Agence Française de Sécurité Sanitaire des Aliments Alfort, Laboratoire Central de Recherches Vétérinaires, 22 rue Pierre Curie, 94703 Maisons-Alfort, France1
Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK2
Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort 0110, Republic of South Africa3
Author for correspondence: Corinne Sailleau. Fax +33 1 43 68 97 62. e-mail c.sailleau{at}alfort.afssa.fr
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Abstract |
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Introduction |
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The AHSV genome is composed of ten double-stranded RNA segments (Oellermann et al., 1970 ; Bremer, 1976
), which encode at least ten viral proteins. The genome segments are numbered 110 in order of their migration in PAGE. Seven of the viral proteins are structural and form the double-shelled virus particle. The outer capsid is composed of two major viral proteins, VP2 and VP5, which determine the antigenic variability of the AHSVs, while the inner capsid is composed of two major (VP3 and VP7) and three minor (VP1, VP4 and VP6) viral proteins (Bremer et al., 1990
; Grubman & Lewis, 1992
). VP3 and VP7 are highly conserved among the nine serotypes (Oellermann et al., 1970
; Bremer et al., 1990
). At least three non-structural proteins, NS1, NS2 and NS3, have been identified (Huismans & Els, 1979
; van Staden & Huismans, 1991
; Mizukoshi et al., 1992
).
Rapid and reliable AHSV serotype differentiation is essential at the start of an outbreak to allow for the early selection of a vaccine to help control the spread of the virus. Traditionally, laboratory confirmation of the AHSV serotype has depended on the isolation of virus by inoculation into suckling mouse brain and tissue culture (BHK21 or Vero cells) and subsequent serotyping by VNT. Confirmation of virus serotype by this route takes a minimum of 2 weeks, but often longer. Such delays can have a significant adverse effect on the extent and duration of an epizootic. This paper reports the first RTPCR method for the rapid identification and differentiation of the nine AHSV serotypes by using nine pairs of primers, each specific for a single AHSV serotype.
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Methods |
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Selection of primers.
Six AHSV intermediate and partial-group-specific primers were designed, based on the nucleotide sequences of segment 2 from one strain of AHSV 3 (Vreede & Huismans, 1994 ) and three strains of AHSV 4 (Sakamoto et al., 1994
; Iwata et al., 1992
; M. Stone-Marschat, unpublished data) (Fig. 1
, step 1; Table 1
). These primers were used to amplify products and identify sequences that were specific for each of the nine AHSV serotypes by using RTPCR methods similar to those described by Zientara et al. (1995)
and Moulay et al. (1995)
(Fig. 1
, step 2). Nine reverse primers (Table 2
) were designed following analysis of the nucleotide sequences of these nine products (Fig. 1
, steps 3 and 4). Primers were named as follows: P represents the forward, positive strand, N represents the reverse, negative strand and the number following P or N corresponds to the starting location on the AHSV genome.
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Serotype-specific RTPCR.
Nine pairs of primers were subsequently designed, each pair corresponding to a separate AHSV serotype but including a serogroup-specific (forward) primer and a serotype-specific (reverse) primer (Table 2). The forward primer P20 used with the reverse primers 1N229, 3N228, 4N353, 6N355, 7N359 and 9N228 amplifies segment 2 of AHSV serotypes 1, 3, 4, 6, 7 and 9, respectively. The forward primer P95 used with the reverse primers 2N228 and 5N358 amplifies segment 2 of AHSV serotypes 2 and 5, respectively, and the forward primer P2100 used with the reverse primer 8N2778 amplifies segment 2 of AHSV serotype 8.
Four µl denatured RNA obtained from AHSV was added to 50 µl reaction mix containing 10 mM TrisHCl, pH 8·8, 50 mM KCl, 200 µM of each dNTP, 1·5 mM MgCl2, 0·1 mg/ml gelatine and 5 pmol of each primer pair, 5 U avian myeloblastosis virus reverse transcriptase and 3·5 U Taq DNA polymerase. The conditions were optimized for each pair of primers (Table 3). RNA was reverse-transcribed at 42 °C for 45 min and the cDNA was amplified for either 30, 35 or 40 cycles of 95 °C for 1 min, either 48, 50 or 52 °C for 1 min and 70 °C for 2 min, with a terminal extension at 70 °C for 8 min. The amplified products from each of the reaction mixtures were electrophoresed across a 2·0% agarose slab gel and stained with ethidium bromide.
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The sensitivity of the RTPCR was determined by amplifying each dilution of a tenfold dilution series prepared for each of the nine AHS vaccine viruses.
A coded panel of 46 infectious and 10 formalin-inactivated AHSV reference strains and field isolates supplied by different Office Internationale des Épizooties-designated reference laboratories and from veterinary services from widely separated countries was used to validate the RTPCR.
Virus titrations.
Fifty µl of a tenfold dilution series prepared for each AHS vaccine virus (10-110-7) was added to separate wells of microtitre plates. Vero cells (ATTC* CCL81) were added at a concentration of 2x105 cells/ml in RPMI 1640 medium supplemented with 5% foetal calf serum, 100 µg/ml streptomycin and 100 IU/ml penicillin. Microplates were sealed, incubated at 37 °C for 7 days and examined microscopically each day for the appearance of a cytopathic effect (CPE) characteristic of a virus infection. Virus titres were calculated by using the methods of Reed & Muench (1938) .
Virus neutralization test (VNT).
The 46 live virus samples were inoculated onto confluent monolayers of Vero cells grown in 25 cm2 flasks with RPMI 1640 medium supplemented with 5% foetal calf serum, 100 µg/ml streptomycin and 100 IU/ml penicillin. Flasks were incubated for 5 days and examined microscopically each day for 5 days for CPE.
VNTs were carried out in microtitre plates where constant amounts of each AHSV type-specific antiserum were added to a tenfold dilution series of each virus sample. The serumvirus mixtures were incubated for 1 h at 37 °C. Vero cells were then added at a concentration of 2x105 cells/ml in RPMI 1640 medium supplemented with 5% foetal calf serum, 100 µg/ml streptomycin and 100 IU/ml penicillin. Microplates were sealed, incubated at 37 °C for 7 days and examined microscopically each day for CPE. Virus titres were calculated by using methods described by Reed & Muench (1938) . The serotype of the type-specific antiserum that reduced the virus titre by at least two log10 compared with the virus control in the absence of any AHSV type-specific antiserum was designated the virus serotype.
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Results |
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Validation
Table 5 shows the RTPCR serotyping results obtained for the 46 live and 10 formalin-inactivated samples. The origin and date of isolation and the confirmation of serotype by VNT are also included for each sample. Each sample was tested by using the nine different primer sets and only one product was amplified for each sample. The size of these amplified products always corresponded to that expected for a particular AHSV serotype-specific primer set. There was a perfect correlation between the serotype designations obtained by RTPCR and by VNT. Unlike the results obtained with the AHSV 2 vaccine strain (Table 4
; Fig. 2
, lanes 2 and 10), the AHSV 4-specific primer set did not amplify any visually detectable product from the three samples of AHSV 2, numbered 13, 29 and 35 (Table 5
).
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Discussion |
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Laboratory confirmation of a clinical diagnosis can be achieved rapidly and directly by the serogroup-specific ELISA (Hamblin et al., 1991 ) from necropsy tissue and by the serogroup-specific RTPCR (Zientara et al., 1995
) from either necropsy tissue or blood. This paper describes the development and validation of the first serotype-specific RTPCR for AHSV, which can be used with either infectious or non-infectious laboratory-adapted viruses and necropsy tissues (Sailleau et al., 1999
). By using these assays concurrently, AHSV can be identified and serotyped within 24 h, thereby providing the opportunity to begin monovalent vaccination with minimum delay.
Genome segment 2 was chosen as the target for the development of the serotype-specific RTPCR as this segment encodes the serotype-specific AHSV protein VP2. The published nucleotide sequences of segment 2 from one strain of AHSV 3 (Vreede & Huismans, 1994 ) and three strains of AHSV 4 (Iwata et al., 1992
; Sakamoto et al., 1994
; M. Stone-Marschat, unpublished data) were compared and then used to select primers that were specific for conserved genome regions. These primers amplified nine products, each specific for one of the nine AHSV serotypes. Analysis of the nucleotide sequences of these nine products enabled the selection of nine primers. Nine pairs of primers were subsequently designed for use in the serotype-specific RTPCR, such that each pair included one partial-group-specific and one serotype-specific primer (Tables 2
and 4
). These primer pairs were shown to be specific for the nine AHSV serotypes, although the primer pair directed against AHSV 4 did amplify a product weakly from the high mouse brain passage AHSV 2 vaccine virus (Table 4
). However, no cross-reaction was recorded with the other three low-passage strains of AHSV 2 isolated between 1961 and 1998. The sensitivity of the serotype-specific RTPCR for the identification of each AHSV serotype was in accordance with previously published values (Stone-Marschat et al., 1994
; Zientara et al., 1994
). However, the sensitivity of the RTPCR is undoubtedly greater than reported in this study, as these results do not take account of the non-infectious virus component of the samples.
Comparative testing by serotype-specific RTPCR and by VNT of 56 AHSV samples showed total agreement. These included 46 infectious samples isolated between 1961 and 1998 in a number of different countries, where epidemiological links would often be unlikely. The success of the RTPCR in identifying correctly the serotype of isolates of eight AHSV serotypes that were isolated 3137 years apart emphasizes the genetic stability of the sequenced regions from which the primers were selected. Isolates of AHSV 5, the remaining serotype, were only available from 1993 to 1995, but were also correctly identified. It was also noted that differences in passage history did not appear to interfere with the ability of the RTPCR to serotype the viruses.
The remaining 10 samples used to validate the RTPCR were duplicates of some of the infectious samples that had been inactivated with formalin. Being able to identify and serotype AHSV in formalin-treated tissues is of particular importance if these are the only types of sample submitted for diagnosis. This advantage could be exploited further for the retrospective epidemiological study of previous AHSV outbreaks or suspect cases by using stored histological tissues.
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Acknowledgments |
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References |
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Received 16 July 1999;
accepted 9 November 1999.