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ISSN : 2287-7991(Print)
ISSN : 2287-8009(Online)
Journal of the Preventive Veterinary Medicine Vol.36 No.4 pp.180-185
DOI :

Evaluation of Akabane vaccine strains based on molecular characterization

Dong-Kun Yang, Ha-Hyun Kim, Jin-Ju Nah, Sung-Suk Choi, Kum-Ok Seok, Sun-Young Kim, Jae-Jo Kim, Jae-Young Song
Animal, Plant and Fishery Quarantine Inspection and Agency
Received 26 April 2012, revised 10 October 2012, accepted 7 December 2012.

Abstract

Akabane virus causes congenital abnormalities of the central nervous system in the fetus or calf of an infectedruminant and classified into an arthropod-borne viral disease. For the purpose of quality control of an Akabane live vaccine,we identified the vaccine strains and investigated the nucleotide sequence similarity of the N gene derived from the commerciallyavailable five Akabane vaccines in Korea. The Vero cells infected with the Akabane vaccines showed specificcytopathic effects, which were characterized by the aggregation and detachment of cells. Four of the five commercial Akabanevaccine strains had identical nucleotide and amino acid sequences but the last vaccine strain had one point mutation in theamino acid sequence of the N gene. Alignment of the nucleotide and amino acid sequences showed 99.9 to 100% similarityamong the five commercial vaccine strains. The Akabane vaccine strains also had high nucleotide similarity ranging from 99.0to 99.6% with the Korean isolates, KV0505, K9 and 93FMX, respectively. Even though the live attenuated Akabane vaccineshave been used in South Korea since the 1980’s, the genetic characteristics of all the commercial vaccine strains have notchanged over the last 20 some years. Therefore, our results indicate that the current Akabane vaccine strains are still geneticallysecure and stable and well controlled during vaccine production.

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INTRODUCTION

Akabane disease can be affected by climate change and global warming because the Akabane virus (AKAV) is transmitted via vectors that are active in the summer season [10]. The mosquito species transmitting AKAV depend on the geographical region and this may include C. brebitarsis, C. oxystoma, and C. nebechulsus [20]. Since AKAV was first isolated in Japan in 1959, several Asian countries including Korea have reported outbreaks of AKAV infection in ruminants [2, 3, 5, 12, 14, 17]. AKAV infections are mainly associated with abortion, stillbirth and congenital defects in pregnant cattle, sheep and goats, which cause considerable economic loss in the livestock industry [4, 9]. Therefore, there have been efforts to develop a new inactivated vaccine containing AKAV to protect against AKAV infection, recently [9].  

AKAV belongs to the Simbu serogroup, genus Orthobunyavirus, family Bunyaviridae, and is composed of a three-segmented RNA genome designated L (large), M (medium), and S (small). The L segment RNA encodes the polymerase gene, which plays an important role in the transcriptase activity [6], whereas the M segment RNA encodes two envelop glycoproteins, Gn and Gc, which are responsible for viral neutralization. The S segment RNA contains nucleotides encoding two proteins, nucleoprotein (N) and a nonstructural protein (NS) [2, 6].  

Although the Akabane live vaccine has been used in cattle since the 1980’s in Korea, the vaccine strains have not been verified by molecular biology techniques such as reverse transcription polymerase chain reaction (RT-PCR) and nucleotide sequencing to improve quality control of the vaccines. Several kinds of vaccine strains including AKAV in other counties have been identified by recent genetic techniques [7, 9, 13]. The evaluation of Akabane vaccine strains based on molecular analysis is needed using advanced methods. In addition, the sequences of the N gene from AKAV have been studied for making diagnoses and for epidemiological studies by many researchers [1, 20]. However, the nucleotide sequence similarity among the commercial vaccine strains in Korea has not been properly investigated yet. Therefore, it is necessary to evaluate the vaccine strains based on their molecular characterization. 

In this study, we identified the Akabane vaccine strains using cytopathic effects (CPE) in cells, the indirect fluorescence assay (FA) test and RT-PCR technique, and analyzed the nucleotide sequence of the complete N gene from AKAV among the five commercial vaccines produced by veterinary pharmaceutical companies in Korea.  

MATERIALS AND METHODS

Identification of the vaccine strains

 For the identification of the Akabane vaccine strains, the five commercial Akabane vaccines were inoculated into Vero cells grown in alpha minimum essential medium (α- MEM: Gibco BRL, USA) with 10% heat-inactivated fetal bovine serum (Gibco BRL, USA). The cells were incubated in a CO2 incubator for 7 days and CPE was observed in the cells and fixed with 80% cold acetone. The fixed cells were incubated with an AKAV-specific monoclonal antibody (QIA, Anyang-si, Korea) at 37℃ for 1 h in a humid chamber and then stained with FITC conjugated anti-mouse IgG (KPL, USA). After washing in phosphate buffered saline, the cells were examined by fluorescence microscopy (Nikon, Japan). 

Vaccines

 The five Korean commercial Akabane vaccines produced by Korean animal vaccine companies and used in this study were as follows: Bovine Akabane Live vaccine (Green-cross Co.); Akabane cattle Vac (Daesung Co.); Bobishot Akabane (ChoongAng Co.); PRO-VAC AKAV (Komipharm Co.); and Bovine Akabane Live vaccine (Korea BNP Co.). The AKAV vaccines were subjected to RNA extraction.  

RNA extraction and RT-PCR

Viral RNAs were extracted from the five commercial vaccines using an RNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The extracted RNAs were eluted in 50 ㎕of RNase- and DNasefree water. RT-PCR was carried out using specific primers (AKAD1F, AKAD1R, AKAD2F and AKAD2R) that amplify the S regions of AKAV (Table 1). RT-PCR was performed in a reaction mixture containing 10 ㎕of denatured RNA, 1 ㎕of each primer (50 pmol), 10 ㎕of 5' buffer (12.5 mM MgCl2), 2 ㎕of dNTP mix, 2 ㎕of enzyme mix (reverse transcriptase and Taq polymerase), and 24 ㎕of distilled water (Qiagen, Hilden, Germany). The cycling profile consisted of cDNA synthesis at 42℃ for 30 min, followed by 35 cycles of 95℃ for 45 sec, 50℃ for 45 sec, and 72℃ for 1 min, with a final extension at 72℃ for 5 min. The PCR products were visualized using electrophoresis on 1.8% agarose gels containing ethidium bromide. 

Table 1. Oligonucleotide primers used for RT-PCR against Akabane virus

Cloning and sequencing

All of the PCR products that were purified using the gel extraction kit were ligated into the pGEM-T easy vector (Promega, USA) according to the manufacturer’s instructions. Plasmid DNA was amplified and isolated from Escherichia coli (DH5α), and recombinant plasmids were identified with EcoRI digestion (Bioneer, Daejeon, Korea). The sequences of the purified plasmids were analyzed with an MJ Research PTC-225 Peltier Thermal Cycler and ABI PRISM BigDyeTM Terminator Cycle Sequencing kits with AmpliTaq DNA polymerase (FS enzyme; Applied Biosystems, USA) according to the manufacturers’ instructions. Single-pass sequencing was performed for each template using universal primers (e.g., SP6 and T7). The fluorescent- labeled fragments were purified from the unincorporated terminators using an ethanol precipitation protocol. The samples were resuspended in distilled water and subjected to electrophoresis in an ABI 3730xl sequencer (Applied Biosystems, USA). Both DNA strands were sequenced to verify the sequences. 

Phylogenetic analysis

The nucleotide sequences, accession numbers, and names of the strains used for the phylogenetic analysis in this study were obtained from the GenBank database. Each N gene sequence of the five commercial vaccines was compared with that of the other known AKAV strains using Clustal W 2 [11]. Genetic distances were calculated using the Kimura-2 correction parameter and a phylogenetic tree was constructed using the neighbor-joining method with 1,000 bootstrap replicates in MEGA4 [18].  

Mouse inoculation test

To check the pathogenicity of the vaccine strain, 4 weekold mice were inoculated using the intracranial and peritoneal route with 0.03 ㎖and 0.5 ㎖of AKAVs, respectively. Clinical signs of the mice were observed for 17 days. 

RESULTS

Identification of Akabane virus

The Vero cells infected with the Akabane vaccine strains showed specific cytopathic effect, which was characterized by the aggregation and detachment of the cells (Fig. 1). In the indirect FA test using an AKAV specific monoclonal antibody, the fluorescence appeared in the infected cells (Fig. 1). RT-PCR with Akabane diagnostic specific primers amplified the N genes from the five vaccine strains. The PCR products of the N genes were detected as a 354 base pair amplicon on the 1.8% agarose gel (Fig. 2).  

Fig. 1 Akabane live vaccines produced by five Korean biological companies were inoculated in Vero cell and showed cytopathic effects. The fluorescence appeared in the Vero cells infected with the Akabane vaccines by immunofluorescence analysis. The abbreviations of the companies are as follows: A, CompanyA; B, CompanyB; C, CompanyC; D, CompanyD; E, CompanyE.

Fig. 2 Amplification of the Akabane virus N gene with the RT-PCR method using Akabane virus-specific primers. M: 1Kb DNA ladder, lane 1~5: Akabane live vaccine A to E, lane 6: KV0505 strain, lane 7: negative.

Sequence analysis of the Akabane vaccine strains

The complete N genes (702 bp) encoding the nucleoprotein from the five Akabane vaccines were cloned into the pGEM-T easy vector and sequenced and their amino acid sequences were deduced. Four of the five commercial Akabane vaccine strains (Company A, B, C and D) had identical nucleotide and amino acid sequences. Only the last Akabane vaccine strain (Company E) had one point mutation in the N protein when compared with the amino acid sequences of the other 4 Akabane vaccine strains (Fig. 3). The N gene sequences of 41 AKAVs retrieved from GenBank (NCBI) were compared with those of the vaccine strains. Alignment with the nucleotide sequences showed 99.9 to 100% similarity among the five commercial vaccine strains. The Akabane vaccine strains had also a high nucleotide similarity ranging from 99.0 to 99.6% with the Korean AKAV isolates, 93FMX, K9, and KV0505, which were isolated in 1993 and 2005, respectively (Fig. 4). In addition, alignment of the deduced amino acid sequences showed 99.9 to 100% similarities among the five commercial vaccine strains. The Akabane vaccine strains possessed high nucleotide similarity with other strains of group Ic between 97.3 and 99.7%. The nucleotide similarities of the Akabane vaccine strains with group Ⅱ, Ⅲ, and Ⅳ strains ranged from 96.7 to 83.5%, respectively.  

Fig. 3 Multiple sequence alignment of the deduced amino acids of the nucleoprotein among the five commercial Akabane vaccine strains. The amino acids that are identical to company A are indicated by dots, while the diferent one is indicated by an abbreviated letter. *: Numbers indicate the amino acid sequence of the Akabane virus nucleoproteins.

Fig. 4 Phylogenetic tree based on the complete N genes (702 bp) of the Akabane virus vaccine strains. Bootstrap values above 50 are shown. The abbreviations of countries are as follows: AUS, Australia; ISR, Israel; JPN, Japan; KEN, Kenya; KOR, Korea; TAI, Thailand.

Pathogenicity of the Akabane vaccine strains in mice

To check the pathogenicity of the Akabane vaccine strains, the vaccines containing 106.0 TCID50/㎖AKAV were diluted 10 fold and evaluated in 4-week-old Balb/c mice via intracranial and intraperitoneal inoculation. All mice were observed for 17 days. None of the mice inoculated with the Akabane vaccine strains intraperitoneally showed any symptoms. On the other hand, the mice inoculated with the Akabane vaccine strains intracranially showed a decreased body weight at 7 days post-inoculation without any clinical signs such as paralysis or death (Fig. 5).  

Fig. 5 Change in body weight in mice. The mice inoculated with the AKAV vaccine strains via the intraperitoneal route did not show any weight lost, but those inoculated via the intracranial route showed a decrease in weight at 7 days post- inoculation.

DISCUSSION

The AKAV live attenuated vaccines manufactured by the five veterinary vaccine companies in Korea have been used in domestic cattle for the prevention of Akabane disease [1, 21]. It is important to verify the vaccine strains because the Akabane vaccine strains approved at the time of licensing will be used for the production of commercial vaccines. Based on the nucleotide and deduced amino acid sequences for the complete N gene of AKAV, epidemiological studies have been reported by several scientists [1, 4, 8, 19, 21].  

We also analyzed the 702 bp sequences of the N genes obtained from five vaccine strains and compared them with those of the reference AKAV strains. Four commercially available vaccine strains (Company A~D) were 100% identical in nucleotide and deduced amino acid sequences when compared with each other (Fig. 3, Fig. 4). Only the last vaccine strain (Company E) had a single amino acid mutation (Methionine→Threonine) at position 177 in the N protein. A mutation in a nucleotide sequence in a conserved position can help to distinguish vaccine strains from other wild type strains. It is well known that RNA viruses have a high mutation rate during replication due to both the lack of proofreading and post-replication error correction by RNA polymerase [16]. The AKAV seed strain used to make vaccines is regulated by Animal, Plant and Fisheries Quarantine and Inspection Agency (QIA) and changes in the seed strain are not allowed without permission. The deduced amino acid sequences of the complete N gene of the AKAV vaccine strains were compared with those of the AKAV strains (Fig. 3) and a single nucleotide substitution was identified in one vaccine strain, suggesting that nucleotide change may be involved in disturbing the fidelity of the viral polymerase during the manufacturing process. The phylogenetic tree based on the nucleotide sequences of the N gene showed that the five commercial vaccine strains were closely related with the Korean isolates but diverged distantly from foreign isolates (Fig. 4).  

AKAV causes reproductive disorders during the first trimester of pregnancy in cattle [8] and leads to encephalitis in suckling mice [15]. The Korean isolate KV0505 was virulent in suckling mice in a previous report [21]. However, the 4 week-old mice inoculated with the vaccine strain via the intracranial or intraperitoneal route, respectively, did not show any symptoms such as paralysis and death, suggesting that the Akabane vaccine strain does not cause encephalitis in 4-week-old mice.  

In conclusion, our results indicate that the current Akabane vaccines strains are still genetically secure and stable and well controlled during vaccine production. However, further studies are required on new strains, which have been isolated recently, and recombinant proteins may be required in order to elevate the immune status in cattle.  

ACKNOWLEDGMENTS

We would like to thank Dr. Kyung-Woo Lee for the critical review of the manuscript. This work was financially supported by a grant from the Korean Institute of Planning & Evaluation for Technology in Food, Agriculture, Forestry & Fisheries (IPET).  

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