- Email: [email protected]
Serodiagnosis of African swine fever using the recombinant protein p30 expressed in insect larvae
Journal of Virological Methods 89 (2000) 129 – 136 www.elsevier.com/locate/jviromet
Serodiagnosis of African swine fever using the recombinant protein p30 expressed in insect larvae M.G. Barderas a, A. Wigdorovitz b, F. Merelo c, F. Beitia c, C. Alonso a, M.V. Borca b, J.M. Escribano a,* a b
Departamento de Mejora Gene´tica y Biotecnologı´a, INIA, Ctra A Corun˜a Km 7, 28040 Madrid, Spain Instituto de Virologı´a, C.I.C.V., Inta-Castelar, CC77, Moro´n, (1708) Pcia. De Buenos Aires, Argentina c Departamento de Proteccio´n Vegetal, INIA, Ctra A Corun˜a Km 7, 28040 Madrid, Spain Received 4 April 2000; received in revised form 7 June 2000; accepted 9 June 2000
Abstract African swine fever (ASF) has a substantial economic impact in many African developing countries and its eradication is based only on an efficient diagnosis program because of the absence of an available vaccine. Previous data suggested the convenience of using the highly antigenic virus protein p30 as ELISA antigen for serological diagnosis of this disease. A simple and efficient method is described for producing the recombinant protein p30 from ASF virus in Trichoplusia ni larvae (cabbage looper) in order to facilitate the large-scale production of this recombinant protein in the absence of fermentation procedures. A baculovirus encoding the virus protein p30 was used to infect insect larvae, showing that recombinant protein production had a sharp optimal peak with a time of occurrence dependent on the initial virus dose inoculated to the larvae. Crude lysates of infected larvae were used without further purification as coating antigen in ELISA to analyse a limited number of sera from natural or experimentally ASF virus infected pigs. Remarkably, the recombinant protein obtained from a single infected larva was sufficient for serological diagnosis of at least 3750 serum samples. Recombinant p30 obtained by this procedure was also used in a confirmatory immunoblotting, reacting with all positive sera tested previously by ELISA. In conclusion, production of the recombinant ASF virus protein p30 in larvae should be applicable to large-scale production of diagnostic reagents for this disease in developing countries, eliminating the need for specialised facilities for tissue culture. © 2000 Elsevier Science B.V. All rights reserved. Keywords: African swine fever; p30; Trichoplusia ni larvae
1. Introduction
* Corresponding author. Fax: +34-91-3573107. E-mail address: [email protected] (J.M. Escribano).
African swine fever (ASF) virus is an icosahedral cytoplasmic deoxyvirus that infects domestic pigs and soft ticks of the Ornithodoros genus. This virus is presently the sole member of the As-
0166-0934/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S0166-0934(00)00209-3
130
M.G. Barderas et al. / Journal of Virological Methods 89 (2000) 129–136
farviridae family, which produces different forms of a disease, ranging from highly lethal to a subclinical illness (DeTray, 1957; Plowright et al., 1969). The lack of a vaccine makes diagnostic procedures the only methodology that can help to plan the complete eradication of the disease in affected countries. In most cases ASF disease is diagnosed by the detection of antibody, due to the presence of strains of reduced virulence (Hess, 1971; Bech-Nielsen et al., 1993). In addition, there is a high incidence of inapparent carriers in enzootic areas (Bech-Nielsen et al., 1993), as well as long-term ASF virus persistent infections in a large percentage of pigs surviving acute infections (DeTray, 1957; Carrillo et al., 1994). Serological diagnostic tests for ASF based on the use of recombinant proteins have been described previously (Alcaraz et al., 1995; Oviedo et al., 1997). The use of recombinant proteins as reagents in the serological tests provide many advantages when compared to antigen production based on obtaining antigens from infected cell extracts. These recombinant antigens provide simpler interpretation of the tests, reduce false positive reactions produced by cellular culture compounds that contaminate the antigens (Escribano et al., 1989), avoid the use of potentially dangerous live virus in antigen production, and allow for a better standardisation in antigen production. ELISA and immunoblotting tests have been adapted for this disease using recombinant antigenic virus proteins produced in Escherichia coli or baculovirus. However, most affected African countries lack the facilities applicable to large-scale production of such recombinant proteins at a low cost for use in serodiagnosis. The expression is described of the product encoded by the CP204L gene from ASF virus, the highly antigenic protein p30, by a recombinant baculovirus (Bacp30) in larvae of Trichoplusia ni. A highly specific and inexpensive diagnostic ELISA and immunoblotting tests for ASF were developed using crude lysates of Bacp30-infected larvae as antigen. A single larva infected with the recombinant baculovirus was adequate to coat enough ELISA plates for the diagnosis of more than 3750 serum samples or to prepare 400 nitrocellulose strips for immunoblotting assays.
2. Methods
2.1. Virus, sera and lar6ae The recombinant baculovirus Bacp30, expressing the ASF virus protein p30 (Oviedo et al., 1997), and a baculovirus expressing green fluorescent protein (BacGFP) were used to infect Sf9 insect cells or T. ni (cabbage looper) larvae. Eleven field pig sera diagnosed previously as ASF positive sera by ELISA and collected between 1991 and 1994, were obtained from inapparent carrier pigs from the southwest of Spain (enzootic areas during these years). Five additional sera from pigs inoculated oronasally with 105 50% tissue culture infectious doses (TCID50) of the attenuated virus E75 CV1-4) and collected on day 20 post-inoculation, were also used as positive ASF control sera in serological tests. Three normal pig sera were used as negative controls.
2.2. Cell culture and lar6al growth conditions Sf9 cells, grown as a monolayer in 75-cm2 tissue culture flasks, were inoculated with virus stocks by co-incubating virus and Sf9 cells in culture medium (lacking foetal bovine serum, FBS) for 1 h at 27°C. The inoculum was aspirated and replaced with medium supplemented with 10% FBS. The infected cells were then cultured at 27°C and collected for assays when a cytopathic effect was evident (about 72 h post-infection). Insect larvae were obtained from a laboratory rearing of T. ni. The eggs were put into larvae developmental cages, containing artificial insect diet (Medin et al., 1995). The eggs hatched and about 90% grew to the appropriate size (3 cm in length, 120–150 mg) in 10–14 days at 249 1°C in a climatic chamber. Fourth instar larvae were sedated by incubation on ice for 15 min and then injected near the proleg (forward along the body cavity) with 20 ml of medium containing different infectious doses of recombinant baculoviruses. When larvae became pale, swollen and lethargic (72–96 h after infection), they were harvested and frozen immediately at −70°C.
M.G. Barderas et al. / Journal of Virological Methods 89 (2000) 129–136
2.3. Lar6a sample preparation Infected larvae were homogenised on ice with a tissue disrupter in the presence of 0.75 ml/larva of extraction buffer (2.5 mM dithiothreitol, 0.01% Triton X-100, 1 mM phenylmethylsulfonyl fluoride). The resulting crude extract was centrifuged at 12 000× g for 30 min. In the case that oxidation of supernatants was evident (discoloration to brown or black) 10 mM b-mercaptoethanol was added. Lipids were discarded from larvae extracts and centrifuged again. Using this method, about 3–4 mg of total protein were obtained per homogenised larva.
2.4. Enzyme-linked immunosorbent assay (ELISA) The ELISA was carried out as described elsewhere (Pastor et al., 1990). Briefly, 100 ml of different dilutions of crude lysates of larvae in 0.05 M carbonate/bicarbonate buffer were used to coat wells of microtitration plates. After 12-h incubation at 4°C, plates were washed with PBS – 0.05% Tween 20 and used immediately or stored at − 20°C until use. Plates were blocked with 10% fetal calf serum in PBS for 1 h at 37°C, washed, and then incubated with 100 ml/well of the test serum at different dilutions for 1 h at 37°C. Wells were washed again, and protein A conjugated with horseradish peroxidase (Sigma) was added at 1:1000 dilution. Plates were incubated for 1 h at 37°C, then washed again and developed with orthophenylenediamine (OPD) as substrate. The reaction was stopped by addition of 100 ml of 3 N H2SO4. Finally, the reactions were read spectrophotometrically at OD492.
2.5. Confirmatory immunoblotting assay Proteins from crude larvae extracts quantified previously were resolved in 17% acrylamide gels and transferred to a nitrocellulose filter. The portion of the filter containing the proteins with molecular weights between 20 and 40 K was cut and divided into 4.0-mm wide strips (as described previously for diagnostic purposes in Oviedo et al., 1997). Filters were incubated with 2% nonfat
131
dry milk as blocking solution for 1 h and reacted with test sera diluted at 1:40 for 1 h. The presence of immunocomplexes was detected using peroxidase-labelled protein A and 4-chloronaphtol as substrate.
3. Results
3.1. Production of recombinant p30 in insect lar6ae The successful expression of the ASF virus protein p30 in cultured insect cells infected by recombinant baculovirus Bacp30 was demonstrated previously. Thus, we attempted to express this protein using the recombinant baculovirus in T. ni insect as an inexpensive medium for accumulation of large quantities of the recombinant protein. The kinetics of recombinant protein expression in infected larvae was examined. Bacp30 baculovirus (1.5× 106 infectious doses in 20 ml of PBS) was injected to different groups of larvae (three larvae per time point) and they were homogenised at 24, 48, 72, 96 and 120 h post-infection. Whole insect homogenate resolved by SDS-PAGE and analysed by Western blot by a pool of sera from infected ASF virus pigs showed maximum expression levels at 72–96 h post-infection (Fig. 1A). A single band of 30 kDa reacted with the antiserum, presenting identical mobility of the recombinant protein expressed in larvae to that observed in Sf9 cells infected by Bacp30 baculovirus or the protein p30 induced during ASF virus infection (not shown). Absorbance values obtained with 1 mg of larva extract to coat ELISA plates and the anti-ASF virus serum confirmed the highest expression levels at 72 and 96 h post-infection (Fig. 1A). Apparently, at 120 h post infection, when most larvae presented severe signs of infection, the recombinant protein accumulation was dramatically reduced. Analyses of recombinant protein yield production by T. ni larvae infected using different virus doses revealed important differences in the p30 accumulation in infected larvae tissues at 96 h post-infection, depending of the amount of virus inoculated. Groups of three larvae each were in-
132
M.G. Barderas et al. / Journal of Virological Methods 89 (2000) 129–136
jected with 20 ml of culture medium containing different doses of Bacp30 virus, ranging from 5 × 105 to 2×106 infectious doses. Extracts from every group of inoculated larvae were analysed by Western blot and the recombinant protein was quantified by ELISA (Fig. 1B). The results showed almost no detectable expression of p30 when 5 ×105 infectious doses were inoculated to the larvae. However, a dramatic increase of recombinant expression was detected when injecting 106 or higher virus infectious doses.
3.2. Use of baculo6irus-expressed p30 in lar6ae for ASF 6irus antibody detection by ELISA and immunoblotting Once the conditions of recombinant protein p30 expression in larvae were established, we analysed the optimal antigen concentration to coat ELISA microtiter plates for serodiagnosis of ASF. A comparative analysis of the reactivity of a pool of positive ASF sera using recombinant p30 pro-
duced in larvae and Sf9 cells as antigen (Fig. 2A), established that 1.25 mg of total larvae protein per well was the optimal concentration to coat ELISA plates. At this antigen concentration, there was a greater than 10 times difference in absorbance between positive and negative pools of sera in ELISA. In addition to standardising the antigen concentration, an identical curve of reactivity was observed with the pool of sera in ELISA between the p30 produced by both methods. As a control of specificity, no reactivity of the positive pool of sera was shown when an extract from larvae infected with the baculovirus expressing the GFP protein was used as antigen or when a negative serum was incubated with the larva-derived p30 (Fig. 2A). A panel of five sera from pigs inoculated with the attenuated virus E75CV1-4, showing antibody titres between 1:320 and 1:640 determined by a conventional ELISA, were then analysed using crude larvae extracts as coating antigen (1.25 mg per well). The absorbance results obtained were
Fig. 1. Expression of the ASF virus protein p30 in larvae of T. ni. (A) Kinetics of recombinant protein p30 expression in larvae inoculated with 1.5 ×106 infectious doses. Larvae protein extracts were analysed at different times after infection by Western blot (WB), using 50 mg of total larva protein per strip, and by ELISA, using 1 mg of total larva protein per well to coat the plates. (B) Analysis of recombinant protein p30 expression in larvae at 96 h post-infection in relation to the baculovirus-inoculated dose. Protein extracts (50 mg of total protein) from larvae inoculated with different virus doses were analysed by Western blot (WB) and quantified by ELISA (1 mg of total protein per well).
M.G. Barderas et al. / Journal of Virological Methods 89 (2000) 129–136
133
Fig. 2. Analysis of sera from ASF virus-infected pigs by ELISA, using recombinant p30 protein expressed in larvae. (A) Comparative ELISA absorbance values obtained with a pool of ASF virus-positive sera using the recombinant p30 as antigen encoded by Bacp30 virus produced in larvae ( ) or in insect cells (). Controls correspond to the reactivity of the pool of sera with an extract from larvae infected with the baculovirus encoding the GFP protein ( ) and an ASF virus-negative pool of sera reacting with a larva extract containing recombinant p30 ( ). Numbers 1 to 11 correspond to serial 1:4 dilutions of the antigens beginning from 100 mg/ml of total larvae extract or the antigen obtained from 2.5× 106 infected Sf9 cells. Sera were tested at 1:30 dilution. (B) Absorbance values obtained with five sera (1–5) from experimentally infected pigs with the attenuated ASF virus E75CV1-4 and with three sera (6 – 8) from uninfected control pigs. Serum 9 corresponds to a pool of sera from experimentally inoculated pigs with ASF virus reacting with an extract from larvae inoculated with the baculovirus expressing the GFP protein. (C) Absorbance values obtained with 11 field sera (1–11) from inapparent carrier pigs naturally infected and three sera (12 – 14) from negative pigs. Serum 15 corresponds to a pool of inapparent carrier pig sera reacting with an extract from larvae inoculated with the baculovirus expressing the GFP protein. All sera were analysed at 1:100 dilution on microtitration ELISA plates coated with 1 mg of crude larvae extracts containing the recombinant p30 or GFP. Results show the mean 9S.E.M. for three independent experiments.
similar to that obtained with the p30 antigen derived from the cell line (not shown), presenting absorbance values more than 15 times higher than negative pig sera used as controls (Fig. 2B). To examine the reactivity of sera from inapparent carrier pigs with the larvae extracts containing the p30, a panel of 11 positive field pig sera recovered during the last years of the ASF virus outbreaks in Spain were also tested by ELISA using this antigen at the concentration determined previously. All sera were positive by ELISA, showing differences in absorbance values greater than eight times with respect to negative control sera (Fig. 2C). Interestingly, sera 8 and 11 presenting low antibody titres (lower than 1:320 in
ELISA) were clearly discriminated as positives, indicating the high sensitivity conferred by the larva-produced p30 to ELISA. For years, immunoblotting has been the confirmatory test of choice for eradication programs in affected countries, due to its sensitivity and technical simplicity. In order to analyse the possibility of using the protein p30 derived from larvae in this serological test, the antigen concentration to be used for conferring the maximum sensitivity to the technique was determined. Different larvae protein amounts were resolved in SDS-PAGE and transferred to nitrocellulose strips before reacting with a pool of three sera from experimentally inoculated pigs (Fig. 3A). The results demon-
134
M.G. Barderas et al. / Journal of Virological Methods 89 (2000) 129–136
strated that the best recombinant p30 reactivity results with control positive pool of sera by this technique were obtained using 25 mg of larvae extracts. No protein from uninfected larvae reacted with the pool of sera. Once the optimal protein concentration needed for immunoblotting serodiagnosis had been established, 10 field sera from naturally infected pigs were examined. All sera reacted with a unique protein band corresponding to the recombinant p30 present in the larvae extracts (Fig. 3B). A pool of three normal pig sera neither reacted with the recombinant p30 nor any larva or baculovirus protein present in the strips (Fig. 3B). Based on Coomassie blue staining analysis, it was estimated that from a single larva (weighing 120 – 150 mg before protein extraction), using the method described above, about 4.5 mg of total protein containing approximately 135 mg of recombinant p30 could be extracted. This means that a single infected larva provides sufficient antigen for coating at least 3750 ELISA wells or sufficient antigen to carry out at least 360 confirmatory immunoblotting assays.
4. Discussion The above results demonstrate that a specific and sensitive ELISA and immunoblotting assays for the serodiagnosis of ASF can be carried out
using as antigen the recombinant protein p30 produced in insect larvae of T. ni. These larvae were reared easily, inexpensive and an easily processed source of antigen. The larval homogenates functioned in ELISA and immunoblotting techniques reproducibly without further purification of antigen. A wide variety of recombinant proteins had been expressed in insect larvae using insect viruses as vectors, including enzymes (Medin et al., 1990; Tremblay et al., 1993), antibodies (Reis et al., 1992), hormones (Mathavan et al., 1995; Sumathy et al., 1996), vaccines (Kuroda et al., 1989; Zhou et al., 1995), cytokines (Deng et al., 1995; Shi et al., 1996; Pham et al., 1999) and diagnostic proteins (Ahmad et al., 1993; Ismail et al., 1995; Katz et al., 1995). Most of these antigens were processed correctly after synthesis, their activities remaining intact in the larvae protein extracts. The potential advantages of antigen production in insect larvae for serodiagnosis are related mainly to the relative simple technology that requires larvae production and infection once the recombinant baculovirus expressing the antigen of diagnostic interest is obtained and the massive amount of recombinant protein produced by this methodology. The efficient, low cost system of production of protein p30 described above allows production of specific antigen for diagnosis in the absence of high-tech fermentation procedures and in the
Fig. 3. Adaptation of recombinant p30 expressed in larvae to a confirmatory serodiagnosis by an immunoblotting technique. (A) Immunoblotting analysis of different amounts of total protein from larvae infected or uninfected with the recombinant baculovirus expressing p30, transferred to nitrocellulose strips and reacted with a pool of sera from ASF virus-infected pigs. (B) Analysis by immunoblotting of sera from inapparent carrier pigs (1–10), using 25 mg of total larvae protein per strip. A negative serum was used as control for the reaction (C).
M.G. Barderas et al. / Journal of Virological Methods 89 (2000) 129–136
absence of a sterile environment. It is of special interest for ASF virus-affected African countries where sophisticated laboratory conditions are not readily available. Only a few reports described the expression of recombinant proteins in larvae for diagnostic purposes. However, the method of production of recombinant proteins in larvae can be used for most proteins that can be expressed efficiently in insect cell lines by recombinant baculoviruses. We have expressed a variety of recombinant proteins from different pathogens and there is a good correlation between recombinant expression levels obtained in insect cell cultures and baculovirus-infected larvae. In addition, due to the high levels of expression obtained in larvae, further purification of the recombinant proteins from the insect extracts is not necessary before being used in antibody determination by ELISA. Nevertheless, in a comparative analysis by ELISA using the larvae-derived or the cell culture-derived p30, lower backgrounds were observed when larvae extracts were used to coat the plates. This reduction in background, considering similar OD values for the positive sera (independently of the source of the recombinant protein), is probably due to the absence of culture medium compounds contaminating the antigens. Those contaminants frequently show reactivities with antibodies present in sera from pigs treated with vaccines produced in cell lines (Escribano et al., 1989). The yield of recombinant protein production in larvae is comparable to the yields obtained in insect cell cultures, and the recombinant p30 produced from a single larvae was equivalent to the antigen produced by 5.6×108 infected insect cells. This equivalence, determined by ELISA titration of the antigen produced by both methods, makes the use of larvae for large-scale antigen production quite efficient.
Acknowledgements This work was supported by Grant Petri 950294-OP and BIO98-0307 from Ministerio de Educacio´n y Cultura.
135
References Ahmad, S., Bassiri, M., Banerjee, A.K., Yilma, T., 1993. Immunological characterization of the VSV nucleocapsid (N) protein expressed by recombinant baculovirus in Spodoptera exigua larva: use in differential diagnosis between vaccinated and infected animals. Virology 192, 207 – 216. Alcaraz, C., Rodrı´guez, F., Oviedo, J.M., Eiras, A., De Diego, M., Alonso, C., Escribano, J.M., 1995. Highly specific confirmatory Western blot test for African swine fever virus antibody detection using the recombinant virus protein p54. J. Virol. Methods 52, 111 – 119. Bech-Nielsen, S., Arias, M.L., Panadero, J., Escribano, J.M., Go´mez-Tejedor, C., Pe´rez-Bonilla, Q., Sa´nchez-Vizcaı´no, J.M., 1993. Laboratory diagnosis and disease occurrence in the current African swine fever eradication program in Spain, 1989 – 1991. Prevent. Vet. Med. 17, 225 – 234. Carrillo, C., Borca, M.V., Afonso, C.L., Onisk, D.V., Rock, D.L., 1994. Long-term persistent infection of swine monocyte/macrophages with African swine fever virus. J. Virol. 68, 580 – 583. Deng, J., Wang, S., Yang, Q., Cheng, X., Li, L., 1995. High-level expression of human beta-interferon gene in the silkworm with new constructed BmNPV vector. Chin. J. Biotechnol. 11, 109 – 117. DeTray, D.E., 1957. Persistence of viremia and immunity in African swine fever. Am. J. Vet. Res. 18, 299 – 333. Escribano, J.M., Pastor, M.J., Sa´nchez-Vizcaı´no, J.M., 1989. Antibodies to bovine serum albumin in swine sera: implication for false-positive reactions in the serodiagnosis of African swine fever. Am. J. Vet. Res. 50, 1118 – 1122. Hess, W.R., 1971. African swine fever virus. In: Gard, S., Hallaner, G., Meyer, K.F. (Eds.), Virology Monographs, vol. 9. Springer, Berlin, pp. 1 – 3. Ismail, T.M., Yamanaka, M.K., Saliki, J.T., el-Kholy, A., Mebus, C., Yilma, T., 1995. Cloning and expression of the nucleoprotein of peste des petits ruminants virus in baculovirus. Virology 208, 776 – 778. Katz, J.B., Shafer, A.L., Eernisse, K.A., 1995. Construction and insect larval expression of recombinant vesicular stomatitis nucleocapsid protein and its use in competitive ELISA. J. Virol. Methods 54, 145 – 157. Kuroda, K., Groner, A., Fres, K., Drenckhahn, D., Hauser, C., Rott, R., Doerfler, W., Klenk, H.D., 1989. Synthesis of biologically active influenza virus hemagglutinin in insect larvae. J. Virol. 63, 1677 – 1685. Mathavan, S., Gautvik, V.T., Rokkones, E., Olstad, O.K., Kareem, B.N., Maeda, S., Gautvik, K.M., 1995. High-level production of human parathyroid hormone in Bombyx mori larvae and BmN cells using recombinant baculovirus. Gene 167, 33 – 39. Medin, J.A., Hunt, L., Gathy, K., Evans, R.K., Coleman, M.S., 1990. Efficient, low-cost protein factories: expression of human adenosine deaminase in baculovirus-infected insect larvae. Proc. Natl. Acad. Sci. USA 87, 2760 – 2764.
136
M.G. Barderas et al. / Journal of Virological Methods 89 (2000) 129–136
Medin, J.A., Gathy, K., Coleman, M.S., 1995. Expression of foreign proteins in Trichoplusia ni larvae. In: Richardson, C.D. (Ed.), Methods in Molecular Biology, Vol. 39: Baculovirus Expression Protocols, pp. 265–275. Oviedo, J.M., Rodrı´guez, F., Go´mez-Puertas, P., Brun, A., Go´mez, N., Alonso, C., Escribano, J.M., 1997. High level expression of the major antigenic African swine fever virus proteins p54 and p30 in baculovirus and their potential use as diagnostic reagents. J. Virol. Methods 64, 27–35. Pastor, M.J., Arias, M., Escribano, J.M., 1990. Comparison of two antigens for use in an enzyme-linked immunosorbent assay to detect African swine fever antibody. Am. J. Vet. Res. 51, 1540 – 1543. Pham, M.Q., Naggie, S., Wier, M., Cha, H.J., Bentley, W.E., 1999. Human interleukin-2 production in insect (Trichoplusia ni ) larvae: effects and partial control of proteolysis. Biotechnol. Bioeng. 62, 175–182. Plowright, W., Parker, J., Pierce, M.A., 1969. The epizootiology of African swine fever in Africa. Vet. Rec. 85, 668– 674.
.
Reis, U., Blum, B., von Specht, B.U., Domdey, H., Collins, J., 1992. Antibody production in silkworm cells and silkworm larvae infected with a dual recombinant Bombyx mori nuclear polyhedrosis virus. Biotechnology 10, 910 – 912. Shi, X., Qin, J., Zhu, J., Zhu, D., 1996. Expression of biologically active human granulocyte-macrophage colony-stimulating factor in the silkworm (Bombyx mori ). Biotechnol. Appl. Biochem. 24, 245 – 249. Sumathy, S., Palhan, V.B., Gopinathan, K.P., 1996. Expression of human growth hormone in silkworm larvae through recombinant Bombyx mori nuclear polyhedrosis virus. Protein Expr. Purif. 7, 262 – 268. Tremblay, N.M., Kennedy, B.P., Street, I.P., Kaupp, W.J., Laliberte, F., Weech, P.K., 1993. Human group II phospholipase A2 expressed in Trichoplusia ni larvae: isolation and kinetic properties of the enzyme. Protein Expr. Purif. 4, 490 – 498. Zhou, N., Zhang, Y., Jing, W., Li, Z., Wu, X., 1995. High expression of HBV S gene in Bombyx mori cell culture and in silkworms. Chin. J. Biotechnol. 11, 149 – 156.
Serodiagnosis of African swine fever using the recombinant protein p30 expressed in insect larvae M.G. Barderas a, A. Wigdorovitz b, F. Merelo c, F. Beitia c, C. Alonso a, M.V. Borca b, J.M. Escribano a,* a b
Departamento de Mejora Gene´tica y Biotecnologı´a, INIA, Ctra A Corun˜a Km 7, 28040 Madrid, Spain Instituto de Virologı´a, C.I.C.V., Inta-Castelar, CC77, Moro´n, (1708) Pcia. De Buenos Aires, Argentina c Departamento de Proteccio´n Vegetal, INIA, Ctra A Corun˜a Km 7, 28040 Madrid, Spain Received 4 April 2000; received in revised form 7 June 2000; accepted 9 June 2000
Abstract African swine fever (ASF) has a substantial economic impact in many African developing countries and its eradication is based only on an efficient diagnosis program because of the absence of an available vaccine. Previous data suggested the convenience of using the highly antigenic virus protein p30 as ELISA antigen for serological diagnosis of this disease. A simple and efficient method is described for producing the recombinant protein p30 from ASF virus in Trichoplusia ni larvae (cabbage looper) in order to facilitate the large-scale production of this recombinant protein in the absence of fermentation procedures. A baculovirus encoding the virus protein p30 was used to infect insect larvae, showing that recombinant protein production had a sharp optimal peak with a time of occurrence dependent on the initial virus dose inoculated to the larvae. Crude lysates of infected larvae were used without further purification as coating antigen in ELISA to analyse a limited number of sera from natural or experimentally ASF virus infected pigs. Remarkably, the recombinant protein obtained from a single infected larva was sufficient for serological diagnosis of at least 3750 serum samples. Recombinant p30 obtained by this procedure was also used in a confirmatory immunoblotting, reacting with all positive sera tested previously by ELISA. In conclusion, production of the recombinant ASF virus protein p30 in larvae should be applicable to large-scale production of diagnostic reagents for this disease in developing countries, eliminating the need for specialised facilities for tissue culture. © 2000 Elsevier Science B.V. All rights reserved. Keywords: African swine fever; p30; Trichoplusia ni larvae
1. Introduction
* Corresponding author. Fax: +34-91-3573107. E-mail address: [email protected] (J.M. Escribano).
African swine fever (ASF) virus is an icosahedral cytoplasmic deoxyvirus that infects domestic pigs and soft ticks of the Ornithodoros genus. This virus is presently the sole member of the As-
0166-0934/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S0166-0934(00)00209-3
130
M.G. Barderas et al. / Journal of Virological Methods 89 (2000) 129–136
farviridae family, which produces different forms of a disease, ranging from highly lethal to a subclinical illness (DeTray, 1957; Plowright et al., 1969). The lack of a vaccine makes diagnostic procedures the only methodology that can help to plan the complete eradication of the disease in affected countries. In most cases ASF disease is diagnosed by the detection of antibody, due to the presence of strains of reduced virulence (Hess, 1971; Bech-Nielsen et al., 1993). In addition, there is a high incidence of inapparent carriers in enzootic areas (Bech-Nielsen et al., 1993), as well as long-term ASF virus persistent infections in a large percentage of pigs surviving acute infections (DeTray, 1957; Carrillo et al., 1994). Serological diagnostic tests for ASF based on the use of recombinant proteins have been described previously (Alcaraz et al., 1995; Oviedo et al., 1997). The use of recombinant proteins as reagents in the serological tests provide many advantages when compared to antigen production based on obtaining antigens from infected cell extracts. These recombinant antigens provide simpler interpretation of the tests, reduce false positive reactions produced by cellular culture compounds that contaminate the antigens (Escribano et al., 1989), avoid the use of potentially dangerous live virus in antigen production, and allow for a better standardisation in antigen production. ELISA and immunoblotting tests have been adapted for this disease using recombinant antigenic virus proteins produced in Escherichia coli or baculovirus. However, most affected African countries lack the facilities applicable to large-scale production of such recombinant proteins at a low cost for use in serodiagnosis. The expression is described of the product encoded by the CP204L gene from ASF virus, the highly antigenic protein p30, by a recombinant baculovirus (Bacp30) in larvae of Trichoplusia ni. A highly specific and inexpensive diagnostic ELISA and immunoblotting tests for ASF were developed using crude lysates of Bacp30-infected larvae as antigen. A single larva infected with the recombinant baculovirus was adequate to coat enough ELISA plates for the diagnosis of more than 3750 serum samples or to prepare 400 nitrocellulose strips for immunoblotting assays.
2. Methods
2.1. Virus, sera and lar6ae The recombinant baculovirus Bacp30, expressing the ASF virus protein p30 (Oviedo et al., 1997), and a baculovirus expressing green fluorescent protein (BacGFP) were used to infect Sf9 insect cells or T. ni (cabbage looper) larvae. Eleven field pig sera diagnosed previously as ASF positive sera by ELISA and collected between 1991 and 1994, were obtained from inapparent carrier pigs from the southwest of Spain (enzootic areas during these years). Five additional sera from pigs inoculated oronasally with 105 50% tissue culture infectious doses (TCID50) of the attenuated virus E75 CV1-4) and collected on day 20 post-inoculation, were also used as positive ASF control sera in serological tests. Three normal pig sera were used as negative controls.
2.2. Cell culture and lar6al growth conditions Sf9 cells, grown as a monolayer in 75-cm2 tissue culture flasks, were inoculated with virus stocks by co-incubating virus and Sf9 cells in culture medium (lacking foetal bovine serum, FBS) for 1 h at 27°C. The inoculum was aspirated and replaced with medium supplemented with 10% FBS. The infected cells were then cultured at 27°C and collected for assays when a cytopathic effect was evident (about 72 h post-infection). Insect larvae were obtained from a laboratory rearing of T. ni. The eggs were put into larvae developmental cages, containing artificial insect diet (Medin et al., 1995). The eggs hatched and about 90% grew to the appropriate size (3 cm in length, 120–150 mg) in 10–14 days at 249 1°C in a climatic chamber. Fourth instar larvae were sedated by incubation on ice for 15 min and then injected near the proleg (forward along the body cavity) with 20 ml of medium containing different infectious doses of recombinant baculoviruses. When larvae became pale, swollen and lethargic (72–96 h after infection), they were harvested and frozen immediately at −70°C.
M.G. Barderas et al. / Journal of Virological Methods 89 (2000) 129–136
2.3. Lar6a sample preparation Infected larvae were homogenised on ice with a tissue disrupter in the presence of 0.75 ml/larva of extraction buffer (2.5 mM dithiothreitol, 0.01% Triton X-100, 1 mM phenylmethylsulfonyl fluoride). The resulting crude extract was centrifuged at 12 000× g for 30 min. In the case that oxidation of supernatants was evident (discoloration to brown or black) 10 mM b-mercaptoethanol was added. Lipids were discarded from larvae extracts and centrifuged again. Using this method, about 3–4 mg of total protein were obtained per homogenised larva.
2.4. Enzyme-linked immunosorbent assay (ELISA) The ELISA was carried out as described elsewhere (Pastor et al., 1990). Briefly, 100 ml of different dilutions of crude lysates of larvae in 0.05 M carbonate/bicarbonate buffer were used to coat wells of microtitration plates. After 12-h incubation at 4°C, plates were washed with PBS – 0.05% Tween 20 and used immediately or stored at − 20°C until use. Plates were blocked with 10% fetal calf serum in PBS for 1 h at 37°C, washed, and then incubated with 100 ml/well of the test serum at different dilutions for 1 h at 37°C. Wells were washed again, and protein A conjugated with horseradish peroxidase (Sigma) was added at 1:1000 dilution. Plates were incubated for 1 h at 37°C, then washed again and developed with orthophenylenediamine (OPD) as substrate. The reaction was stopped by addition of 100 ml of 3 N H2SO4. Finally, the reactions were read spectrophotometrically at OD492.
2.5. Confirmatory immunoblotting assay Proteins from crude larvae extracts quantified previously were resolved in 17% acrylamide gels and transferred to a nitrocellulose filter. The portion of the filter containing the proteins with molecular weights between 20 and 40 K was cut and divided into 4.0-mm wide strips (as described previously for diagnostic purposes in Oviedo et al., 1997). Filters were incubated with 2% nonfat
131
dry milk as blocking solution for 1 h and reacted with test sera diluted at 1:40 for 1 h. The presence of immunocomplexes was detected using peroxidase-labelled protein A and 4-chloronaphtol as substrate.
3. Results
3.1. Production of recombinant p30 in insect lar6ae The successful expression of the ASF virus protein p30 in cultured insect cells infected by recombinant baculovirus Bacp30 was demonstrated previously. Thus, we attempted to express this protein using the recombinant baculovirus in T. ni insect as an inexpensive medium for accumulation of large quantities of the recombinant protein. The kinetics of recombinant protein expression in infected larvae was examined. Bacp30 baculovirus (1.5× 106 infectious doses in 20 ml of PBS) was injected to different groups of larvae (three larvae per time point) and they were homogenised at 24, 48, 72, 96 and 120 h post-infection. Whole insect homogenate resolved by SDS-PAGE and analysed by Western blot by a pool of sera from infected ASF virus pigs showed maximum expression levels at 72–96 h post-infection (Fig. 1A). A single band of 30 kDa reacted with the antiserum, presenting identical mobility of the recombinant protein expressed in larvae to that observed in Sf9 cells infected by Bacp30 baculovirus or the protein p30 induced during ASF virus infection (not shown). Absorbance values obtained with 1 mg of larva extract to coat ELISA plates and the anti-ASF virus serum confirmed the highest expression levels at 72 and 96 h post-infection (Fig. 1A). Apparently, at 120 h post infection, when most larvae presented severe signs of infection, the recombinant protein accumulation was dramatically reduced. Analyses of recombinant protein yield production by T. ni larvae infected using different virus doses revealed important differences in the p30 accumulation in infected larvae tissues at 96 h post-infection, depending of the amount of virus inoculated. Groups of three larvae each were in-
132
M.G. Barderas et al. / Journal of Virological Methods 89 (2000) 129–136
jected with 20 ml of culture medium containing different doses of Bacp30 virus, ranging from 5 × 105 to 2×106 infectious doses. Extracts from every group of inoculated larvae were analysed by Western blot and the recombinant protein was quantified by ELISA (Fig. 1B). The results showed almost no detectable expression of p30 when 5 ×105 infectious doses were inoculated to the larvae. However, a dramatic increase of recombinant expression was detected when injecting 106 or higher virus infectious doses.
3.2. Use of baculo6irus-expressed p30 in lar6ae for ASF 6irus antibody detection by ELISA and immunoblotting Once the conditions of recombinant protein p30 expression in larvae were established, we analysed the optimal antigen concentration to coat ELISA microtiter plates for serodiagnosis of ASF. A comparative analysis of the reactivity of a pool of positive ASF sera using recombinant p30 pro-
duced in larvae and Sf9 cells as antigen (Fig. 2A), established that 1.25 mg of total larvae protein per well was the optimal concentration to coat ELISA plates. At this antigen concentration, there was a greater than 10 times difference in absorbance between positive and negative pools of sera in ELISA. In addition to standardising the antigen concentration, an identical curve of reactivity was observed with the pool of sera in ELISA between the p30 produced by both methods. As a control of specificity, no reactivity of the positive pool of sera was shown when an extract from larvae infected with the baculovirus expressing the GFP protein was used as antigen or when a negative serum was incubated with the larva-derived p30 (Fig. 2A). A panel of five sera from pigs inoculated with the attenuated virus E75CV1-4, showing antibody titres between 1:320 and 1:640 determined by a conventional ELISA, were then analysed using crude larvae extracts as coating antigen (1.25 mg per well). The absorbance results obtained were
Fig. 1. Expression of the ASF virus protein p30 in larvae of T. ni. (A) Kinetics of recombinant protein p30 expression in larvae inoculated with 1.5 ×106 infectious doses. Larvae protein extracts were analysed at different times after infection by Western blot (WB), using 50 mg of total larva protein per strip, and by ELISA, using 1 mg of total larva protein per well to coat the plates. (B) Analysis of recombinant protein p30 expression in larvae at 96 h post-infection in relation to the baculovirus-inoculated dose. Protein extracts (50 mg of total protein) from larvae inoculated with different virus doses were analysed by Western blot (WB) and quantified by ELISA (1 mg of total protein per well).
M.G. Barderas et al. / Journal of Virological Methods 89 (2000) 129–136
133
Fig. 2. Analysis of sera from ASF virus-infected pigs by ELISA, using recombinant p30 protein expressed in larvae. (A) Comparative ELISA absorbance values obtained with a pool of ASF virus-positive sera using the recombinant p30 as antigen encoded by Bacp30 virus produced in larvae ( ) or in insect cells (). Controls correspond to the reactivity of the pool of sera with an extract from larvae infected with the baculovirus encoding the GFP protein ( ) and an ASF virus-negative pool of sera reacting with a larva extract containing recombinant p30 ( ). Numbers 1 to 11 correspond to serial 1:4 dilutions of the antigens beginning from 100 mg/ml of total larvae extract or the antigen obtained from 2.5× 106 infected Sf9 cells. Sera were tested at 1:30 dilution. (B) Absorbance values obtained with five sera (1–5) from experimentally infected pigs with the attenuated ASF virus E75CV1-4 and with three sera (6 – 8) from uninfected control pigs. Serum 9 corresponds to a pool of sera from experimentally inoculated pigs with ASF virus reacting with an extract from larvae inoculated with the baculovirus expressing the GFP protein. (C) Absorbance values obtained with 11 field sera (1–11) from inapparent carrier pigs naturally infected and three sera (12 – 14) from negative pigs. Serum 15 corresponds to a pool of inapparent carrier pig sera reacting with an extract from larvae inoculated with the baculovirus expressing the GFP protein. All sera were analysed at 1:100 dilution on microtitration ELISA plates coated with 1 mg of crude larvae extracts containing the recombinant p30 or GFP. Results show the mean 9S.E.M. for three independent experiments.
similar to that obtained with the p30 antigen derived from the cell line (not shown), presenting absorbance values more than 15 times higher than negative pig sera used as controls (Fig. 2B). To examine the reactivity of sera from inapparent carrier pigs with the larvae extracts containing the p30, a panel of 11 positive field pig sera recovered during the last years of the ASF virus outbreaks in Spain were also tested by ELISA using this antigen at the concentration determined previously. All sera were positive by ELISA, showing differences in absorbance values greater than eight times with respect to negative control sera (Fig. 2C). Interestingly, sera 8 and 11 presenting low antibody titres (lower than 1:320 in
ELISA) were clearly discriminated as positives, indicating the high sensitivity conferred by the larva-produced p30 to ELISA. For years, immunoblotting has been the confirmatory test of choice for eradication programs in affected countries, due to its sensitivity and technical simplicity. In order to analyse the possibility of using the protein p30 derived from larvae in this serological test, the antigen concentration to be used for conferring the maximum sensitivity to the technique was determined. Different larvae protein amounts were resolved in SDS-PAGE and transferred to nitrocellulose strips before reacting with a pool of three sera from experimentally inoculated pigs (Fig. 3A). The results demon-
134
M.G. Barderas et al. / Journal of Virological Methods 89 (2000) 129–136
strated that the best recombinant p30 reactivity results with control positive pool of sera by this technique were obtained using 25 mg of larvae extracts. No protein from uninfected larvae reacted with the pool of sera. Once the optimal protein concentration needed for immunoblotting serodiagnosis had been established, 10 field sera from naturally infected pigs were examined. All sera reacted with a unique protein band corresponding to the recombinant p30 present in the larvae extracts (Fig. 3B). A pool of three normal pig sera neither reacted with the recombinant p30 nor any larva or baculovirus protein present in the strips (Fig. 3B). Based on Coomassie blue staining analysis, it was estimated that from a single larva (weighing 120 – 150 mg before protein extraction), using the method described above, about 4.5 mg of total protein containing approximately 135 mg of recombinant p30 could be extracted. This means that a single infected larva provides sufficient antigen for coating at least 3750 ELISA wells or sufficient antigen to carry out at least 360 confirmatory immunoblotting assays.
4. Discussion The above results demonstrate that a specific and sensitive ELISA and immunoblotting assays for the serodiagnosis of ASF can be carried out
using as antigen the recombinant protein p30 produced in insect larvae of T. ni. These larvae were reared easily, inexpensive and an easily processed source of antigen. The larval homogenates functioned in ELISA and immunoblotting techniques reproducibly without further purification of antigen. A wide variety of recombinant proteins had been expressed in insect larvae using insect viruses as vectors, including enzymes (Medin et al., 1990; Tremblay et al., 1993), antibodies (Reis et al., 1992), hormones (Mathavan et al., 1995; Sumathy et al., 1996), vaccines (Kuroda et al., 1989; Zhou et al., 1995), cytokines (Deng et al., 1995; Shi et al., 1996; Pham et al., 1999) and diagnostic proteins (Ahmad et al., 1993; Ismail et al., 1995; Katz et al., 1995). Most of these antigens were processed correctly after synthesis, their activities remaining intact in the larvae protein extracts. The potential advantages of antigen production in insect larvae for serodiagnosis are related mainly to the relative simple technology that requires larvae production and infection once the recombinant baculovirus expressing the antigen of diagnostic interest is obtained and the massive amount of recombinant protein produced by this methodology. The efficient, low cost system of production of protein p30 described above allows production of specific antigen for diagnosis in the absence of high-tech fermentation procedures and in the
Fig. 3. Adaptation of recombinant p30 expressed in larvae to a confirmatory serodiagnosis by an immunoblotting technique. (A) Immunoblotting analysis of different amounts of total protein from larvae infected or uninfected with the recombinant baculovirus expressing p30, transferred to nitrocellulose strips and reacted with a pool of sera from ASF virus-infected pigs. (B) Analysis by immunoblotting of sera from inapparent carrier pigs (1–10), using 25 mg of total larvae protein per strip. A negative serum was used as control for the reaction (C).
M.G. Barderas et al. / Journal of Virological Methods 89 (2000) 129–136
absence of a sterile environment. It is of special interest for ASF virus-affected African countries where sophisticated laboratory conditions are not readily available. Only a few reports described the expression of recombinant proteins in larvae for diagnostic purposes. However, the method of production of recombinant proteins in larvae can be used for most proteins that can be expressed efficiently in insect cell lines by recombinant baculoviruses. We have expressed a variety of recombinant proteins from different pathogens and there is a good correlation between recombinant expression levels obtained in insect cell cultures and baculovirus-infected larvae. In addition, due to the high levels of expression obtained in larvae, further purification of the recombinant proteins from the insect extracts is not necessary before being used in antibody determination by ELISA. Nevertheless, in a comparative analysis by ELISA using the larvae-derived or the cell culture-derived p30, lower backgrounds were observed when larvae extracts were used to coat the plates. This reduction in background, considering similar OD values for the positive sera (independently of the source of the recombinant protein), is probably due to the absence of culture medium compounds contaminating the antigens. Those contaminants frequently show reactivities with antibodies present in sera from pigs treated with vaccines produced in cell lines (Escribano et al., 1989). The yield of recombinant protein production in larvae is comparable to the yields obtained in insect cell cultures, and the recombinant p30 produced from a single larvae was equivalent to the antigen produced by 5.6×108 infected insect cells. This equivalence, determined by ELISA titration of the antigen produced by both methods, makes the use of larvae for large-scale antigen production quite efficient.
Acknowledgements This work was supported by Grant Petri 950294-OP and BIO98-0307 from Ministerio de Educacio´n y Cultura.
135
References Ahmad, S., Bassiri, M., Banerjee, A.K., Yilma, T., 1993. Immunological characterization of the VSV nucleocapsid (N) protein expressed by recombinant baculovirus in Spodoptera exigua larva: use in differential diagnosis between vaccinated and infected animals. Virology 192, 207 – 216. Alcaraz, C., Rodrı´guez, F., Oviedo, J.M., Eiras, A., De Diego, M., Alonso, C., Escribano, J.M., 1995. Highly specific confirmatory Western blot test for African swine fever virus antibody detection using the recombinant virus protein p54. J. Virol. Methods 52, 111 – 119. Bech-Nielsen, S., Arias, M.L., Panadero, J., Escribano, J.M., Go´mez-Tejedor, C., Pe´rez-Bonilla, Q., Sa´nchez-Vizcaı´no, J.M., 1993. Laboratory diagnosis and disease occurrence in the current African swine fever eradication program in Spain, 1989 – 1991. Prevent. Vet. Med. 17, 225 – 234. Carrillo, C., Borca, M.V., Afonso, C.L., Onisk, D.V., Rock, D.L., 1994. Long-term persistent infection of swine monocyte/macrophages with African swine fever virus. J. Virol. 68, 580 – 583. Deng, J., Wang, S., Yang, Q., Cheng, X., Li, L., 1995. High-level expression of human beta-interferon gene in the silkworm with new constructed BmNPV vector. Chin. J. Biotechnol. 11, 109 – 117. DeTray, D.E., 1957. Persistence of viremia and immunity in African swine fever. Am. J. Vet. Res. 18, 299 – 333. Escribano, J.M., Pastor, M.J., Sa´nchez-Vizcaı´no, J.M., 1989. Antibodies to bovine serum albumin in swine sera: implication for false-positive reactions in the serodiagnosis of African swine fever. Am. J. Vet. Res. 50, 1118 – 1122. Hess, W.R., 1971. African swine fever virus. In: Gard, S., Hallaner, G., Meyer, K.F. (Eds.), Virology Monographs, vol. 9. Springer, Berlin, pp. 1 – 3. Ismail, T.M., Yamanaka, M.K., Saliki, J.T., el-Kholy, A., Mebus, C., Yilma, T., 1995. Cloning and expression of the nucleoprotein of peste des petits ruminants virus in baculovirus. Virology 208, 776 – 778. Katz, J.B., Shafer, A.L., Eernisse, K.A., 1995. Construction and insect larval expression of recombinant vesicular stomatitis nucleocapsid protein and its use in competitive ELISA. J. Virol. Methods 54, 145 – 157. Kuroda, K., Groner, A., Fres, K., Drenckhahn, D., Hauser, C., Rott, R., Doerfler, W., Klenk, H.D., 1989. Synthesis of biologically active influenza virus hemagglutinin in insect larvae. J. Virol. 63, 1677 – 1685. Mathavan, S., Gautvik, V.T., Rokkones, E., Olstad, O.K., Kareem, B.N., Maeda, S., Gautvik, K.M., 1995. High-level production of human parathyroid hormone in Bombyx mori larvae and BmN cells using recombinant baculovirus. Gene 167, 33 – 39. Medin, J.A., Hunt, L., Gathy, K., Evans, R.K., Coleman, M.S., 1990. Efficient, low-cost protein factories: expression of human adenosine deaminase in baculovirus-infected insect larvae. Proc. Natl. Acad. Sci. USA 87, 2760 – 2764.
136
M.G. Barderas et al. / Journal of Virological Methods 89 (2000) 129–136
Medin, J.A., Gathy, K., Coleman, M.S., 1995. Expression of foreign proteins in Trichoplusia ni larvae. In: Richardson, C.D. (Ed.), Methods in Molecular Biology, Vol. 39: Baculovirus Expression Protocols, pp. 265–275. Oviedo, J.M., Rodrı´guez, F., Go´mez-Puertas, P., Brun, A., Go´mez, N., Alonso, C., Escribano, J.M., 1997. High level expression of the major antigenic African swine fever virus proteins p54 and p30 in baculovirus and their potential use as diagnostic reagents. J. Virol. Methods 64, 27–35. Pastor, M.J., Arias, M., Escribano, J.M., 1990. Comparison of two antigens for use in an enzyme-linked immunosorbent assay to detect African swine fever antibody. Am. J. Vet. Res. 51, 1540 – 1543. Pham, M.Q., Naggie, S., Wier, M., Cha, H.J., Bentley, W.E., 1999. Human interleukin-2 production in insect (Trichoplusia ni ) larvae: effects and partial control of proteolysis. Biotechnol. Bioeng. 62, 175–182. Plowright, W., Parker, J., Pierce, M.A., 1969. The epizootiology of African swine fever in Africa. Vet. Rec. 85, 668– 674.
.
Reis, U., Blum, B., von Specht, B.U., Domdey, H., Collins, J., 1992. Antibody production in silkworm cells and silkworm larvae infected with a dual recombinant Bombyx mori nuclear polyhedrosis virus. Biotechnology 10, 910 – 912. Shi, X., Qin, J., Zhu, J., Zhu, D., 1996. Expression of biologically active human granulocyte-macrophage colony-stimulating factor in the silkworm (Bombyx mori ). Biotechnol. Appl. Biochem. 24, 245 – 249. Sumathy, S., Palhan, V.B., Gopinathan, K.P., 1996. Expression of human growth hormone in silkworm larvae through recombinant Bombyx mori nuclear polyhedrosis virus. Protein Expr. Purif. 7, 262 – 268. Tremblay, N.M., Kennedy, B.P., Street, I.P., Kaupp, W.J., Laliberte, F., Weech, P.K., 1993. Human group II phospholipase A2 expressed in Trichoplusia ni larvae: isolation and kinetic properties of the enzyme. Protein Expr. Purif. 4, 490 – 498. Zhou, N., Zhang, Y., Jing, W., Li, Z., Wu, X., 1995. High expression of HBV S gene in Bombyx mori cell culture and in silkworms. Chin. J. Biotechnol. 11, 149 – 156.