an alternative to the current native GP extracted from VSV-NJ. With the cut-off value of 73% inhibition, the R-GP ELISA exhibited 99.1% specificity for naive sera from cattle and horses. It did not cross-react with VSV-Indiana (VSV-IN) positive sera and differentiated from foot-and-mouth disease and swine vesicular disease. Taken together, this is the first report that the R-GP has a potential to be used as a diagnostic antigen in place of the native GP for the detection of antibodies to VSV-NJ in cattle and horses.

Agar gel immunodiffusion analysis using baculovirus-expressed recombinant bovine leukemia virus envelope glycoprotein (gp51/gp30(T-)).

Bovine leukemia virus (BLV) envelope glycoprotein (gp51/ gp30(T-)), consisting of BLV gp51 and BLV gp30 that lacked its C-terminal transmembrane domain, was expressed in insect cells under the control of the baculovirus polyhedron promoter. Recombinant BLV gp51/gp30(T-) secreted from insect cells was determined by immunofluorescence, enzyme-linked immunosorbent and western blot assays using a BLV-specific monoclonal antibody and BLV-positive bovine antibodies. An agar gel immunodiffusion (AGID) test using gp51/gp30(T-) as the antigen for the detection of BLV antibodies in serum was developed and compared to traditional AGID, which uses wild type BLV antigen derived from fetal lamb kidney cells. AGID with the recombinant BLV gp51/gp30(T-) was relatively more sensitive than traditional AGID. When the two methods were tested with bovine sera from the field, the recombinant BLV gp51/gp30(T-) and traditional antigen had a relative sensitivity of 69.8% and 67.4%, respectively, and a relative specificity of 93.3% and 92.3%. These results indicated that the recombinant BLV gp51/gp30(T-) is an effective alternative antigen for the diagnosis of BLV infection in cattle.

A recombinant protein-based ELISA for detecting antibodies to foot-and-mouth disease virus serotype Asia 1.

A recombinant protein-based ELISA was evaluated for detecting antibodies to foot-and-mouth disease virus (FMDV) serotype Asia 1. The recombinant protein (rP13C) was derived from the P1 precursor and 3C protease genes that were cloned into a single expression vector and expressed in insect cells. This protein elicited a low titer of FMDV neutralizing antibodies in pigs. Its utility as a diagnostic antigen was explored in a blocking ELISA using monoclonal antibodies. The rP13C ELISA yielded higher endpoint titers than the liquid phase blocking (LPB) ELISA and virus neutralization test performed on sera from goats challenged with FMDV post-vaccination. The rP13C ELISA correctly scored the FMD international reference weak positive serum. The relative sensitivity between the rP13C ELISA and LPB ELISA was equivalent for vaccinated sera. With this comparable sensitivity, the rP13C ELISA exhibited a specificity of 99.7% for domestic naive swine, bovine and caprine sera. This report demonstrates that an ELISA using recombinant proteins has the potential to replace the LPB ELISA using an inactivated FMDV antigen as a simple and robust serological tool for screening antibodies to FMDV serotype Asia 1.