isolation and molecular characterisation of a ...

Acta Veterinaria Hungarica 61 (2), pp. 270–280 (2013) DOI: 10.1556/AVet.2013.007 First published online 6 February 2013

ISOLATION AND MOLECULAR CHARACTERISATION OF A PESTIVIRUS FROM GOATS IN EGYPT Ahmed O. ABDEL-LATIF1,2, Sagar M. GOYAL1*, Yogesh CHANDER1, Ahmed S. ABDEL-MONEIM2,3, Sabry M. TAMAM2 and Hanafy M. MADBOULY2 1

Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, Saint Paul, Minnesota, 55108, USA; 2Department of Virology, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef, Egypt; 3 Division of Virology, Department of Microbiology, College of Medicine, Taif University, Al-Taif, Saudi Arabia (Received 21 December 2011; accepted 4 June 2012) Nine fetuses and neonates from sheep and goats in Egypt were screened for pestiviruses using immunohistochemistry (IHC), virus isolation, and reverse transcription-polymerase chain reaction (RT-PCR). Two goat kids with typical border disease (BD) were positive for pestivirus infection by immunohistochemistry (IHC) using polyclonal anti-BDV serum but not when four different monoclonal antibodies (MAbs) were used. On inoculation in MDBK cells, a cytopathic bovine viral diarrhoea virus (BVDV) was isolated from one of the two kids. PCR amplification followed by sequencing of the 5′-UTR region confirmed it as BVDV subtype 1b. Although the circulating virus in Egypt is considered to be BVDV 1a, this report confirms the existence of BVDV 1b in addition to BVDV 1a. To our knowledge, this is the first report of isolation of a pestivirus from goats in Egypt and is probably the second report worldwide of a goat kid showing central nervous signs associated with border disease. Key words: Bovine viral diarrhoea virus, goats, border disease

Pestiviruses are important animal pathogens and cause significant economic losses to the livestock industry worldwide (Paton et al., 1995; Sullivan et al., 1997). The genus Pestivirus in the family Flaviviridae currently contains four established species: bovine viral diarrhoea virus type 1 (BVDV-1), bovine viral diarrhoea virus type 2 (BVDV-2), border disease virus (BDV), and classical swine fever virus (CSFV) (Thiel et al., 2005). Historically, pestiviruses were named according to the species from which they were isolated: CSFV in pig, BVDV in cattle, and BDV in sheep. However, it is now known that sheep and goats can be infected not only with BDV but also with BVDV-1 and BVDV-2 (Marco et al., 2008; Mishra et al., 2008). In fact, transmission of BVDV and *

Corresponding author; E-mail: [email protected]; Phone: 001 (612) 625-2714; Fax: 001 (612) 624-8707 0236-6290/$ 20.00 © 2013 Akadémiai Kiadó, Budapest

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BDV between cattle and small ruminants has been demonstrated (Carlsson, 1991; Loken et al., 1991). Border disease (BD) is a congenital disease of sheep and goats. Clinical manifestations in sheep include barren ewes, abortions, stillbirths, and birth of small and weak lambs and persistent infection (PI) of the offspring. Affected lambs show tremors, abnormal body conformation, poor growth rate and hairy fleece (Osburn et al., 1972). Infection in goats is characterised by reproductive failure and weak-born kids (Loken, 2000). Signs of central nervous system illness in a newborn goat kid have been reported in one study (Loken et al., 1982). Goat kids are considered generally resistant to the establishment of persistent infection but the existence of persistently infected goats has recently been reported (Krametter-Froetscher et al., 2008). Serological evidence of pestivirus infection in goats has been reported from many countries (Krametter-Froetscher et al., 2006; Zaghawa, 1998), but reports on clinical disease in goats are scarce (Krametter-Froetscher et al., 2006; Mishra et al., 2007). This paper describes the occurrence of pestivirus-induced central nervous system illness in a goat kid in Upper Egypt. Materials and methods Source of animals. A total of three ovine fetuses, two ovine neonates and four caprine neonates were examined from individual rearing systems in different regions of Beni-Suef and El-Minia Governorates in Upper Egypt. The three ovine fetuses aborted at about 110 days of pregnancy. The two lambs showed mild shaking, were unable to stand or suckle, and died 3–5 days after birth. One of the four goat kids was very weak, showed mild tremors, and died on day 5 after birth. Another kid was weak but did not show tremors or shaking. However, its dam died after giving birth and the kid died one week later. The remaining two goat kids were from a single flock and showed BD-like signs: congenital anomalies in the form of aggressive tremors all over the body, convulsions, inability to stand or to suckle, corneal opacity, and conjunctivitis. These goat kids were humanely killed at one and two weeks of age. Sampling. Brain, spleen, liver, kidney, lung, mesenteric lymph node and spinal cord specimens were collected under sterile conditions from all nine fetuses and neonates. Each organ was divided into two pieces; one piece was fixed in 10% buffered formalin and then embedded in paraffin, and the other piece was stored at –20 °C until processed for virus isolation. For virus isolation, brain specimens were processed separately while the remaining organs were pooled and then processed. The tissues were minced in a pestle and mortar along with sterile sea sand and enough phosphate buffered saline (pH 7.4) to produce 20% homogenates. After centrifugation at 780 × g for 15 min, the supernatants were collected and passed through 0.45-µm filters followed by storage at –80 °C. Acta Veterinaria Hungarica 61, 2013

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Immunohistochemistry. The method described by Mahlum et al. (2002) was used. An HRP kit, LSAB2-HRP system (DAKO A/S, Glostrup, Denmark), was used with a polyclonal antibody (rabbit anti-BDV-1 ‘Moredun’ serum) or with four different MAbs; D89, BA-2, 348 (all from VMRD, WA, USA) which target the E2 glycoprotein, and 15C5 (kindly provided by Edward Dubovi, Cornell University, Ithaca, NY) which targets the Erns glycoprotein. Detection of immune reaction was done by covering the tissue sections with diaminobenzidine (DAB) (Sigma, St. Louis, MO) for 10 min followed by a 5-min rinse in water. Following a 2- to 5-min counterstaining with Mayer’s hematoxylin (Sigma, St. Louis, MO), the slides were rinsed in running tap water. Positive and negative controls were included in the test. Pestivirus antigen was visualised by dark brown staining under a light microscope. Virus isolation and detection. Filtrates of brain and pooled tissue homogenates were inoculated into duplicate wells of a 48-well cell culture plate containing monolayers of Madin-Darby bovine kidney cells (MDBK). The cells were maintained in Eagle’s Minimum Essential Medium (EMEM) (Invitrogen, Carlsbad, CA) containing 4% fetal calf serum (FCS). The cells, the media and the FCS were found to be free from adventitious pestivirus contamination when tested by RT-PCR. After 5 days of incubation at 37 °C, the cells were frozen and thawed thrice and the clarified supernatant was passaged once more on confluent monolayers of MDBK cells. After incubation for 5 days, the cells were examined for the presence of pestivirus by DFA (Goyal, 2005) using fluorescein isothiocyanate (FITC)-conjugated anti-BVDV (VMRD, WA) antibody. Samples found positive were inoculated in confluent monolayers of MDBK seeded in 25-cm2 tissue culture flasks. Blind passages were made at 5-day intervals until consistent cytopathic effects were seen after the 5th passage. Reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from 250 µl of 5th passage cell culture fluid and original homogenates of brain and pooled tissues using TRIzol reagent (Invitrogen, Carlsbad, CA). Total RNA was also extracted from known isolates of BVDV-1 and BVDV-2 as positive controls. The RNA was recovered in 30 µl of RNase-free water and kept at –20 °C until used. The OneStep RT-PCR kit (Qiagen, Valencia, CA) was used for the amplification of two genomic regions. A 288-bp fragment of the genomic region encoding the highly conserved 5′-UTR of the pestivirus genome was amplified using panpestivirus primers 324 and 326 (Vilcek et al., 1994). The sense primer 390F and antisense primer 1400R, amplifying a 1080-bp fragment from the open reading frame (ORF) covering the entire Npro, C, and a part of Erns were used (Mishra et al., 2008). For amplification of the E2 gene, primers targeting a 1160-bp region between 2274 and 3434 nucleotides were used (Nagai et al., 2004). Amplified products were detected by electrophoresis on ethidium bromide-stained 1.2% agarose gel in Tris-acetate-ethylenediamine tetraacetic acid (TAE) buffer and visualised on a UV transilluminator. Acta Veterinaria Hungarica 61, 2013


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Nucleotide sequencing and phylogenetic analysis. PCR products from positive samples were sequenced to identify the pestivirus genotype. For this purpose, PCR products were purified using QIAquick PCR purification kit (Qiagen, Valencia, CA) and then sequenced at the BioMedical Genomics Center, University of Minnesota. Sequencing was performed in both directions using the corresponding primers. Using Sequencher software (www.msi.umn.edu), the forward and reverse nucleotide sequences were edited, curated, and aligned to make one sequence. The GenBank database (www.ncbi.nlm.nih.gov) was searched for related sequences and sequences representative of every known member of the genus Pestivirus. Multisequence alignment was performed for 5′-UTR (244 bp) and part of the Npro gene (470 bp) and partially for the E2 gene (1160 bp) with other published sequences using Clustal W Multiple Sequence Alignment Program, version 1.83 (GenomeNet: http://www.genome.jp). Phylogenetic trees were generated by the neighbour-joining method using MEGA 4.1 software (Tamura et al., 2007). Nucleotide sequence accession numbers. The nucleotide sequence for the goat strain described in this work has been deposited in the GenBank with the following accession numbers: fragment of 5′-UTR (GU085506), the entire Npro, C, a part from the Erns coding regions (GU991550) and a partial E2 region (JF968611). Results Of the nine animals, only two goat kids (with neurological signs and from the same flock) showed positive staining for pestivirus in IHC using polyclonal anti-BDV serum but none was found positive with any of the four MAbs used. Both animals showed positive staining in the brain tissue (Fig. 1). Positive staining was also seen in the spinal cord of one goat kid; spinal cord from the other kid was not available for examination. No other organ showed positive staining. On virus isolation, the brain homogenate of only one of the positive kids showed positive staining with DFA after the 2nd passage. After the 5th passage, this isolate showed consistent cytopathic effects on MDBK. RT-PCR was successful only from one goat kid but negative for all other animals. Amplification products of the expected sizes, 288 bp (5′-UTR), 1080 bp (Npro, C and a part of Erns) and 1160 bp (E2), were detected only in the original brain homogenate and the clarified tissue culture fluid after the 5th passage. The 5′-UTR sequences obtained from the brain homogenate and the isolated virus were identical, indicating that it was not due to contamination. Phylogenetic analysis of 244 nucleotides from the 5′-UTR (Fig. 2) typed the Egyptian goat isolate (BSU1) as BVDV type 1 and subtype b. Although located in a different clade than the reference strains 24/15 and Osloss of BVDV-1 subtype b, BSU1 shared high nucleotide sequence identity of 95% to 96% and 95%, respectively, with these two strains. However, BSU1 shared only 90% identity with the Egyptian strain Iman, which is the strain used in the BVDV vaccine for cattle in Egypt. Acta Veterinaria Hungarica 61, 2013

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A

C

E

B

D

F

Fig. 1. Immunohistochemical staining of cerebellar tissue obtained from the two goat kids (sections A and B are from the kid from which BSU1 was isolated, while sections C and D are from the virus-negative kid) and from ear notch tissue from a positive control animal (sections E and F). Sections A, C and E were subjected to staining with primary polyclonal antibody (rabbit anti-BDV-1 ‘Moredun’ serum), while sections B, D and F were maintained as negative controls. Sections A and C showed positive staining in the granular cell layer of the cerebellum. Positive control showed positive staining of the fixed macrophages (histiocytes) of the dermal layer of the skin

To further characterise the new BSU1 isolate, genetic analysis of the partial Npro gene encoding the viral autoprotease was done. The phylogenetic analysis (Fig. 3) revealed that BSU1 belonged to BVDV-1b and was located on the same clade as the German goat isolate ‘Goat-G1’. While this isolate shared 93% nucleotide sequence identity with the German goat strain, the similarity with other BVDV-1 strains such as NADL and Osloss was only 68–71% and 70%, respectively. Phylogenetic analysis based on alignment of 420 nucleotides from the N-terminus of E2 also confirmed the isolate BSU1 to be BVDV-1b. This conclusion was supported by the nucleotide and amino acid identity when 960 nucleotides and their coding amino acids in the 5′-end of the E2 were analysed. BSU1 was found to be closely related to the German BVDV-1b isolate KE9 (89.5% amino acid similarity, 91% nucleotide similarity). Alignment of the deduced amino acid sequences of the partial E2 gene of BSU1 isolates with those of other BVDV-1b strains (Fig. 4) showed amino acid substitutions throughout the E2 protein where BSU1 was found to contain 19 unique amino acids, six of Acta Veterinaria Hungarica 61, 2013


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which were shared with KE9 while the remaining 13 were unique to BSU1. In addition, the Egyptian isolate was found to contain five glycosylation sites, three of which were shared with most of the other selected sequences, one was shared with the German isolate KE9 and one was unique to BSU1.

Fig. 2. Genetic typing of the Egyptian BVDV1 isolate (BSU1) in the 5′-UTR region. The tree was prepared using the neighbour-joining method and bootstrap testing. Numbers over branches indicate the percentage of 1000 bootstrap replicates that support each phylogenetic branch. The GenBank accession numbers of reference strains are as follow: NADL (M31182), Singer (L32875), SD1 (M96751), 28/1 (AF298061), ncp2 (AY443027), Oregon (AF091605), 2204/82 (AJ304377), Iman (AY960485), Osloss (M96687), VEDEVAC (AJ585412), CP7 (AF220247), P (AF298070), Draper (L32880), 24/15 (AF298060), NY-1 (FJ387232), B68/05 (EU224254), B145/05 (EU224245), B216/05 (EU224223), B425/06 (EU224235), 890 (U18059), New York 93 (AF502399), BD31 (U70263), X818 (AF037405), BSU1 (GU085506, this study)

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Fig. 3. Phylogenetic tree showing the relationship between the Egyptian goat BVDV1 (BSU1) isolate and other pestiviruses based on the analysis of 470 nucleotides derived from the Npro gene. The tree was prepared using the neighbour-joining method and bootstrap testing. Numbers over branches indicate the percentage of 1000 bootstrap replicates that support each phylogenetic branch. The GenBank accession numbers of pestivirus reference strains in Npro region (accession numbers being the same as those in 5′-UTR are omitted) are as follow: Singer (AF145364), V360 (AF144471), P (AF287288), 24/15 (AF287280), B75/05 (EU224254), B216/05 (EU224248), RIT (AF144465), Goat-G1 (U80898), BSU1 (GU991550, this study)

Discussion Abortion, stillbirths and neonatal infections in Egyptian sheep and goats are widespread. These manifestations can be caused by many infectious agents. In this study, nine caprine and ovine fetuses and/or neonates were screened for pestiviruses since they are considered responsible for many of these manifestations in sheep and goats (Pratelli et al., 1999). A pestivirus was isolated from one goat kid with BD-like signs in the form of central nervous system signs. Two goat kids from the same flock were positive for pestivirus by IHC but RT-PCR and virus isolation were successful in only one of them. Positive IHC Acta Veterinaria Hungarica 61, 2013


BSU1 KE9 Osloss VEDEVAC CP7 NY1

1 1 1 1 1 1

YPSCKPDFSYAIAKNDEIGPLGATGLTTQWYEYSDRMRLQDSAVVVWCKDGEFRHLTICE ..D............................K...E.....TV.T....N......IT.. L.V...G.Y......N...................G.....TG......G..IKY.IT.. L.A............N...................G.....TE.........IKY.IT.. ..D...G............................G......V.E....N..IKY.IR.G L.D............E...Q...............G.....ET.....E..K..Y.....


61 61 61 61 61 61

REARYLAILHTRALATSVVFEKIINGIKQEDVVEMDDNFELGLCPCDAKPLVKGKFNVTL ..............P...........KE...I...S....F...........R....... ..............P.........D.KE........................R....T.. ..............P.........K.KE....L....D..F...........R....T.. .......V......P........FD.KE...I........F.......R..IR....T.. .......A......P.....T..L..K-...M........F.......R.VIR....T..


121 121 121 121 121 120

LNGPAFQMVCPIGWTGTVS-CTLANKDTLATTVVRTYKRHKPFPYRQGCITQKVIGEDLY ...................-...................D.............T...... ...................L.HWS......M.........R...F...........G... ..................RL.HWP......L......T..Q...F........T...... ...................-...........I.......VR......D.V...T...... ...................-.........S.........VR........V...I......


180 180 181 181 180 180

DCMLGGNWTCVPGDQLRYVDGPIESCKWCGYKFDVSEGLPHFPIGKCKLKNESGYRQVDE ..V..........N.....G..V...........K......................... ..A...........I.......V..........HK......................... N.D.......I...........V........N.YK......................... ..A...........A....A..V...E......LK............R............ ..A...........E....G..V..........LK..............E..........


240 240 240 241 240 240

TSCNRDGVAIVPSGLVKCKIGNTVVQVVAMDDKLGPMPCRPYEIIPSEGPVEKTACTFNY ...........................I.............H...S.............. ............T.S......D.....I.............H...S.............. ...........LH.R......D.....I....R......I.H.................. .....N........T......D.....I...........K.H...S.............. ...........Q..R.R....D.....I.L.........K....................

BSU1 KE9 Osloss VEDEVAC CP7 NY

320 320 320 321 320 320

TRTLKNKYFEPRDNYFNNT A...............QQY .K......Y.......QQY .K......Y.......QQY ................QQY S...............QQY

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Fig. 4. Alignment of the deduced amino acid sequences from the E2 region of Egyptian isolate BSU1 in comparison with reference strains of BVDV-1. Dots indicate identical sequences. Unique amino acids for BSU1 are underlined and shown in bold format. Gaps indicate deletions. Potential glycosylation sites (NXS or NXT, except where X = P) are shaded. The published sequences of BVDV strain KE9 (EF101530), Osloss (M96687), VEDEVAC (AJ585412), CP7 (U63479), NY-1 (AY027671), BSU1 (JF968611, this study) were obtained from the GenBank database

staining in the other kid may have been due to a false staining reaction or due to the presence of a pestivirus variant that was able to escape the antibody used in DFA (Belknap et al., 2000). When MAbs were used in the IHC test, no positive staining was seen which is not surprising because monoclonal antibodies typically do not detect all BVDV strains (Goyal et al., 2002). Although clone 15C5, which targets the Erns glycoprotein of BVDV, has been found to recognise all BVDV strains tested (Goyal et al., 2002), it could not detect the Egyptian isolate, indicating that there may be a variation in the Erns glycoprotein of this isolate (Gripshover et al., 2007). Acta Veterinaria Hungarica 61, 2013

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Deregt et al. (1998) reported escape mutants of BVDV that could not be detected with clone 157 and 348, which target epitopes on the immunodominant E2 protein of BVDV indicating variation in the E2 gene. Multiple alignments for partial sequences of nucleotides and amino acids for the Egyptian isolate BSU1 showed mutations in the E2 gene, which might be responsible for the escape of this isolate from Mab clones targeting E2 epitopes. Mutations in that gene are plausible, since the E2 gene encoding envelope protein is the gene with a high mutation rate in other pestivirus isolates as well (Tajima et al., 2001). Previous studies in llamas and alpacas have indicated that in certain cases, virus isolation and/or RT-PCR may detect BVDV infection, while DFA or IHC may not (Belknap et al., 2000; Goyal et al., 2002). Many genomic regions are used for the genetic typing of pestiviruses, including the 5′-UTR, Npro and E2 (Vilcek et al., 2001; Dubois et al., 2008). All analyses in this study confirmed that BSU1 belonged to BVDV-1b. We expected this isolate to be located in the BDV clade since the goat kid showed typical signs of BD. However, it was located far from the BDV clade and there was only a 66% nucleotide sequence identity with reference strain BD31 of BDV. To our knowledge, this is the first report of isolation of a pestivirus from goats in Egypt and the second report worldwide on the isolation of pestivirus from a goat kid showing typical central nervous signs of BD (Loken et al., 1982). The use of several genetic regions for phylogenetic analysis is fruitful in molecular epidemiological studies and in tracing newly introduced viruses (Nagai et al., 2004). The origin of BVDV-1b in Egypt could not be traced as there is no control on the movement of animals between markets and purchased animals are not tagged. In addition to animal movement between markets, there is always contact between cattle and sheep as all ruminants are reared closely in Egypt. BVD-1a (NADL-like) is the dominant circulating genotype in Egypt, which is believed to have been introduced to Egypt by importation of foreign cattle breeds in the 1970s (El-Kholy et al., 2005). Although the local vaccine strain used in Egypt (Iman strain) is BVDV-1a (El-Kholy et al., 2005) and goats are not vaccinated against BVDV, the BSU1 isolate in this study showed high nucleotide sequence identity with the European BVDV-1b isolates. Although not proven, it is possible that this strain was introduced to Egypt through historical trade contacts or through importation of contaminated biological materials used in local vaccine production. Pestivirus antibodies in Egyptian goats have been demonstrated; in one serological survey, 31.4% of Egyptian goats were positive for BVDV antibodies (Zaghawa, 1998). However, the pathogenesis of pestiviruses in goats has not been thoroughly investigated in Egypt. In conclusion, our results indicate that the role of goats in the epidemiology of BVDV should be considered when formulating a national control programme in Egypt. Regular genotyping of pestiviruses in Egypt will prove to be beneficial in this regard. Acta Veterinaria Hungarica 61, 2013


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Acknowledgements We thank Jeremy Schefers and Jan Shivers for their help and Professor Horst Schirrmeier for supplying pestivirus antiserum. We also thank the Egyptian Cultural and Educational Bureau in Washington D.C., the Beni-Suef University and the Egyptian Ministry of Higher Education and State for Scientific Research for partial funding of this study.

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