Pestivirus
20170342387 · 2017-11-30
Assignee
Inventors
- Ad De Groof (Groesbeek, NL)
- Lars Guelen (Nijmegen, NL)
- Carla Christina Schrier (Boxmeer, NL)
- Martin Deijs (Huizen, NL)
- Cornelia Maria van der Hoek (Amsterdam, NL)
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
C12N2770/24321
CHEMISTRY; METALLURGY
C12N2770/24334
CHEMISTRY; METALLURGY
A61K39/00
HUMAN NECESSITIES
C12N2770/24322
CHEMISTRY; METALLURGY
G01N2333/183
PHYSICS
International classification
C12N7/00
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a novel porcine pestivirus, to proteins of the virus and to vaccines based upon the virus and proteins thereof. The invention also relates to DNA fragments comprising a gene of the virus and to DNA vaccines based upon genes of the virus. Furthermore the invention relates to antibodies that are reactive with the novel virus and to diagnostic tests for the detection of the virus or antibodies against the virus.
Claims
1. An isolated virus which is a member of the pestiviruses, wherein said virus: a) is the causative agent of Group A-II congenital tremors in pigs and b) has a viral genome comprising a gene encoding an envelope protein E.sup.rns, a gene encoding an envelope protein E2 and a gene encoding an envelope protein E1, wherein the nucleotide sequence of the E.sup.rns gene has a level of identity of at least 80% to the nucleotide sequence of SEQ ID NO: 1 and/or the nucleotide sequence of the E2 gene has a level of identity of at least 80% to the nucleotide sequence of SEQ ID NO: 3 and/or the nucleotide sequence of the E1 gene has a level of identity of at least 80% to the nucleotide sequence of SEQ ID NO: 5.
2. The isolated virus of claim 1, wherein the nucleotide sequence of the E.sup.rns gene has a level of identity of at least 80% to the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of the E2 gene has a level of identity of at least 80% to the nucleotide sequence of SEQ ID NO: 3 and the nucleotide sequence of the E1 gene has a level of identity of at least 80% to the nucleotide sequence of SEQ ID NO: 5.
3. An isolated virus which is a member of the pestiviruses, wherein said virus: a) is the causative agent of Group A-II congenital tremors in pigs and b) the cDNA reverse-transcribed from the viral RNA genome reacts in a PCR reaction with a primer set of SEQ ID NO: 7 and 8 to give a PCR product of 156+/−10 base pairs and/or reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 9 and 10 to give a PCR product of 335+/−10 base pairs and/or reacts in a PCR reaction with a primer set of SEQ ID NO: 11 and 12 to give a PCR product of 896+/−10 base pairs and/or reacts in a PCR reaction with a primer set of SEQ ID NO: 13 and 14 to give a PCR product of 896+/−10 base pairs and/or reacts in a PCR reaction with a primer set of SEQ ID NO: 15 and 16 to give a PCR product of 182+/−10 base pairs and/or reacts in a PCR reaction with a primer set of SEQ ID NO: 17 and 18 to give a PCR product of 182+/−10 base pairs.
4. The isolated virus of claim 3, wherein the cDNA reverse-transcribed from the viral RNA genome reacts in a PCR reaction with a primer set of SEQ ID NO: 7 and 8 to give a PCR product of 156+/−10 base pairs and reacts in a PCR reaction with a primer set of SEQ ID NO: 9 and 10 to give a PCR product of 335+/−10 base pairs and reacts in a PCR reaction with a primer set of SEQ ID NO: 11 and 12 to give a PCR product of 896+/−10 base pairs and reacts in a PCR reaction with a primer set of SEQ ID NO: 13 and 14 to give a PCR product of 896+/−10 base pairs and reacts in a PCR reaction with a primer set of SEQ ID NO: 15 and 16 to give a PCR product of 182+/−10 base pairs and reacts in a PCR reaction with a primer set of SEQ ID NO: 17 and 18 to give a PCR product of 182+/−10 base pairs.
5. The isolated virus of claim 1, wherein the nucleotide sequence of the E.sup.rns gene has a level of identity of at least 80% to the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of the E2 gene has a level of identity of at least 80% to the nucleotide sequence of SEQ ID NO: 3 and the nucleotide sequence of the E1 gene has a level of identity of at least 80% to the nucleotide sequence of SEQ ID NO: 5 and in that the cDNA reverse-transcribed from the viral RNA genome reacts in a PCR reaction with a primer set of SEQ ID NO: 7 and 8 to give a PCR product of 156+/−10 base pairs and reacts in a PCR reaction with a primer set of SEQ ID NO: 9 and 10 to give a PCR product of 335+/−10 base pairs and reacts in a PCR reaction with a primer set of SEQ ID NO: 11 and 12 to give a PCR product of 896+/−10 base pairs and reacts in a PCR reaction with a primer set of SEQ ID NO: 13 and 14 to give a PCR product of 896+/−10 base pairs and reacts in a PCR reaction with a primer set of SEQ ID NO: 15 and 16 to give a PCR product of 182+/−10 base pairs and reacts in a PCR reaction with a primer set of SEQ ID NO: 17 and 18 to give a PCR product of 182+/−10 base pairs.
6. A cell culture that comprises the virus of claim 1.
7. A gene encoding an E.sup.rns protein, wherein the nucleotide sequence of said gene has a level of identity of at least 80% to the nucleotide sequence of SEQ ID NO: 1.
8. An E.sup.rns protein that is encoded by the gene of claim 7.
9. A gene encoding an E2 protein, wherein the nucleotide sequence of said gene has a level of identity of at least 80% to the nucleotide sequence of SEQ ID NO: 3.
10. An E2 protein that is encoded by the gene of claim 9.
11. A gene encoding an E1 protein, wherein the nucleotide sequence of said gene has a level of identity of at least 80% to the nucleotide sequence of SEQ ID NO: 5.
12. An E1 protein that is encoded by the gene of claim 11.
13. A DNA fragment comprising the E.sup.rns gene of claim 7, wherein the E.sup.rns gene is under the control of a functional heterologous promoter.
14. A DNA fragment comprising the E2 gene of claim 9, wherein the E2 gene is under the control of a functional heterologous promoter.
15. A DNA fragment comprising the E1 gene of claim 11, wherein the E1 gene is under the control of a functional heterologous promoter.
16. A live recombinant vector virus comprising the DNA fragment of claim 13.
17. A pseudo-particle that comprises an E.sup.rns protein encoded by a gene that has a level of identity of at least 80% to the nucleotide sequence of SEQ ID NO: 1, an E2 protein encoded by a gene that has a level of identity of at least 80% to the nucleotide sequence of SEQ ID NO: 3, and the E1 protein of claim 12.
18. A vaccine for combating Group A-II CT in pigs, wherein said vaccine comprises an immunogenically effective amount of the virus of claim 1 and a pharmaceutically acceptable carrier.
19. The vaccine of claim 18, wherein said virus is in a live attenuated form.
20. The vaccine of claim 18, wherein said virus is in an inactivated form.
21. A vaccine for combating Group A-II CT in pigs, wherein said vaccine comprises an immunogenically effective amount of the E.sup.rns protein of claim 8 and a pharmaceutically acceptable carrier.
22. A vaccine for combating Group A-II CT in pigs, wherein said vaccine comprises an immunogenically effective amount of the E2 protein of claim 10 and a pharmaceutically acceptable carrier.
23. A vaccine for combating Group A-II CT in pigs, wherein said vaccine comprises an immunogenically effective amount of the E1 protein of claim 12 and a pharmaceutically acceptable carrier.
24. A vaccine for combating Group A-II CT in pigs, wherein said vaccine comprises an immunogenically effective amount of the pseudo-particle of claim 17 and a pharmaceutically acceptable carrier.
25. A vaccine for combating Group A-II CT in pigs, wherein said vaccine comprises the live recombinant vector virus of claim 16 and a pharmaceutically acceptable carrier.
26. An antibody or antiserum reactive with the virus of claim 1.
27. A diagnostic test kit for the detection of antibodies reactive with Group A-II congenital tremor associated porcine pestivirus, wherein said test kit comprises the virus of claim 1 or antigenic material thereof.
28. A diagnostic test kit for the detection of Group A-II congenital tremor associated porcine pestivirus, wherein said test kit comprises antibodies reactive with the virus of claim 1 or with antigenic material thereof.
29. A diagnostic test kit for the detection of Group A-II congenital tremor associated porcine pestivirus, wherein said test kit comprises a PCR primer set that is specifically reactive with the genome of the virus of claim 1.
Description
LEGEND TO THE FIGURES
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EXAMPLES
Example 1
Discovery of New Virus, CTAPV 1, on a Pig Farm in the Netherlands.
[0261] On a pig farm located in the Netherlands, an outbreak of congenital tremor type A-II was diagnosed in early 2012. Piglets born from gilt, first parity animals, were primarily affected but also higher parity sows were occasionally affected. Diagnosis was based on clinical observations and subsequent exclusion of congenital tremor types A-I, A-III, A-IV and A-V as the possible cause for disease. Clinically, affected piglets showed tremor in different grades, due to excessive muscle contractions during activity. The symptoms diminished when sleeping. Piglet loss was a secondary effect caused by the inability of affected animals to feed themselves, especially during the first week after birth. Histologically, the brain and the spinal cord were characterized by hypomyelinization. As further described below, not all affected pigs survived. In those that survived, the tremor diminished and finally disappeared as pigs grew older.
[0262] Based on the outbreak information, an infectious origin of the disease was suspected. In the first 20 weeks of the year 2012, a total of 48 μlitters with symptoms of congenital tremor were born from gilts, out of 231 litters born from gilts in total. This equals 21% of all litters born from gilts. At the peak of infection, 8 weeks after the initial outbreak, 85% of the gilt litters showed piglets with congenital tremor type A-II. The percentage piglet loss (piglet death) till weaning was 26% in affected litters, compared to 11% in non-affected litters. In affected litters, 60% of piglet death was attributable to congenital tremor. The total number of piglets born per litter was not affected. Congenital tremor affected both sexes, and prevalence within the litter varied between <10%-100%.
[0263] Prior to the outbreak in 2012, congenital tremor was observed in a few litters in November 2009 and December 2010.
[0264] Problems with outbreaks of congenital tremor have continued on this farm since 2012, and affected piglets were obtained in 2013 and 2014 (see below). However, the incidence rate decreased.
[0265] Blood plasma samples were obtained in March 2012 (6 samples, all piglets with symptoms of CT type A-II) and April 2012 (5 samples, all piglets with symptoms of CT type A-II). The new virus CTAPV 1 was detected in 11/11 samples.
[0266] More blood plasma samples were obtained from the same farm in July 2012. A total of 16 serum samples from piglets born from 2 sows and 1 gilt were analyzed. None of these piglets showed congenital tremor. CTAPV was found in 1/16 samples.
[0267] A new outbreak of the disease was diagnosed in January 2013. Four newborn pre-colostral piglets were obtained for necropsy, all showed CT type A-II. This virus was named CTAPV 1A because it originated from the same farm, but significant time had elapsed between the original outbreak and the occurrence of new clinical problems. The new virus CTAPV 1A was detected in 4/4 piglets.
[0268] A new outbreak of the disease was diagnosed in March 2013. Three newborn pre-colostral piglets were obtained for necropsy, all showed CT type A-II. This virus was named CTAPV 1B. The new virus CTAPV 1 was detected in 3/3 samples.
[0269] A new outbreak of the disease was diagnosed in January 2014. Four newborn pre-colostral piglets were obtained (rectal swabs), all showed CT type A-II. This virus was named CTAPV 1C. The new virus CTAPV 1 was detected in 4/4 samples. Necropsy on an additional 3 piglets was performed in February 2014, again all 3 piglets showed CT type A-II, and CTAPV was detected in 3/3 samples.
[0270] Post mortem examination was performed on piglets from outbreaks in January 2013, March 2013 and February 2014. Brains and spinal cord showed signs of demyelinization (see Example 2).
[0271] Seven piglets (6 pre-partus, last week of gestation; 1 newborn) from a farm with no history of congenital tremor type A-II were used as negative control for PCR and for post mortem examination. All plasma samples were negative for CTAPV virus, and no histological abnormalities were observed in these piglets.
Collection of Serum and Feces Samples
[0272] Feces and serum samples were obtained at farms in the Netherlands that have problems with CT type A-II in newborn pigs. Blood was collected in a tube (type: Vacuolette 8 ml Sep Clot Activator ref: 455071) and serum was isolated by centrifuging 20 minutes at 3000×g at 4° C. Feces were collected using a dry cotton-swab and put in a sterile tube containing 2 ml Phosphate-buffered saline solution (PBS). Then cotton swabs with feces were stirred strongly and discarded. Both serum and feces samples were stored at −70° C. until analysis.
Viral RNA Isolation with Optional DNAse Treatment
[0273] For viral RNA isolation, the QIAamp Viral RNA mini Kit (Qiagen) was used in combination with RNase free DNase kit (Qiagen).
[0274] In short, 1% solution of carrier-RNA/AVE in AVL buffer was prepared. 560 μl carrier-RNA/AVE in AVL was mixed with 140 μl sample and incubated 10 minutes at room temperature. Then 560 μl ethanol (>99%) was added and samples were transferred to a QIAamp mini spin column. Columns were centrifuged for 1 minute at 6000×g. Columns were washed by adding 250 μl AW1 and spinning the columns 30 seconds at 6000×g. DNase-mix was prepared by mixing 10 μL DNase with 70 μl RDD buffer per sample. 80 μl DNase-mix was incubated on the membrane during 15 minutes at room temperature. Washing was continued by putting 250 μl AW1 on the column and spinning it 30 seconds at 6000×g, followed by adding 500 μl AW2 to the columns and centrifuging 3 minutes at 13000×g. Collection tubes were replaced and columns were centrifuged for another minute. Spin columns were transferred into a 1.5 ml Eppendorf tube, where 65 μl AVE buffer was added on membranes and centrifuged 1 minute at 6000×g. The RNA samples were preceded to the Reverse Transcriptase-reaction immediately.
Reverse Transcriptase-Reaction
[0275] RNA was transcribed into cDNA using SuperScript® III First-Strand Synthesis System for RT-PCR (Invitrogen). The manufacturer's protocol was followed with some minor modifications. In summary, 1 μl random hexamers and 1 μl 10 mM dNTPs were mixed with 8 μl RNA. This was first incubated 5 minutes at 65° C., then chilled on ice. Then 10 μl cDNA synthesis mix, consisting of 2 μl 10×RT buffer, 4 μl MgCL.sub.2, 2 μl DTT, 1 μl RNaseOUT and 1 μl Superscript®III RT, was added to the samples. The samples were first incubated 10 minutes at 25° C., then 50 minutes at 50° C., followed by 5 minutes at 85° C. and finally chilled on ice. 1 μl RNase H was added to the samples and this was incubated 20 minutes at 37° C. The obtained cDNA samples were stored at −20° C. until use.
PCR
[0276] A. Primer combination CTAPV-PAN2-F1R1, -F2R1, -F1R2, -F2R2, Table 1,2
[0277] Each PCR reaction contained 27 μl WFI, 1 μl Super Taq Plus 5 μl 10× Super Taq PCR buffer, 5 μl dNTPs, 5 μl forward primer and 5 μl reverse primer. Overview of used primers is depicted in Table 1. The PCR program used to detect CTAPV consisted of a 4 minute initialization-phase, at 95° C. This was followed by 35 cycles of sequentially denaturation for 30 seconds at 95° C., annealing for 30 seconds at the appropriate annealing temperature for the primer pair (see Table 1) and extension for 30 seconds at 72° C. A final extension at 72° C. was maintained for 10 minutes. All PCR products were analyzed with 1.5% agarose-gel electrophoresis. See
[0278] B. Primer combination CTAPV-PAN-FW-RV, PANdeg-FW-PANdeg-REV, Table 1,2
[0279] Each PCR reaction contained 27 μl WFI, 1 μl Super Taq Plus 5 μl 10× Super Taq PCR buffer, 5 μl dNTPs, 5 μl forward primer and 5 μl reverse primer. Overview of used primers is depicted in Table 2. The PCR program used to detect CTAPV consisted of a 4 minute initialization-phase, at 95° C. This was followed by 40 cycles of sequentially denaturation for 30 seconds at 95° C., annealing for 30 seconds at the appropriate annealing temperature for the primer pair (see Table 2) and extension for 60 seconds at 72° C. A final extension at 72° C. was maintained for 10 minutes. All PCR products were analyzed with 1.5% agarose-gel electrophoresis.
[0280] C. Primer combination CTAPV-PAN2-F3R3, -F4R4, Table 1,2
[0281] Each PCR reaction contained 27 μl WFI, 1 μl Super Taq Plus 5 μl 10× Super Taq PCR buffer, 5 μl dNTPs, 5 μl forward primer and 5 μl reverse primer. Overview of used primers is depicted in Table 1. The PCR program used to detect CTAPV consisted of a 4 minute initialization-phase, at 95° C. This was followed by 35 cycles of sequentially denaturation for 30 seconds at 95° C., annealing for 30 seconds at the appropriate annealing temperature for the primer pair (see Table 1) and extension for 30 seconds at 72° C. A final extension at 72° C. was maintained for 10 minutes. All PCR products were analyzed with 1.5% agarose-gel electrophoresis.
TABLE-US-00003 TABLE 1 Overview of Primers Primer name Short name DNA Sequence CTAPV-PAN2-F2 F2 5′-CGGATACAGAAATACTAC-3′ CTAPV-PAN2-R2 R2 5′-CCGAATGCAGCTARCAGAGG-3′ CTAPV-PAN2-F1 F1 5′-GCCATGATGGAGGAAGTG-3′ CTAPV-PAN2-R1 R1 5′-GGGCAGRTTTGTGGATTCAG-3′ CTAPV-PAN-FW PAN-FW 5′-GAAACAGCCATGCCAAAAAATGAG-3′ CTAPV-PAN-REV PAN-RV 5′-AGTGGGTTCCAGGGGTAGATCAG-3′ CTAPV-PANdeg-FW PANdeg-FW 5′-GAAACAGCCATGCCMAARAATGAG-3′ CTAPV-PANdeg-REV PANdeg-RV 5′-AGTGGGTTCCAGGRGTAGATYAG-3′ CTAPV-PAN2-F3 F3 5′-GAGTACGGGGCAGACGTCAC-3′ CTAPV-PAN2-R3 R3 5′-CATCCGCCGGCACTCTATCAAGCAG-3′ CTAPV-PAN2-F4 F4 5′-ATGCATAATGCTTTGATTGG-3′ CTAPV-PAN2-R4 R4 5′-GTGACGTCTGCCCCGTACTC-3′
TABLE-US-00004 TABLE 2 Overview of primer combinations used, and characteristics of targets Anneal PCR temperature product Primer combination (° C.) size (bp) Target F1-R1 60.2 156 NS5B F1-R2 60.2 277 NS5B F2-R1 50.9 213 NS5B F2-R2 50.9 335 NS5B PAN-FW - PAN-RV 58.0 896 NS5B PANdeg-FW - PANdeg-RV 58.0 896 NS5B F3-R3 50.0 182 5′-UTR F4-R4 50.0 182 5′-UTR
D. SYBR Green Quantitative PCR
[0282] Standard Line for Quantification of qPCR Results
[0283] To obtain a standard for qPCR, a 155 bp PCR product of the CTAPV sequence containing the qPCR target sequence was cloned into a TOPO4 plasmid vector (Life Technologies) according to the manufacturer's instructions. The 155 bp CTAPV PCR product for cloning was obtained by performing a PCR with CTAPV-PAN2-F1 and CTAPV-PAN2-R1 primers, see Table 3. Subsequently, the PCR-product was electrophoresed on a 1.5% agarose-gel. The 155 bp band was cut out and DNA was extracted from the agarose-gel prior to cloning in the TOPO4 vector.
[0284] The TOPO TA Cloning Kit (Invitrogen) was used to ligate the PCR product into a pCR 4-TOPO4 vector and to transform this into One Shot TOP10 Chemically Competent E. Coli. In summary, 4 μl of DNA was mixed with 1 μl salt solution and 1 μl of TOPO vector. This ligation was incubated for 5 minutes at room temperature and then placed on ice. 2 μl ligation mix was added to One Shot® TOP10 Chemically Competent E. Coli. After 30 minutes incubation on ice, the mixture was heat shocked in a 42° C. water bath during 30 seconds and placed back on ice. Now 250 μl warm SOC medium was added and the mixture was incubated 1 hour at 37° C. in a shaking incubator, after which 100 μl mixture was spread out over an agar-LB+100 μg/ml ampicillin plate. The plate was incubated overnight in a 37° C. incubator.
[0285] Correctly cloned colonies were identified using colony-PCR using M13 Primers (see Table 3 below; (SEQ ID NO: 30 and 31)) in standard PCR assays, followed by gel electrophoresis. The correct colonies were grown in LBACF medium (MSD AH Media Production lot. No. 318781; Luria-Bertani medium, animal component free) with ampicillin, from which plasmid DNA was isolated using a QIAGEN® Plasmid Midi kit (Qiagen) according to manufacturer's protocol. To check for mutations, the plasmid DNA was sequenced using M13 primers.
TABLE-US-00005 TABLE 3 Overview of primer combinations used for qPCR analysis Annealing Primer name Primer DNA sequence Temperature CTAPV-PAN2- 5′-GCCATGATGGAGGAAGTG-3′ 60.0° C. F1 CTAPV-PAN2- 5′-GGGCAGRTTTGTGGATTCAG-3′ 60.0° C. R1 M13 Fw 5′-GTAAAACGACGGCCAG-3′ 55.0° C. M13 Rv 5′-CAGGAAACAGCTATGAC-3′ 55.0° C.
[0286] Standard dilutions of the target sequence were calculated by measuring plasmid DNA concentrations of the vector. The formula for calculating plasmid copies/μl is depicted below (Formula 1). The DNA concentration (ng/μl) was measured using spectrophotometry. A, G, T and C are counts of the homonymous nucleotides in the plasmid. 6.02*10.sup.23 is the number of Avogadro. The multiplication by 2 converts ssDNA concentration into dsDNA concentration, and the multiplication by 10.sup.9 converts gram into nanogram. For qPCR reactions, eight dilutions were made containing 10.sup.8-10.sup.1 copies/2 μl.
qPCR
[0287] A SYBR green based qPCR was developed. Each reaction contained 10 μl KAPA SYBR Fast qPCR master mix, 0.4 μl 10M forward primer, 0.4 μl 10M reverse primer, 7.2 μl WFI and 2 μl template. Primers CTAPV-PAN-F1 and CTAPV-PAN-R1 were used (See Table 4). The following program was used: 3 minutes at 95° C., followed by 39 cycles of sequentially 10 seconds at 95° C., 10 seconds at 60° C. and plate read in a Biorad CFX system. Results were analyzed using Biorad CFX software. Results were compared with a standard line as described above; a 10-fold dilution series of the 155 bp CTAPV product, cloned into a TOPO4 plasmid. A melting curve analysis between 65° C. ->95° C.; per 0.5° C. 0.05 seconds was included in the qPCR program.
[0288] Specificity of the qPCR reaction was validated by gel electrophoresis of the amplified PCR product. The calibration curve slope and y-intercept were calculated by the CFX software. The r.sup.2 was >0.99. The PCR efficiency calculated from the slope was between 95-105%.
TABLE-US-00006 TABLE 4 qPCR reaction mix volume User solution (μl)/reaction KAPA SYBR Fast qPCR mastermix 2x 10 CTAPV-PAN2-F1 10 μM 0.4 CTAPV-PAN2-R1 10 μM 0.4 WFI n.a. 7.2 Template (cDNA) n.a. 2
Nucleotide Sequencing
[0289] Sanger sequencing was performed according to methods described in literature. Sequences were analyzed using Sequencer 5.0 and Clone Manager 9.
Phylogenetic Analysis
[0290] Phylogenetic analysis was performed to categorize CTAPV 1 as a pestivirus.
[0291] The amino acid sequences of the entire gene of the novel virus were used to make phylogenetic trees based on the Neighbor-Joining Maximum Likelyhood method, the Poisson correction model and bootstrap analysis (500 replicates).
[0292] These trees were made using the program MEGA, version 5, using standard settings. (MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Koichiro Tamura, Daniel Peterson, Nicholas Peterson, Glen Stecher, Masatoshi Nei and Sudhir Kumar. Mol. Biol. Evol. 28(10): 2731-2739. 2011 doi:10.1093/molbev/msr121 Advance Access publication May 4, 2011).
Example 2
Virus CTAPV can be Found in Organs and PBLs; Histology Indicative for Demyelination in Brain and Spinal Cord
[0293] PCR analysis of the following organs of the necropsied pre-colostral new-born piglets (CTAPV 1A/1B, 2013) with congenital tremor type A-II indicated presence of CTAPV virus.
[0294] CTAPV could be detected in blood, serum, plasma, and PBLs (peripheral blood leukocytes), heart, small intestine, large intestine, brain, thoracic spinal cord, lumbar spinal cord, liver, inguinal lymph node, lung, gall bladder, bladder, kidney, tonsil and spleen. Highest quantities were detected in serum and tonsils.
[0295] The same organs were samples from pre-partus (last week of gestation) control piglets from a farm with no history of CT type A-II. All organs were negative in the PCR.
[0296] Brains and spinal cords of control and CTAPV-infected piglets were necropsied, formalin fixed and hematoxyline-eosine stained. Histological examination revealed indications for demyelination exclusively in CTAPV-infected piglets (
CTAPV Variants from Farms at Different Geographical Locations.
[0297] CTAPV variants 2-9 were obtained from pig farms in the Netherlands from outbreaks in 2013 and onwards.
[0298] Table 5 shows the number of piglets tested on each farm, and the number of CTAPV PCR positive piglets (serum/rectal swabs).
TABLE-US-00007 TABLE 5 Overview of CTAPV variants from different farms in The Netherlands. Results of PCR analysis of CTAPV in serum and/or rectal samples. CTAPV pos. CTAPV neg. CTAPV pos. with with without Total number Variant Farm symptoms symptoms symptoms of samples Date CTAPV 1 1 6 0 0 6 15-mrt-2012 CTAPV 1 1 5 0 0 5 5-apr-2012 CTAPV 1 1 0 0 1 15 20-jul-2012 CTAPV 1A 1 4 0 0 4 28-jan-2013 CTAPV 1B 1 3 0 0 3 5-mrt-2013 CTAPV 1C 1 4 0 0 4 31-jan-2014 CTAPV 1C 1 3 0 0 3 12-feb-2014 CTAPV 2 2 8 0 0 8 14-aug-2013 CTAPV 3 3 8 0 0 8 11-okt-2013 CTAPV 4 3 0 0 4 8 11-okt-2013 CTAPV 5 4 5 0 0 5 31-mei-2013 CTAPV 6 5 10 0 0 10 4-dec-2013 CTAPV 7 6 15 0 0 15 8-jan-2014 CTAPV 7 6 4 0 0 4 24-jan-2014 CTAPV 8 7 4 0 0 4 6-mrt-2014 CTAPV 9 8 4 0 0 4 12-feb-2014 NEG. CONT. 9 0 0 0 1 5-mrt-2013 NEG. CONT. 9 0 0 0 6 18-dec-2014 TOTAL 83 0 5 113
[0299] The disease association is 100% for piglets showing CT type A-II. CTAPV virus was detected in all piglets with congenital tremor type II, and not in control samples taken on a farm with no history of CT type A-II.
[0300] CTAPV 1 was found in one piglet that did not show congenital tremor. This piglet originated from Farm 1, a farm with history of CT type A-II.
[0301] CTAPV 4 was found in piglets that did not show congenital tremor. CTAPV 4 was found at the same farm where CTAPV 3 was found (Farm 3). Thus, CTAPV 4 was present on a farm with history of CT type A-II.
[0302] A total of 12 variants from 8 geographical different locations were found. [0303] Variants CTAPV 1, 1A, 1B, 1C originate from the same farm at different points in time. [0304] Variants CTAPV 3 and 4 originate from the same farm [0305] Although found at different geographical locations, Variants CTAPV 5 and 8 are identical at the nucleotide level
[0306] Table 6 shows reactivity of primer pairs.
TABLE-US-00008 TABLE 6 Reactivity of primer pairs. PAN-FW - PANdeg-FW - Variant F1R1 F1R2 F2R1 F2R2 F3R3 F4R4 PAN-RV PANdeg-RV CTAPV 1 + + + + + + + + CTAPV 1A + + + + + + + + CTAPV 1B + + + + + + + − CTAPV 1C + na na na + + − − CTAPV2 + + + + + na + na CTAPV 3 − − + + + na na na CTAPV 4 na na na na + na na na CTAPV 5 + + + + + na + na CTAPV 6 + + + + + na na na CTAPV 7 + na na + + + + + CTAPV 8 + na na + + + + + CTAPV 9 na na na na + na na na
[0307] All variants can be detected using PCR primer pair F3R3
[0308] All variants can be detected using one of the PCR primer combinations F1R1, F1R2, F2R1, F2R2, however, Variant CTAPV 9 was not tested.
Genome Sequencing
[0309] The complete genome sequence of CTAPV 1 was obtained by Sanger sequencing.
[0310] Of other variants, CTAPV 1A, 1B, 1C, 2, 3, 4, 6, 8 and 9, the first 5000 bp including the coding sequences for E.sup.rns, E1 and E2 were obtained.
[0311] Only a limited nucleotide sequence of 1073 nt is available for M7
[0312] Based on genome sequencing, it was concluded that CTAPV 5=CTAPV 8
Example 3
Phylogenetic Analysis of CTAPV and CTAPV Variants
[0313] The phylogenetic tree of the CTAPV 1 and other known pestiviruses is presented in
[0314] The phylogenetic tree of 10 of the CTAPV variants described in this patent application is presented in
[0315] CTAPV 7 was not included because only 1073 nt are available for M7.
[0316] CTAPV 5 is not included, because CTAPV 5=CTAPV 8
Example 4
[0317] Analysis of the Predicted E2 Protein/Nucleotide Sequence Shows that CTAPV 1B E2 Protein=CTAPV 1 E2 Protein. CTAPV 8 Protein Shows 14 Amino Acid Substitutions Compared to CTAPV 1.
[0318] Necropsied organs that could serve as starting material for infection experiments were available for CTAPV 1B, but not for CTAPV 1. We analyzed the nucleotide and amino acid sequence of the E.sup.rns-E1-E2 genes/proteins of CTAPV 1 and 1B. The amino acid sequence is 100% identical (
[0319] Necropsied organs that could serve as starting material for infection experiments were also available for CTAPV 8. We analyzed the nucleotide and amino acid sequence of the E.sup.rns-E1-2 genes/proteins of CTAPV 1B and 8 (amino acid comparison in
Example 5
Preparation of Challenge Material
[0320] Challenge material was obtained from necropsied organs (field material) of piglets affected by CTAPV 1B (2013) and CTAPV 8 (2014). Necropsied organs were stored at −70° C. until use.
CTAPV 1B
[0321] Brains of 3 piglets of the affected litter were pooled prior to homogenization.
Spinal cord of 3 piglets of the affected litter were pooled prior to homogenization
Spleens of 3 piglets of the affected litter were pooled prior to homogenization
Tonsils of 3 piglets of the affected litter were pooled prior to homogenization
CTAPV 8
[0322] Brains of 4 piglets of the affected litter were pooled prior to homogenization
Spinal cord of 4 piglets of the affected litter were pooled prior to homogenization
Spleens of 4 piglets of the affected litter were pooled prior to homogenization
Tonsils of 4 piglets of the affected litter were pooled prior to homogenization
Pooled tissues were weighted after thawing. Subsequently, 9 times tissue-weight PBS (CTAPV 1B) or M6B8 medium with 1 μM HEPES (Sigma H3375-250G, CTAPV 8) was added to the tissue material. The tissue was homogenized using a blender, followed by shaking with small glass beads for 5 minutes. During homogenizing organ-pulp was kept on ice. The organ-pulp was centrifuged 1 hour at 3200×g. Supernatant was first passed over a 0.45 μm filter, and subsequently over a 0.22 μm filter. The filtered homogenate was stored at −70° C. until use.
Example 6
Infection Experiment in Weaner Aged Piglets to Obtain Infectious Material:
[0323] Challenge experiments with CTAPV 1B and CTAPV 8 organ homogenates originating from field isolates were conducted in 4 to 8 week old weaning-aged SPF/high health piglets of a commercial finisher pig breed.
[0324] At the time of placing in the test facility, CPDA (citrate phosphate dextrose adenine) blood samples, rectal swabs, oropharynx swabs and nasal swabs were obtained from the animals. Animals were housed in two separate experiment rooms: group A 8 animals and group B 8 animals. There was no physical contact or indirect contact via animal caretakers between the rooms.
[0325] In group A, six pigs were inoculated with CTAPV 1B homogenates via the intramuscular (IM), subcutaneous (SC), intranasal (N) and oral (OR) routes.
Two pigs received inoculum from mixed spleen+spinal cord+brain homogenate
Two pigs received inoculum from mixed spleen+tonsil+brain homogenate
Two pigs received inoculum from mixed brain+spinal cord homogenate
Two pigs served as contact sentinels
[0326] IM, SC and N volumes were 1.0 ml per dose, left and right. OR volume was 4 ml. Nasal dose was sprayed. Challenge doses are given in Table 7.
[0327] After inoculation, all pigs were observed daily for clinical signs, but the animals remained asymptomatic during the course of the experiment.
[0328] CPDA-blood, nose swabs, oropharynx swabs and rectal swabs were taken on day 0, day 3, day 7, day 10 and day 14 after inoculation to monitor infection and excretion of CTAPV 1B via qPCR analysis. Plasma was obtained from CPDA blood using the Leucosep® kit (Greiner Mat. no. 163 288). The results of qPCR analysis on plasma samples are presented in Table 7.
[0329] All inoculated animals showed a positive CTAPV qPCR result in blood plasma at day 10. Based on excretion of virus, animals were sacrificed at different time points to obtain fresh infectious material for subsequent in vitro and in vivo studies.
[0330] At the time of necropsy, brain, spinal cord, spleen, tonsils, and blood were taken from the animals.
TABLE-US-00009 TABLE 7 Challenge doses and Results challenge CTAPV 1B CTAPV 1B: Challenge challenge T = 3 d p T = 7 d p T = 10 d p T = 14 d p load RNA T = 0 chall chall chall chall copies/ml Plasma Plasma Plasma Plasma Plasma in 10% RNA RNA RNA RNA RNA Animal Material Route homogenate copies/ml copies/ml copies/ml copies/ml copies/ml 326 sentinels n.d. n.d. n.d. n.d. n.d. 365 n.d. n.d. n.d. n.d. n.d. 366 spleen + IM, 4 ml oral; 6.15E+05 n.d. n.d. n.d. 2.38E+05 N/A 367 spinal nasal 2 x 1 ml IM n.d. n.d. n.d. 3.24E+04 2.00E+06 c + brain oral + 2 x 1 ml nasal; SC 2 x 1 ml SC 368 spleen + IM, 4 ml oraal; 8.65E+05 n.d. n.d. n.d. 3.50E+05 N/A 369 tonsil + brain nasal 2 x 1 ml IM n.d. n.d. n.d. 2.16E+05 2.67E+06 oral + 2 x 1 ml nasal; SC 2 x 1 ml SC 370 brain + IM, 4 ml oral; 3.91E+05 n.d. n.d. n.d. 3.24E+05 3.31E+06 371 spinal cord nasal 2 x 1 ml IM n.d. n.d. 4.06E+04 5.23E+05 N/A oral + 2 x 1 ml nasal; SC 2 x 1 ml SC n.d.: not detectable N/A: not analysed (animal already sacrificed)
[0331] In group B, six pigs were inoculated with CTAPV 8 homogenates via the intramuscular (IM), subcutaneous (SC), Intranasal (N) and oral (OR) routes.
Two pigs received inoculum from spleen+tonsil+brain+spinal cord homogenate
Two pigs received inoculum from spleen+tonsil homogenate
Two pigs received inoculum from brain+spinal cord homogenate
Two pigs served as contact sentinels.
[0332] IM, SC and N volumes were 2.0 ml per dose, left and right. OR volume was 3 or 4 ml. Nasal dose was sprayed. Challenge doses are given in Table 8.
[0333] After inoculation, all pigs were observed daily for clinical signs, but the animals remained asymptomatic during the course of the experiment.
[0334] CPDA-blood, nose swabs, oropharynx swabs and rectal swabs were taken on day 0, day 3, day 7 and day 14 after inoculation to monitor infection and excretion of CTAPV-8 via qPCR analysis. Plasma was obtained from CPDA blood using the Leucosep® kit (Greiner Mat. no. 163 288). The results of qPCR analysis on plasma samples are presented in Table 8.
[0335] All inoculated animals showed a positive CTAPV qPCR result in blood plasma at day 3 and/or day 7. Based on excretion of virus, animals were sacrificed at different time points to obtain fresh infectious material for subsequent in vitro and in vivo studies.
[0336] At the time of necropsy, brain, spinal cord, spleen, tonsils, and blood were taken from the animals. The organ materials were used as challenge material in the vaccination-challenge study as described in Example 8/9
TABLE-US-00010 TABLE 8 Challenge doses and Results challenge CTAPV 8 CTAPV 8: Challenge challenge T = 3 d p T = 7 d p T = 14 d p load RNA T = 0 chall chall chall copies/ml in Plasma Plasma Plasma Plasma 10% RNA RNA RNA RNA Animal Material Route homogenate copies/ml copies/ml copies/ml copies/ml 394 sentinels n.d. n.d. n.d. n.d. 395 n.d. n.d. n.d. n.d. 397 mix 4 IM, 3 ml oral; 1.04E+06 n.d. 5.50E+03 2.55E+06 N/A 398 organs nasal 2 x 2 ml IM n.d. 5.22E+03 8.35E+04 N/A oral + 2 x 2 ml nasal; SC 2 x 2 ml SC 399 spleen + IM, 4 ml oral; 1.03E+06 n.d. 7.92E+03 N/A N/A 400 tonsil nasal 2 x 2 ml IM n.d. 2.46E+03 1.57E+05 N/A oral + 2 x 2 ml nasal; SC 2 x 2 ml SC 401 brain + IM, 4 ml oral; 4.02E+05 n.d. 3.28E+03 1.73E+04 N/A 402 spinal nasal 2 x 2 ml IM n.d. 5.07E+03 4.77E+06 N/A cord oral + 2 x 2 ml nasal; SC 2 x 2 ml SC n.d.: not detectable N/A: not analysed (animal already sacrificed)
Example 7
Preparation of Challenge Material for Vaccination-Challenge Experiment
[0337] Challenge material was obtained from Example 6.
CTAPV 1B
[0338] Brains, spinal cord, spleen and tonsils of 1 necropsied animal of example 6, group A
CTAPV 8
[0339] Brains, spinal cord, spleen and tonsils of 1 necropsied animal of example 6, group B Pooled tissues were weighted after thawing. Subsequently, 9 times tissue-weight M6B8 medium with 10 μM HEPES (Sigma H3375-250G) was added to the tissue material. The tissue was homogenized using a blender, followed by shaking with small glass beads for 5 minutes. During homogenizing organ-pulp was kept on ice. The organ-pulp was centrifuged 1 hour at 3200×g. Supernatant was first passed over a 0.45 μm filter, and subsequently trough a 0.22 μm filter with exception of the material for oral administration. The filtered homogenate was stored at −70° C. until use.
Example 8
Vaccination-Challenge Experiment
Vaccine Design: Expression of E2 Protein:
[0340] The amino acids sequence of CTAPV 1 virus was analyzed. The start and stop of the E2 gene were determined using an alignment of the CTAPV virus genome with Classical Swine Fever virus (CSF) E2 protein (Genbank: AAS 20412.1) and Bovine Virus Diarrhea virus (BVDV) E2 protein (Genbank: AGN03787.1), and predicted cleavage sites of the E2 protein were determined using SignalP4.1 software (http://www.cbs.dtu.dk/services/SignalP/)
[0341] The predicted amino acid sequence of CTAPV 1 E2 (SEQ ID NO: 32):
TABLE-US-00011 SCHKRQDYYSIQLVVDGKTGVEKRSIVGKWTVITREGREPRLMEQISMVS NDSLSETYCYNRLNTSSWGRQPARQRGCGQTVPFWPGDNVLEEQYYSTGY WVNATGGCQLREGVWLSRKGNVQCQRNGSSLILQLAIKEENDTMEIPCDP VETESMGPVTQGTCVYSWAFAPRGWYYNRKDGYWLQYVKKNDYQYWTKMP TASSATTMYRH
[0342] Subsequently, the CTAPV E2 nucleotide sequence for expression of CTAPV E2 protein in the Baculovirus expression system in insect cells was optimized using the Genscript OptimumGene™ algorithm (www.genscript.com) (SEQ ID NO: 33).
TABLE-US-00012 CGCGGATCCAAATATGTCATGTCACAAGCGTCAAGACTACTACTCTATCC AACTGGTGGTGGACGGAAAAACTGGCGTGGAAAAGCGTTCTATCGTGGGC AAGTGGACGGTCATCACCAGGGAGGGCAGAGAACCGCGCCTAATGGAGCA AATTTCGATGGTATCTAACGACTCTCTTTCAGAAACCTACTGCTATAACC GTCTCAATACTAGCTCTTGGGGTCGTCAACCTGCCCGTCAGCGCGGATGT GGGCAAACCGTCCCCTTCTGGCCTGGTGACAACGTACTCGAGGAACAGTA CTATAGCACCGGATACTGGGTTAACGCTACTGGCGGTTGCCAACTACGCG AGGGAGTTTGGTTATCTCGTAAGGGGAACGTGCAATGTCAGCGTAATGGC TCATCGCTGATCCTTCAACTCGCTATTAAAGAGGAAAACGACACCATGGA AATCCCGTGCGATCCAGTCGAGACTGAATCAATGGGCCCCGTTACTCAAG GCACGTGTGTGTACAGCTGGGCTTTCGCCCCTAGGGGATGGTACTATAAC CGTAAGGACGGCTACTGGCTTCAATACGTGAAGAAAAACGATTACCAGTA CTGGACCAAAATGCCCACTGCATCCAGCGCGACCACTATGTACCGTCACC ATCACCATCACCATCACTAAGAATTCTCGAG
[0343] The restriction sites BamHI and EcoRI are underlined. The start codon is indicated in Italic and the stop codon is indicated in bold.
Transformation and Expression:
[0344] The E2 gene of CTAPV was synthesized at Genscript and directly cloned in a plasmid vector (pFastbac1) using the BamHI and EcoRI restriction sites. The plasmid was transformed to E. coli using standard transformation techniques, and subsequently plasmid DNA was purified and used for transfection of SF9 insect cells. The transfection was carried out as follows:
[0345] 2 ml cell suspension of 5*10.sup.5 cells/ml was added to each well of a 6 well plate. The cells were allowed to attach to the plate for 1 hour at 27° C. The following transfection solution (200 μl medium without antibiotics, 5 μl miniprep DNA and 6 μl cellfectin (Invitrogen)) was prepared and incubated at room temperature for 45 minutes. After 45 minutes 0.8 ml medium was added to the transfection solution and this was added to the attached cells. The transfected cells were incubated for 4 hours at 27° C. After 5 hours another 1 ml of medium (supplemented with gentamycin and natamycin) was added to the cells. Cells were grown for 3 days at 27° C. The supernatant was stored at −70° C. as P1 virus stock.
[0346] The expression of the CTAPV E2 protein in the SF9 cultures was checked by SDS-page gel electrophoresis. The obtained samples from the SF9 cultures were diluted 1:1 with Bio-Rad Laemmli sample buffer with 5% P3-mercaptoethanol, and subsequently samples were heated to 99° C. for 10 minutes. All samples and a Precision Plus Protein™ All Blue (Bio-Rad) marker were loaded into a Bio-Rad CriterionMTGX™ precast gel (any kD™) and electrophoresed at 200 V for 42 minutes. The electrophoresis buffer used was 1× Tris/Glycine/SDS. After electrophoresis, the gel was stained for 1 hour in InstantBlue™ (Expedeon) protein staining buffer.
Purification:
[0347] After expression in SF9 cells, the E2 protein was purified in two different ways. The first purification method was by making a whole cell lysate. A SF9 culture expressing E2 of CTAPV was pelleted, resuspended in PBS and sonicated using a Branson sonifier (2 times 30 pulses, output 5, duty cycle 55%). After sonication the lysate was centrifuged for 10 minutes at 8,000 rpm. The pellet containing the overexpressed E2 was resuspended in PBS. Another way of purifying the E2 protein was by a purification method using IMAC and anionic detergents. This method is described in BMC Biotechnology 2012, 12:95. (BMC Biotechnology 2012, 12:95; Use of anionic denaturing detergents to purify insoluble proteins after overexpression; Benjamin Schlager, Anna Straessle and Ernst Hafen). A lysis buffer containing an anionic denaturing detergent (SDS) was used to lyse the overexpressed E2 culture. The excess of detergent was removed by cooling and purification, prior to affinity purification.
[0348] E2 proteins expressed in SF9 cells and purified as describe above were run on SDS-page gel together with Bovine Serum Albumin standards with known protein concentration. Protein concentration was estimated by comparison of band intensities using Genetools software (Syngene version 3.08.07).
Formulation
[0349] The final vaccine was formulated in a water-in-oil emulsion based on mineral oil. The water: oil ratio based on weight was 45:55. Droplet size of the emulsion was mainly smaller than 1 m and viscosity was about 80-150 mPa.Math.sec.
[0350] Vaccine 1: water phase consisted of purified E2 protein (estimated E2 concentration 60 μg/ml)
[0351] Vaccine 2: water phase consisted of whole cell lysate (estimated E2 concentration 62 μg/ml)
Vaccination-Booster
[0352] For this experiment, 48 weaner-aged piglets at 5 weeks of age were available. 3×8 animals per group were housed in stable 1, and 3×8 animals per group were housed in stable 2. No contact between animals was possible between stables.
[0353] Per group of 8 animals, 6 piglets receive a primo vaccination with vaccine 1, the other 2 piglets were not vaccinated at the beginning of the study.
[0354] At t=21 days, 5 out of 6 primo-vaccinated animals in each of the groups received a booster vaccination with vaccine 2.
[0355] Blood samples were collected prior to primo vaccination, at day 21 after infection prior to booster vaccination, and at day 39, prior to challenge
[0356] Of each group, 4 animals that received primo and booster vaccination, plus 2 non-vaccinated animals were moved to the challenge facilities prior to challenge.
[0357] Of each group, 1 animal that received only primo vaccination, and 1 animal that received both primo and booster vaccination were monitored for an additional two weeks.
Challenge
[0358] The 36 animals for this experiment were housed in stable 3, 3×6 animals per group, group 1-3, and in stable 4, 3×6 animals per group, group 4-6, were housed. The animals in stable 3 originated form stable 1, the animals in stable 4 originated from stable 2.
[0359] No contact between animals was possible between stables. No physical contact was possible between animals of different groups within a stable, but air-contact was possible.
[0360] Animals were challenged with live virus material on day 39 after primo vaccination.
[0361] In stable 3, 3×6 piglets (group 1-3) were challenged with CTAPV 1 challenge material (see above).
Group 1: 10.0 ml oral and 2×2.0 ml nasal
Group 2: 2×1.0 ml IM
Group 3: 2×1.0 ml IM
[0362] In stable 4 3×6 piglets (group 4-6) were challenged with CTAPV 8 challenge material (see above).
Group 1: 10.0 ml oral and 2×2.0 ml nasal
Group 2: 2×1.0 ml IM
Group 3: 2×1.0 ml IM
[0363] Serum blood samples and nasal, rectal and oropharynx swabs were collected prior to challenge, and at 3, 6, 9, 13, 16, 20, 23 and 27 days post challenge to monitor infection and excretion of CTAPV viruses via qPCR analysis. Three animals (two vaccinated, one non-vaccinated) per group were necropsied at day 13 post challenge, the other 3 animals (two vaccinated, one non-vaccinated) were necropsied at day 27 post challenge. Inguinal lymph nodes, mesenteric lymph nodes and tonsils were sampled at the time of necropsy.
Example 9
Antibodies to CTAPV E2 Protein
Expression of E2 Protein in E. Coli:
[0364] The amino acids sequence of CTAPV 1 virus was analyzed. The start and stop of the E2 gene were determined using an alignment of the CTAPV virus genome with Classical Swine Fever virus (CSF) E2 protein (Genbank: AAS 20412.1) and Bovine Virus Diarrhea virus (BVDV) E2 protein (Genbank: AGN03787.1), and predicted cleavage sites of the E2 protein were determined using SignalP4.1 software (http://www.cbs.dtu.dk/services/SignalP/)
[0365] The predicted amino acid sequence of CTAPV 1 E2 (SEQ ID NO: 32):
TABLE-US-00013 SCHKRQDYYSIQLVVDGKTGVEKRSIVGKWTVITREGREPRLMEQISMVS NDSLSETYCYNRLNTSSWGRQPARQRGCGQTVPFWPGDNVLEEQYYSTGY WVNATGGCQLREGVWLSRKGNVQCQRNGSSLILQLAIKEENDTMEIPCDP VETESMGPVTQGTCVYSWAFAPRGWYYNRKDGYWLQYVKKNDYQYWTKMP TASSATTMYRH
Protein Sequence for Expression in E. Coli (Includes a HIS-Tag)
[0366]
TABLE-US-00014 SCHKRQDYYSIQLVVDGKTGVEKRSIVGKWTVITREGREPRLMEQISMVS NDSLSETYCYNRLNTSSWGRQPARQRGCGQTVPFWPGDNVLEEQYYSTGY WVNATGGCQLREGVWLSRKGNVQCQRNGSSLILQLAIKEENDTMEIPCDP VETESMGPVTQGTCVYSWAFAPRGWYYNRKDGYWLQYVKKNDYQYWTKMP TASSATTMYRHHHHHHH
[0367] Subsequently, the CTAPV E2 nucleotide sequence for expression of CTAPV E2 protein in E. Coli was optimized using the Genscript OptimumGene™ algorithm (www.genscript.com) (SEQ ID NO: 34).
TABLE-US-00015 CATATGTCGTGTCACAAACGCCAAGATTATTATTCTATTCAACTGGTCGT GGATGGTAAAACGGGTGTCGAAAAACGCTCTATCGTCGGTAAATGGACCG TGATTACGCGTGAAGGCCGCGAACCGCGTCTGATGGAACAGATCAGTATG GTTTCCAACGATAGCCTGTCTGAAACCTATTGCTACAACCGCCTGAATAC GAGCTCTTGGGGTCGTCAGCCGGCACGTCAACGCGGCTGTGGTCAGACCG TCCCGTTTTGGCCGGGCGACAACGTGCTGGAAGAACAATATTACAGTACC GGTTATTGGGTGAATGCAACGGGCGGTTGCCAGCTGCGTGAAGGCGTTTG GCTGTCTCGTAAGGGTAACGTCCAGTGTCAACGCAATGGCAGTTCCCTGA TTCTGCAACTGGCGATCAAAGAAGAAAACGATACCATGGAAATCCCGTGC GACCCGGTCGAAACCGAATCAATGGGCCCGGTGACCCAGGGCACGTGTGT TTATTCGTGGGCATTCGCACCGCGCGGCTGGTATTACAACCGTAAAGATG GTTATTGGCTGCAGTACGTGAAGAAAAACGACTATCAATACTGGACCAAA ATGCCGACGGCATCATCGGCTACCACGATGTACCGTCATCACCATCACCA TCACCATTAACTCGAG
[0368] Restriction sites added (in bold) are NdeI and XhoI.
Transformation and Expression:
[0369] The E2 gene of CTAPV was synthesized at Genscript and directly cloned in a plasmid vector (pET22b) using the NdeI and XhoI restriction sites. The plasmid was transformed to E. coli BL21star+pLysS using standard transformation techniques, and expression was induced.
[0370] Expression was achieved by growing the expression strains in autoinducing media for 18 hours at 37° C.
[0371] Expression was verified by running SDS-page gel electrophoresis.
[0372] E2 was found to be in the insoluble fraction. The E2 protein was purified by applying a purification method using IMAC and anionic detergents. This method is described in BMC Biotechnology 2012, 12:95. (BMC Biotechnology 2012, 12:95; Use of anionic denaturing detergents to purify insoluble proteins after overexpression; Benjamin Schlager, Anna Straessle and Ernst Hafen). A lysis buffer containing an anionic denaturing detergent (SDS) was used to lyse the overexpressed E2 culture. The excess of detergent was removed by cooling and purification, prior to affinity purification.
[0373] The purified protein was checked on SDS-page as described in Example 8. The purified protein was formulated in GNE and used for injection of rabbits to generate antibodies. The estimated concentration of the protein in the water phase was 0.5 mg/ml.
[0374]
Example 10
[0375] SYBR Green One-Step qRT-PCR
Animal Samples
[0376] Swine serum and spleen samples were collected from experimentally infected and control pigs. Blood was collected (Vacuolette 8 ml Sep Clot Activator ref: 455071; Greiner Bio-one) and serum was obtained by centrifugation 20 minutes at 3,000×g at 4° C. Sperm samples were obtained from a commercial breeding company and tested without pretreatment.
[0377] 10% Tissue homogenates were prepared in PBS on ice. Homogenization was performed in Gentle Macs M tubes with the Gentle Macs Dissociator (Miltenyi Biotec). This homogenized material was then centrifuged twice, first at 3,200×g for 30 minutes and subsequently at 10,000×g for 10 minutes. Subsequently a DNase treatment was done: 24 μl 10× Turbo DNase buffer and 20 μl Turbo DNase (AMbion) was added to 250 μl supernatant and this mixture was incubated at 37° C. for 10 minutes.
RNA Extraction
[0378] RNA was extracted from these samples with the Magnapure 96 instrument (Roche) with external lysis. This system purifies DNA, RNA, and viral nucleic acids using magnetic glass particle technology. 200 μl sample was mixed with 250 μl magnapure total nucleic acid isolation kit lysis/binding buffer and the extraction was performed in the Magnapure instrument using the external lysis protocol. RNA samples were stored at −70° C. until further use.
SYBR Green One-Step qRT-PCR
Specific Primer Design
[0379] Oligonucleotide primers were used to amplify the 5′ UTR genome of the CTAPV genome. This part of the viral genome was chosen based on conserved nucleotide sequence between CTAPV variants 1-9 (based on alignment of the nucleotide sequences). The primer sequences were as follows: CTAPV-PAN2-F3-B: CGTGCCCAAAGAGAAATCGG (SEQ ID NO: 35) and CTAPV-PAN2-R3-B (SEQ ID NO: 36): CCGGCACTCTATCAAGCAGT.
qRT-PCR Protocol
[0380] A SYBR green based one step qRT-PCR was developed using the Superscript III Platinum SYBR Green One-Step qRT-PCR kit (ThermoFisher). Each reaction contained 25 μl 2×SYBR Green Reaction Mix, 1 μl Superscript III RT/Platinum Taq Mix, 1 μl 10 μM CTAPV-PAN2-F3-B primer, 1 μl 10 μM CTAPV-PAN2-R3-B primer, 17 μl RNAse free water and 5 μl RNA template. All reaction were performed on a BioRad CFX96 with the following cycling parameters; a RT reaction at 55° C. for 3 min, Pre-denaturation at 95° C. for 5 min and then 40 cycles of 95° C. for 15 sec, 60° C. for 30 sec followed by a melting curve program from 60° C. until 95° C. with 0.5° C./5 sec.
Standard Line Creation
[0381] For quantification of the detected RNA in the SYBR Green One-Step qRT-PCR a standard line was constructed containing the q-PCR target sequence of which standard dilutions can be calculated. A 177 base pairs long sequence from the 5′UTR part (162-338) of the CTAPV genome was synthesized (Genscript) and ligated in a pUC57 vector that was subsequently transfected in E. coli. Plasmid DNA was isolated by midiprep.
[0382] The formula for calculating plasmids copies/μl is:
Plasmid copies/μl=DNA concentration(ng/μl)/((A×328.4+G×344.24+T×303.22+C×304.16)/(6.02×10.sup.23))×2×10.sup.9).
[0383] The DNA concentration of the plasmid was 100 ng/l. Eight dilutions were made containing 10.sup.8 until 10.sup.1 copies/2 μl.
Results
[0384] Validation of the qRT-PCR.
[0385] A standard line with eight dilutions containing 10.sup.8 until 10.sup.1 copies/5 μl and a negative control sample were included in an experiment to validate the qRT-PCR.
[0386] These data show that the developed qRT-PCR meets the requirements for the detection and quantification of CTAPV. The qRT-PCR was subsequently used for sample analysis of suspected CTAPV positive samples and control samples. Interpretation of the data was based on the RFU per cycle plus the characteristics of the melting curve. Aberrant melting curves would be indicative for non-specificity of the amplicon.
Detection and Quantification of CTAPV RNA in Serum, Spleen and Sperm Samples.
[0387] Serum and spleen from experimentally infected and from control gilts were tested in duplicate for CTAPV RNA presence. Also, sperm samples were tested. The (average) results are presented in Table 9. In the assays performed, the standard lines were confirmed to be within the quality range for an accurate qPCR (
TABLE-US-00016 TABLE 9 CTAPV RNA quantification of swine serum, sperm and spleen samples. Ct values* RNA copies/5 μl* RNA copies/ml* Serum CTAPV 27.41 9.99E+02 5.00E+04 positive gilt 1 Serum CTAPV ND — — negative gilt 2 Spleen CTAPV 29.45 7.93E+02 3.97E+04 positive gilt 3 Spleen CTAPV ND — — negative gilt 4 Sperm CTAPV 29.75 5.39E+02 2.70E+04 positive boar 1 Sperm CTAPV ND — — negative boar 2 *Means of duplicate experiments; ND: not detectable; spleen refers to 10% (w/v) homogenate sample. Column RNA copies/5 μl shows the number of copies of the virus in 5 μl extracted RNA sample obtained from 200 μl of the original sample. Column RNA copies/mL shows the number of copies of the virus in the original sample (serum, sperm) or the 10% homogenate (spleen).
Example 11
CTAPV Positive Sperm Infects Gilts and Offspring
Animals
[0388] Six gilts were obtained from an SPF/High Health farm. Sperm from a CTAPV-positive boar was used for artificial insemination of the gilts.
Methods
[0389] Blood was collected from gilts and offspring (Vacuolette 5/8 ml Sep Clot Activator ref: 455071; Greiner Bio-one) and serum was obtained by centrifugation 20 minutes at 3,000×g at 4° C. Sperm samples were tested without pretreatment.
[0390] RNA extraction and qRT-PCR were performed as described in the section “SYBR Green One-Step qRT-PCR” of Example 10.
Results
[0391] Tested gilts were serum-negative for CTAPV prior to insemination (qRT-PCR). Boar sperm was positive for CTAPV as analysed by qRT-PCR. At t=+4 weeks after insemination, gilts 4 and 5 contained detectable levels of CTAPV in serum. At the day of farrowing, gilts 1, 2 and 6 contained detectable levels of CTAPV in serum. Piglets with detectable levels of CTAPV in serum were born out of 5 of 6 gilts (see Table 10 for results). Piglets were healthy and showed no clinical tremor or increased incidence of other clinical symptoms related to congenital tremor type AII such as splay legs.
TABLE-US-00017 TABLE 10 CTAPV positive sperm infects gilts and offspring RNA copies t = 4 w RNA copies/mL Results qRT-PCR serum gilt gestation at farrowing* piglets: clinical score: 52 1.65E+02 6 out of 10 10 x no CTAPV positive congenital tremor 53 4.15E+01 6 out of 11 11 x no CTAPV positive congenital tremor 54 ND** 0 out of 16 16 x no CTAPV positive congenital tremor 55 2.47E+02 ND 1 out of 15 15 x no CTAPV positive congenital tremor 56 8.70E+01 ND 3 out of 17 17 x no CTAPV positive congenital tremor 57 ND 3.19E+04 11 out of 18 18 x no CTAPV positive congenital tremor *Column RNA copies shows the number of RNA copies of the virus per mL in the original sample (serum) **ND: not detected/below detection level
Example 12
[0392] Infection of Pregnant Gilts with CTAPV Variant 1B Obtained from “Shaking Piglets” and Effect on Newborn Piglets: CTAPV Positive Sperm
Animals
[0393] Six gilts were obtained from a SPF/high health farm. Gilts were inseminated via artificial insemination with CTAPV positive sperm. Pregnancy was confirmed at day 28 of gestation using ultrasound. All gilts gave birth to a litter of piglets on day 115 or day 116 of gestation.
Infection
[0394] Three of the gilts were infected on day 32 after insemination with a CTAPV1B inoculum consisting of organ homogenates of spleen and brain obtained from necropsied pig 371 at t=11 days after infection with CTAPV1B infected material. This experiment was described in Example 6/Table 7. The homogenate was prepared as follows. To 14 grams of spleen and 8 grams of brain, 9 times tissue-weight M6B8 medium (MSD AH) with 10 μM HEPES (Sigma H3375-250G) was added. The tissue was homogenized using a blender, followed by shaking with small glass beads for 5 minutes. During homogenizing and subsequent processing, organ-pulp was kept on ice. The organ-pulp was centrifuged 1 hour at 3200×g at 4° C. Supernatant was passed over a 0.22 μm filter. The filtered homogenate was stored at −70° C. until use. These three gilts received an intramuscular injection of 5 mL inoculum (two injections of 2.5 mL each in the left and right neck).
[0395] The other three gilts were infected with an inoculum of serum obtained from the same pig at the time of necropsy. The serum was filtered over a 0.22 μm filter prior to injection. These three gilts received an intramuscular injection of 5 mL inoculum (two injections of 2.5 mL each in the left and right neck).
[0396] The quantitative amount of CTAPV in the inoculums was determined by qRT-PCR as described in Example 10.
Serum Collection
[0397] Serum was collected prior to infection of the gilts, and at t=10 days after insemination. Serum was also collected from newborn piglets within hours after birth. Blood was collected (Vacuolette 5/8 ml Sep Clot Activator ref: 455071; Greiner Bio-one) and serum was obtained by centrifugation 20 minutes at 3,000×g at 4° C. RNA extraction and qRT-PCR were performed as described in the section “SYBR Green One-Step qRT-PCR”, Example 10.
Results
[0398] The mixed homogenate of spleen and brain used for infection of the first three gilts contained 4.5 E+02 genomes copies per 5 μl of the extracted RNA. This equals 2.3 E+04 genome copies per mL in the homogenate that was used for infection of the gilts.
[0399] The serum inoculum used for infection of the other three gilts contained 1.2 E+04 genomes per 5 μl of the extracted RNA, which equals 6.0E+05 genome copies per mL that was used for infection of gilts. Table 11 presents the quantitative amount (genomes per mL serum) at day 10 post infection as determined by qRT-PCR results. Five out of six gilts gave birth to piglets with severe congenital tremor type A-II. One gilt, the gilt with a relatively low virus quantity in the serum at t=10 days after infection, gave birth to a relatively healthy litter where only 2 piglets with mild symptoms were observed. Litter information scored after farrowing is presented in Table 11. An increased incidence of splay legs was associated with clinical tremor, as described by M. White (http://www.nadis.org.uk/bulletins/congenital-tremor.aspx?altTemplate=PDF).
[0400] Presence of CTAPV in three piglets per litter (those with severe clinical tremor, except those piglets born from gilt 44 which showed no clinical tremor) was tested by the qRT-PCR test described in Example 10. The number of CTAPV positive piglets is depicted in Table 11
TABLE-US-00018 TABLE 11 RNA quantitation in gilt serum samples on day 10 after inoculation, and litter information. CT type A-II piglets born (live RNA copies/ piglets - # severe/# mild/# CTAPV presence in piglets Gilt Infection mL* no symptoms) (piglets tested/piglets positive) 42 Organ 1.1E+05 9 - 4/4/1 3/3 homogenate 43 Organ 4.5E+04 15 - 7/7/1 3/3 homogenate 44 Organ 5.5E+02 14 - 0/2/12 3/2 homogenate 45 Serum 1.2E+05 18 - 9/8/0 3/3 (1 not scored) 47 Serum 1.3E+05 16 - 11/4/1 3/3 48 Serum 1.2E+05 15 - 8/7/0 3/3 *Means of duplicate experiments; Column RNA copies/mL shows the number of copies of the virus in the original sample (serum).
Example 13
[0401] Infection of Pregnant Gilts with CTAPV Variant 1B Obtained from “Shaking Piglets” and Effect on Newborn Piglets: CTAPV Negative Sperm
Animals
[0402] Three gilts were obtained from a SPF/high health farm. Gilts were inseminated via artificial insemination with CTAPV negativeregnancy was confirmed at day 28 of gestation using ultrasound. All gilts gave birth to a litter of piglets on day 114 or day 115 of gestation.
Infection
[0403] The three gilts were infected with an inoculum of serum obtained from pig 371 at the time of necropsy (see example 12). The serum was filtered over a 0.22 μm filter prior to injection. Three gilts received an intramuscular injection of 5 mL inoculum (two injections of 2.5 mL each in the left and right neck) at 32 days of gestation.
[0404] The quantitative amount of CTAPV in the inoculum was determined by qRT-PCR as described in Examples 10 and 12.
Serum Collection
[0405] Serum was collected prior to infection of the gilts, and at t=10 days after insemination. Serum was also collected from newborn piglets within hours after birth. Blood was collected (Vacuolette 5/8 ml Sep Clot Activator ref: 455071; Greiner Bio-one) and serum was obtained by centrifugation 20 minutes at 3,000×g at 4° C. RNA extraction and qRT-PCR were performed as described in the section “SYBR Green One-Step qRT-PCR”, Example 10.
Results
[0406] Table 12 presents the quantitative amount (genomes per mL serum) at day 10 post infection as determined by qRT-PCR results. Two of three gilts gave birth to piglets with mild congenital tremor type A-II. One gilt, the gilt with a relatively low virus quantity in the serum at t=10 days after infection, gave birth to a healthy litter. Litter information scored after farrowing is presented in Table 11.
[0407] Presence of CTAPV in piglets with CT type A-II was confirmed by the qRT-PCR test described in Example 10. The number of CTAPV positive piglets is depicted in Table 12. An increased incidence of splay legs was associated with clinical tremor.
TABLE-US-00019 TABLE 12 RNA quantitation in gilt serum samples on day 10 after inoculation, and litter information. CT type A-II piglets born (live RNA copies/ piglets -# severe/# mild/# CTAPV presence in piglets Gilt Infection mL* no symptoms) (piglets tested/piglets positive) 49 Serum 5.85E+02 13 - 0/0/13 13/0 50 Serum 1.39E+04 13 - 3/8/2 13/11 51 Serum 2.32E+04 15 - 1/12/2 15/15 *Means of duplicate experiments; Column RNA copies/mL shows the number of copies of the virus in the original sample (serum).