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Serogrouping and topotyping of Sudanese and United States strains of epizootic hemorrhagic disease virus using PCR
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Pergamon
Comp. lmmun. Microbiol. in/ect. Dis. Vol. 20, No. 3, pp. 211 218, 1997 ~) 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain S0147-9571(97)00002-7 0147-9571/97 $17.00 + 0.00
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S E R O G R O U P I N G A N D T O P O T Y P I N G OF S U D A N E S E A N D U N I T E D STATES S T R A I N S OF EPIZOOTIC HEMORRHAGIC DISEASE VIRUS USING PCR IMADELDIN E. A R A D A I B , I M O H A M M E D E. H. M O H A M M E D , 2 M U A W I A M . M U K H T A R , 2 H A S H I M W . G H A L I B 3 a n d B E N N I E I. O S B U R N ~ ~Department of Veterinary Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616, U.S.A.; 2Department of Preventive Medicine and Veterinary Public Health, Faculty of Veterinary Science, University of K h a r t o u m , K h a r t o u m , Sudan; 3Department of Microbiology, Faculty of Medicine, University of K h a r t o u m , K h a r t o u m , Sudan
(Received for publication 26 January 1997)
Abstract -The potential use of the recently reported polymerase chain reaction (PCR) protocol for detection of United States epizootic hemorrhagic disease virus (EHDV) serotype 1 (EHDV-I) and serotype 2 (EHDV-2) ribonucleic acid in cell culture and clinical specimens was evaluated for detection of Sudanese E H D V strains. E H D V serotype 5 (EHDV-5) and EHDV, isolate 318 (untyped) designated (EHDV-318), recovered from sentinel calves at the K h a r t o u m University farm (Sudan) were studied. R N A from EHDV-5 and EHDV-318 and a n u m b e r of E H D V field isolates, propagated in cell cultures, were detected by the described PCR-based assay. The specific 387 bp P C R products were visualized on ethidium bromide stained agarose gel. Specificity of the P C R products was confirmed by chemiluminescent hybridization with non-radiolabeled internal probe. Amplification product was not detected when the PCR-based assay was applied to R N A from bluetongue virus (BTV) prototypes serotypes 2, 10, 11, 13, 16 and 17; total nucleic acid extracts from uninfected BHK-21 cells. The results of this study indicated that the previously described E H D V P C R assay could be applied for detection of Sudanese as well as United States strains of E H D V serogroup. In addition, the described E H D V - P C R assay could be used as a supportive diagnostic assay to the current conventional virus isolation procedures used for detection of E H D V infection in susceptible ruminants. ((_2 1997 Elsevier Science Ltd
Key words: EHDV, PCR, serogroup, topotype, Sudanese.
R~sum~5~L'utilit6 d ' u n protocole bas6 sur la r6action en cha~ne de la polym6rase (RCP), mis au point pour la d6tection de I ' A R N de souches am~ricaines (s6rotypes 1 et 2) du virus de la maladie h6morrhagique ~pizootique (VMHE), fut 6valu6e pour la d6tection de souches soudanaises du V M H E . L'&ude porta sur le V M H E s~rotype 5 (VMHE-5) et le V M H E isolat 318 (non s6rotyp6), d6sign6 VMHE-318, qui furent isol6s de veaux sentinelles de la ferme de l'Universit~ de K h a r t o u m (Soudan). Le protocole permit la d&ection de I'ARN du VMHE-5, du VMHE-318 et d'autres isolats du V M H E , propag~s en culture cellulaire. Les produits d'acides nucleiques obtenus par RCP, de 387 bp, furent mis en 6vidence sur gel agar, color~ au bromure d'&hidium. La sp6cificit6 de ces produits rut confirm~e par hybridation chimioluminescente, utilisant une sonde non radio-marqu&. Ces produits ne furent pas d&ect& lorsque la technique RCP fur appliqu& 5. I'ARN des s6rotypes 2, 10, 11, 13, 16 et 17 du virus du Blue Tongue, ou 5_des extraits d'acides nucl6iques de cellules B H K non-infect6es. Ces r&ultats indiquent que la technique R C P peut d&ecter les souches soudanaises du V M H E , en plus des souches am&icaines. De plus, cette technique pourrait avoir une utilit6 diagnostique, compl6mentant les m6thodes conventionnelles d'isolation virale du V M H E chez les ruminants. (c) 1997 Elsevier Science Ltd
Mots c&[~: V M H E , RCP, s6rotype, topotype, soudan. 211
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1. E. Aradaib et al. INTRODUCTION
Epizootic hemorrhagic disease virus (EHDV) is a double stranded (ds) RNA orbivirus of the family Reoviridae and is related to bluetongue virus (BTV)[1-3]. The virus has a worldwide distribution and exists in at least 10 distinct serotypes [4]. In central Sudan, previous studies have shown that the seasonal incidence of E H D V is a predictable event related to the rainy season [5, 6]. E H D V serotype 5 (EHDV-5) and an untyped E H D V isolate designated EHDV-318 were recovered from a sentinel calf herd at the Khartoum University farm, Shambat[5]. EHDV-318 was also recovered from sentinel calves in Sultanate of Oman [7]. Recently, EHDV-318 was reported to be a contaminant of genetically engineered Chinese hamster ovary (CHO) cells in Bahrain [8]. EHDV serotype 1 (EHDV-1) and serotype 2 (EHDV-2), which are known to be enzootic in the United States, cause an often fatal hemorrhagic disease in North American white-tailed deer (Odocoileus virginianus) [9 15]. There is no information available on the impact of E H D V on the wild ruminant species in the Sudan including the deer population. Infections caused by EHDV in local Sudanese breeds of sheep, goats and cattle is usually unapparent and no evidence of clinical hemorrhagic disease has been reported. However, indirect losses associated with loss of body weight and condition, drop in milk production and poor subsequent reproductive performance were thought to have greater economic effect than occasional overt disease [5]. In addition, because of unfamiliarity with the biology and ecology of the virus, there is the potential of restriction on the international movement of livestock and associated germplasm from countries suspected to harbor the disease to E H D V free countries unless the animals are certified free of infection by conventional virus isolation or serology [16]. Such a restriction could lead to economic losses for EHDV-endemic countries, like Sudan, which rely on the sale of livestock for foreign exchange. Current diagnosis of E H D V infection by conventional virus isolation and serology is time-consuming, labor intensive and expensive [17 20]. The development of a rapid, sensitive, specific and inexpensive method for serogrouping and topotyping of EHDV from different geographical locations would be advantageous for a variety of circumstances including clinical disease investigations, molecular epidemiological studies for understanding the biology of the virus, and enhancement of international trade of livestock and associated germplasm [21, 6]. Recently, we have described a simple, rapid, sensitive and specific E H D V PCR assays for detection of United States serogroup-specific detection and serotype specific identification of EHDV-1 and EHDV~2 in cell culture and a variety of clinical samples from naturally and experimentally infected animals [21-25]. In the present study, the potential of the recently described E H D V - P C R assay [21] was evaluated for serogrouping and topotyping of United States and Sudanese strains of E H D V using well-characterized primers derived from segment 6 of EHDV-2 (Alberta strain). MATERIALS AND METHODS Cell culture and virus propagation
The 2 prototype serotypes of E H D V present in the Sudan (EHDV-5 and EHDV-318), 5 E H D V field isolates, BTV serotype 16 (School of Veterinary Medicine, University of Khartoum, Sudan), E H D V serotype 1 (EHDV-1) and 2 (EHDV-2) and the BTV prototypes serotypes 2, 10, 11, 13, and 17 (Arthropod-Borne Animal Disease Research
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Laboratory, Laramie, WY, U.S.A.) were used in this study. The viruses were isolated and processed as described previously [2 l, 22, 26, 27]. All viruses were propagated on confluent monolayers of BHK-21 cells. The infectious material was harvested and centrifuged at 1500 g for 30 min and the cell pellet was used for the dsRNA extraction. Extraction of t,iral nucleic acid frorn infected cell culture The E H D V and the BTV dsRNA were extracted from the infected cells as previously described [21]. Total nucleic acid was ethanol-precipitated. Viral dsRNA was purified by differential lithium chloride precipitation, and resuspended in 100/d double distilled water, and quantified using a spectrophotometer at 260 nm wavelength. Primer selection and synthesis of the probe Primer selection and synthesis of the probe were described in details in a previous report [21]. Briefly, primers (20 mer each) were selected from the published sequence of genome segment 6 of EHDV-2 (Alberta strain)[28] and used in these PCR assays. E H D V PCR using primer P1 and P2 would result in a 387 bp product. For synthesis of a probe complementary to the predicted amplified viral sequences generated by P1 and P2, oligonucleotide primers (P3 and P4) were selected from the same published sequence cited above. PCR amplification using P3 and P4 would result in 224 bp PCR product internal to the annealing sites of PI and P2. All primers were synthesized on a DNA synthesizer (Milligen/Biosearch, A division of Millipore, Burlington, MA, U.S.A.) and purified using oligo-pak oligonucleotide purification columns (Glen Research Corporation, Sterling, VA, U.S.A.) as per the manufacturer's instructions. The probe was purified using D N A binding beads (Mermaid Kit, Bio 101, La Jolla, CA, U.S.A.) according to the manufacturer's instructions and used for chemiluminescent hybridization. Polymerase chain reaction The PCR protocol used in this study was basically as previously described [21, 22]. Taq DNA polymerase 5 units//d (Perkin-Elmer Cetus, The Perkin Elmer Corporation, Norwalk, CT, U.S.A.) were used in each reaction. The thermal cycling profiles were as follows: a 2 min incubation at 95~C, followed by 40 cycles of 9 5 C for 1 min, 5 5 C for 30 s and 72-C for 45 s and a final incubation at 62~C for 10 min. Thermal profiles were performed on a Techne PHC-2 thermal cycler (Techne, Princeton, N J, U.S.A.). From each PCR reaction containing amplified product 20/tl were loaded onto 2% SeaKem agarose (FMC Bioproducts, Rockland, ME, U.S.A.) and electrophoresed. The gels were stained with ethidium-bromide, and the specific 387 bp PCR products were visualized under UV light. Southern blot hybridization Southern blot hybridization was performed basically as previously described [21]. Briefly, agarose gels from PCR reaction were denatured, neutralised and transferred to nylon membranes (Schleicher and Schvell, Inc., Woburn, MA, U.S.A.) in vacuo at 50 mm Hg (Vacugene XL Vacuum Blotting System, Pharmacia LKB Biotechnology, Piscataway, N J, U.S.A.). Southern blotted nucleic acid was UV cross linked to the nylon membrane. Chemiluminescent hybridization was performed using a kit (ECL direct nucleic acid labeling and detection system, Amersham Corporation, Arlington Heights, IL, U.S.A.) according to the manufacturers instructions. The probe prepared by PCR was labeled with
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p e r o x i d a s e in the presence o f g l u t a r a l d e h y d e . S o u t h e r n blots were p r e h y b r i d i z e d with h y b r i d i z a t i o n buffer c o n t a i n i n g 5% b l o c k i n g agent a n d 0.85 M NaC1 at 4 2 C for 1 h. The labeled p r o b e was a d d e d to the h y b r i d i z a t i o n buffer a n d the m e m b r a n e s were h y b r i d i z e d overnight. A f t e r p o s t h y b r i d i z a t i o n w a s h i n g d e t e c t i o n reagents were a p p l i e d to the m e m b r a n e s for 1 rain. The m e m b r a n e s were then sealed in plastic w r a p a n d exposed to X - r a y film for 1 30 min with an intensifying screen. RESULTS All E H D V isolates t h a t were E H D V - p o s i t i v e by c o n v e n t i o n a l virus i s o l a t i o n were also positive to E H D V P e R assay. The described E H D V - P C R b a s e d assay afforded sensitive a n d specific detection o f E H D V serotypes a n d field isolates. T h e specific 387 bp P C R p r o d u c t was visualized on e t h i d i u m - b r o m i d e stained gel from 1.0 pg R N A o f the U n i t e d States E H D V serotypes 1 ( E H D V - 1 ) a n d 2 ( E H D V - 2 ) ; the S u d a n e s e E H D V s e r o t y p e 5 ( E H D V - 5 ) , the u n t y p e d isolate o f E H D V - 3 1 8 used in this study [Fig. (1A)]. The identity o f the amplified p r o d u c t s was c o n f i r m e d by s o u t h e r n blot with c h e m i l u m i n e s c e n t h y b r i d i z a t i o n [Fig. (1B)]. U s i n g 1.0 pg E H D V R N A , the 387 bp specific P C R p r o d u c t was detected in the five
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Fig. 1. Detection of the United states and the Sudanese epizootic hemorrhagic disease virus serotypes by the polymerase chain reaction (PCR) based assay. (A) Visualization of the 387 bp specific EHDV PCR product on ethidium bromide stained agarose gel from 1.0 pg of RNA of the United states prototypes serotypes 1 and 2; and the Sudanese EHDV serotype 5 (EHDV-5) and EHDV, isolate 318 (untyped). Lane MW: molecular weight marker: Lane 1: EHDV-I; Lane 2: EHDV-2; Lane 3: EHDV-5; Lane 4: EHDV-318; Lane 5: BHK cell control. (B) Southern blot with chemiluminescent hybridization of the above gel.
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Serogrouping and topotying of EHDV
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Fig. 2. (A) Visualization of the 387 bp specific EHDV PCR product on ethidium-bromide stained agarose gel from 1.0 pg of RNA of 5 different EHDV field isolates. Lane MW: molecular weight marker; Lane 1:EHDV-5 field isolate; Lanes 2-5:EHDV-318 isolated from sentinel calves; Lane 6:BHK-21 cell total nucleic acid extract. (B) Southern blot with chemiluminescent hybridization of the above gel.
Sudanese E H D V field isolates with both ethidium-bromide stained agarose gel [Fig. (2A)] and chemiluminescent hybridization [Fig. (2B)]. The amount of 1.0 ng RNA from BTV serotypes 2, 10, 11, 13, 16 and 17; and total nucleic acid extracts from uninfected BHK-21 cells failed to demonstrate PCR products (Fig. 3). DISCUSSION Sporadic outbreaks of clinical hemorrhagic disease among susceptible deer were observed in different regions of the Sudan. These oubreaks were first attributed to EHDV infection without confirmatory data. Subsequently, the virus was first isolated from sentinel calves in a herd in Khartoum [6]. At least 2 serotypes of E H D V designated EHDV-5 and EHDV-318 are known to be enzootic in the Sudan[5]. Very little information is available about orbiviruses originally isolated in Africa. Further studies on these orbiviruses is necessary to determine their biology, ecology and molecular epidemiology. The E H D V field isolates used in this study represented a range of topotype viruses, isolated from a diverse geographic locations in North America and Central Africa, recovered from calves and deer. The described E H D V - P C R assay using well-characterized serogrouping primers derived from segment 6 of EHDV, which codes for NS1 [12, 28, 29], reproducibly and specifically detected E H D V RNA in infected cell cultures. Selection of the
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Fig. 3. Specificity of the polymerase chain reaction for EHDV RNA. Amplification product was not detected from a high concentration of 1.0 ng RNA different BTV serototypes or total nucleic acid extracts from BHK-21 cells Lane MW: molecular weight marker: Lane l: 1 pg EHDV-2 isolated from a deer (positive control); Lane: 2 7 BLU virus prototypes serotypes 2, 10, 11, 13, 16 and 17, respectively; Lane 8:BHK-21 total nucleic acid extract. primers was based on the observation that the NS1 genome specific probe is the best o f all E H D V g e n o m e segments to produce positive hybridization signal c o m p a r e d to other nucleic acid probes [30]. The specific 387 bp P C R products, visualized on ethidium bromide stained agarose gel or detected with chemilumiescent hybridization, were obtained from all E H D V R N A samples tested. The E H D V - P C R assay was a simple procedure that efficiently detected all E H D V strains under the stringency condition used in this study. In the present study, the use of non-radioactive chemiluminescent hybridization removes the hazardous and c u m b e r s o m e radioactive l a b o r a t o r y procedures o f working with 32p or 33p. In a previous report, the sensitivity studies o f the described E H D V P C R protocol indicated that the P C R assay was capable of detecting the a m o u n t o f 0.1 fg o f total E H D V - 1 or E H D V - 2 genomic d s R N A . The total molecular weight of the E H D V genome is 11.44 x 106 Da, and 0.1 fg o f E H D V R N A corresponds to 6 viral particles [21]. The EHDV-PCR amplification technology with chemiluminiscent hybridization was approximately a million times more sensitive than the d o t - b l o t hybridization using E H D V c D N A probe, where at least a few n a n o g r a m s o f the d s R N A are required to produce a positive hybridization signal [30]. The specificity studies indicated that the specific 387 bp P C R p r o d u c t was not amplified from a relatively high concentration o f 1.0 ng o f R N A from BTV serotypes 2, 10, l 1, 13 and 17 or total nucleic acid extracts from BHK-21 cell controls under the same stringency conditions described in this study. Temperature and time for denaturation, primer annealing and extension, enzyme and MgC12 concentration, and n u m b e r o f cycles o f the three temperature per time segments were very important for maintaining sensitivity and specificity o f the P C R reaction. Chemiluminescence is necessary to confirm the identity o f the amplified p r o d u c t and to increase the sensitivity o f the assay [21, 24, 25].
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T h e s a m p l e size used in this s t u d y represents all E H D V strains, so far, i s o l a t e d in the S u d a n . T h e s a m p l e s were e n o u g h to v a l i d a t e o u r p r e v i o u s l y d e s c r i b e d E H D V - P C R assay for d e t e c t i o n o f two m o r e s e r o t y p e s o f E H D V s e r o g r o u p ( E H D V - 5 a n d E H D V - 3 1 8 ) f r o m different g e o g r a p h i c a l origin in C e n t r a l Africa. T h e E H D V - P C R assays p r o v i d e s u p p o r t i v e d i a g n o s t i c techniques to the lengthy c u m b e r s o m e c o n v e n t i o n a l virus i s o l a t i o n p r o c e d u r e s . T h e time r e q u i r e d f r o m s a m p l e s u b m i s s i o n to i n t e r p r e t a t i o n o f the final results was consistently 50 consecutive hours, which is less t h a n three w o r k i n g d a y s [21]. T h e r a p i d i t y , sensitivity a n d specificity o f the P C R assay w o u l d greatly facilitate d e t e c t i o n o f E H D V infection in an o u t b r e a k a m o n g susceptible r u m i n a n t s . P r e v i o u s studies s h o w e d t h a t several o t h e r sets o f serogroup-specific o l i g o n u c l e o t i d e p r i m e r s were e x a m i n e d a n d were f o u n d to be specific only for a p a r t i c u l a r BTV o r E H D V t o p o t y p e a n d thus were o n l y useful for s e r o g r o u p i n g viruses f r o m a p a r t i c u l a r g e o g r a p h i c a l region [31]. In the p r e s e n t study, o f the 10 serotypes o f E H D V , we v a l i d a t e d the d e t e c t i o n o f E H D V serotypes 1, 2, 5, a n d E H D V , isolate 318 ( u n t y p e d ) b y the d e s c r i b e d E H D V - P C R b a s e d d e t e c t i o n assay. All E H D V strains used in this s t u d y were positive to the P C R assay using the same o l i g o n u c l e o t i d e primers. T h e d e s c r i b e d P C R assay will p r o b a b l y detect the r e m a i n i n g serotypes a n d t o p o t y p e s o f E H D V s e r o g r o u p . H o w e v e r , a d d i t i o n a l research is necessary to c o n f i r m t h a t a s s u m p t i o n . T h e d e v e l o p m e n t o f a P C R b a s e d assay for d e t e c t i o n o f C e n t r a l A f r i c a n a n d N o r t h A m e r i c a n strains o f E H D V , d e s c r i b e d in this study, p r o v i d e s the basis for future s e r o g r o u p i n g a n d t o p o t y p i n g o f E H D V . F u r t h e r studies are in p r o g r e s s to d e t e r m i n e the c a p a b i l i t y o f the d e s c r i b e d E H D V - P C R assay to detect t o p o t y p e viruses a n d a d d i t i o n a l s e r o t y p e s o f E H D V s e r o g r o u p , a n d to e v a l u a t e its p o t e n t i a l as a sensitive a n d specific d i a g n o s t i c assay t h r o u g h c o m p a r i s o n with c u r r e n t d i a g n o s t i c techniques used for d e t e c t i o n o f E H D V infection. In conclusion, the recently d e s c r i b e d serogroup-specific E H D V - P C R assay using well c h a r a c t e r i z e d s e r o g r o u p i n g p r i m e r s d e r i v e d f r o m g e n o m e segment 6 o f E H D V - 2 p r o v i d e s r a p i d c h a r a c t e r i s a t i o n o f a n u n k n o w n strain o f E H D V f r o m S u d a n or U n i t e d states. Acknowledgements--We are very grateful to Dr Hans Riemann for critical review of the draft manuscript. This work was supported by funds from the University of Khartoum, Sudan; and Livestock Disease Research Laboratory (LDRL), School of Veterinary Medicine, University of California, Davis; USDA/APHIS Cooperative Agreement USDA 12-34-93-0234-CA and the USDA/CSRS Animal Health and Disease 1433 Program.
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(1988) Arbovirus recovered from sentinel cattle using several virus isolation methods. Vet. Mierobiol. 18, 119. 18. Gibbs E. P. and Lawman M. J. P. (1977) Infection of British deer and farm animals with epizootic hemorrhagic disease of deer virus. J. Comp. Pathol. 87, 335 343. 19. Pearson, J. E., Gustafson, G. A.. Sharer, A. L. and Alstad, A. D. (1992) Diagnosis of bluetongue virus and epizootic hemorrhagic disease. In: Proceedings oil the 2nd International Symposium on Bluetongue, A/?ican Horse Siekness and Related Orbiviruses (Edited by T. E. Walton and B. I. Osburn), pp. 533 546. C R C Press, Boca Raton, FL. 20. Work T. M., Jessup D. A. and Sawyer M. M. (1992) Experimental bluetongue and epizootic hemorrhagic disease virus infection in California black-tailed deer. J. Wild/. Dis. 4, 623-628. 21. Aradaib 1. E., Akita G. Y. and Osburn B. I. (1994) Detection of epizootic hemorrhagic disease virus serotypes 1 and 2 in cell culture and clinical samples using polymerase chain reaction. ,I. Vel. Diag. Invest. 6, 143 147. 22. Aradaib I. E., Sawyer M. M. and Osburn B. 1. (1994) Experimental epizootic hemorrhagic disease virus infection in calves: virologic and serologic studies. J. Vet. Diag. h,~est. 6, 489 492. 23. Aradaib 1. E., Akita G. Y., Pearson J. E. and Osburn B. I. (1995) Comparison of polymerase chain reaction and virus isolation for detection of epizootic hemorrhagic disease virus in clinical samples from clinically infected deer. J. Vet Diag. hTvest. 7, 196 200. 24. Aradaib I. E., Wilson W. C., Cheney W., Pearson J. E. and Osburn B. 1. (1995) Application of the polymerase chain reaction for specific identification of epizootic hemorrhagic disease virus serotype 2. J. Vet Diagn. Incest. 7, 388 392. 25. Aradaib I. E. and Osburn B. 1. (1995) Detection of epizootic hemorrhagic disease virus in blood samples by PCR. Sudan J. Vet. Sei. Anita. Hush. (in press). 26. H a m m a m i S. and Osburn B. I. (1992) Analysis of genetic variation of epizootic hemorrhagic disease virus and bluetongue virus field isolates by co-electrophoresis of their double stranded RNA. Am. J. Vel. Res. 53, 636 642. 27. Stott J. L., Else K. C., M c G w a n B., Wilson L. K. and Osburn B. 1. (1981 ) Epizootiology of bluetongue Virus in western United States. In: Proceedings (?[' the 85th annual oleel#lg Of the United Stases Animal Health Association, pp. 170 180. 28. Nel L. H., Picard L. A. and Huismans H. (1990) A characterization of the non structural protein from which the virus-specified tubules in epizootic hemorrhagic disease virus infected cells are composed. Virus Res. 18, 219 230. 29. Huismans H., Bremer C, W. and Barber L. T. (1979) The nucleic acid and proteins ofepizootic hemorrhagic disease virus. Onderstepoort J. l@t. Res. 46, 95 104. 30. Venter E. H., Viljoen G. J., Nel L. H., Huismans H. and Van Dijk A. A. (1991) A comparison of different genomic probe in the detection of virus-specified R N A in orbivirus-infected cell. J. 1tirol. Method~ 32, 171 180. 31. Gould, A. R., McColl, K. A. and Prichchord, L. 1. (1992) Phylogenetic relationship between bluetongue virus and other orbiviruses, In: Bluetongue A/?ican Horse Sickness and Related Orhiviruses (Edited by Walton T. E. and Osburn B. l.), pp. 452 460. C R C Press, Boca Raton, FL.
Pergamon
Comp. lmmun. Microbiol. in/ect. Dis. Vol. 20, No. 3, pp. 211 218, 1997 ~) 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain S0147-9571(97)00002-7 0147-9571/97 $17.00 + 0.00
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S E R O G R O U P I N G A N D T O P O T Y P I N G OF S U D A N E S E A N D U N I T E D STATES S T R A I N S OF EPIZOOTIC HEMORRHAGIC DISEASE VIRUS USING PCR IMADELDIN E. A R A D A I B , I M O H A M M E D E. H. M O H A M M E D , 2 M U A W I A M . M U K H T A R , 2 H A S H I M W . G H A L I B 3 a n d B E N N I E I. O S B U R N ~ ~Department of Veterinary Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616, U.S.A.; 2Department of Preventive Medicine and Veterinary Public Health, Faculty of Veterinary Science, University of K h a r t o u m , K h a r t o u m , Sudan; 3Department of Microbiology, Faculty of Medicine, University of K h a r t o u m , K h a r t o u m , Sudan
(Received for publication 26 January 1997)
Abstract -The potential use of the recently reported polymerase chain reaction (PCR) protocol for detection of United States epizootic hemorrhagic disease virus (EHDV) serotype 1 (EHDV-I) and serotype 2 (EHDV-2) ribonucleic acid in cell culture and clinical specimens was evaluated for detection of Sudanese E H D V strains. E H D V serotype 5 (EHDV-5) and EHDV, isolate 318 (untyped) designated (EHDV-318), recovered from sentinel calves at the K h a r t o u m University farm (Sudan) were studied. R N A from EHDV-5 and EHDV-318 and a n u m b e r of E H D V field isolates, propagated in cell cultures, were detected by the described PCR-based assay. The specific 387 bp P C R products were visualized on ethidium bromide stained agarose gel. Specificity of the P C R products was confirmed by chemiluminescent hybridization with non-radiolabeled internal probe. Amplification product was not detected when the PCR-based assay was applied to R N A from bluetongue virus (BTV) prototypes serotypes 2, 10, 11, 13, 16 and 17; total nucleic acid extracts from uninfected BHK-21 cells. The results of this study indicated that the previously described E H D V P C R assay could be applied for detection of Sudanese as well as United States strains of E H D V serogroup. In addition, the described E H D V - P C R assay could be used as a supportive diagnostic assay to the current conventional virus isolation procedures used for detection of E H D V infection in susceptible ruminants. ((_2 1997 Elsevier Science Ltd
Key words: EHDV, PCR, serogroup, topotype, Sudanese.
R~sum~5~L'utilit6 d ' u n protocole bas6 sur la r6action en cha~ne de la polym6rase (RCP), mis au point pour la d6tection de I ' A R N de souches am~ricaines (s6rotypes 1 et 2) du virus de la maladie h6morrhagique ~pizootique (VMHE), fut 6valu6e pour la d6tection de souches soudanaises du V M H E . L'&ude porta sur le V M H E s~rotype 5 (VMHE-5) et le V M H E isolat 318 (non s6rotyp6), d6sign6 VMHE-318, qui furent isol6s de veaux sentinelles de la ferme de l'Universit~ de K h a r t o u m (Soudan). Le protocole permit la d&ection de I'ARN du VMHE-5, du VMHE-318 et d'autres isolats du V M H E , propag~s en culture cellulaire. Les produits d'acides nucleiques obtenus par RCP, de 387 bp, furent mis en 6vidence sur gel agar, color~ au bromure d'&hidium. La sp6cificit6 de ces produits rut confirm~e par hybridation chimioluminescente, utilisant une sonde non radio-marqu&. Ces produits ne furent pas d&ect& lorsque la technique RCP fur appliqu& 5. I'ARN des s6rotypes 2, 10, 11, 13, 16 et 17 du virus du Blue Tongue, ou 5_des extraits d'acides nucl6iques de cellules B H K non-infect6es. Ces r&ultats indiquent que la technique R C P peut d&ecter les souches soudanaises du V M H E , en plus des souches am&icaines. De plus, cette technique pourrait avoir une utilit6 diagnostique, compl6mentant les m6thodes conventionnelles d'isolation virale du V M H E chez les ruminants. (c) 1997 Elsevier Science Ltd
Mots c&[~: V M H E , RCP, s6rotype, topotype, soudan. 211
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1. E. Aradaib et al. INTRODUCTION
Epizootic hemorrhagic disease virus (EHDV) is a double stranded (ds) RNA orbivirus of the family Reoviridae and is related to bluetongue virus (BTV)[1-3]. The virus has a worldwide distribution and exists in at least 10 distinct serotypes [4]. In central Sudan, previous studies have shown that the seasonal incidence of E H D V is a predictable event related to the rainy season [5, 6]. E H D V serotype 5 (EHDV-5) and an untyped E H D V isolate designated EHDV-318 were recovered from a sentinel calf herd at the Khartoum University farm, Shambat[5]. EHDV-318 was also recovered from sentinel calves in Sultanate of Oman [7]. Recently, EHDV-318 was reported to be a contaminant of genetically engineered Chinese hamster ovary (CHO) cells in Bahrain [8]. EHDV serotype 1 (EHDV-1) and serotype 2 (EHDV-2), which are known to be enzootic in the United States, cause an often fatal hemorrhagic disease in North American white-tailed deer (Odocoileus virginianus) [9 15]. There is no information available on the impact of E H D V on the wild ruminant species in the Sudan including the deer population. Infections caused by EHDV in local Sudanese breeds of sheep, goats and cattle is usually unapparent and no evidence of clinical hemorrhagic disease has been reported. However, indirect losses associated with loss of body weight and condition, drop in milk production and poor subsequent reproductive performance were thought to have greater economic effect than occasional overt disease [5]. In addition, because of unfamiliarity with the biology and ecology of the virus, there is the potential of restriction on the international movement of livestock and associated germplasm from countries suspected to harbor the disease to E H D V free countries unless the animals are certified free of infection by conventional virus isolation or serology [16]. Such a restriction could lead to economic losses for EHDV-endemic countries, like Sudan, which rely on the sale of livestock for foreign exchange. Current diagnosis of E H D V infection by conventional virus isolation and serology is time-consuming, labor intensive and expensive [17 20]. The development of a rapid, sensitive, specific and inexpensive method for serogrouping and topotyping of EHDV from different geographical locations would be advantageous for a variety of circumstances including clinical disease investigations, molecular epidemiological studies for understanding the biology of the virus, and enhancement of international trade of livestock and associated germplasm [21, 6]. Recently, we have described a simple, rapid, sensitive and specific E H D V PCR assays for detection of United States serogroup-specific detection and serotype specific identification of EHDV-1 and EHDV~2 in cell culture and a variety of clinical samples from naturally and experimentally infected animals [21-25]. In the present study, the potential of the recently described E H D V - P C R assay [21] was evaluated for serogrouping and topotyping of United States and Sudanese strains of E H D V using well-characterized primers derived from segment 6 of EHDV-2 (Alberta strain). MATERIALS AND METHODS Cell culture and virus propagation
The 2 prototype serotypes of E H D V present in the Sudan (EHDV-5 and EHDV-318), 5 E H D V field isolates, BTV serotype 16 (School of Veterinary Medicine, University of Khartoum, Sudan), E H D V serotype 1 (EHDV-1) and 2 (EHDV-2) and the BTV prototypes serotypes 2, 10, 11, 13, and 17 (Arthropod-Borne Animal Disease Research
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Laboratory, Laramie, WY, U.S.A.) were used in this study. The viruses were isolated and processed as described previously [2 l, 22, 26, 27]. All viruses were propagated on confluent monolayers of BHK-21 cells. The infectious material was harvested and centrifuged at 1500 g for 30 min and the cell pellet was used for the dsRNA extraction. Extraction of t,iral nucleic acid frorn infected cell culture The E H D V and the BTV dsRNA were extracted from the infected cells as previously described [21]. Total nucleic acid was ethanol-precipitated. Viral dsRNA was purified by differential lithium chloride precipitation, and resuspended in 100/d double distilled water, and quantified using a spectrophotometer at 260 nm wavelength. Primer selection and synthesis of the probe Primer selection and synthesis of the probe were described in details in a previous report [21]. Briefly, primers (20 mer each) were selected from the published sequence of genome segment 6 of EHDV-2 (Alberta strain)[28] and used in these PCR assays. E H D V PCR using primer P1 and P2 would result in a 387 bp product. For synthesis of a probe complementary to the predicted amplified viral sequences generated by P1 and P2, oligonucleotide primers (P3 and P4) were selected from the same published sequence cited above. PCR amplification using P3 and P4 would result in 224 bp PCR product internal to the annealing sites of PI and P2. All primers were synthesized on a DNA synthesizer (Milligen/Biosearch, A division of Millipore, Burlington, MA, U.S.A.) and purified using oligo-pak oligonucleotide purification columns (Glen Research Corporation, Sterling, VA, U.S.A.) as per the manufacturer's instructions. The probe was purified using D N A binding beads (Mermaid Kit, Bio 101, La Jolla, CA, U.S.A.) according to the manufacturer's instructions and used for chemiluminescent hybridization. Polymerase chain reaction The PCR protocol used in this study was basically as previously described [21, 22]. Taq DNA polymerase 5 units//d (Perkin-Elmer Cetus, The Perkin Elmer Corporation, Norwalk, CT, U.S.A.) were used in each reaction. The thermal cycling profiles were as follows: a 2 min incubation at 95~C, followed by 40 cycles of 9 5 C for 1 min, 5 5 C for 30 s and 72-C for 45 s and a final incubation at 62~C for 10 min. Thermal profiles were performed on a Techne PHC-2 thermal cycler (Techne, Princeton, N J, U.S.A.). From each PCR reaction containing amplified product 20/tl were loaded onto 2% SeaKem agarose (FMC Bioproducts, Rockland, ME, U.S.A.) and electrophoresed. The gels were stained with ethidium-bromide, and the specific 387 bp PCR products were visualized under UV light. Southern blot hybridization Southern blot hybridization was performed basically as previously described [21]. Briefly, agarose gels from PCR reaction were denatured, neutralised and transferred to nylon membranes (Schleicher and Schvell, Inc., Woburn, MA, U.S.A.) in vacuo at 50 mm Hg (Vacugene XL Vacuum Blotting System, Pharmacia LKB Biotechnology, Piscataway, N J, U.S.A.). Southern blotted nucleic acid was UV cross linked to the nylon membrane. Chemiluminescent hybridization was performed using a kit (ECL direct nucleic acid labeling and detection system, Amersham Corporation, Arlington Heights, IL, U.S.A.) according to the manufacturers instructions. The probe prepared by PCR was labeled with
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p e r o x i d a s e in the presence o f g l u t a r a l d e h y d e . S o u t h e r n blots were p r e h y b r i d i z e d with h y b r i d i z a t i o n buffer c o n t a i n i n g 5% b l o c k i n g agent a n d 0.85 M NaC1 at 4 2 C for 1 h. The labeled p r o b e was a d d e d to the h y b r i d i z a t i o n buffer a n d the m e m b r a n e s were h y b r i d i z e d overnight. A f t e r p o s t h y b r i d i z a t i o n w a s h i n g d e t e c t i o n reagents were a p p l i e d to the m e m b r a n e s for 1 rain. The m e m b r a n e s were then sealed in plastic w r a p a n d exposed to X - r a y film for 1 30 min with an intensifying screen. RESULTS All E H D V isolates t h a t were E H D V - p o s i t i v e by c o n v e n t i o n a l virus i s o l a t i o n were also positive to E H D V P e R assay. The described E H D V - P C R b a s e d assay afforded sensitive a n d specific detection o f E H D V serotypes a n d field isolates. T h e specific 387 bp P C R p r o d u c t was visualized on e t h i d i u m - b r o m i d e stained gel from 1.0 pg R N A o f the U n i t e d States E H D V serotypes 1 ( E H D V - 1 ) a n d 2 ( E H D V - 2 ) ; the S u d a n e s e E H D V s e r o t y p e 5 ( E H D V - 5 ) , the u n t y p e d isolate o f E H D V - 3 1 8 used in this study [Fig. (1A)]. The identity o f the amplified p r o d u c t s was c o n f i r m e d by s o u t h e r n blot with c h e m i l u m i n e s c e n t h y b r i d i z a t i o n [Fig. (1B)]. U s i n g 1.0 pg E H D V R N A , the 387 bp specific P C R p r o d u c t was detected in the five
MW 1
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1,636-
1,018506-
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Fig. 1. Detection of the United states and the Sudanese epizootic hemorrhagic disease virus serotypes by the polymerase chain reaction (PCR) based assay. (A) Visualization of the 387 bp specific EHDV PCR product on ethidium bromide stained agarose gel from 1.0 pg of RNA of the United states prototypes serotypes 1 and 2; and the Sudanese EHDV serotype 5 (EHDV-5) and EHDV, isolate 318 (untyped). Lane MW: molecular weight marker: Lane 1: EHDV-I; Lane 2: EHDV-2; Lane 3: EHDV-5; Lane 4: EHDV-318; Lane 5: BHK cell control. (B) Southern blot with chemiluminescent hybridization of the above gel.
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Serogrouping and topotying of EHDV
MW123456
1,018506-
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1
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Fig. 2. (A) Visualization of the 387 bp specific EHDV PCR product on ethidium-bromide stained agarose gel from 1.0 pg of RNA of 5 different EHDV field isolates. Lane MW: molecular weight marker; Lane 1:EHDV-5 field isolate; Lanes 2-5:EHDV-318 isolated from sentinel calves; Lane 6:BHK-21 cell total nucleic acid extract. (B) Southern blot with chemiluminescent hybridization of the above gel.
Sudanese E H D V field isolates with both ethidium-bromide stained agarose gel [Fig. (2A)] and chemiluminescent hybridization [Fig. (2B)]. The amount of 1.0 ng RNA from BTV serotypes 2, 10, 11, 13, 16 and 17; and total nucleic acid extracts from uninfected BHK-21 cells failed to demonstrate PCR products (Fig. 3). DISCUSSION Sporadic outbreaks of clinical hemorrhagic disease among susceptible deer were observed in different regions of the Sudan. These oubreaks were first attributed to EHDV infection without confirmatory data. Subsequently, the virus was first isolated from sentinel calves in a herd in Khartoum [6]. At least 2 serotypes of E H D V designated EHDV-5 and EHDV-318 are known to be enzootic in the Sudan[5]. Very little information is available about orbiviruses originally isolated in Africa. Further studies on these orbiviruses is necessary to determine their biology, ecology and molecular epidemiology. The E H D V field isolates used in this study represented a range of topotype viruses, isolated from a diverse geographic locations in North America and Central Africa, recovered from calves and deer. The described E H D V - P C R assay using well-characterized serogrouping primers derived from segment 6 of EHDV, which codes for NS1 [12, 28, 29], reproducibly and specifically detected E H D V RNA in infected cell cultures. Selection of the
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MW
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et al.
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Fig. 3. Specificity of the polymerase chain reaction for EHDV RNA. Amplification product was not detected from a high concentration of 1.0 ng RNA different BTV serototypes or total nucleic acid extracts from BHK-21 cells Lane MW: molecular weight marker: Lane l: 1 pg EHDV-2 isolated from a deer (positive control); Lane: 2 7 BLU virus prototypes serotypes 2, 10, 11, 13, 16 and 17, respectively; Lane 8:BHK-21 total nucleic acid extract. primers was based on the observation that the NS1 genome specific probe is the best o f all E H D V g e n o m e segments to produce positive hybridization signal c o m p a r e d to other nucleic acid probes [30]. The specific 387 bp P C R products, visualized on ethidium bromide stained agarose gel or detected with chemilumiescent hybridization, were obtained from all E H D V R N A samples tested. The E H D V - P C R assay was a simple procedure that efficiently detected all E H D V strains under the stringency condition used in this study. In the present study, the use of non-radioactive chemiluminescent hybridization removes the hazardous and c u m b e r s o m e radioactive l a b o r a t o r y procedures o f working with 32p or 33p. In a previous report, the sensitivity studies o f the described E H D V P C R protocol indicated that the P C R assay was capable of detecting the a m o u n t o f 0.1 fg o f total E H D V - 1 or E H D V - 2 genomic d s R N A . The total molecular weight of the E H D V genome is 11.44 x 106 Da, and 0.1 fg o f E H D V R N A corresponds to 6 viral particles [21]. The EHDV-PCR amplification technology with chemiluminiscent hybridization was approximately a million times more sensitive than the d o t - b l o t hybridization using E H D V c D N A probe, where at least a few n a n o g r a m s o f the d s R N A are required to produce a positive hybridization signal [30]. The specificity studies indicated that the specific 387 bp P C R p r o d u c t was not amplified from a relatively high concentration o f 1.0 ng o f R N A from BTV serotypes 2, 10, l 1, 13 and 17 or total nucleic acid extracts from BHK-21 cell controls under the same stringency conditions described in this study. Temperature and time for denaturation, primer annealing and extension, enzyme and MgC12 concentration, and n u m b e r o f cycles o f the three temperature per time segments were very important for maintaining sensitivity and specificity o f the P C R reaction. Chemiluminescence is necessary to confirm the identity o f the amplified p r o d u c t and to increase the sensitivity o f the assay [21, 24, 25].
Serogrouping and topotying of EHDV
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T h e s a m p l e size used in this s t u d y represents all E H D V strains, so far, i s o l a t e d in the S u d a n . T h e s a m p l e s were e n o u g h to v a l i d a t e o u r p r e v i o u s l y d e s c r i b e d E H D V - P C R assay for d e t e c t i o n o f two m o r e s e r o t y p e s o f E H D V s e r o g r o u p ( E H D V - 5 a n d E H D V - 3 1 8 ) f r o m different g e o g r a p h i c a l origin in C e n t r a l Africa. T h e E H D V - P C R assays p r o v i d e s u p p o r t i v e d i a g n o s t i c techniques to the lengthy c u m b e r s o m e c o n v e n t i o n a l virus i s o l a t i o n p r o c e d u r e s . T h e time r e q u i r e d f r o m s a m p l e s u b m i s s i o n to i n t e r p r e t a t i o n o f the final results was consistently 50 consecutive hours, which is less t h a n three w o r k i n g d a y s [21]. T h e r a p i d i t y , sensitivity a n d specificity o f the P C R assay w o u l d greatly facilitate d e t e c t i o n o f E H D V infection in an o u t b r e a k a m o n g susceptible r u m i n a n t s . P r e v i o u s studies s h o w e d t h a t several o t h e r sets o f serogroup-specific o l i g o n u c l e o t i d e p r i m e r s were e x a m i n e d a n d were f o u n d to be specific only for a p a r t i c u l a r BTV o r E H D V t o p o t y p e a n d thus were o n l y useful for s e r o g r o u p i n g viruses f r o m a p a r t i c u l a r g e o g r a p h i c a l region [31]. In the p r e s e n t study, o f the 10 serotypes o f E H D V , we v a l i d a t e d the d e t e c t i o n o f E H D V serotypes 1, 2, 5, a n d E H D V , isolate 318 ( u n t y p e d ) b y the d e s c r i b e d E H D V - P C R b a s e d d e t e c t i o n assay. All E H D V strains used in this s t u d y were positive to the P C R assay using the same o l i g o n u c l e o t i d e primers. T h e d e s c r i b e d P C R assay will p r o b a b l y detect the r e m a i n i n g serotypes a n d t o p o t y p e s o f E H D V s e r o g r o u p . H o w e v e r , a d d i t i o n a l research is necessary to c o n f i r m t h a t a s s u m p t i o n . T h e d e v e l o p m e n t o f a P C R b a s e d assay for d e t e c t i o n o f C e n t r a l A f r i c a n a n d N o r t h A m e r i c a n strains o f E H D V , d e s c r i b e d in this study, p r o v i d e s the basis for future s e r o g r o u p i n g a n d t o p o t y p i n g o f E H D V . F u r t h e r studies are in p r o g r e s s to d e t e r m i n e the c a p a b i l i t y o f the d e s c r i b e d E H D V - P C R assay to detect t o p o t y p e viruses a n d a d d i t i o n a l s e r o t y p e s o f E H D V s e r o g r o u p , a n d to e v a l u a t e its p o t e n t i a l as a sensitive a n d specific d i a g n o s t i c assay t h r o u g h c o m p a r i s o n with c u r r e n t d i a g n o s t i c techniques used for d e t e c t i o n o f E H D V infection. In conclusion, the recently d e s c r i b e d serogroup-specific E H D V - P C R assay using well c h a r a c t e r i z e d s e r o g r o u p i n g p r i m e r s d e r i v e d f r o m g e n o m e segment 6 o f E H D V - 2 p r o v i d e s r a p i d c h a r a c t e r i s a t i o n o f a n u n k n o w n strain o f E H D V f r o m S u d a n or U n i t e d states. Acknowledgements--We are very grateful to Dr Hans Riemann for critical review of the draft manuscript. This work was supported by funds from the University of Khartoum, Sudan; and Livestock Disease Research Laboratory (LDRL), School of Veterinary Medicine, University of California, Davis; USDA/APHIS Cooperative Agreement USDA 12-34-93-0234-CA and the USDA/CSRS Animal Health and Disease 1433 Program.
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