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Reactivity and prevalence of neutralizing antibodies against Japanese strains of bovine viral diarrhea virus subgenotypes
Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 35–39
Contents lists available at ScienceDirect
Comparative Immunology, Microbiology and Infectious Diseases journal homepage: www.elsevier.com/locate/cimid
Reactivity and prevalence of neutralizing antibodies against Japanese strains of bovine viral diarrhea virus subgenotypes Fujiko Minami a, Makoto Nagai b, Mika Ito a, Tatsuhiko Matsuda c, Hikaru Takai c, Yoshiko Jinkawa c, Takeshi Shimano a, Michiko Hayashi b, Yoshihisa Seki d, Yoshihiro Sakoda e, Katsuaki Sugiura f, Hiroomi Akashi g,* a
Ishikawa Nanbu Livestock Hygiene Service Center, Kanazawa, Ishikawa 920-3101, Japan Ishikawa Prefectual Livestock Research Center, Hodatsushimizu-cho, Ishikawa 929-1325, Japan Ishikawa Hokubu Livestock Hygiene Service Center, Nanao, Ishikawa 929-2126, Japan d Iwate Prefecture Central Livestock Hygiene Service Center, Takizawa-mura, Iwate 020-0173, Japan e Laboratory of Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido 060-0818, Japan f Food and Agricultural Materials Inspection Center, 2-1 Shintoshin, Chuo-ku, Saitama, Saitama 330-9731, Japan g Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan b c
A R T I C L E I N F O
A B S T R A C T
Article history: Accepted 9 October 2009
Bovine viral diarrhea virus (BVDV) field isolates show genetic and antigenic diversity. At least 14 subgenotypes of BVDV-1 and 4 of BVDV-2 have been identified in Artiodactyla worldwide. Of these, 6 subgenotypes of BVDV-1 and 1 of BVDV-2 have been isolated in Japan. Previously, we reported that each subgenotype virus expresses different antigenic characteristics. Here we investigated the reactivity of neutralizing antibodies against representative strains of Japanese BVDV subgenotypes using sera from 266 beef cattle to estimate the prevalence of this epidemic virus among cattle in Japan. Antibody titers at concentrations at least 4-fold higher than antibodies against other subgenotype viruses were considered subgenotype specific. Subgenotype-specific antibodies were detected from 117 (80.7%) of 145 sera samples (69.7% against BVDV-1a, 1.4% against BVDV-1b, 8.3% against BVDV-1c, and 1.4% against BVDV-2a). The results suggest that neutralization tests are useful in estimating currently epidemic subgenotypes of BVDV in the field. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Antigenic difference Bovine viral diarrhea virus Epidemic virus Neutralization test Subgenotype
1. Introduction Bovine viral diarrhea virus (BVDV) is an important bovine pathogen because it causes significant economic losses in the cattle industry [1]. BVDV is classified based on differences in genomic sequences [2]. Two genotypes, BVDV-1 and BVDV-2, were recently reported [3,4], and have been recognized as virus species [5]. Investigations have revealed that BVDV-1 and BVDV-2 are subdivided
* Corresponding author. Tel.: +81 3 5841 5396; fax: +81 3 5841 8184. E-mail address: [email protected] (H. Akashi). 0147-9571/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.cimid.2009.10.007
into at least 14 and 4 subgenotypes, respectively [6–8]. Although BVDVs share epitopes of a common pestiviral antigen, antigenic differences among BVDV isolates have been demonstrated using cross-neutralization tests [2,7]. Because the antigenic diversity among BVDVs has an impact on disease diagnosis and control via vaccines [2,9,10], it is important to identify the antigenic properties of field isolates for BVDV prevention and control. New subgenotypes of BVDV-1 were recently reported [6,11]. We thereby confirmed the subgenotypes of Japanese BVDV using phylogenetic analyses of these new subgenotypes. Furthermore, to estimate the currently epidemic subgenotypes of BVDV among the
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F. Minami et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 35–39
Japanese cattle population, we investigated the reactivity of neutralizing antibodies against the representative strains of subgenotypes. 2. Materials and methods 2.1. Representative strains of Japanese BVDV subgenotypes and cells BVDV-1a, -1b, -1c, and BVDV-2a are the predominant subgenotypes of BVDV in Japan [12]. Nose [13], IS27CP/01 [7], SoCP/75 [7], IS25CP/01 [7], and KZ91CP [14] were selected as the representative strains of BVDV-1a, -1b, -1n, -1o, and BVDV-2a, respectively. IW56/05/CP isolated from a cow that had developed mucosal disease in 2005 [15] was selected as a representative strain of BVDV-1c. In addition, 7 BVDV-1 isolates, IS16NCP/99, IS22NCP/00, IS17NCP/99, IS36NCP/04, IS14NCP/99, IS37NCP/05 and IS26NCP/01[0], isolated from persistently infected cattle were subjected to cross-neutralization tests and phylogenetic analyses together with above strains. All viruses used for sera testing of field cattle were purified using limiting dilution methods as described previously [14] except nonpathogenic (NCP) strains. Bovine fetal muscle (BFM) cell cultures within 20 passages [16] were used for virus preparation and neutralization tests. BFM cells were grown in Eagle’s minimum essential medium (EMEM) containing 10% horse serum, 10% tryptose phosphate broth and 0.015% sodium bicarbonate. The cells and horse serum supplemented into the media were confirmed absence of contamination with BVDV by virus isolation and RT-PCR before use. 2.2. Nucleotide sequencing and phylogenetic analyses A portion of the 50 -untranslated region and Erns, and the full genomic region of Npro and Core, were amplified by RTPCR using primers 324 (position in BVDV SD-1: 108-128) [17] and 1400R (position in BVDV SD-1: 1448-1430) [18]. The nucleotide sequences of the genomic region encoding Npro were determined by direct sequencing as described previously [7]. Of the nucleotide sequences obtained, 385 bp of the Npro gene were subjected to phylogenetic analyses using MEGA4 [19], employing neighbor-joining (NJ) [20] and maximum-parsimony (MP) [21] methods. The distances between sequences were estimated by using the three-parameter method [22]. The reliability of the phylogenetic tree was evaluated by running 1000 replicas in the bootstrap test [23].
wells of the microplate, and incubated at 37 8C. SN titers were expressed as the reciprocal of the highest serum dilution that neutralized the virus. Antisera against the representative Japanese strains were prepared using BVDV-free sheep as previously described [24]. To characterize the antigenic relatedness among BVDV subgenotypes, antigenic similarity (R) values were calculated according to the formula reported by Archetti and Horsfall [25]. 2.4. Sera testing and subgenotype-specific antibodies A total of 266 serum samples from breeding Japanese Black beef cattle unvaccinated against BVDV infection were collected from 33 farms in Ishikawa Prefecture between 2004 and 2006. The movement history of the animals was traced using the beef traceability system (http://www.id.nlbc.go.jp/top.html) of the National Livestock Breeding Center in Japan. Based on results from cross-SN tests, antibody titers at concentrations at least 4-fold higher than antibodies against other subgenotype viruses were considered subgenotype specific. 3. Results 3.1. Phylogenetic analyses Fig. 1 shows the phylogenetic tree, which was constructed using the NJ method with 385 bp of the Npro gene nucleotide sequences of 17 Japanese BVDV isolates together with those of 26 reference strains obtained from the DDBJ databank. Nucleotide sequence data for BVDV-1 strains 71-03, 71-15, and 71-16 were kindly provided by Dr. S. Vilcek (University of Veterinary Medicine, Slovakia). In all, 16 clusters of BVDV-1 and 1 cluster of BVDV-2 were identified. Japanese isolates branched into 7 clusters: BVDV-1a, -1b, -1c, -1j, -1n, -1o, and BVDV-2a, as previously reported. Bootstrap analysis can be applied to a phylogenetic tree in order to estimate the frequency with which particular branch occurs and values of greater than 70% are considered as supporting the grouping. BVDV-1l reported by Jackova et al. [6] and Kadir et al. [11] were independently placed in different clusters and distinct from the Japanese subgenotypes. The phylogenetic tree constructed by the MP method showed almost the same topology as that constructed by the NJ method (data not shown). 3.2. Cross-SN test
2.3. Cross-neutralization tests and antisera Serum neutralization (SN) tests were performed in BFM cell cultures grown in 96-well microplates as described by Shimizu and Satou [16]. Briefly, serial 2fold dilutions of heat-inactivated sera were made in volume of 0.025 ml in a microplate with the maintenance medium, an equal volume of the virus suspension containing 200 TCID50/0.1 ml was added, and incubated at 37 8C for 1 h. Then 0.1 ml of BFM cell suspension (about 2 105 cells/ml) was added into all
Neutralizing antibody titers against each virus subgenotype were tested and are shown in Table 1. The crossSN titers of the antisera against homologous BVDV-1 were 4–64-fold higher than those against the heterologous viruses. The maximum antigenic differences within BVDV-1 were observed between So CP/75 (BVDV-1n) and either Nose (BVDV-1a) or IS25CP/01 (BVDV-1m) using antiserum against So CP/75. The minimum antigenic difference was found between Nose (BVDV-1a) and IW56/ 05 (BVDV-1c) using antiserum against Nose. The SN titers
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Fig. 1. Phylogenetic analysis of the 385 nt sequence in the Npro region of 17 Japanese BVDV isolates (bold and italics) with the reported nucleotide sequences of 26 reference strains obtained from the DDBJ/EMBL/GenBank databases and personal communication. The phylogenetic tree was constructed using the neighbor-joining method and bootstrap analysis (n = 1000) in MEGA4. The accession numbers of nucleotide sequences are as follows: NADL, M31182; SD-1, M96751; Deer GB1, U80902; 23-15, AF287279; G-Au, AF287285; J-Au, AF287286; W-Au, AF287290; A-Au, AF287283; L-Au, AF287283; ZM-95, AF526381; 519, AF144464; Deer NZ1, U80903; Bega, AF049221; CP7, U63479; Osloss, M96687; TR16, EU163964; TR1, EU163950; TR29, EU163977; 3186V6, AF287282; CH-Suwa, AF117699; 721, AF144463; F-Au, AF287284; 890, U18059.
of antiserum against BVDV-2 and BVDV-1 viruses were 8– 512-fold lower than that of the homologous virus. R values calculated for each pair of subgenotypes are shown in Table 2. The maximum and minimum R values among BVDV-1 were found between BVDV-1a and BVDV-1c (19.8), and between BVDV-1b and BVDV-1o (3.3), respectively.
3.3. Reactivity of neutralizing antibody detected from cattle against Japanese subgenotype viruses Next we investigated the neutralizing reactivity of serum samples obtained from unvaccinated breeding Japanese Black beef cattle against representative strains of the Japanese subgenotypes. A total of 145 out of 266
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Table 1 Cross-neutralization titers of antisera raised against different strains of Japanese BVDV subgenotypes. Nuetralization titers to strain of the antisera againsta
Viruses
BVDV-1a
BVDV-1b
BVDV-1c
BVDV-1n
BVDV-1o
BVDV-2a
Nose
IS27CP/01
IS8NCP/97
So CP/75
IS25CP/01
KZ 91 CP
BVDV-1a Nose BVDV-1a IS16NCP/99 BVDV-1a IS22NCP/00
256 256 256
32 32 32
128 256 256
16 64 32
128 128 256
8 4 4
BVDV-1b IS27CP/01 BVDV-1b IS17NCP/99 BVDV-1b IS36NCP/04
32 64 64
512 1024 512
128 256 128
64 64 64
64 64 64
64 4 4
BVDV-1c IW56/05 BVDV-1c IS14NCP/99 BVDV-1c IS37NCP/05
64 64 64
64 16 128
1024 2048 1024
64 16 128
64 32 128
8 4 16
BVDV-1n So CP/75
16
64
64
1024
128
16
BVDV-1o IS25CP/01 BVDV-1o IS26NCP/01
64 64
16 16
128 128
16 32
2048 1024
4 4
2
<2
16
2
4
512
BVDV-2a KZ 91 CP a
Reciprocal of the highest dilution that inhibits 50% viral growth, homologous titers are in bold.
Table 2 Antigenic similarity (R) values of BVDV subgenotypes.
BVDV-1a BVDV-1b BVDV-1c BVDV-1n BVDV-1o BVDV-2a
BVDV-1a
BVDV-1b
BVDV-1c
BVDV-1n
BVDV-1o
BVDV-2a
100
9.9 100
19.8 9.9 100
4.4 7.9 5.0 100
16.7 3.3 6.6 4.4 100
0.9 0.6 1.4 0.8 0.5 100
(54.5%) calves had antibodies against at least one strain. The rest of the serum samples did not contain antibodies against any of the strains. Subgenotype-specific antibodies at concentrations at least 4-fold higher than those against other subgenotype viruses were detected in 117 out of 145 calves (80.7%). Subgenotype-specific antibody rates were as follows: 69.6% against BVDV-1a, 1.4% against BVDV-1b, 8.3% against BVDV-1c, and 1.4% against BVDV-2a (Table 2). The highest incidence of BVDV infection was found against BVDV-1a. 4. Discussion Genetic and antigenic diversity of BVDV occurs not only between but also among subgenotypes of BVDV-1 [2,7]. Genetic typing is important for the precise classification and molecular epidemiology of BVDV-1 [26], and epidemiological information on currently epidemic viruses is also important for BVDV prevention and control. Therefore, we compared nucleotide sequences of Japanese isolates with those of newly reported subgenotype viruses of BVDV-1. We clearly demonstrated that new Japanese subgenotypes, such as BVDV-1n and -1o, are different from French-1l [6] and Turkey-1l, which were recently reported by Kadir et al. [11]. Antigenic differences among subgenotypes of BVDV-1 have been reported [2,27–30]. In particular, significant antigenic differences between BVDV-1a and -1b have been found using a cross-SN assay [2,7,9]. Furthermore, studies
indicate that vaccines containing BVDV-1a induce lower antibody responses against BVDV-1b [10,31]. In this study, cross-SN assay found 8–16-fold antigenic differences between BVDV-1a and -1b. Since R value of <25 indicates significant antigenic differences [32], the R values among Japanese subgenotypes strongly support their antigenic diversity. Although 6 subgenotypes of BVDV-1 and 1 of BVDV-2 have been isolated in Japan to date, BVDV-1a, -1b, -1c, and BVDV-2a are most common. Because it is difficult to determine the subgenotypes of BVDV currently circulating in the field using virus isolation methods, we attempted to identify the virus subgenotype by measuring the reactivity of neutralizing antibodies against each subgenotype virus. The minimal antibody titer difference as determined using a cross-SN assay in this study was 4-fold; this difference was found between BVDV-1a and BVDV-1c. Thus, the subgenotype-specific antibody was at least 4-fold higher against one subgenotype than the others. The subgenotype-specific antibody was found in approximately 80% of cattle that had antibodies against at least one subgenotype. It is hypothesized that the remainder of the cattle (approximately 20%), which had less than 4-fold difference in antibody levels, were infected with two or more subgenotype viruses or with another subgenotype virus outside of BVDV-1a, -1b, -1c, or BVDV-2a, or have low or undetectable specific antibody because of previous infection that have been acquired long time ago. We also performed neutralization tests using representative strains of BVDV-1n (So CP/75) and -1o
F. Minami et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 35–39
(IS25CP/01) but did not detect specific antibodies against BVDV-1n or -1o (data not shown). Reports on the molecular epidemiology of Japanese BVDVs indicate that BVDV-1a and -1b are the predominant subgenotypes among dairy cattle in Japan [11,23]. Little is known regarding the epidemiology of BVDV infection among beef cattle in Japan because it is difficult to obtain milk, skin, and blood samples from the animals. In this study, we estimated the predominant subgenotypes present in beef cattle within Ishikawa Prefecture. The predominant subgenotype was BVDV-1a except in one farm, in which it was BVDV-1c. We detected these subgenotypes by measuring antibodies against the representative strains of Japanese subgenotypes. Two cattle in different farms possessed BVDV-1bspecific antibodies, and were introduced from the same breeding station at almost the same time (data not shown). Similar data were found for the other two cattle, which had BVDV-2a-specific antibodies. It is suggested that BVDV-1b and BVDV-2a might be prevalent at each of these breeding stations. In this study, we could estimate currently epidemic subgenotypes of BVDV in the field by investigation of the reactivity of neutralizing antibodies against the representative strains of subgenotypes. Furthermore, the epidemic subgenotypes of BVDV in the breeding stations were presumed by tracking study of cattle possessing BVDV subgenotype-specific antibodies. These results suggest that neutralization tests with representative strains of BVDV subgenotypes are useful for estimating the subgenotypes of currently epidemic viruses without isolating the viruses. Future epidemiological analyses of disease prevalence should consider this information together with data on cattle movement history. Acknowledgments We would like to express our appreciation to Dr. Stefan Vilcek for providing the nucleotide sequence data for BVDV strains 17-3, 17-15 and 17-16 and for giving helpful advice. We also thank Ms. H. Yoshida for technical help with the genetic analyses. References [1] Baker JC. Bovine viral diarrhea virus: a review. J Am Vet Med Assoc 1987;190:1449–58. [2] Becher P, Avalos-Ramirez R, Orlich M, Rosales SC, Ko¨nig M, Schweizer M, et al. Genetic and antigenic characterization of novel pestivirus genotypes: implications for classification. Virology 2003;311:96–104. [3] Pellerin C, van den H, Lecomte J, Tussen P. Identification of a new group of bovine viral diarrhea virus strains associated with severe outbreaks and high mortalities. Virology 1994;203:260–8. [4] Ridpath JF, Bolin SR, Dubovi EJ. Segregation of bovine viral diarrhea virus into genotypes. Virology 1994;205:66–74. [5] Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA. Family Flaviviridae. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA, editors. Virus taxonomy. Eighth report of the international committee on taxonomy of viruses. San Diego, USA: Elsevier Academic Press; 2005. [6] Jackova A, Novackova M, Pelletier C, Audeval C, Gueneau E, Haffer A, et al. The extended genetic diversity of BVDV-1. Typing of BVDV isolates from France. Vet Res Commun 2008;32:7–11. [7] Nagai M, Hayashi M, Itou M, Fukutomi T, Akashi H, Kida H, et al. Identification of new genetic subtypes of bovine viral diarrhea virus genotype 1 isolated in Japan. Virus Genes 2008;36:135–9.
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[8] Giangaspero M, Harasawa R. Numerical taxonomy of the genus Pestivirus based on palindromic nucleotide substitutions in the 50 -untranslated region. J Virol Methods 2007;146:375–88. [9] Bolin SR, Grooms DL. Origination and consequences of bovine viral diarrhea virus diversity. Vet Clin North Am Food Anim Pract 2004;20:51–68. [10] Fulton RW, Ridpath JF, Confer AW, Saliki JT, Burge LJ, Payton ME. Bovine viral diarrhea virus antigenic diversity, impact on disease and vaccination programmes. Biologicals 2003;31:89–95. [11] Kadir Y, Christine F, Barbara B-W, Zeki Y, Feray A, Aykut O, et al. Heterogeneity of bovine viral diarrhea virus (BVDV) isolates from Turkey. Identification of a new subgroup in BVDV-1. Vet Microbiol 2008;130:258–67. [12] Matsuno K, Sakoda Y, Kaneyama K, Tamai K, Ito A, Kida H. Genetic and pathobiological characterization of bovine viral diarrhea viruses recently isolated from cattle in Japan. J Vet Med Sci 2007;69:515–20. [13] Kodama K, Sasaki N, Fukuyama S, Izumida A, Ishii F. Studies on cytopathogenic bovine diarrhea virus recovery, identification, and properties of the isolated virus. Bull Nippon Vet Zootech Coll 1974;23:51–60. [14] Nagai M, Sato M, Nagano H, Pang H, Kong X, Murakami T, et al. Nucleotide sequence homology to bovine viral diarrhea virus 2 (BVDV 2) in the 50 -untranslated region of BVDVs from cattle with mucosal disease or persistent infection in Japan. Vet Microbiol 1998;60:271–6. [15] Seki Y, Seimiya Y, Motokawa M, Yaegashi G, Nagai M, Hayashi M. Application of restriction fragment length polymorphism analysis to simple and rapid genotyping of bovine viral diarrhea virus strains isolated in Japan. J Vet Med Sci 2008;70:393–5. [16] Shimizu M, Satou K. Frequency of persistent infection of cattle with bovine viral diarrhea-mucosal disease virus in epidemic areas. Jpn J Vet Sci 1987;49:1045–51. [17] Vilcek S, Herring AJ, Herring JA, Nettleton PF, Lowings JP, Paton DJ. Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis. Arch Virol 1994;136:309–23. [18] Becher P, Orlich M, Shannon AD, Horner G, Ko¨nig M, Thiel HJ. Phylogenetic analysis of pestiviruses from domestic and wild ruminants. J Gen Virol 1997;78:1357–66. [19] Tamura K, Dudley J, Nei M, Kumar S. MEGA4 molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596–9. [20] Saitou N, Nei M. The neighbor-joining method. A new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406–25. [21] Fitch WM. On the problem of discovering the most parsimonious tree. Am Nat 1977;111:223–57. [22] Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 1980;16:111–20. [23] Felsenstein J. Confidence limits on phylogenies, an approach using the bootstrap. Evolution 1985;39:783–91. [24] Nagai M, Ito T, Sugita S, Genno A, Takeuchi K, Ozawa T, et al. Genomic and serological diversity of bovine viral diarrhea virus in Japan. Arch Virol 2001;146:685–96. [25] Archetti I, Horsfall FL. Persistent antigenic variation of influenza A viruses after incomplete neutralization in ovo with heterologous immune serum. J Exp Med 1950;92:441–62. [26] Vilcek S, Durkovic B, Kolesarova M, Paton DJ. Genetic diversity of BVDV, consequences for classification and molecular epidemiology. Prev Vet Med 2005;72:31–5. [27] Couvreur B, Letellier C, Collard A, Quenon P, Dehan P, Hamers C, et al. Genetic and antigenic variability in bovine viral diarrhea virus (BVDV) isolates from Belgium. Virus Res 2002;85:17–28. [28] Dekker A, Wensvoort G, Terpstra C. Six antigenic groups within the genus pestivirus as identified by cross-neutralization assays. Vet Microbiol 1995;47:317–29. [29] Howard CJ, Brownlie J, Clarke MC. Comparison by the neutralization assay of pairs of non-cytopathogenic and cytopathogenic strains of bovine virus diarrhea virus isolated from cases of mucosal disease. Vet Microbiol 1987;19:13–21. [30] Avalos-Ramirez R, Orlich M, Thiel HJ, Becher P. Evidence for the presence of two novel pestivirus species. Virology 2001;286:456– 65. [31] Jones L, Van Campen H, Zu ZC, Schnackel JA. Comparison of neutralizing antibodies to type 1a, 1b, and 2 bovine viral diarrhea virus from experimentally infected and vaccinated cattle. Bovine Pract 2001;35:137–40. [32] Hubalek Z. Numerical comparative serology—the methods. J Appl Bacteriol 1982;52:307–18.
Contents lists available at ScienceDirect
Comparative Immunology, Microbiology and Infectious Diseases journal homepage: www.elsevier.com/locate/cimid
Reactivity and prevalence of neutralizing antibodies against Japanese strains of bovine viral diarrhea virus subgenotypes Fujiko Minami a, Makoto Nagai b, Mika Ito a, Tatsuhiko Matsuda c, Hikaru Takai c, Yoshiko Jinkawa c, Takeshi Shimano a, Michiko Hayashi b, Yoshihisa Seki d, Yoshihiro Sakoda e, Katsuaki Sugiura f, Hiroomi Akashi g,* a
Ishikawa Nanbu Livestock Hygiene Service Center, Kanazawa, Ishikawa 920-3101, Japan Ishikawa Prefectual Livestock Research Center, Hodatsushimizu-cho, Ishikawa 929-1325, Japan Ishikawa Hokubu Livestock Hygiene Service Center, Nanao, Ishikawa 929-2126, Japan d Iwate Prefecture Central Livestock Hygiene Service Center, Takizawa-mura, Iwate 020-0173, Japan e Laboratory of Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido 060-0818, Japan f Food and Agricultural Materials Inspection Center, 2-1 Shintoshin, Chuo-ku, Saitama, Saitama 330-9731, Japan g Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan b c
A R T I C L E I N F O
A B S T R A C T
Article history: Accepted 9 October 2009
Bovine viral diarrhea virus (BVDV) field isolates show genetic and antigenic diversity. At least 14 subgenotypes of BVDV-1 and 4 of BVDV-2 have been identified in Artiodactyla worldwide. Of these, 6 subgenotypes of BVDV-1 and 1 of BVDV-2 have been isolated in Japan. Previously, we reported that each subgenotype virus expresses different antigenic characteristics. Here we investigated the reactivity of neutralizing antibodies against representative strains of Japanese BVDV subgenotypes using sera from 266 beef cattle to estimate the prevalence of this epidemic virus among cattle in Japan. Antibody titers at concentrations at least 4-fold higher than antibodies against other subgenotype viruses were considered subgenotype specific. Subgenotype-specific antibodies were detected from 117 (80.7%) of 145 sera samples (69.7% against BVDV-1a, 1.4% against BVDV-1b, 8.3% against BVDV-1c, and 1.4% against BVDV-2a). The results suggest that neutralization tests are useful in estimating currently epidemic subgenotypes of BVDV in the field. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Antigenic difference Bovine viral diarrhea virus Epidemic virus Neutralization test Subgenotype
1. Introduction Bovine viral diarrhea virus (BVDV) is an important bovine pathogen because it causes significant economic losses in the cattle industry [1]. BVDV is classified based on differences in genomic sequences [2]. Two genotypes, BVDV-1 and BVDV-2, were recently reported [3,4], and have been recognized as virus species [5]. Investigations have revealed that BVDV-1 and BVDV-2 are subdivided
* Corresponding author. Tel.: +81 3 5841 5396; fax: +81 3 5841 8184. E-mail address: [email protected] (H. Akashi). 0147-9571/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.cimid.2009.10.007
into at least 14 and 4 subgenotypes, respectively [6–8]. Although BVDVs share epitopes of a common pestiviral antigen, antigenic differences among BVDV isolates have been demonstrated using cross-neutralization tests [2,7]. Because the antigenic diversity among BVDVs has an impact on disease diagnosis and control via vaccines [2,9,10], it is important to identify the antigenic properties of field isolates for BVDV prevention and control. New subgenotypes of BVDV-1 were recently reported [6,11]. We thereby confirmed the subgenotypes of Japanese BVDV using phylogenetic analyses of these new subgenotypes. Furthermore, to estimate the currently epidemic subgenotypes of BVDV among the
36
F. Minami et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 35–39
Japanese cattle population, we investigated the reactivity of neutralizing antibodies against the representative strains of subgenotypes. 2. Materials and methods 2.1. Representative strains of Japanese BVDV subgenotypes and cells BVDV-1a, -1b, -1c, and BVDV-2a are the predominant subgenotypes of BVDV in Japan [12]. Nose [13], IS27CP/01 [7], SoCP/75 [7], IS25CP/01 [7], and KZ91CP [14] were selected as the representative strains of BVDV-1a, -1b, -1n, -1o, and BVDV-2a, respectively. IW56/05/CP isolated from a cow that had developed mucosal disease in 2005 [15] was selected as a representative strain of BVDV-1c. In addition, 7 BVDV-1 isolates, IS16NCP/99, IS22NCP/00, IS17NCP/99, IS36NCP/04, IS14NCP/99, IS37NCP/05 and IS26NCP/01[0], isolated from persistently infected cattle were subjected to cross-neutralization tests and phylogenetic analyses together with above strains. All viruses used for sera testing of field cattle were purified using limiting dilution methods as described previously [14] except nonpathogenic (NCP) strains. Bovine fetal muscle (BFM) cell cultures within 20 passages [16] were used for virus preparation and neutralization tests. BFM cells were grown in Eagle’s minimum essential medium (EMEM) containing 10% horse serum, 10% tryptose phosphate broth and 0.015% sodium bicarbonate. The cells and horse serum supplemented into the media were confirmed absence of contamination with BVDV by virus isolation and RT-PCR before use. 2.2. Nucleotide sequencing and phylogenetic analyses A portion of the 50 -untranslated region and Erns, and the full genomic region of Npro and Core, were amplified by RTPCR using primers 324 (position in BVDV SD-1: 108-128) [17] and 1400R (position in BVDV SD-1: 1448-1430) [18]. The nucleotide sequences of the genomic region encoding Npro were determined by direct sequencing as described previously [7]. Of the nucleotide sequences obtained, 385 bp of the Npro gene were subjected to phylogenetic analyses using MEGA4 [19], employing neighbor-joining (NJ) [20] and maximum-parsimony (MP) [21] methods. The distances between sequences were estimated by using the three-parameter method [22]. The reliability of the phylogenetic tree was evaluated by running 1000 replicas in the bootstrap test [23].
wells of the microplate, and incubated at 37 8C. SN titers were expressed as the reciprocal of the highest serum dilution that neutralized the virus. Antisera against the representative Japanese strains were prepared using BVDV-free sheep as previously described [24]. To characterize the antigenic relatedness among BVDV subgenotypes, antigenic similarity (R) values were calculated according to the formula reported by Archetti and Horsfall [25]. 2.4. Sera testing and subgenotype-specific antibodies A total of 266 serum samples from breeding Japanese Black beef cattle unvaccinated against BVDV infection were collected from 33 farms in Ishikawa Prefecture between 2004 and 2006. The movement history of the animals was traced using the beef traceability system (http://www.id.nlbc.go.jp/top.html) of the National Livestock Breeding Center in Japan. Based on results from cross-SN tests, antibody titers at concentrations at least 4-fold higher than antibodies against other subgenotype viruses were considered subgenotype specific. 3. Results 3.1. Phylogenetic analyses Fig. 1 shows the phylogenetic tree, which was constructed using the NJ method with 385 bp of the Npro gene nucleotide sequences of 17 Japanese BVDV isolates together with those of 26 reference strains obtained from the DDBJ databank. Nucleotide sequence data for BVDV-1 strains 71-03, 71-15, and 71-16 were kindly provided by Dr. S. Vilcek (University of Veterinary Medicine, Slovakia). In all, 16 clusters of BVDV-1 and 1 cluster of BVDV-2 were identified. Japanese isolates branched into 7 clusters: BVDV-1a, -1b, -1c, -1j, -1n, -1o, and BVDV-2a, as previously reported. Bootstrap analysis can be applied to a phylogenetic tree in order to estimate the frequency with which particular branch occurs and values of greater than 70% are considered as supporting the grouping. BVDV-1l reported by Jackova et al. [6] and Kadir et al. [11] were independently placed in different clusters and distinct from the Japanese subgenotypes. The phylogenetic tree constructed by the MP method showed almost the same topology as that constructed by the NJ method (data not shown). 3.2. Cross-SN test
2.3. Cross-neutralization tests and antisera Serum neutralization (SN) tests were performed in BFM cell cultures grown in 96-well microplates as described by Shimizu and Satou [16]. Briefly, serial 2fold dilutions of heat-inactivated sera were made in volume of 0.025 ml in a microplate with the maintenance medium, an equal volume of the virus suspension containing 200 TCID50/0.1 ml was added, and incubated at 37 8C for 1 h. Then 0.1 ml of BFM cell suspension (about 2 105 cells/ml) was added into all
Neutralizing antibody titers against each virus subgenotype were tested and are shown in Table 1. The crossSN titers of the antisera against homologous BVDV-1 were 4–64-fold higher than those against the heterologous viruses. The maximum antigenic differences within BVDV-1 were observed between So CP/75 (BVDV-1n) and either Nose (BVDV-1a) or IS25CP/01 (BVDV-1m) using antiserum against So CP/75. The minimum antigenic difference was found between Nose (BVDV-1a) and IW56/ 05 (BVDV-1c) using antiserum against Nose. The SN titers
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Fig. 1. Phylogenetic analysis of the 385 nt sequence in the Npro region of 17 Japanese BVDV isolates (bold and italics) with the reported nucleotide sequences of 26 reference strains obtained from the DDBJ/EMBL/GenBank databases and personal communication. The phylogenetic tree was constructed using the neighbor-joining method and bootstrap analysis (n = 1000) in MEGA4. The accession numbers of nucleotide sequences are as follows: NADL, M31182; SD-1, M96751; Deer GB1, U80902; 23-15, AF287279; G-Au, AF287285; J-Au, AF287286; W-Au, AF287290; A-Au, AF287283; L-Au, AF287283; ZM-95, AF526381; 519, AF144464; Deer NZ1, U80903; Bega, AF049221; CP7, U63479; Osloss, M96687; TR16, EU163964; TR1, EU163950; TR29, EU163977; 3186V6, AF287282; CH-Suwa, AF117699; 721, AF144463; F-Au, AF287284; 890, U18059.
of antiserum against BVDV-2 and BVDV-1 viruses were 8– 512-fold lower than that of the homologous virus. R values calculated for each pair of subgenotypes are shown in Table 2. The maximum and minimum R values among BVDV-1 were found between BVDV-1a and BVDV-1c (19.8), and between BVDV-1b and BVDV-1o (3.3), respectively.
3.3. Reactivity of neutralizing antibody detected from cattle against Japanese subgenotype viruses Next we investigated the neutralizing reactivity of serum samples obtained from unvaccinated breeding Japanese Black beef cattle against representative strains of the Japanese subgenotypes. A total of 145 out of 266
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Table 1 Cross-neutralization titers of antisera raised against different strains of Japanese BVDV subgenotypes. Nuetralization titers to strain of the antisera againsta
Viruses
BVDV-1a
BVDV-1b
BVDV-1c
BVDV-1n
BVDV-1o
BVDV-2a
Nose
IS27CP/01
IS8NCP/97
So CP/75
IS25CP/01
KZ 91 CP
BVDV-1a Nose BVDV-1a IS16NCP/99 BVDV-1a IS22NCP/00
256 256 256
32 32 32
128 256 256
16 64 32
128 128 256
8 4 4
BVDV-1b IS27CP/01 BVDV-1b IS17NCP/99 BVDV-1b IS36NCP/04
32 64 64
512 1024 512
128 256 128
64 64 64
64 64 64
64 4 4
BVDV-1c IW56/05 BVDV-1c IS14NCP/99 BVDV-1c IS37NCP/05
64 64 64
64 16 128
1024 2048 1024
64 16 128
64 32 128
8 4 16
BVDV-1n So CP/75
16
64
64
1024
128
16
BVDV-1o IS25CP/01 BVDV-1o IS26NCP/01
64 64
16 16
128 128
16 32
2048 1024
4 4
2
<2
16
2
4
512
BVDV-2a KZ 91 CP a
Reciprocal of the highest dilution that inhibits 50% viral growth, homologous titers are in bold.
Table 2 Antigenic similarity (R) values of BVDV subgenotypes.
BVDV-1a BVDV-1b BVDV-1c BVDV-1n BVDV-1o BVDV-2a
BVDV-1a
BVDV-1b
BVDV-1c
BVDV-1n
BVDV-1o
BVDV-2a
100
9.9 100
19.8 9.9 100
4.4 7.9 5.0 100
16.7 3.3 6.6 4.4 100
0.9 0.6 1.4 0.8 0.5 100
(54.5%) calves had antibodies against at least one strain. The rest of the serum samples did not contain antibodies against any of the strains. Subgenotype-specific antibodies at concentrations at least 4-fold higher than those against other subgenotype viruses were detected in 117 out of 145 calves (80.7%). Subgenotype-specific antibody rates were as follows: 69.6% against BVDV-1a, 1.4% against BVDV-1b, 8.3% against BVDV-1c, and 1.4% against BVDV-2a (Table 2). The highest incidence of BVDV infection was found against BVDV-1a. 4. Discussion Genetic and antigenic diversity of BVDV occurs not only between but also among subgenotypes of BVDV-1 [2,7]. Genetic typing is important for the precise classification and molecular epidemiology of BVDV-1 [26], and epidemiological information on currently epidemic viruses is also important for BVDV prevention and control. Therefore, we compared nucleotide sequences of Japanese isolates with those of newly reported subgenotype viruses of BVDV-1. We clearly demonstrated that new Japanese subgenotypes, such as BVDV-1n and -1o, are different from French-1l [6] and Turkey-1l, which were recently reported by Kadir et al. [11]. Antigenic differences among subgenotypes of BVDV-1 have been reported [2,27–30]. In particular, significant antigenic differences between BVDV-1a and -1b have been found using a cross-SN assay [2,7,9]. Furthermore, studies
indicate that vaccines containing BVDV-1a induce lower antibody responses against BVDV-1b [10,31]. In this study, cross-SN assay found 8–16-fold antigenic differences between BVDV-1a and -1b. Since R value of <25 indicates significant antigenic differences [32], the R values among Japanese subgenotypes strongly support their antigenic diversity. Although 6 subgenotypes of BVDV-1 and 1 of BVDV-2 have been isolated in Japan to date, BVDV-1a, -1b, -1c, and BVDV-2a are most common. Because it is difficult to determine the subgenotypes of BVDV currently circulating in the field using virus isolation methods, we attempted to identify the virus subgenotype by measuring the reactivity of neutralizing antibodies against each subgenotype virus. The minimal antibody titer difference as determined using a cross-SN assay in this study was 4-fold; this difference was found between BVDV-1a and BVDV-1c. Thus, the subgenotype-specific antibody was at least 4-fold higher against one subgenotype than the others. The subgenotype-specific antibody was found in approximately 80% of cattle that had antibodies against at least one subgenotype. It is hypothesized that the remainder of the cattle (approximately 20%), which had less than 4-fold difference in antibody levels, were infected with two or more subgenotype viruses or with another subgenotype virus outside of BVDV-1a, -1b, -1c, or BVDV-2a, or have low or undetectable specific antibody because of previous infection that have been acquired long time ago. We also performed neutralization tests using representative strains of BVDV-1n (So CP/75) and -1o
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(IS25CP/01) but did not detect specific antibodies against BVDV-1n or -1o (data not shown). Reports on the molecular epidemiology of Japanese BVDVs indicate that BVDV-1a and -1b are the predominant subgenotypes among dairy cattle in Japan [11,23]. Little is known regarding the epidemiology of BVDV infection among beef cattle in Japan because it is difficult to obtain milk, skin, and blood samples from the animals. In this study, we estimated the predominant subgenotypes present in beef cattle within Ishikawa Prefecture. The predominant subgenotype was BVDV-1a except in one farm, in which it was BVDV-1c. We detected these subgenotypes by measuring antibodies against the representative strains of Japanese subgenotypes. Two cattle in different farms possessed BVDV-1bspecific antibodies, and were introduced from the same breeding station at almost the same time (data not shown). Similar data were found for the other two cattle, which had BVDV-2a-specific antibodies. It is suggested that BVDV-1b and BVDV-2a might be prevalent at each of these breeding stations. In this study, we could estimate currently epidemic subgenotypes of BVDV in the field by investigation of the reactivity of neutralizing antibodies against the representative strains of subgenotypes. Furthermore, the epidemic subgenotypes of BVDV in the breeding stations were presumed by tracking study of cattle possessing BVDV subgenotype-specific antibodies. These results suggest that neutralization tests with representative strains of BVDV subgenotypes are useful for estimating the subgenotypes of currently epidemic viruses without isolating the viruses. Future epidemiological analyses of disease prevalence should consider this information together with data on cattle movement history. Acknowledgments We would like to express our appreciation to Dr. Stefan Vilcek for providing the nucleotide sequence data for BVDV strains 17-3, 17-15 and 17-16 and for giving helpful advice. We also thank Ms. H. Yoshida for technical help with the genetic analyses. References [1] Baker JC. Bovine viral diarrhea virus: a review. J Am Vet Med Assoc 1987;190:1449–58. [2] Becher P, Avalos-Ramirez R, Orlich M, Rosales SC, Ko¨nig M, Schweizer M, et al. Genetic and antigenic characterization of novel pestivirus genotypes: implications for classification. Virology 2003;311:96–104. [3] Pellerin C, van den H, Lecomte J, Tussen P. Identification of a new group of bovine viral diarrhea virus strains associated with severe outbreaks and high mortalities. Virology 1994;203:260–8. [4] Ridpath JF, Bolin SR, Dubovi EJ. Segregation of bovine viral diarrhea virus into genotypes. Virology 1994;205:66–74. [5] Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA. Family Flaviviridae. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA, editors. Virus taxonomy. Eighth report of the international committee on taxonomy of viruses. San Diego, USA: Elsevier Academic Press; 2005. [6] Jackova A, Novackova M, Pelletier C, Audeval C, Gueneau E, Haffer A, et al. The extended genetic diversity of BVDV-1. Typing of BVDV isolates from France. Vet Res Commun 2008;32:7–11. [7] Nagai M, Hayashi M, Itou M, Fukutomi T, Akashi H, Kida H, et al. Identification of new genetic subtypes of bovine viral diarrhea virus genotype 1 isolated in Japan. Virus Genes 2008;36:135–9.
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