Journal of General Virology (1995), 76, 260~2611.
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Proteolytic cleavage of VP2, an outer capsid protein of African horse sickness virus, by species-specific serum proteases enhances infectivity in Culicoides P. R. Marchi, P. Rawlings, J. N. Burroughs, M. Wellby, P. P. C. Mertens, P. S. Mellor and A. M . Wade-Evans* Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 ONF, UK
Purified African horse sickness virus (AHSV) was fed, as part of a blood meal, to adult females from a susceptible colony of Culicoides variipennis, established in the insectories at the Institute for Animal Health, Pirbright Laboratory, UK. The meal consisted of heparinized blood obtained from ovine, bovine, equine (horse and donkey) or canine sources spiked with AHSV serotype 9 (AHSV9). The infectivity levels observed for C. variipennis varied significantly, according to the source
of the blood sample. Comparison of the protein profiles obtained from AHSV9 incubated with the individual serum of plasma samples indicated that some speciesspecific serum proteases were able to cleave the outer capsid protein, VP2. The blood samples containing serum proteases that were able to cleave VP2 also showed an increase in infectivity for the insect vector when spiked with purified AHSV.
African horse sickness virus (AHSV) is the causative agent of a non-contagious, arthropod-borne disease of equines. The major vectors that have been identified in the field are biting midges of the genus Culicoides, particularly C. imicola (du Toit, 1944). Nine AHSV serotypes have been identified to date, which have been classified as a single serogroup of viruses within the Orbivirus genus, in the family Reoviridae. AHSV has many properties that are similar to the prototype Orbivirus, bluetongue virus (BTV). The rate of infection of the vector by the pathogen is an important parameter in all epidemiological models of vector-borne disease (Anderson & May, 1991). Therefore any factors which affect or influence the infectivity of arthropod-borne viruses for their insect vector are important. Studies on oral infection of C. variipennis and C. nubeculosus with BTV (Mertens et al., 1993) have shown that purified BTV virus particles and cores (lacking VP2 and VP5) have similar infectivity levels in this vector species, whereas infectious sub-viral particles (ISVP), where VP2 has been cleaved enzymatically, are 100-1000-fold more infectious. The ability of speciesspecific, serum proteases to cleave AHSV structural proteins and the possible effect of such cleavage on
infectivity for the insect vector have been investigated in this paper. Both AHSV and BTV replicate in the North American midge C. variipennis (Mellor et al., 1975), colonies of which are maintained in the insectories at the Institute for Animal Health (1.A.H.), Pirbright (as described by Boorman, 1974). These colonies have been used as a model for the epidemiological studies of AHSV and BTV in the absence of any colonies of C. imicola. A colony of midges (Pir-s-3), shown to be highly susceptible to infection by AHSV, was selected from the parent colony. Females from this colony were fed on heparinized blood spiked with tissue culture-harvested AHSV, serotype 9 (5 x 107 TCIDs0/ml of isolate 90/61 - MB3, BHK4 kindly provided by Dr B, J. Erasmus, Onderstepoort, South Africa) using glass feeder units (Mellor et al., 1974) over which a membrane of Parafilm ' M ' (American National Can) had been stretched. The blood meal, consisting of 1 ml of ovine, bovine, equine (horse or donkey) or canine blood mixed with 1 ml of AHSV9 (10 s TCIDs0/ml), was pipetted onto the upper surface of the membrane (preheated to 37 °C with a bypass waterbath). The midges were given the opportunity to feed for about 30 rain, then anaesthetized using CO~. Engorged females (approx. 40 per experiment) were selected and maintained for at least 5 days at 25 °C in 6 cm diameter, waxed, paper pill boxes capped with nylon netting. After this period the midges were removed from the holding
*Author for correspondence. Fax +44 1483 232448. e-mail
[email protected] 0001-3245 © 1995 SGM
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Table 1. Infection of C. variipennis fed on different mammalian bloods containing purified, intact A H S V particles of serotype 9
Mammalian blood Equine (horse) Canine Equine (donkey) Ovine Bovine Bovine + chymotrypsin treated virus (40 ng/ml)
No. of expts
Total no. of engorged females
Percentage positive females ± standard error
21 6 4 4 4 2
933 209 160 160 160 60
51 ± 3 41 ± 12 28 ± 5 26 ± 9 11 ± 5 51.5 ± 15
cage and sacrificed by exposure to - 7 0 °C for 10 min. Each female was homogenized individually in 400 gl of Eagle's medium (100 U/ml penicillin, 10 ng/ml streptomycin and 2.5 lag/ml fungizone). The homogenate was centrifuged for 1 min at 6500 r.p.m, and 100 gl of the supernatant added, in triplicate, to a 96-well microtitre plate containing a confluent BHK-21 cell monolayer and 100 gl of Eagle's medium supplemented with 2% fetal calf serum. The plates were incubated at 37 °C for 5 days. The presence of AHSV was detected by the appearance of a characteristic cytopathic effect (CPE) in the BHK cell monolayer and confirmed using a sandwich ELISA, specific for AHSV (Hamblin et al., 1991). Tested females were recorded as either positive or negative. Heparinized horse blood containing tissue culturepurified, intact AHSV9 (108 TCID~o, final conch 5 × 107 TCIDs0/ml) infected the largest percentage (mean 51% - s e e Table 1) of female C. variipennis. The least infectious dose was observed when the virus was fed to the midges in heparinized bovine blood (11% positive see Table 1), but this could be increased to the levels observed with horse blood by pretreating the purified AHSV added to the blood sample with chymotrypsin, i.e. by adding ISVPs to the blood as opposed to virus particles. The second highest infectivity levels were achieved using heparinized canine blood plus AHSV (41%), whereas the use of donkey or ovine blood resulted in similarly low infectivities (28 % and 26 %, respectively). The fact that the use of ovine blood resulted in an equivalent level of infectivity as donkey blood suggests that cleavage of VP2 is not the only factor involved in determining infectivity levels of AHSV for the insect host. It is possible since the ISVPs of both AHSV and BTV do not haemagglutinate (P. P. C. Mertens, unpublished data) that release of the virus from the red blood cells (RBCs) in the blood sample is an important factor in determining infectivity levels in the Culicoides. It is known that the ability of BTV to haemagglutinate RBCs is dependent on the mammalian
source of the blood sample (Eaton & Crameri, 1989), but these data are not yet available for AHSV. Possibly AHSV does not bind to ovine RBCs, but does bind to bovine RBCs and consequently the infectivity of the virus is slightly higher in ovine than in bovine blood. In order to determine whether incubation with the different blood samples had any effect on the integrity of the virus particle or the viral proteins, 50 pl of purified AHSV (Burroughs et al., 1994), labelled with [a~S]methionine (1000 c.p.m./gl), was incubated at 37 °C, for 1 h or overnight, with 2 gl of either ovine, bovine, equine or canine serum or plasma; or with chymotrypsin (40 gg/ml or 40 ng/ml) for 1 h only. An equal volume of loading buffer was added to each sample, which was then boiled for 5 min and electrophoresed on a 10% SDSpolyacrylamide gel (Laemmli, 1970). The resulting gel was fixed, dried and then exposed to autoradiographic film for 4-28 days. The results obtained from serum or plasma were indistinguishable (see Figs 1 and 2). When purified AHSV was incubated in either canine or horse serum or plasma for 1 h (see Fig. 1, lanes 3 and 11 and lanes 5 and 13, respectively) two new, but distinctly different protein products were detected on a 10% SDS-polyacrylamide gel. These protein cleavage products (marked with an * on Fig. 1) migrated faster than VP1 (approx. 150 kDa), VP2 (approx. 98 kDa) and VP3 (approx. 86 kDa), but not any of the other viral protein products. These new protein products, therefore, appear to be the result of cleavage of one of these three viral proteins. Other species of serum or plasma (ovine, bovine and donkey) did not appear to affect any viral proteins during the 1 h incubation. The distortion of migration patterns on SDS-PAGE prevented the use of higher concentrations of serum or plasma. This is the reason for not using the same concentration of serum or plasma that would be equivalent to the dilution of heparinized blood used in the virus blood meal. Heparinized blood was only diluted 2-fold whereas the serum or plasma used together with purified, intact virus particles was diluted 25-fold. Overnight incubation of the purified virus with canine, horse or donkey plasma (see Fig. 2, lanes 3 and 10, lanes 5 and 12 and lanes 4 and 11, respectively) yielded further, more extensive cleavage, indicating that only partial proteolysis had occurred after 1 h. In all of these cases it appeared that VP2 had been cleaved due to the absence or significant decrease in the band migrating at this position. Canine serum and plasma produced the most extensive cleavage of VP2 (Fig. 2, lanes 3 and 10). The partial cleavage of VP2 by horse and donkey serum and plasma resulted in some polypeptide fragments that migrated differently (Fig. 2, lanes 5 and 12) with approximate molecular masses of 78 kDa and 70 kDa respectively. No proteolytic activity was observed with
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1
3
2
4
5
6
7
8
9
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10 11 12 13 14 15
kDa VP2
97
VP3 ..~..-. ,
69 VP5 46 VP7
30
3
4
5
6
8
9
Fig. I. Partial cleavage products from intact AHSV9 particles incubated for 1 h with serum or plasma samples. The products resulting from a 1 h incubation of [35S]methionine-labelledpurified, intact virus with several different species of serum or plasma were analysed on a 10% SDS~olyacrylamide gel. The gel was fixed and dried, then autoradiographed. Lanes 1 5 are serum samples and lanes 8 15 plasma samples. Lanes 1, 8 and 9, bovine; lanes 2 and 10, ovine; lanes 3 and 11, canine; lanes 4 and 12, equine (donkey); lanes 5 and 13, equine (horse). Lane 14 is goat plasma and lane 15 porcine plasma. Lane 6 is AHSV incubated with 40 gg/ml chymotrypsin and lane 7 is virus alone. The major AHSV structural proteins, VP2, VP3, VP5 and VP7, are marked with arrows. The two asterisks mark the position of the partial cleavage products from VP2.
10 11 12 13 14
kDa 97
VP2 VP3
69 VP5
46 VP7
30
b o v i n e or ovine sera or p l a s m a (Fig. 2, lanes 1 a n d 8 a n d lanes 2 a n d 9 respectively) even after overnight incubations. I n c u b a t i o n with serum from four i n d i v i d u a l horses gave identical proteolytic cleavage p a t t e r n s in each case, i n d i c a t i n g that the results were species-specific a n d n o t i n d i v i d u a l characteristics or a n o m a l i e s (data n o t shown). The p a t t e r n of c h y m o t r y p s i n cleavage of A H S V structural proteins f r o m intact virus particles (which results in ISVP f o r m a t i o n ) was d e p e n d e n t u p o n the c o n c e n t r a t i o n o f e n z y m e used: 40 ~ g / l n l [the c o n c e n -
Fig. 2. Cleavage products from overnight incubation of purified, intact AHSV9 particles with serum or plasma samples. The products obtained from overnight incubation of [35S]methioninelabelled purified, intact virus particles with either serum or plasma samples were analysed on a 10 % SDS-polyacrylamide gel, fixed, dried and then autoradiographed. Lanes 1 5 are serum samples and lanes 8-14 plasma samples. Lanes 1 and 8, bovine; lanes 2 and 9, ovine; lanes 3 and 10, canine; lanes 4 and 11 equine (donkey); lanes 5 and 12, equine (horse). Lane 13 is goat plasma and lane 14 porcine plasma. A white arrow highlights the faint band that corresponds to that seen in the partial digestion pattern with horse plasma or serum. The major AHSV structural proteins, VP2, VP3, VP5 and VP7, are labelled with arrows. Complete cleavage products of approx. 28 and 29 kDa are marked with asterisks. Lane 6 is untreated virus and lane 7 shows viral proteins after incubation for 1 h with 40 gg/ml chymotrypsin.
t r a t i o n used for ISVP purification from cellular m a t e r i a l (Burroughs et aL, 1994)] a p p e a r e d to cleave the m a j o r i t y of the A H S V structural proteins from purified virus particles, with the exception o f VP7 (Fig. 1, lane 6), whereas 40 n g / m l resulted in m o r e specific cleavage of VP2 alone (Fig. 2, lane 7). T h e p r o t e i n profile p r o d u c e d by c h y m o t r y p s i n cleavage of A H S V was different from that p r o d u c e d by a n y of the serum proteases. E n d o p r o t e o l y t i c cleavage, usually at arginine residues, is a c o m m o n p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n observed with m a n y viral m e m b r a n e proteins a n d in some
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instances has proved to be a crucial factor in determining organ and host tropism, spread of infection and pathogenicity of viruses. (Klenk & Garten, 1994). Previous studies with BTV and AHSV have demonstrated proteolytic cleavage of the outer capsid protein, VP2, by treatment with either trypsin or chymotrypsin (Mertens et al., 1987; Burroughs et al., 1994). The AHSV-spiked blood samples (horse and canine) that gave the highest infectivity levels in C. variipennis also contained proteases that cleaved VP2 of AHSV. The ability of serum proteases to cleave VP2 of AHSV9 appears to be species-specific. Horses and dogs are the only two domestic species known to be highly susceptible to AHSV infection and suffer high mortality rates during epidemics of the disease (Dardiri & Salama, 1988; Van Rosenburg et al., 1981). AHSV9 particles may be circulating in these species (horses and dogs) as infectious sub-viral particles (ISVP), in which the outer capsid protein VP2 has been selectively cleaved through the action of chymotrypsin-like proteases, possibly plasmin. However, to date, there is no direct evidence that cleavage alters the infectivity of either AHSV or BTV particles for their mammalian hosts. The production of ISVPs is already known to be highly important in the initial stages of infection (Dimmock, 1982). In many viruses proteolytic cleavage of the virus particle (usually involving the viral protein responsible for haemagglutination) enhances viral infectivity. Strain-specific differences in the susceptibility of the haemagglutinin to cleavage by trypsin-like proteases can account for differences in host-range, organ tropism and pathogenicity. The initiation of a productive infection in mammalian cells by direct plasma membrane penetration has been demonstrated for icosahedral viruses such as rotavirus (Suzuki et al., 1986; Kaljot et al., 1988), adenovirus 2 (Morgan et at., 1969; Brown & Burlingham, 1973), poliovirus (Dunnebacke et al., 1969) and reovirus (Borsa et al., 1979). The ISVP of BTV appear to have a reduced eclipse period on infection compared to either intact virus particles or cores, suggesting that ISVPs may also utilize a non-endosomal route of entry (P. P. C. Mertens and others, unpublished data). Therefore the production of ISVP from intact Orbiviruses may be highly significant in determining infection rates in insect vectors as well as mammalian hosts. Specific protease inhibitors are at present being used to identify the serum protease(s) involved in the cleavage of AHSV VP2. Recent studies (O'Hara, 1995) have shown that VP2 of AHSV has a controlling influence on pathogenicity in a mouse model. Therefore, other serotypes of AHSV, including attenuated virus isolates (vaccine strains) are currently being analysed to determine whether cleavage of VP2 occurs in all AHSV
isolates, or correlates with either serotype or virulence characteristics. The authors would like to thank B. Clarke for his photographic expertise, J. Eveleigh for the blood samples, E. Denisen for maintenance of the Culicoides colony, and the Ministry of Agriculture, Fisheries and Food (MAFF) and EC contract no. 8001-CT91-0211 (AHSV in Europe) for financial support.
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O'HARA, R.S. (1995). Identification of the genome segments and proteins controlling the virulence of AHSV. PhD thesis, University of Reading, UK. SuzuKI, H., KITAOKA, S., SATO, T., KoNNO, T., IWASAKI, Y., NUmAZA~:I, Y. & ISmDA, N. (1986). Further investigation on the mode of entry of human rotavirus into cells. Archives of Virology 91, 135-144.
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VAN ROSENBURG, L. B.J., DE CLERK, J., GROENEWALD, H.B. & BOTnA, W. S. (1981). An outbreak of African horse sickness in dogs. Journal of the South African Veterblary Association 52, 323-325.
(Received 1 March 1995; Accepted 23 May 1995)
