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1 Department of Epidemiology, Institute for Animal Health (IAH), Pirbright Laboratory, Ash Road, Woking, Surrey GU24 0NF, UK
2 Department of Biotechnology, CCS Haryana Agricultural University, Hisar 125 004, Haryana, India
3 Virology Division, Onderstepoort Veterinary Institute, 0110 Onderstepoort, South Africa
Correspondence
P. P. C. Mertens
peter.mertens{at}bbsrc.ac.uk
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Supplementary figures showing electrophoretic analysis of cDNA products are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). BTV can infect most ruminant species, but causes economically important disease (known as bluetongue; BT) primarily in species of deer, sheep and cattle (Mertens, 1999; Osburn, 2000; Mertens & Mellor, 2003; Purse et al., 2005). The severity of clinical signs caused by BTV can vary depending on the virus strain, breed or species of host and their level of immunity. BTV can cause significant mortality, which was estimated at approximately 50 and 15 % in clinically affected sheep and cattle, respectively, during the northern European outbreak in 2006–2007. Although BTV has been linked to severe outbreaks of disease in deer in the USA (Dubay et al., 2006; Johnson et al., 2006), the severity and epidemiological significance of BTV infection in European deer populations are largely unknown. Economic losses associated with BTV infection are also caused by reductions in productivity and the costs of implementing control measures, including restricting animal movements/trade and diagnostic testing. The losses caused by BT worldwide were estimated at US$ 3 billion year–1 in 1996 (Tabachnick, 1996). BT is an Office International des Epizooties (OIE)-listed disease, which was included in the previous OIE list A because of its economic importance (OIE, 2000).
Before 1998, BTV had caused only periodic and relatively short-lived epizootics in southern Europe, involving a single serotype on each occasion (Mellor & Boorman, 1995; Mellor et al., 2000; Mellor & Wittmann, 2002). However, between 1998 and 2006, there have been at least ten separate introductions of BTV into Europe, involving a total of eight distinct virus strains of six different serotypes (types 1, 2, 4, 8, 9 and 16) via at least four distinct entry routes (Purse et al., 2005). In 2006 alone, BTV-8 arrived in northern Europe, BTV-1 emerged in north Africa (OIE, 2006a) and then spread to Sardinia, BTV-16 was detected in Cyprus, with BTV-4 and -15 in Israel, and serological evidence (only) for BTV-8 in Bulgaria (OIE, 2006b). Since 1998, BTV has extended its range gradually northwards into southern and central Europe (Mellor & Wittmann, 2002; Purse et al., 2005). In August 2006, BT occurred for the first time in northern Europe, with outbreaks of BTV-8 in Holland, Belgium, Germany, Luxembourg and north-east France (European Commission, 2006; IAH, 2006; OIE, 2006c), demonstrating that the whole of Europe is now at risk from BTV and possibly other arbovirus diseases, including those caused by other Culicoides-transmitted orbiviruses [e.g. African horse sickness virus (AHSV) and epizootic hemorrhagic disease virus] (Anthony et al., 2007; Maan et al., 2007).
Changes in the distribution of BTV in Europe reflect changes in the distribution of vector species (particularly Culicoides imicola) in the Mediterranean region, caused by climate change (Purse et al., 2005). However, C. imicola is absent from certain areas of the eastern Mediterranean region and northern Europe, where BTV transmission appears to depend on novel vector species [Culicoides obsoletus, and Culicoides pulicaris groups (particularly Culicoides dewolfi)]. The recent detection of additional BTV serotypes in the USA (Johnson et al., 2006) suggests that similar changes are occurring in other parts of the world.
Live-attenuated vaccine strains of BTV serotypes 2, 4, 9 and 16 have been used in the Mediterranean region (primarily in Italy, Iberia and the eastern Mediterranean islands) and there is evidence that the BTV-2 and BTV-16 vaccine strains have been transmitted in the field (Ferrari et al., 2005; Veronesi et al., 2005; Monaco et al., 2006). Group B (containing serotypes 3, 8, 9, 10 and 11) of the South African live-attenuated multivalent vaccines was also used in Bulgaria, although there is no evidence that any of these strains have persisted in the region. An annual vaccination campaign has been carried out in Israel, involving BTV serotypes 2, 4, 6, 10 and 16. Serotypes 2, 4, 15 and 16 have also been detected in diagnostic field samples from Israel, and may represent further threats to southern Europe.
In an area (e.g. Europe since 1998) where multiple BTV strains of several distinct serotypes are co-circulating with repeated new introductions, a full epidemiological understanding inevitably depends on assay systems that can rapidly detect and identify the different viruses involved (Davies et al., 1992; Mertens et al., 2005).
The BTV genome is composed of ten linear segments of double-stranded RNA (dsRNA), which encode ten viral proteins. Twenty-four distinct BTV serotypes have been recognized, based on the specificity of interactions between neutralizing antibodies and the outer surface of the virus particle (neutralization types; Howell, 1960; Howell & Verwoerd, 1971; Erasmus, 1990; Gould & Eaton, 1990; Mertens, 1999; Maan et al., 2007), which is composed of viral proteins VP2 and VP5. Most of the neutralization epitopes involved are present on VP2 (DeMaula et al., 2000), encoded by genome segment 2 (Seg-2) (Mertens et al., 1987b, 1989). The nucleotide sequence of Seg-2 therefore exerts a primary control of BTV serotype (Huismans & Erasmus, 1981; Mertens et al., 1987a, 1989; Roy et al., 1994; Maan et al., 2007). The majority of the remaining core structural proteins and non-structural proteins are relatively conserved and serologically cross-reactive within the BTV serogroup.
Conclusive diagnosis of BT currently involves detection and identification of BTV, usually by virus isolation and serological assays to determine virus serogroup and serotype. Orbivirus serogroup reflects antigens that are cross-reactive between members of the individual virus species (e.g. most of the virus non-structural and core proteins, particularly VP7; Gumm & Newman, 1982); consequently, serogroup-specific assays can detect any BTV strain (e.g. by ELISA; Afshar, 1994) and distinguish it from other orbiviruses. In contrast, BTV serotype is determined primarily by outer capsid protein VP2, which controls the specificity of interactions with neutralizing antibodies in serum neutralization assays (Maan et al., 2007). However, the viral genome can be detected by RT-PCR (assays that are both rapid and very sensitive) in the nucleic acid extracted from blood or tissues sent for diagnostic testing, removing the need for virus isolation. Serogroup-specific RT-PCR assays for BTV, targeting the more conserved regions of the virus genome (e.g. Seg-1, 5 and 7), have been designed and evaluated (Wade-Evans et al., 1990; Pearson et al., 1992; Afshar, 1994; Tabachnick et al., 1996; Bonneau et al., 2000; Billinis et al., 2001; Breard et al., 2003; Anthony et al., 2007; Shaw et al., 2007).
Conventional procedures for typing BTV isolates involve virus isolation, adaptation to cell culture and serological neutralization assays that may take several weeks to complete. They also require stocks of well-characterized and standardized antisera for all 24 BTV serotypes. These are expensive to produce and are in short supply. These serological assays can also give inconclusive results, particularly if the sample contains more than one BTV serotype (Howell et al., 1970; Davies & Blackburn, 1971; Afshar, 1994; Blacksell & Lunt, 1996). BTV serotype can also be identified by the specificity of neutralizing antibodies generated by a specific strain during infection. However, sequential infection (or vaccination) with multiple BTV serotypes generates antibodies that neutralize serotypes not encountered previously (Jeggo et al., 1983, 1986), potentially making typing by this method unreliable, particularly in areas (such as southern Europe) where multiple serotypes are co-circulating.
RT-PCR assays (targeting Seg-2) have been used to detect and identify a limited range of BTV serotypes within certain geographical regions (including Australia and the USA; Gould & Pritchard, 1990; McColl & Gould, 1991; Wilson & Chase, 1993; Pritchard & Gould, 1995; Johnson et al., 2000). However, these studies were hampered by the absence of Seg-2 sequences for multiple isolates of each serotype from geographically distinct origins. The specificity of these assays was not evaluated widely by using multiple strains of all 24 BTV serotypes and, although the primers and assays are effective within certain geographical limits, they may not identify all isolates of the target serotype reliably.
Recent sequencing studies of Seg-2 from multiple isolates of different BTV types have revealed the level of sequence variation within and between different BTV serotypes (Maan et al., 2004, 2007). These studies show significant variation between strains of the same serotype from different geographical areas (different Seg-2 topotypes). In most cases, different isolates of the same serotype could be divided into an eastern topotype (from Australia, Indonesia and India) and a western topotype (from Africa and North or South America). A maximum of 13 % nucleotide sequence variation was detected in Seg-2 within each topotype, with up to 31 % variation between topotypes of the same serotype (Maan, 2004; Maan et al., 2007).
This paper describes the design and evaluation of RT-PCR-based assays (and primers) to detect members of individual BTV serotypes and to distinguish eastern and western Seg-2 topotypes within each serotype. Attempts were also made to design primers that would distinguish the European field strains from the live-attenuated vaccines of the same serotype that have been used in southern Europe.
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and are stored in a reference collection at IAH Pirbright. More information concerning each isolate is available on the IAH website (http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/BTV-isolates.htm).
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). These viruses were grown in BHK-21 cells and harvested when 100 % cytopathic effect was observed. RNA was extracted from cell-free supernatants by using a QIAamp Viral RNA Mini kit (Qiagen) or from infected monolayers by using TRIzol (Invitrogen) in accordance with the manufacturers' instructions (Attoui et al., 2000).
Serotype-specific primers.
Seg-2-specific primers were designed for intratypic conservation and heterotypic variation, by comparisons of sequences of multiple BTV isolates of different serotypes (Maan et al., 2004, 2007). Multiple primer pairs (identified by the letter A) were selected to detect any isolate of the European serotypes. Other primers were selected to target eastern or western topotypes of Seg-2 for each European BTV serotype (identified by the letters E and W, respectively; Tables 2–7). Each primer pair was evaluated by using multiple isolates of the homologous serotype and most closely related heterologous serotypes (including, wherever possible, different topotypes) (Maan et al., 2007), as well as reference strains of the other serotypes. Primer footprints were compared (in silico) with Seg-2 sequences from many other BTV isolates (over 300 in total) of different serotypes, to test type specificity.
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–7). Primers designed to be specific for the vaccine strains are identified by the letter V.
RNA denaturation and RT-PCR.
RNA test samples were denatured in 5 µl 0.1 mM methyl mercuric hydroxide (MMOH) for 10 min at room temperature as described previously (Wade-Evans et al., 1990; Anthony et al., 2007), followed by addition of 1 µl 0.7 M 2-mercaptoethanol.
Two RT-PCR protocols were evaluated for amplification of BTV Seg-2. These include a single-tube method using a One-Step RT-PCR kit (Qiagen), essentially as described by Anthony et al. (2007), with the exception that 30 amplification cycles were used (at 94 °C for 1 min, 50 °C for 1 min and 72 °C for 2.5 min), followed by a terminal extension step at 72 °C for 10 min.
The other RT-PCR protocol uses a kit from Amersham Biosciences, consisting of ready-to-use beads that include all reagents required for reverse-transcription and PCR-amplification steps. The primer–template mix was heated to 95 °C for 3 min or denatured with MMOH (as described above), then added to the RT-PCR beads reconstituted with nuclease-free water, making a final volume of 50 µl. Samples were subjected to reverse transcription at 37 °C for 60 min, then PCR amplification using the following thermal profile: initial denaturation at 95 °C for 5 min; 94 °C for 30 s, annealing at 50 °C for 30 s and extension at 72 °C for 2.5 min, for 30 cycles. There was a final extension step of 72 °C for 10 min. The cDNA products were analysed by 1 % agarose gel electrophoresis and visualized under UV light after staining with ethidium bromide.
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RESULTS |
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) and Australian isolates of BTV-1, 9 and 16 (McColl & Gould, 1991) were tested for their ability to detect isolates of the homologous serotype from different geographical areas (particularly those from Europe) and to distinguish them from reference strains of the other 24 BTV serotypes. The primers for BTV-2 (North American) and BTV-16 (Australian) generated multiple, incorrectly sized PCR products with reference strains of the homologous serotype. In contrast, the BTV-1 and 9 primers failed to amplify Seg-2 of the homologous reference strain (results not shown). The unsatisfactory nature of these results prompted attempts to design other primers that could detect and distinguish different strains of current European BTV serotypes reliably.
Design of serotype-specific primers
Sequence data for Seg-2 of the vaccine and field strains of BTV serotypes that have affected Europe since 1998 (types 1, 2, 4, 8, 9 and 16) (Maan et al., 2004, 2007; Potgieter et al., 2005) were compared and used to design novel, serotype-specific primers. Wherever possible, these analyses included multiple isolates from different geographical origins, to ensure that the primers would amplify different Seg-2 topotypes within the same serotype (Maan et al., 2004, 2007). These primers are listed in Tables 2–7.
BTV-1.
Nine pairs of BTV-1 forward and reverse primers (Table 2) were evaluated by using BTV-1 isolates from the eastern and western topotypes identified by Maan et al. (2004) (listed in Table 1). Four primer pairs (1A1, 1A2, 1A3 and 1A4) worked well, generating products of the expected sizes (Table 2) from Seg-2 of all 24 BTV-1 isolates. Other combinations of these forward and reverse primers were not evaluated widely, but proved to be effective (and type-specific) in other limited studies (data not shown). Previous sequencing studies of Seg-2 showed that BTV-2 is the most closely related heterologous serotype to BTV-1 (Maan et al., 2007). The four primer pairs (1A1, 1A2, 1A3 and 1A4) were therefore also tested with multiple strains of BTV-2, including the reference strain (RSArrrr/02). However, in each case, cDNA amplification was not detected (see Supplementary Fig. S1, available in JGV Online).
Primer pairs 1E1 and 1E2 or 1W1 and 1W2 were designed to be complementary to regions of Seg-2 showing consistent differences between eastern and western topotypes of BTV-1. These primers generated cDNA products of the expected sizes (Table 2) from either the eastern or western BTV-1 isolates tested, respectively, but did not cross-react with isolates from the heterologous BTV-1 topotype.
Two regions of Seg-2 were also identified that are unique (among the isolates sequenced) to the BTV-1 vaccine strain (Table 2). A primer pair was designed (pair 1V) that amplified cDNAs of the expected size (2115 bp) from Seg-2 of the vaccine strain (Table 2), but not from other western or eastern strains of BTV-1 (see Supplementary Fig. S1, available in JGV Online).
BTV-2.
Six pairs of forward and reverse primers (pairs 2A, 2W1, 2W2, 2W3, 2W4 and 2E) (Table 3) were designed and evaluated for amplification of Seg-2 from BTV-2. Primer pair 2A amplified a product of the expected size from RNA samples of all 33 BTV-2 isolates tested. These included an Indian isolate (IND1982/01) from an eastern BTV-2 topotype (Table 1). Primer pairs 2W1 and 2W2 also generated products of the expected sizes from the western BTV-2 isolates, but gave no cDNA products with the Indian isolate (IND1982/01), confirming that they are specific for the western Seg-2 topotype of BTV-2. One of the western primer sets (2W3) amplified Seg-2 of each European and African isolate, except for the vaccine and reference strains of BTV-2. This pair could therefore be used to differentiate European and African strains of BTV from the South African vaccine strain. In contrast, primer set 2E amplified Seg-2 from the Indian isolate of BTV-2 (IND1982/01), but not from any of the western isolates. Although primer pair 2W4 amplified a 1806 bp region from Seg-2 of the BTV-2 vaccine strain, it also amplified sequences from the reference strain and some of the other African isolates of BTV-2 (see Supplementary Fig. S2, available in JGV Online). This primer pair is not therefore entirely specific for the BTV-2 vaccine strain. As stated previously, BTV-1 is the most closely related heterologous serotype to BTV-2. However, no cross-amplification was detected by using any of these primer sets with the isolates of BTV-1 that were tested, including the reference strain (RSArrrr/01) (see Supplementary Fig. S2, available in JGV Online).
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). Three pairs (4W1, 4W2 and 4W3) generated products of the expected sizes with all 26 of the western-topotype isolates of BTV-4 (listed in Table 1; see Supplementary Fig. S3, available in JGV Online). Primer pairs 4W1 and 4W2 generated smaller amounts of cDNA from BTV-4 isolates from the Balearic Islands. Sequence analyses showed that all three primers (in sets 4W1 and 4W2) have one nucleotide mismatch in their respective footprints in Seg-2 of the Balearic isolates of BTV-4, explaining their lower priming efficiency. None of these primer pairs amplified Seg-2 from reference strains of the more closely related serotypes in ‘nucleotype A’, which includes types 10, 11, 17, 20 and 24, as defined by Maan et al. (2007).
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) was designed to amplify a 1233 bp region of Seg-2 from the western BTV-4 strains (from Europe or Argentina), but not from the vaccine strains from South Africa or Turkey (see Supplementary Fig. S3, available in JGV Online). Primer pair 4V (Table 4) targets the same primer footprint positions, but has six base changes unique to the vaccine strain. These primers amplified a 1249 bp product from Seg-2 of the South African and Turkish BTV-4 vaccines, but not from the other BTV-4 isolates tested (see Supplementary Fig. S3, available in JGV Online).
BTV-8.
Only western BTV-8 isolates were available for this study (from Africa and Europe). Four pairs of BTV-8 Seg-2-specific primers (8W1, 8W2, 8W3 and 8W4; Table 5) were designed and generated products of the expected size with most of the ten field isolates of BTV-8 tested (Table 1). However, primer pair 8W2 did not amplify sequences from Seg-2 of the South African isolate of BTV-8 (RSA1998/01), suggesting that there are nucleotide differences in the 8W2 primer footprints that are unique to this isolate. No amplification was detected with reference isolates of BTV-18 and 23, representing the other more closely related serotypes, from the same nucleotype as BTV-8 (nucleotype D; Maan et al., 2007) (see Supplementary Fig. S4, available in JGV Online).
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; Tlotleng, 2004; http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/BTV-8-Seg-2-tree.htm). It may also be possible to design RT-PCR primers targeting other genome segments that could be used to distinguish these BTV-8 strains.
BTV-9.
Six pairs of BTV-9 Seg-2-specific primers were designed and evaluated (9A1, 9A2, 9E1, 9E2, 9W1 and 9W2; Table 6) with 25 BTV-9 isolates (Table 1). The 9A1 and 9A2 pairs amplified products of the expected sizes from all BTV-9 isolates, from both eastern and western topotypes (see Supplementary Fig. S5, available in JGV Online). Primer pairs 9W1 and 9W2 amplified products of the expected sizes (Table 6) from the western isolates (from Africa and the USA), but not from eastern isolates of BTV-9. Primer pairs 9E1 and 9E2 also amplified cDNA products (1241 and 2330 bp) only from the eastern isolates of BTV-9 (from India, Australia and Europe). None of these primer pairs amplified Seg-2 sequences of BTV-5, the most closely related heterologous serotype to BTV-9, both of which belong to nucleotype E (Maan et al., 2007). As the European BTV-9 isolates are derived from an eastern lineage, whereas the vaccine strain is derived from the South African reference strain, it possible to distinguish them simply by using the BTV-9 east–west-specific primers described above.
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) and evaluated with 22 isolates of BTV-16 (Table 1). Primer pair 16A amplified a product of the expected size from all of these isolates (which are mainly eastern or European isolates from an eastern lineage, but include a single western strain from Nigeria). Primer pairs 16W1 and 16W2 amplified the expected cDNA products from Seg-2 of BTV-16 from Nigeria (NIG1982/02), but not from any of the eastern BTV-16 viruses. Primer pairs 16E1, 16E2, 16E3 and 16E4 also worked well with each eastern/European BTV-16 isolate, but not with the Nigerian isolate, and were therefore confirmed as specific for the eastern Seg-2 topotype of BTV-16.
None of the BTV-16 primer pairs generated cDNA products from BTV-3 or BTV-13, the other BTV serotypes belonging to nucleotype B (Maan et al., 2007) (see Supplementary Fig. S6, available in JGV Online).
The European field and vaccine strains of BTV-16 are from the eastern group (Seg-2 topotype) and it was therefore difficult to design primers that would distinguish between them reliably. However, it is possible to differentiate these strains by sequence comparisons of Seg-2, although this is inevitably slower than by RT-PCR (Maan et al., 2004; Potgieter et al., 2005; http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/btv16-segment2-tree.htm). It may also be possible to design RT-PCR primers targeting other genome segments that could be used to distinguish field and vaccine strains of BTV-16.
Confirmation of type specificity
Serotype-specific primers for each of the European BTV serotypes (described above) were evaluated for type specificity by comparisons to Seg-2 sequence data for multiple isolates of the 24 BTV serotypes (including a total of over 300 Seg-2 sequences from the western and eastern strains that were available). These comparisons indicate that there were no high-similarity binding sites in Seg-2 of heterologous serotypes that would result in mispriming under the reaction conditions used. The primers listed in Tables 2–7 are therefore thought to be serotype-specific, although novel strains may appear later on that are cross-reactive.
Field samples
The primers described here were used for primary (or confirmatory) identification of different field strains of BTV. These include BTV-1 from Greece in 2001 (GRE2001/01) and from Morocco and Algeria in 2006 (MOR2006/03 and ALG2006/01), and BTV-2 from Florida, USA (USA2003/01). Seg-2 of the early Greek field strains was amplified by using primer pairs 1E1 and 1E2, neither of which amplifies the western strains, including the vaccine strain. However, the primers designed to amplify Seg-2 of the western BTV-1 strains from North Africa also work with the vaccine strain (which has a western origin). The European strains of BTV-2 are all derived from a western origin and can be differentiated from eastern viruses by using the east–west-specific BTV-2 primers described earlier (Table 3; see Supplementary Fig. S2, available in JGV Online). BTV-4 isolates from Israel in 2002 (ISR2002/11) and 2006 (ISR2006/12) were initially identified by using the primers described here. BTV-8 from northern Europe (isolate NET2006/01) was initially identified by using the methods and primers described here. Subsequent 2006 isolates from Belgium, the Netherlands and Germany were also confirmed in this way; BTV-9 (IND2005/01) and BTV-16 (IND2004/05) from India were identified by using these methods. In many of these cases, the RNA samples tested were extracted directly from blood or tissues from infected animals. In each case, the serotype and east–west-specific assays worked well, identifying both serotype and geographical origins of the virus lineage.
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; Mertens et al., 1984; Yamaguchi et al., 1988a, b). Sequencing studies (of all 24 types) have confirmed that variations in the nucleotide sequence in Seg-2 correlate with differences in virus serotype (Maan et al., 2007). Seg-2 therefore represented a potential target for the development of serotype-specific RT-PCR assays. However, primers designed previously to target Seg-2 of American (Wilson & Chase, 1993) or Australian (McColl & Gould, 1991) BTV isolates gave, in most cases, multiple or partial products when tested with reference strains of the homologous BTV serotype, most of which are from Africa. These primers are therefore unsuitable for global BTV-typing assays. However, comparisons of full-length sequence data for Seg-2 from multiple, geographically distinct isolates of the European BTV serotypes (Breard et al., 2003; Maan et al., 2004; Potgieter et al., 2005; Zientara et al., 2006) have supported the design of primers and diagnostic assays that show greater serotype specificity (for types 1, 2, 4, 8, 9 and 16). Multiple primer sets were evaluated for each of the European serotypes, using different field isolates of the homologous serotype and the most closely related heterologous serotypes, as well as the 24 BTV reference strains. Although some variation in amplification efficiency was detected (depending largely on geographical origins), primer sets were identified that that were serotype-specific for Seg-2 of each of the European types.
Primers were also developed to distinguish European field and vaccine strains of BTV types 1, 2, 4 and 9. These primers identify strains on the basis of variations in Seg-2 that appear to at least partially reflect the geographical origins of the virus. Primer pairs were identified that could be used to distinguish the European strains of BTV-1, which were derived from either eastern or western lineages, whereas the BTV-1 vaccine is derived from an African virus isolate (western group). One primer pair (1V) was also identified that only amplified Seg-2 from the vaccine strain and could therefore be used to distinguish it from the other BTV-1 strains tested.
Although primer sets were developed to distinguish eastern and western strains of BTV-2, there are insufficient nucleotide differences between the vaccine strain and reference strain (from which it is derived) to distinguish between them by RT-PCR. However, primer pair 2W3 could amplify Seg-2 from any of the western BTV-2 strains apart from the reference or vaccine strains, and could therefore be used to distinguish recent European BTV-2 field strains from the vaccine strain. Attempts to design a primer pair that would only amplify Seg-2 of the BTV-2 vaccine were unsuccessful and, although primer pair 2W4 worked efficiently with RNA templates from the vaccine strain, it also gave some amplification of Seg-2 from other African isolates of BTV-2.
A real-time PCR was reported that could be used to distinguish Seg-2 of the original Italian field strain of BTV-2 from the vaccine that was used in the region (Orrù et al., 2004). Although this assay was not evaluated with other BTV-2 topotypes, it was used successfully to detect RNA from both the live BTV-2 vaccine and the field strain of this serotype in clinical samples from the same animals in Italy. A subsequent report (Ferrari et al., 2005) indicated that the BTV-2 vaccine strain was itself circulating in the field in Italy. Although it is difficult to distinguish Seg-2 of the BTV-2 vaccine strain from other African strains of this serotype by RT-PCR, they can be distinguished by sequence analysis and phylogenetic comparisons (Breard et al., 2003; Maan et al., 2004, 2007; Potgieter et al., 2005) (http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/BTV2-segment-2-tree.htm). The similarity between Seg-2 of the BTV-2 vaccine and field strains suggests that attenuation may be due to changes in other regions of the genome. Full-genome comparisons may identify other targets for RT-PCR primers that could be used to distinguish these field and vaccine strains. Indeed, Breard et al. (2003) suggested that variations in Seg-10 could be used to differentiate vaccine and field strains and therefore distinguish animals that had been vaccinated from those infected naturally with the field strain of BTV-2. Earlier reassortment studies with AHSV (O'Hara et al., 1998) have also indicated a role for the NS3 protein, encoded by Seg-10, in determination of virulence characteristics.
Although a partial sequence (GenBank accession no. AF135220) is published for Seg-2 of a Chinese isolate of BTV-4 (Bonneau et al., 1999), no eastern strains of BTV-4 were available for this study. The European field and vaccine strains of BTV-4 all belong to a western group. However, it was possible to design primers that would distinguish any of the field or reference strains tested (from Europe, Africa, Argentina or Israel) from the South African or Turkish vaccine strains.
The European field strains of BTV-9 are all from an eastern origin, whilst the vaccine is derived from the African (western) reference strain. The primers designed to distinguish eastern and western strains of BTV-9 can therefore also be used to differentiate European field and vaccine strains, but would not distinguish the reference and vaccine strains.
The European field strains and South African BTV-16 vaccine are both derived from an eastern lineage (from Pakistan). In contrast, the northern European strain of BTV-8 (from 2006) and the BTV-8 vaccine strain are both from an African (western) lineage. Attempts to design Seg-2-specific primers to distinguish vaccine and field strains of either serotype were hampered by a high level of nucleotide identity (99.9 %) (Maan, 2004; Tlotleng, 2004). Indeed, it was only possible to identify potential primer sites in Seg-2 that had a maximum of three nucleotide differences between vaccine and field strains of either type. This was insufficient to give full specificity.
Comparisons of the Seg-5 (NS1 gene) sequences of the BTV-16 vaccine strain and a 2002 BTV-16 field isolate (isolate number 94280/02) from Italy showed a much higher level of sequence variation (17.3 %) than was detected in Seg-2 (Monaco et al., 2006). Although this would make it possible to design primers targeting Seg-5 that would distinguish these strains, subsequent sequencing studies (C. A. Batten, A. E. Shaw, N. S. Maan, S. Maan & P. P. C. Mertens, unpublished data) indicate that the Italian field strain had acquired its NS1 gene by reassortment with the BTV-2 vaccine strain. Assays based on this segment alone would not therefore identify serotype or distinguish European vaccine and field strains reliably. The high level of similarity in Seg-2 of BTV-16 (96 %) suggests that the field and vaccine strains were derived from a relatively recent common ancestor (Maan, 2004). Indeed, the use of the BTV-16 vaccine strain as part of an annual vaccination campaign in Israel provides an obvious potential source for the virus that initially invaded Europe from the east (via Turkey) in 1999 (Potgieter et al., 2005; Monaco et al., 2006).
The RT-PCR assays and primers described here, supported by sequencing of Seg-2, have already confirmed the identity of BTV-1 from Greece, the identity of BTV-2 and the absence of BTV-9 in field samples from Corsica and vice versa in field samples from Serbia. In July 2001, an outbreak occurred in Corsica, despite vaccination against BTV-2. The results of RT-PCR assays provided data that helped the French veterinary authorities to make decisions concerning further sheep vaccination during the winter of 2001–2002. The sequences of the BTV-2 isolates demonstrated that wild-type BTV-2 (and not the vaccine strain) was the cause of the outbreak. The assays and primers described here were used by the European Community Reference Laboratory during August–September 2006 to provide primary identification of BTV-8 in clinical samples from the Netherlands, and identified BTV-1 in Algeria and Morocco.
The primers that were designed to amplify Seg-2 of BTV-4 from the eastern Mediterranean region (e.g isolates from Greece 1999–2000) failed to amplify the BTV-4 strain that subsequently arrived in north Africa, the western Mediterranean islands and the Iberian Peninsula (in 2003), although both strains are from a western lineage. These results demonstrate a need for monitoring to identify any changes in field strains (that might involve mutations in individual primer footprints). Multiple primer pairs were therefore developed for each BTV type, listed here and on the dsRNA virus website (http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm). By making these data available via the web, primer and assay designs can be updated to take into account any significant changes in the identity of the dominant field strains.
There is also a need for serotype-specific primers against the other 18 BTV serotypes. However, for at least some of these types, there are very few isolates and, consequently, Seg-2 sequences available. In order to develop fully effective, type-specific primers, additional isolates and sequence data will therefore be required.
The RT-PCR assays and primers described, together with associated sequencing studies, allow individual BTV serotypes to be identified with greater accuracy, sensitivity and more rapidly than is possible by conventional serological typing methods. It is now also possible to identify individual strains and lineages within a single BTV serotype in a manner that is impossible by conventional serological typing methods.
As serotype-specific primers are readily available, their use is likely to reduce the need for highly characterized BTV serotype-specific antisera for serological typing methods. As stated above, these antisera are in short supply, are expensive to produce in animals and are not generally available. The RT-PCR methods described here have the potential to replace or reduce the use of experimental animals to make antisera.
These diagnostic PCRs may also help to differentiate between natural infection and live vaccination by identifying the virus (or viral RNA) that persists in the animal's bloodstream following infection (Singer et al., 2001; Bonneau et al., 2002). These assays are helping to provide more information concerning the persistence and movements of individual virus strains in the field, improving our understanding of the epidemiology and spread of BTV in Europe and elsewhere. This information will help to inform the design of control strategies and the rapid implementation of appropriate vaccination programmes.
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REFERENCES |
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Anthony, S., Jones, H., Darpel, K. E., Elliott, H., Maan, S., Samuel, A., Mellor, P. S. & Mertens, P. P. C. (2007). A duplex RT-PCR assay for detection of genome segment 7 (VP7 gene) from 24 BTV serotypes. J Virol Methods 141, 188–197.[CrossRef][Medline]
Attoui, H., Billoir, F., Cantaloube, J. F., Biagini, P., de Micco, P. & de Lamballerie, X. (2000). Strategies for the sequence determination of viral dsRNA genomes. J Virol Methods 89, 147–158.[CrossRef][Medline]
Billinis, C., Koumbati, M., Spyrou, V., Nomikou, K., Mangana, O., Panagiotidis, C. A. & Papadopoulos, O. (2001). Bluetongue virus diagnosis of clinical cases by a duplex reverse transcription-PCR: a comparison with conventional methods. J Virol Methods 98, 77–89.[CrossRef][Medline]
Blacksell, S. D. & Lunt, R. A. (1996). A simplified fluorescence inhibition test for the serotype determination of Australian bluetongue viruses. Aust Vet J 73, 33–34.[Medline]
Bonneau, K. R., Zhang, N., Zhu, J., Zhang, F., Li, Z., Zhang, K., Xiao, L., Xiang, W. & MacLachlan, N. J. (1999). Sequence comparison of the L2 and S10 genes of bluetongue viruses from the United States and the People's Republic of China. Virus Res 61, 153–160.[CrossRef][Medline]
Bonneau, K. R., Zhang, N. Z., Wilson, W. C., Zhu, J. B., Zhang, F. Q., Li, Z. H., Zhang, K. L., Xiao, L., Xiang, W. B. & MacLachlan, N. J. (2000). Phylogenetic analysis of the S7 gene does not segregate Chinese strains of bluetongue virus into a single topotype. Arch Virol 145, 1163–1171.[CrossRef][Medline]
Bonneau, K. R., DeMaula, C. D., Mullens, B. A. & MacLachlan, N. J. (2002). Duration of viraemia infectious to Culicoides sonorensis in bluetongue virus-infected cattle and sheep. Vet Microbiol 88, 115–125.[CrossRef][Medline]
Breard, E., Sailleau, C., Coupier, H., Ravaud, K. M., Hammoumi, S., Gicquel, B., Hamblin, C., Dubourget, P. & Zientara, S. (2003). Comparison of genome segments 2, 7 and 10 of bluetongue viruses serotype 2 for differentiation between field isolates and the vaccine strain. Vet Res 34, 777–789.[CrossRef][Medline]
Davies, F. G. & Blackburn, N. K. (1971). The typing of bluetongue virus. Res Vet Sci 12, 181–183.[Medline]
Davies, F. G., Mungai, J. N. & Pini, A. (1992). A new bluetongue virus serotype isolated in Kenya. Vet Microbiol 31, 25–32.[CrossRef][Medline]
DeMaula, C. D., Bonneau, K. R. & MacLachlan, N. J. (2000). Changes in the outer capsid proteins of bluetongue virus serotype ten that abrogate neutralization by monoclonal antibodies. Virus Res 67, 59–66.[Medline]
Dubay, S. A., Rosenstock, S. S., Stallknecht, D. E. & deVos, J. C., Jr (2006). Determining prevalence of bluetongue and epizootic hemorrhagic disease viruses in mule deer in Arizona (USA) using whole blood dried on paper strips compared to serum analyses. J Wildl Dis 42, 159–163.
Erasmus, B. J. (1990). Bluetongue virus. In Virus Infections of Ruminants, vol. 3, pp. 227–237. Edited by Z. Dinter & B. Morein. New York: Elsevier.
European Commission (2006).Bluetongue – Netherlands. Urgent fax 001 (17/08/2006; 19:54), D1 BVG (06) D, Brussels, 17 August 2006. http://www.defra.gov.uk/animalh/diseases/monitoring/pdf/bt-nlsheep180806.pdf
Ferrari, G., De Liberato, C., Scavia, G., Lorenzetti, R., Zini, M., Farina, F., Magliano, A., Cardeti, G., Scholl, F. & other authors (2005). Active circulation of bluetongue vaccine virus serotype-2 among unvaccinated cattle in central Italy. Prev Vet Med 68, 103–113.[CrossRef][Medline]
Gould, A. R. & Eaton, B. T. (1990). The amino acid sequence of the outer coat protein VP2 of neutralizing monoclonal antibody-resistant, virulent and attenuated bluetongue viruses. Virus Res 17, 161–172.[CrossRef][Medline]
Gould, A. R. & Pritchard, L. I. (1990). Relationships amongst bluetongue viruses revealed by comparisons of capsid and outer coat protein nucleotide sequences. Virus Res 17, 31–52.[CrossRef][Medline]
Gumm, I. D. & Newman, J. F. (1982). The preparation of purified bluetongue virus group antigen for use as a diagnostic reagent. Arch Virol 72, 83–93.[CrossRef][Medline]
Howell, P. G. (1960). A preliminary antigenic classification of strains of bluetongue virus. Onderstepoort J Vet Res 28, 357–363.
Howell, P. G. & Verwoerd, D. W. (1971). Bluetongue virus. Virol Monogr 9, 35–74.[Medline]
Howell, P. G., Kumm, N. A. & Botha, M. J. (1970). The application of improved techniques to the identification of strains of bluetongue virus. Onderstepoort J Vet Res 37, 59–66.[Medline]
Huismans, H. & Erasmus, B. J. (1981). Identification of the serotype-specific and group specific antigens of bluetongue virus. Onderstepoort J Vet Res 48, 51–58.[Medline]
Huismans, H., van der Walt, N. T., Cloete, M. & Erasmus, B. J. (1983). The biochemical and immunological characterization of bluetongue virus outer capsid polypeptides. In Double-Stranded RNA Viruses, pp. 165–172. Edited by R. W. Compans & D. H. L. Bishop. New York: Elsevier.
IAH (2006). Isolates of bluetongue virus type 8 (BTV-8) in the dsRNA virus collection at IAH Pirbright. Pirbright, UK: Institute for Animal Health. Accessed 29 August 2006. http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/btv-8.htm.
Jeggo, M. H., Gumm, I. D. & Taylor, W. P. (1983). Clinical and serological response of sheep to serial challenge with different bluetongue virus types. Res Vet Sci 34, 205–211.[Medline]
Jeggo, M. H., Wardley, R. C., Brownlie, J. & Corteyn, A. H. (1986). Serial inoculation of sheep with two bluetongue virus types. Res Vet Sci 40, 386–392.[Medline]
Johnson, D. J., Wilson, W. C. & Paul, P. S. (2000). Validation of a reverse transcriptase multiplex PCR test for the serotype determination of US isolates of bluetongue virus. Vet Microbiol 76, 105–115.[CrossRef][Medline]
Johnson, D. J., Ostlund, E. N., Stallknecht, D. E., Goekjian, V. H., Jenkins-Moore, M. & Harris, S. C. (2006). First report of bluetongue virus serotype 1 isolated from a white-tailed deer in the United States. J Vet Diagn Invest 18, 398–401.
Maan, S. (2004). Complete nucleotide sequence analyses of genome segment 2 from the twenty-four serotypes of bluetongue virus: development of nucleic acid based typing methods and molecular epidemiology. PhD thesis, University of London.
Maan, S., Samuel, A. R., Maan, N., Attoui, H., Rao, S. & Mertens, P. P. C. (2004). Molecular epidemiology of bluetongue viruses from disease outbreaks in the Mediterranean Basin. Vet Ital 40, 489–496.
Maan, S., Maan, N. S., Samuel, A. R., Rao, S., Attoui, H. & Mertens, P. P. C. (2007). Analysis and phylogenetic comparisons of full-length VP2 genes of the twenty-four bluetongue virus serotypes. J Gen Virol 88, 621–630.
McColl, K. A. & Gould, A. R. (1991). Detection and characterisation of bluetongue virus using the polymerase chain reaction. Virus Res 21, 19–34.[CrossRef][Medline]
Mellor, P. S. & Boorman, J. (1995). The transmission and geographical spread of African horse sickness and bluetongue viruses. Ann Trop Med Parasitol 89, 1–15.[Medline]
Mellor, P. S. & Wittmann, E. J. (2002). Bluetongue virus in the Mediterranean Basin 1998–2001. Vet J 164, 20–37.[CrossRef][Medline]
Mellor, P. S., Boorman, J. & Baylis, M. (2000). Culicoides biting midges: their role as arbovirus vectors. Annu Rev Entomol 45, 307–340.[CrossRef][Medline]
Mertens, P. P. C. (1999). Orbiviruses and coltiviruses – general features. In Encyclopaedia of Virology, 2nd edn, pp. 1043–1061. Edited by R. G. Webster & A. Granoff. London: Academic Press.
Mertens, P. P. C. & Mellor, P. S. (2003). Bluetongue. State Vet J 13, 18–25.
Mertens, P. P. C., Brown, F. & Sangar, D. V. (1984). Assignment of the genome segments of bluetongue virus type 1 to the proteins which they encode. Virology 135, 207–217.[CrossRef][Medline]
Mertens, P. P. C., Burroughs, J. N. & Anderson, J. (1987a). Purification and properties of virus particles, infectious subviral particles, and cores of bluetongue virus serotypes 1 and 4. Virology 157, 375–386.[CrossRef][Medline]
Mertens, P. P. C., Pedley, S., Cowley, J. & Burroughs, J. N. (1987b). A comparison of six different bluetongue virus isolates by cross-hybridization of the dsRNA genome segments. Virology 161, 438–447.[CrossRef][Medline]
Mertens, P. P. C., Pedley, S., Cowley, J., Burroughs, J. N., Corteyn, A. H., Jeggo, M. H., Jennings, D. M. & Gorman, B. M. (1989). Analysis of the roles of bluetongue virus outer capsid proteins VP2 and VP5 in determination of virus serotype. Virology 170, 561–565.[CrossRef][Medline]
Mertens, P. P. C., Maan, S., Samuel, A. & Attoui, H. (2005). Genus Orbivirus. In Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses, pp. 466–483. Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & L. A. Ball. London: Elsevier/Academic Press.
Monaco, F., Camma, C., Serini, S. & Savini, G. (2006). Differentiation between field and vaccine strain of bluetongue virus serotype 16. Vet Microbiol 116, 45–52.[CrossRef][Medline]
O'Hara, R. S., Meyer, A. J., Burroughs, J. N., Pullen, L., Martin, L. A. & Mertens, P. P. C. (1998). Development of a mouse model system, coding assignments and identification of the genome segments controlling virulence of African horsesickness virus serotypes 3 and 8. Arch Virol Suppl 14, 259–279.[Medline]
OIE (2000). Manual of Standards for Diagnostic Tests and Vaccines: List A and B Diseases of Mammals, Birds and Bees, 4th edn. Paris: Office International des Epizooties.
OIE (2006a). Bluetongue in Morocco. Office International des Epizooties (OIE) Disease Information, 2 November 2006, vol. 19, no. 44. http://www.oie.int/eng/info/hebdo/AIS_72.HTM#Sec7.
OIE (2006b). Bluetongue in Bulgaria. Office International des Epizooties (OIE) Disease Information, 19 October 2006, vol. 19, no. 42. http://www.oie.int/eng/info/hebdo/AIS_74.HTM#Sec8.
OIE (2006c). Bluetongue in the Netherlands. Office International des Epizooties (OIE) Disease Information, 24 August 2006, vol. 19, no. 34. http://www.oie.int/eng/info/hebdo/AIS_04.HTM#Sec3.
Orrù, G., Santis, P. D., Solinas, F., Savini, G., Piras, V. & Caporale, V. (2004). Differentiation of Italian field and South African vaccine strains of bluetongue virus serotype 2 using real-time PCR. J Virol Methods 122, 37–43.[CrossRef][Medline]
Osburn, B. I. (2000). Bluetongue. In Diseases of Sheep, 3rd edn, pp. 393–397. Edited by B. Martin & I. D. Aitken. Oxford: Blackwell Science.
Pearson, J. E., Gustafson, G. A., Shafer, A. L. & Alstad, A. D. (1992). Diagnosis of bluetongue and epizootic hemorrhagic disease. In Proceedings of the Second International Symposium on Bluetongue, African Horse Sickness and Related Orbiviruses, pp. 533–546. Edited by T. E. Walton & B. I. Osburn. Boca Raton, FL: CRC Press.
Potgieter, A. C., Monaco, F., Mangana, O., Nomikou, K., Yadin, H. & Savini, G. (2005). VP2-segment sequence analysis of some isolates of bluetongue virus recovered in the Mediterranean Basin during the 1998–2003 outbreak. J Vet Med B Infect Dis Vet Public Health 52, 372–379.[Medline]
Pritchard, L. I. & Gould, A. R. (1995). Phylogenetic comparison of the serotype-specific VP2 protein of bluetongue and related orbiviruses. Virus Res 39, 207–220.[CrossRef][Medline]
Purse, B. V., Mellor, P. S., Rogers, D. J., Samuel, A. R., Mertens, P. P. & Baylis, M. (2005). Climate change and the recent emergence of bluetongue in Europe. Nat Rev Microbiol 3, 171–181.[CrossRef][Medline]
Roy, P., Bishop, D. H., LeBlois, H. & Erasmus, B. J. (1994). Long-lasting protection of sheep against bluetongue challenge after vaccination with virus-like particles: evidence for homologous and partial heterologous protection. Vaccine 12, 805–811.[CrossRef][Medline]
Shaw, A. E., Monaghan, P., Alpar, H. O., Anthony, S., Darpel, K. E., Batten, C. A., Guercio, A., Alimena, G., Vitale, M. & other authors (2007). Development and initial evaluation of a real-time RT-PCR assay to detect bluetongue virus genome segment 1. J Virol Methods in press
Singer, R. S., MacLachlan, N. J. & Carpenter, T. E. (2001). Maximal predicted duration of viremia in bluetongue virus-infected cattle. J Vet Diagn Invest 13, 43–49.
Tabachnick, W. J. (1996). Culicoides variipennis and bluetongue-virus epidemiology in the United States. Annu Rev Entomol 41, 23–43.[Medline]
Tabachnick, W. J., MacLachlan, N. J., Thompson, L. E., Hunt, G. J. & Patton, J. F. (1996). Susceptibility of Culicoides variipennis sonorensis to infection by polyermase chain reaction-detectable bluetongue virus in cattle blood. Am J Trop Med Hyg 54, 481–485.
Tlotleng, K. R. (2004). Partial sequencing of live attenuated polyvalent bluetongue viral vaccine from South Africa. MSc thesis, London School of Hygiene and Tropical Medicine.
Veronesi, E., Hamblin, C. & Mellor, P. S. (2005). Live attenuated bluetongue vaccine viruses in Dorset Poll sheep, before and after passage in vector midges (Diptera: Ceratopogonidae). Vaccine 23, 5509–5516.[CrossRef][Medline]
Wade-Evans, A. M., Mertens, P. P. C. & Bostock, C. J. (1990). Development of the polymerase chain reaction for the detection of bluetongue virus in tissue samples. J Virol Methods 30, 15–24.[CrossRef][Medline]
Wilson, W. C. & Chase, C. C. (1993). Nested and multiplex polymerase chain reactions for the identification of bluetongue virus infection in the biting midge, Culicoides variipennis. J Virol Methods 45, 39–47.[CrossRef][Medline]
Yamaguchi, S., Fukusho, A. & Roy, P. (1988a). Complete sequence of VP2 gene of the bluetongue virus Australian serotype 1 (BTV-1). Nucleic Acids Res 16, 2725
Yamaguchi, S., Fukusho, A. & Roy, P. (1988b). Complete sequence of neutralization protein VP2 of the recent US isolate bluetongue virus serotype 2: its relationship with VP2 species of other US serotypes. Virus Res 11, 49–58.[Medline]
Zientara, S., Breard, E. & Sailleau, C. (2006). Bluetongue: characterization of virus types by reverse transcription-polymerase chain reaction. Dev Biol (Basel) 126, 187–196.[Medline]
Received 22 March 2007;
accepted 19 June 2007.
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