- Email: [email protected]
A high-resolution melting (HRM) assay for the differentiation between Israeli field and Neethling vaccine lumpy skin disease viruses
Accepted Manuscript Title: A High-resolution melting (HRM) Assay for the Differentiation between Israeli Field and Neethling Vaccine Lumpy Skin Disease Viruses Author: Sophia Menasherow Oran Erster Marisol Rubinstein-Giuni Anita Kovtunenko Evgeny Eyngor Boris Gelman Evgeny Khinich Yehuda Stram PII: DOI: Reference:
S0166-0934(16)00041-0 http://dx.doi.org/doi:10.1016/j.jviromet.2016.02.008 VIRMET 12961
To appear in:
Journal of Virological Methods
Received date: Revised date: Accepted date:
25-6-2015 17-2-2016 17-2-2016
Please cite this article as: Menasherow, Sophia, Erster, Oran, Rubinstein-Giuni, Marisol, Kovtunenko, Anita, Eyngor, Evgeny, Gelman, Boris, Khinich, Evgeny, Stram, Yehuda, A High-resolution melting (HRM) Assay for the Differentiation between Israeli Field and Neethling Vaccine Lumpy Skin Disease Viruses.Journal of Virological Methods http://dx.doi.org/10.1016/j.jviromet.2016.02.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A High-resolution melting (HRM) Assay for the Differentiation between Israeli Field and Neethling Vaccine Lumpy Skin Disease Viruses Sophia Menasherow#, Oran Erster#, Marisol Rubinstein-Giuni, Anita Kovtunenko, Evgeny Eyngor, Boris Gelman, Evgeny Khinich, Yehuda Stram* [email protected] Virology Division, Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, Israel *
Corresponding author at: Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, Israel.
Tel: +972 3 968 1697; Fax: +972 3 968 1788 #
SM and OE contributed equally to this work
1
Highlights This manuscript is a continuation and extension of our previous paper, describing an assay able to distinguish between field and vaccine Lumpy skin disease viruses. The earlier test was based on; sequencing alignment, RFLP and gradient PCR which are time consuming. This work is based on the finding that the Neethling vaccine virus carry a 27 base deletion in the virus LSDV126 gene. A pair of primers flanking the deleted fragment were designed and used to test the melting profiles of both the Neethling vaccine and field strain. It was found that there is a 0.50C difference in the Tm’s between amplicons originated from the attenuated and virulent viruses which is easily detectable (Fig.2). By applying this newly developed system, it could be shown that viruses isolated from immunize cattle that developed disease symptoms behave in the assay similar to the vaccine virus (Table 1). To validate these findings the LSDV126 gene of these viruses were sequenced (Data not shown) and found to carry the 27 bp deletion whereas virus isolates from naturally infected cattle were found to carry the complete gene.
2
Abstract Lumpy skin disease (LSD) is a constant threat to the Middle East including the State of Israel. During vaccination programs it is essential for veterinary services and farmers to be able to distinguish between animals affected by the cattle-borne virulent viruses and vaccinated animals, subsequently affected by the vaccine strain. This study describes an improved high resolution-melting (HRM) test that exploits a 27 base pair (bp) fragment of the LSDV126 extracellular enveloped virion (EEV) gene that is present in field viruses but is absent from the Neethling vaccine strain. This difference leads to ~ 0.5⁰C melting point change in the HRM assay, when testing the quantitative PCR (qPCR) products generated from the virulent field viruses compared to the attenuated vaccine. By exploiting this difference, it could be shown using the newly developed HRM assay that virus isolated from vaccinated cattle that developed disease symptoms behave similarly to vaccine virus control, indicating that the vaccine virus can induce disease symptoms. This assay is not only in full agreement with the previously published PCR gradient and restriction fragment length polymorphism (RFLP) tests but it is faster with, fewer steps, cheaper and dependable.
Keywords: Lumpy Skin Disease Virus; high-resolution melting; HRM; LSDV126 gene; EEV gene
3
Lumpy skin disease (LSD) is an infectious, eruptive disease of cattle, which is economically important but rarely fatal (Davies, 1991; Coetzer et al., 1994; Fenner, 1996). The clinical onset is characterized by fever, loss of appetite, increased nasal secretions and watery eyes; it is especially severe in cows at the peak of lactation, and causes a sharp drop in milk production. After several days skin nodules appear; they may cover the entire body. Both cows and bulls may exhibit temporary or permanent infertility. Morbidity ranges from 3 to 85%, whereas mortality is usually low, ranging from 1 to 3%, but in rare cases can reach up to as high
as 85% (Prozesky & Barnard, 1982; Kitching & Taylor, 1985; Barnard et al., 1994). The disease is present in southern and eastern Africa, and in recent years it has spread northwestward through the continent into sub-Saharan West Africa and into the Middle East, where it was first detected in Egypt in 1988 and in Israel in 1989 (House et al., 1990). In 2006, the disease reappeared in Egypt and spread into Israel and, probably, to other Middle-Eastern countries (Brenner et al., 2009). LSD outbreaks have been reported in Kuwait in 1991, Lebanon in 1993, Yemen in 1995, the United Arab Emirates in 2000, Bahrain in 2003, Israel in 2006-7, Oman in 2010, Israel 2012-13, Turkey 2013, Cyprus 2014 and Egypt 2014 (Yeruham et al., 1995) LSD Virus (LSDV) is an enveloped virus from the Capripox genus of the Poxviridae family, with a genome of approximately 150,000 bp long, comprising a double-stranded DNA, covalently cross-linked at the ends, and with a typical poxvirus geomorphology. Other closely related viruses of the same genus are sheep pox and goat pox (Bhanuprakasha et al., 2006). The virus is suspected to be transmitted by biting insects, especially blood-feeding ones such as mosquitoes (Chihota et al., 2001) or Stomoxys flies. Recently it was suggested that hard ticks might be involved in LSDV transmission (Tuppurainen et al., 2013; Lubinga et al., 2015). All three Capripoxvirus genome sequences are highly conserved, sharing more than 95% sequence homology (Tulman et al., 2002; Kara et al., 2003).
4
In July 2012, a massive LSD outbreak began in the Northeastern part of Israel. In order to strengthen the efforts to control the disease, the Israeli Veterinary Services initiated the use of a Neethling vaccine strain, LSDV-based vaccine. One of the drawbacks of using the Neethling vaccine stems from its ability to induce mild disease symptoms following vaccination. The present communication describes the development and use of an improved high-resolution melting (HRM) system that provides a fast and easy way to distinguish between field and attenuated LSD viruses, based on the previously published data of (Menasherow et al., 2014). A single HRM test, avoiding the use of multiple steps of sequencing, gradient PCR and RFLP assays, was established. It takes advantage of the presence or absence of a 27-bp segment in the LSDV126 gene, and amplification of this region from the field and vaccine LSDVs will produce fragments that differ in their melting temperatures (Tm) by 0.4-0.50C.
Two primers,
5’-AATCGTTGTTACAACTCAAA-3’ and 5’-ATAGTTTGACTCGGAATTAT-3’, directed at positions 116582 and 116652, respectively, in acc. AF409137.1, flanking the 27 bp fragment in the LSDV126 gene, were selected specially for the HRM assay, using Oligo 6.5 software (MBI, Colorado Springs, USA). To confirm that the primers can recognize most if not all LSDV isolates, BLAST analysis was performed. The analysis indicated that there is 100% homology between each of the primers and all Israeli and published LSDV sequences (data not shown). To evaluate the reaction sensitivity, qPCR of 10-fold dilutions of 1ng of a 755 bp long amplicon from position 116172 to position 116927 at acc.
AF409137.1, which includes the HRM
amplicon, from the field isolate 289 was performed. The result indicated that the reaction can detect ~1x10-7 ng/reaction, equivalent to ~20 copies of viral DNA (Fig. 1). Similar sensitivities (~20 copies) were obtained using the 345 vaccine isolate as template (data not shown). HRM analyses were performed with the Type-it HRM PCR Kit (Qiagen, Hilden, Germany) containing the EVAGreen fluorophore, according to the manufacturer's instructions. Briefly: the reaction mixture comprised 5 l of viral DNA, 10 ml of PCR mix, and 1 l of each
5
primer in a total volume of 20 l. Reaction conditions were: 95⁰C for 4 min, followed by 40 cycles of 95⁰C for 10 s and 55⁰C for 1 min. The melting profiles were established from 70⁰C to 85⁰C, in increments of 0.1⁰C, using the CFX 96 instrument (Bio-Rad, Hercules, USA) and each reaction was run in a duplicate. The DNA samples were from full blood and in some cases, buffy coat, skin and tears, using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany), as well as Viral Gene-Spin Viral DNA/RNA Extraction Kit (iNtRON, Kyungki-Do, South Korea). Statistical analysis of the HRM results was performed using GraphPad Prism 5.03 (GraphPad software, La Jolla. USA). There were distinct differences in the melting peaks between the field and vaccine LSDVs, (Fig. 2a) and in their melting curves (Fig. 2b). This analysis revealed a difference of 0.4-0.50C in the melting points, in the vaccine and virulent strain, respectively. To further validate the method, LSDV DNA isolated from Neethling-vaccinated cattle that developed skin lesions, as well as samples from naturally infected cattle, were examined. Table 1 presents the results of the experiment that included 5 LSDV isolates from naturally infected cattle as well as 6 isolates from Neethling vaccinated cattle. All viruses from the immunized cattle (Table 1) showed a Tm similar to the vaccine virus 75.60C in the range of 75.2-75.60C. In contrast, isolates from diseased, naturally infected but unvaccinated cattle showed a higher Tm in the range of 75.9-76.1 as expected for the virulent virus (Fig. 2a, b). To statistically evaluate the results, t and F tests were performed. The unpaired t-test compared the means of the melt point obtained from the vaccinated animals group versus that of the field virus-infected animals group, assuming that they have similar variances. The P value obtained in this test reflects the likelihood that the difference between the vaccinated and infected groups is due to chance. If the assumption is true, and the P value is <0.05, one can be >95% sure that there is true difference between the Tm of the two virus variants. Since in this case the P value was <0.0001, much smaller than 0.05, the
6
difference is most likely real and reflects a true difference. The above unpaired t-test depends on the assumption that the two Tm groups come from two populations that have equal standard deviations, or the same variances. A P value >0.05 in the F-test indicates that the variances are not significant. The obtained P value in the present F test was 0.4898, indicating that the t test is valid. To further validate the assay, the fragments that carry the LSDV126 gene from two isolates from vaccinated and naturally infected cattle were sequenced and found to be identical to the original Neethling vaccine virus (Data not shown). This study demonstrated the use of the HRM assay for differentiating between LSDV virulent or field strains, and the Neethling vaccine strain. Previously, a set of tests for distinguishing between the two viruses based on 3 different analyses, including LSDV126 gene sequencing, gradient PCR and RFLP,
were developed
(Menasherow et al., 2014). To improve the speed and render the analysis easier to perform, as the published assays are somewhat laborious, an HRM assay was developed, based on the differences in the length of the LSDV126 gene between the field and Neethling vaccine viruses. The design of the primers was based on their ability to amplify fragments that differed only in this deletion. The results presented in Fig. 2 show a mean difference of ~ 0.5⁰C in Tm between the fragments from the two respective sources, thus demonstrating the successful design of the detection system. It could be argued that a 0.50C difference in the Tm revealed by the HRM analysis is not sufficient enough for discriminating between two forms of genes but it is well documented that differences smaller than ~0.50C are satisfactory for virus genotyping, as was done with classical swine fever virus (Titov et al. 2015). Similarly, canine parvovirus strains were also differentiated by HRM and in a few cases, the diversities in the Tm were smaller than ~0.5 0C among the different isolates (Bingga et al. 2014). Moreover, statistical analysis performed on the results shown in Table 1 strongly corroborates the view that the differences are reproducible
7
and reliable. The work presented in this paper validates the HRM methodology: samples that were isolated from symptomatic vaccinated cattle in 2013 proved to be of the vaccine strain as verified by sequencing the LSDV126 gene, whereas samples isolated from naturally infected cattle from 2012-13 were identified as originating from the virulent field virus. Therefore, this assay not only provides an easy test involving a single experimental step, compared with the two or more described previously (Menasherov et al., 2014), but it also provides a reliable mean to distinguish between the two forms of the virus. Given its simplicity, the assay could also be adapted for on-site testing that will speed up the detection of the virus in infected cattle, eliminating the need for sending samples to a central dedicate laboratory.
8
References Barnard, B.J., Munz, E., Dumbell, K. & Prozesky, L. 1994. Lumpy skin disease. In Infectious Diseases of Livestock with Special Reference to Southern Africa, vol. 1. pp. 604–612. Edited by J. A. W. Coetzer, G. R. Thomson & R. C. Tustin. Cape Town: Oxford University Press. Bhanuprakasha, V., Indranib, B.K., Hosamania, M. Singha, R.K. 2006. The current status of sheep pox disease. Comp Immunol Microbiol Infect Dis 29, 27–60. Bingga, G., Liu, Z., Zhang, J., Zhu, Y., Lin, L., Ding, S., Guo, P., 2014. High resolution melting curve analysis as a new tool for rapid identification of canine parvovirus type 2 strains. Molecular and Cellular Probes, 28, 271–278. Brenner, J., Bellaiche, M., Gross, E., Elad, D., Oved, Z., Haimovitz, M., Wasserman, A., Friedgut, O., Stram, Y. & other authors 2009. Appearance of skin lesions in cattle populations vaccinated against lumpy skin disease: statutory challenge. Vaccine 27, 1500–1503. Chihota, C.M., Rennie, L.F., Kitching, R.P., Mellor, P.S., 2001. Mechanical transmission of lumpy skin disease virus by Aedes aegypti (Diptera: Culicidae). Epidemiol Infect 126, 317–321. Coetzer, J.A.W., Thomson, G.R., Tustin, R.C., 1994. Poxviridae. In Infectious Diseases of Livestock, vol. 1. pp. 601–603. Edited by J. A. W. Coetzer, G. R. Thomson & R. C. Tustin. Cape Town: Oxford University Press. Davies, F.G., 1991. Lumpy skin disease of cattle: a growing problem in Africa and the Near East. World Anim. Rev. 68, 37–42. Fenner, F. 1996. Poxviruses. In: Fields Virology. pp. 2673–2702. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: Lippincott-Raven. House, J.A., Wilson, T.M., El Nakashly, S., Karim, I.A., Ismail, I., El Danaf, N., Moussa, A. M., Ayoub, N.N., 1990. The isolation of lumpy skin disease virus and bovine herpesvirus-4 from cattle in Egypt. J. Vet. Diagn. Invest. 2, 111–115. Kara, P.D., Afonso, C.L., Wallace, D.B., Kutish, G. F., Abolnik, C., Lu, Z., Vreede, F. T., Taljaard, L. C. F., Zsak, A. & other authors 2003. Comparative sequence analysis of the South African vaccine strain and two virulent field isolates of lumpy skin disease virus. Arch. Virol. 148, 1335–1356. Kitching, R.P. & Taylor, W.P., 1985. Transmission of capripox viruses. Res. Vet. Sci. 39, 196– 199. Lubinga, J.C., Tuppurainen, E.S., Mahlare, R., Coetzer, J.A., Stoltsz, W.H., Venter, E.H., 2015. Evidence of transstadial and mechanical transmission of lumpy skin disease virus by Amblyomma hebraeum ticks. Transbound Emerg Dis (Epub ahead of print) Menasherow S, Rubinstein-Giuni M, Kovtunenko A, Eyngor Y, Fridgut O, Rotenberg D, Khinich Y, Stram Y., 2014. Development of an assay to differentiate between virulent and vaccine strains of lumpy skin disease virus (LSDV). J. Virol. Meth. 199, 95–101. Prozesky, L. & Barnard, B.J.H., 1982. A study of the pathology of lumpy skin disease in cattle. Onderst J. Vet. Res. 49, 167–175. Roberts, K.L. & Smith, G.L., 2008. Vaccinia virus morphogenesis and dissemination. Trends Microbiol. 16, 472–479. Roberts, K.L. & Smith, G.L., 2008. Vaccinia virus morphogenesis and dissemination. Trends in Microbiology, 16(10), pp.472–479. Smith, G.L., Vanderplasschen, A. Law, M., 2002. The formation and function of extracellular enveloped vaccinia virus. Journal of General Virology, 83, 2915–2931. Titov, I., Tsybanov, S. & Malogolovkin, A., 2015. Genotyping of classical swine fever virus using high-resolution melt analysis. Journal of Virological Methods, 224, pp.53–57. 9
Tulman, E.R., Afonso, C.L., Lu, Z., Zsak, L., Sur, J. H., Sandybaev, N.T., Kerembekova, U.Z., Zaitsev, V.L., Kutish, G.F. & other authors 2002. The genomes of sheeppox and goatpox viruses. J. Virol. 76, 6054–6061. Tuppurainen, E.S., Venter, E. H., Coetzer, J.A., 2005. The detection of lumpy skin disease virus in samples of experimentally infected cattle using different diagnostic techniques. Onderst. J. Vet. Res. 72, 153–164. Tuppurainen, E.S., Oura, C.A., 2012. Lumpy skin disease: an emerging threat to Europe, the Middle East and Asia. Transbound. Emerg. Dis. 59, 40–48. Tuppurainen, E.S., Lubinga, J.C., Stoltsz, W.H., Troskie, M., Carpenter, S.T., Coetzer, J.A., Venter, E.H. & Oura, C.A., 2013. Evidence of vertical transmission of lumpy skin disease virus in Rhipicephalus decoloratus ticks. Ticks. Tick. Borne. Dis. 4, 329–333. Von Backstrom, U.. 1945. Ngamiland cattle disease. Preliminary report on a new disease, the aetiological agent probably being of an infectious nature. J. S. Afr. Vet Med. Assoc 16, 29–35. Yeruham, I., Nir, O., Braverman, Y., Davidson, M., Grinstein, H., Haymovitch, M., Zamir, O., 1995. Spread of lumpy skin disease in Israeli dairy herds. Vet. Rec. 137, 91–93.
10
Figure Captions Figure 1. Sensitivity assay of the HRM reaction. A qPCR test of a tenfold dilution of 1 ng of the LSDV, 755 bp long amplicon, between positions 116172 and 116927, at acc. AF409137.1, carrying the HRM amplicon.
11
Figure 2. HRM analysis conducted on Neethling vaccine strain and Ein Zurim-06 field viruses. a) Melting peaks. b) Normalized melting curve.
12
Tables Table 1. Tm analysis of the HRM fragment performed on field-infected and Neethlingvaccinated isolates cattle. Tm values of field infected cattle isolates, Column A. and Neethling vaccinated cattle, Column B. The t and F-tests were performed using GraphPad Prism software, version 5.03 (www.graphpad.com/).
Sample 112 289 331 378 435 304 630 Ein Zurim-06
Field strain Melt temp. (C0) 76 76.1 76.1 76.1 76 76 76 76
Ct 20 22.3 16.1 29.2 24.4 26.5 25.6 14
Sample 317 332 432 343 345 346 347 VAC H2O
Unpaired t test P value < 0.0001 Are means significant different? (P < 0.05) Yes Field virus vs Vaccine virus F test to compare variances P value P value summary Are variances significantly different?
0.4898 ns No
13
Vaccine strain Melt temp. (C0) 75.5 75.6 75.6 75.6 75.6 75.5 75.5 75.6 None
Ct 23.5 23.9 27.4 24.3 22.4 30.4 29.4 26.3 N/A
S0166-0934(16)00041-0 http://dx.doi.org/doi:10.1016/j.jviromet.2016.02.008 VIRMET 12961
To appear in:
Journal of Virological Methods
Received date: Revised date: Accepted date:
25-6-2015 17-2-2016 17-2-2016
Please cite this article as: Menasherow, Sophia, Erster, Oran, Rubinstein-Giuni, Marisol, Kovtunenko, Anita, Eyngor, Evgeny, Gelman, Boris, Khinich, Evgeny, Stram, Yehuda, A High-resolution melting (HRM) Assay for the Differentiation between Israeli Field and Neethling Vaccine Lumpy Skin Disease Viruses.Journal of Virological Methods http://dx.doi.org/10.1016/j.jviromet.2016.02.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A High-resolution melting (HRM) Assay for the Differentiation between Israeli Field and Neethling Vaccine Lumpy Skin Disease Viruses Sophia Menasherow#, Oran Erster#, Marisol Rubinstein-Giuni, Anita Kovtunenko, Evgeny Eyngor, Boris Gelman, Evgeny Khinich, Yehuda Stram* [email protected] Virology Division, Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, Israel *
Corresponding author at: Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, Israel.
Tel: +972 3 968 1697; Fax: +972 3 968 1788 #
SM and OE contributed equally to this work
1
Highlights This manuscript is a continuation and extension of our previous paper, describing an assay able to distinguish between field and vaccine Lumpy skin disease viruses. The earlier test was based on; sequencing alignment, RFLP and gradient PCR which are time consuming. This work is based on the finding that the Neethling vaccine virus carry a 27 base deletion in the virus LSDV126 gene. A pair of primers flanking the deleted fragment were designed and used to test the melting profiles of both the Neethling vaccine and field strain. It was found that there is a 0.50C difference in the Tm’s between amplicons originated from the attenuated and virulent viruses which is easily detectable (Fig.2). By applying this newly developed system, it could be shown that viruses isolated from immunize cattle that developed disease symptoms behave in the assay similar to the vaccine virus (Table 1). To validate these findings the LSDV126 gene of these viruses were sequenced (Data not shown) and found to carry the 27 bp deletion whereas virus isolates from naturally infected cattle were found to carry the complete gene.
2
Abstract Lumpy skin disease (LSD) is a constant threat to the Middle East including the State of Israel. During vaccination programs it is essential for veterinary services and farmers to be able to distinguish between animals affected by the cattle-borne virulent viruses and vaccinated animals, subsequently affected by the vaccine strain. This study describes an improved high resolution-melting (HRM) test that exploits a 27 base pair (bp) fragment of the LSDV126 extracellular enveloped virion (EEV) gene that is present in field viruses but is absent from the Neethling vaccine strain. This difference leads to ~ 0.5⁰C melting point change in the HRM assay, when testing the quantitative PCR (qPCR) products generated from the virulent field viruses compared to the attenuated vaccine. By exploiting this difference, it could be shown using the newly developed HRM assay that virus isolated from vaccinated cattle that developed disease symptoms behave similarly to vaccine virus control, indicating that the vaccine virus can induce disease symptoms. This assay is not only in full agreement with the previously published PCR gradient and restriction fragment length polymorphism (RFLP) tests but it is faster with, fewer steps, cheaper and dependable.
Keywords: Lumpy Skin Disease Virus; high-resolution melting; HRM; LSDV126 gene; EEV gene
3
Lumpy skin disease (LSD) is an infectious, eruptive disease of cattle, which is economically important but rarely fatal (Davies, 1991; Coetzer et al., 1994; Fenner, 1996). The clinical onset is characterized by fever, loss of appetite, increased nasal secretions and watery eyes; it is especially severe in cows at the peak of lactation, and causes a sharp drop in milk production. After several days skin nodules appear; they may cover the entire body. Both cows and bulls may exhibit temporary or permanent infertility. Morbidity ranges from 3 to 85%, whereas mortality is usually low, ranging from 1 to 3%, but in rare cases can reach up to as high
as 85% (Prozesky & Barnard, 1982; Kitching & Taylor, 1985; Barnard et al., 1994). The disease is present in southern and eastern Africa, and in recent years it has spread northwestward through the continent into sub-Saharan West Africa and into the Middle East, where it was first detected in Egypt in 1988 and in Israel in 1989 (House et al., 1990). In 2006, the disease reappeared in Egypt and spread into Israel and, probably, to other Middle-Eastern countries (Brenner et al., 2009). LSD outbreaks have been reported in Kuwait in 1991, Lebanon in 1993, Yemen in 1995, the United Arab Emirates in 2000, Bahrain in 2003, Israel in 2006-7, Oman in 2010, Israel 2012-13, Turkey 2013, Cyprus 2014 and Egypt 2014 (Yeruham et al., 1995) LSD Virus (LSDV) is an enveloped virus from the Capripox genus of the Poxviridae family, with a genome of approximately 150,000 bp long, comprising a double-stranded DNA, covalently cross-linked at the ends, and with a typical poxvirus geomorphology. Other closely related viruses of the same genus are sheep pox and goat pox (Bhanuprakasha et al., 2006). The virus is suspected to be transmitted by biting insects, especially blood-feeding ones such as mosquitoes (Chihota et al., 2001) or Stomoxys flies. Recently it was suggested that hard ticks might be involved in LSDV transmission (Tuppurainen et al., 2013; Lubinga et al., 2015). All three Capripoxvirus genome sequences are highly conserved, sharing more than 95% sequence homology (Tulman et al., 2002; Kara et al., 2003).
4
In July 2012, a massive LSD outbreak began in the Northeastern part of Israel. In order to strengthen the efforts to control the disease, the Israeli Veterinary Services initiated the use of a Neethling vaccine strain, LSDV-based vaccine. One of the drawbacks of using the Neethling vaccine stems from its ability to induce mild disease symptoms following vaccination. The present communication describes the development and use of an improved high-resolution melting (HRM) system that provides a fast and easy way to distinguish between field and attenuated LSD viruses, based on the previously published data of (Menasherow et al., 2014). A single HRM test, avoiding the use of multiple steps of sequencing, gradient PCR and RFLP assays, was established. It takes advantage of the presence or absence of a 27-bp segment in the LSDV126 gene, and amplification of this region from the field and vaccine LSDVs will produce fragments that differ in their melting temperatures (Tm) by 0.4-0.50C.
Two primers,
5’-AATCGTTGTTACAACTCAAA-3’ and 5’-ATAGTTTGACTCGGAATTAT-3’, directed at positions 116582 and 116652, respectively, in acc. AF409137.1, flanking the 27 bp fragment in the LSDV126 gene, were selected specially for the HRM assay, using Oligo 6.5 software (MBI, Colorado Springs, USA). To confirm that the primers can recognize most if not all LSDV isolates, BLAST analysis was performed. The analysis indicated that there is 100% homology between each of the primers and all Israeli and published LSDV sequences (data not shown). To evaluate the reaction sensitivity, qPCR of 10-fold dilutions of 1ng of a 755 bp long amplicon from position 116172 to position 116927 at acc.
AF409137.1, which includes the HRM
amplicon, from the field isolate 289 was performed. The result indicated that the reaction can detect ~1x10-7 ng/reaction, equivalent to ~20 copies of viral DNA (Fig. 1). Similar sensitivities (~20 copies) were obtained using the 345 vaccine isolate as template (data not shown). HRM analyses were performed with the Type-it HRM PCR Kit (Qiagen, Hilden, Germany) containing the EVAGreen fluorophore, according to the manufacturer's instructions. Briefly: the reaction mixture comprised 5 l of viral DNA, 10 ml of PCR mix, and 1 l of each
5
primer in a total volume of 20 l. Reaction conditions were: 95⁰C for 4 min, followed by 40 cycles of 95⁰C for 10 s and 55⁰C for 1 min. The melting profiles were established from 70⁰C to 85⁰C, in increments of 0.1⁰C, using the CFX 96 instrument (Bio-Rad, Hercules, USA) and each reaction was run in a duplicate. The DNA samples were from full blood and in some cases, buffy coat, skin and tears, using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany), as well as Viral Gene-Spin Viral DNA/RNA Extraction Kit (iNtRON, Kyungki-Do, South Korea). Statistical analysis of the HRM results was performed using GraphPad Prism 5.03 (GraphPad software, La Jolla. USA). There were distinct differences in the melting peaks between the field and vaccine LSDVs, (Fig. 2a) and in their melting curves (Fig. 2b). This analysis revealed a difference of 0.4-0.50C in the melting points, in the vaccine and virulent strain, respectively. To further validate the method, LSDV DNA isolated from Neethling-vaccinated cattle that developed skin lesions, as well as samples from naturally infected cattle, were examined. Table 1 presents the results of the experiment that included 5 LSDV isolates from naturally infected cattle as well as 6 isolates from Neethling vaccinated cattle. All viruses from the immunized cattle (Table 1) showed a Tm similar to the vaccine virus 75.60C in the range of 75.2-75.60C. In contrast, isolates from diseased, naturally infected but unvaccinated cattle showed a higher Tm in the range of 75.9-76.1 as expected for the virulent virus (Fig. 2a, b). To statistically evaluate the results, t and F tests were performed. The unpaired t-test compared the means of the melt point obtained from the vaccinated animals group versus that of the field virus-infected animals group, assuming that they have similar variances. The P value obtained in this test reflects the likelihood that the difference between the vaccinated and infected groups is due to chance. If the assumption is true, and the P value is <0.05, one can be >95% sure that there is true difference between the Tm of the two virus variants. Since in this case the P value was <0.0001, much smaller than 0.05, the
6
difference is most likely real and reflects a true difference. The above unpaired t-test depends on the assumption that the two Tm groups come from two populations that have equal standard deviations, or the same variances. A P value >0.05 in the F-test indicates that the variances are not significant. The obtained P value in the present F test was 0.4898, indicating that the t test is valid. To further validate the assay, the fragments that carry the LSDV126 gene from two isolates from vaccinated and naturally infected cattle were sequenced and found to be identical to the original Neethling vaccine virus (Data not shown). This study demonstrated the use of the HRM assay for differentiating between LSDV virulent or field strains, and the Neethling vaccine strain. Previously, a set of tests for distinguishing between the two viruses based on 3 different analyses, including LSDV126 gene sequencing, gradient PCR and RFLP,
were developed
(Menasherow et al., 2014). To improve the speed and render the analysis easier to perform, as the published assays are somewhat laborious, an HRM assay was developed, based on the differences in the length of the LSDV126 gene between the field and Neethling vaccine viruses. The design of the primers was based on their ability to amplify fragments that differed only in this deletion. The results presented in Fig. 2 show a mean difference of ~ 0.5⁰C in Tm between the fragments from the two respective sources, thus demonstrating the successful design of the detection system. It could be argued that a 0.50C difference in the Tm revealed by the HRM analysis is not sufficient enough for discriminating between two forms of genes but it is well documented that differences smaller than ~0.50C are satisfactory for virus genotyping, as was done with classical swine fever virus (Titov et al. 2015). Similarly, canine parvovirus strains were also differentiated by HRM and in a few cases, the diversities in the Tm were smaller than ~0.5 0C among the different isolates (Bingga et al. 2014). Moreover, statistical analysis performed on the results shown in Table 1 strongly corroborates the view that the differences are reproducible
7
and reliable. The work presented in this paper validates the HRM methodology: samples that were isolated from symptomatic vaccinated cattle in 2013 proved to be of the vaccine strain as verified by sequencing the LSDV126 gene, whereas samples isolated from naturally infected cattle from 2012-13 were identified as originating from the virulent field virus. Therefore, this assay not only provides an easy test involving a single experimental step, compared with the two or more described previously (Menasherov et al., 2014), but it also provides a reliable mean to distinguish between the two forms of the virus. Given its simplicity, the assay could also be adapted for on-site testing that will speed up the detection of the virus in infected cattle, eliminating the need for sending samples to a central dedicate laboratory.
8
References Barnard, B.J., Munz, E., Dumbell, K. & Prozesky, L. 1994. Lumpy skin disease. In Infectious Diseases of Livestock with Special Reference to Southern Africa, vol. 1. pp. 604–612. Edited by J. A. W. Coetzer, G. R. Thomson & R. C. Tustin. Cape Town: Oxford University Press. Bhanuprakasha, V., Indranib, B.K., Hosamania, M. Singha, R.K. 2006. The current status of sheep pox disease. Comp Immunol Microbiol Infect Dis 29, 27–60. Bingga, G., Liu, Z., Zhang, J., Zhu, Y., Lin, L., Ding, S., Guo, P., 2014. High resolution melting curve analysis as a new tool for rapid identification of canine parvovirus type 2 strains. Molecular and Cellular Probes, 28, 271–278. Brenner, J., Bellaiche, M., Gross, E., Elad, D., Oved, Z., Haimovitz, M., Wasserman, A., Friedgut, O., Stram, Y. & other authors 2009. Appearance of skin lesions in cattle populations vaccinated against lumpy skin disease: statutory challenge. Vaccine 27, 1500–1503. Chihota, C.M., Rennie, L.F., Kitching, R.P., Mellor, P.S., 2001. Mechanical transmission of lumpy skin disease virus by Aedes aegypti (Diptera: Culicidae). Epidemiol Infect 126, 317–321. Coetzer, J.A.W., Thomson, G.R., Tustin, R.C., 1994. Poxviridae. In Infectious Diseases of Livestock, vol. 1. pp. 601–603. Edited by J. A. W. Coetzer, G. R. Thomson & R. C. Tustin. Cape Town: Oxford University Press. Davies, F.G., 1991. Lumpy skin disease of cattle: a growing problem in Africa and the Near East. World Anim. Rev. 68, 37–42. Fenner, F. 1996. Poxviruses. In: Fields Virology. pp. 2673–2702. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: Lippincott-Raven. House, J.A., Wilson, T.M., El Nakashly, S., Karim, I.A., Ismail, I., El Danaf, N., Moussa, A. M., Ayoub, N.N., 1990. The isolation of lumpy skin disease virus and bovine herpesvirus-4 from cattle in Egypt. J. Vet. Diagn. Invest. 2, 111–115. Kara, P.D., Afonso, C.L., Wallace, D.B., Kutish, G. F., Abolnik, C., Lu, Z., Vreede, F. T., Taljaard, L. C. F., Zsak, A. & other authors 2003. Comparative sequence analysis of the South African vaccine strain and two virulent field isolates of lumpy skin disease virus. Arch. Virol. 148, 1335–1356. Kitching, R.P. & Taylor, W.P., 1985. Transmission of capripox viruses. Res. Vet. Sci. 39, 196– 199. Lubinga, J.C., Tuppurainen, E.S., Mahlare, R., Coetzer, J.A., Stoltsz, W.H., Venter, E.H., 2015. Evidence of transstadial and mechanical transmission of lumpy skin disease virus by Amblyomma hebraeum ticks. Transbound Emerg Dis (Epub ahead of print) Menasherow S, Rubinstein-Giuni M, Kovtunenko A, Eyngor Y, Fridgut O, Rotenberg D, Khinich Y, Stram Y., 2014. Development of an assay to differentiate between virulent and vaccine strains of lumpy skin disease virus (LSDV). J. Virol. Meth. 199, 95–101. Prozesky, L. & Barnard, B.J.H., 1982. A study of the pathology of lumpy skin disease in cattle. Onderst J. Vet. Res. 49, 167–175. Roberts, K.L. & Smith, G.L., 2008. Vaccinia virus morphogenesis and dissemination. Trends Microbiol. 16, 472–479. Roberts, K.L. & Smith, G.L., 2008. Vaccinia virus morphogenesis and dissemination. Trends in Microbiology, 16(10), pp.472–479. Smith, G.L., Vanderplasschen, A. Law, M., 2002. The formation and function of extracellular enveloped vaccinia virus. Journal of General Virology, 83, 2915–2931. Titov, I., Tsybanov, S. & Malogolovkin, A., 2015. Genotyping of classical swine fever virus using high-resolution melt analysis. Journal of Virological Methods, 224, pp.53–57. 9
Tulman, E.R., Afonso, C.L., Lu, Z., Zsak, L., Sur, J. H., Sandybaev, N.T., Kerembekova, U.Z., Zaitsev, V.L., Kutish, G.F. & other authors 2002. The genomes of sheeppox and goatpox viruses. J. Virol. 76, 6054–6061. Tuppurainen, E.S., Venter, E. H., Coetzer, J.A., 2005. The detection of lumpy skin disease virus in samples of experimentally infected cattle using different diagnostic techniques. Onderst. J. Vet. Res. 72, 153–164. Tuppurainen, E.S., Oura, C.A., 2012. Lumpy skin disease: an emerging threat to Europe, the Middle East and Asia. Transbound. Emerg. Dis. 59, 40–48. Tuppurainen, E.S., Lubinga, J.C., Stoltsz, W.H., Troskie, M., Carpenter, S.T., Coetzer, J.A., Venter, E.H. & Oura, C.A., 2013. Evidence of vertical transmission of lumpy skin disease virus in Rhipicephalus decoloratus ticks. Ticks. Tick. Borne. Dis. 4, 329–333. Von Backstrom, U.. 1945. Ngamiland cattle disease. Preliminary report on a new disease, the aetiological agent probably being of an infectious nature. J. S. Afr. Vet Med. Assoc 16, 29–35. Yeruham, I., Nir, O., Braverman, Y., Davidson, M., Grinstein, H., Haymovitch, M., Zamir, O., 1995. Spread of lumpy skin disease in Israeli dairy herds. Vet. Rec. 137, 91–93.
10
Figure Captions Figure 1. Sensitivity assay of the HRM reaction. A qPCR test of a tenfold dilution of 1 ng of the LSDV, 755 bp long amplicon, between positions 116172 and 116927, at acc. AF409137.1, carrying the HRM amplicon.
11
Figure 2. HRM analysis conducted on Neethling vaccine strain and Ein Zurim-06 field viruses. a) Melting peaks. b) Normalized melting curve.
12
Tables Table 1. Tm analysis of the HRM fragment performed on field-infected and Neethlingvaccinated isolates cattle. Tm values of field infected cattle isolates, Column A. and Neethling vaccinated cattle, Column B. The t and F-tests were performed using GraphPad Prism software, version 5.03 (www.graphpad.com/).
Sample 112 289 331 378 435 304 630 Ein Zurim-06
Field strain Melt temp. (C0) 76 76.1 76.1 76.1 76 76 76 76
Ct 20 22.3 16.1 29.2 24.4 26.5 25.6 14
Sample 317 332 432 343 345 346 347 VAC H2O
Unpaired t test P value < 0.0001 Are means significant different? (P < 0.05) Yes Field virus vs Vaccine virus F test to compare variances P value P value summary Are variances significantly different?
0.4898 ns No
13
Vaccine strain Melt temp. (C0) 75.5 75.6 75.6 75.6 75.6 75.5 75.5 75.6 None
Ct 23.5 23.9 27.4 24.3 22.4 30.4 29.4 26.3 N/A