A novel Bluetongue virus strain related to ... - Wiley Online Library

4 downloads 0 Views 412KB Size Report
Cristina Radaelli3 | Cesare Camm a1,2 | Piet A. van Rijn4,5 | Giovanni Savini1,2 |. Alessio Lorusso1,2. 1OIE Reference Laboratory for Bluetongue,.
Received: 27 November 2017 DOI: 10.1111/tbed.12822

RAPID COMMUNICATION

One after the other: A novel Bluetongue virus strain related to Toggenburg virus detected in the Piedmont region (Northwestern Italy), extends the panel of novel atypical BTV strains Maurilia Marcacci1,2 | Serena Sant3 | Iolanda Mangone1,2 | Maria Goria3 |  G. P. van Gennip4 | Maria Alessandro Dondo3 | Simona Zoppi3 | Rene Cristina Radaelli3 | Cesare Camm a1,2 | Piet A. van Rijn4,5 | Giovanni Savini1,2 | Alessio Lorusso1,2 1 OIE Reference Laboratory for Bluetongue, Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise (IZSAM), Teramo, Italy 2

National Reference Center for Whole Genome Sequencing of microbial pathogens: database and bioinformatic analysis, Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise (IZSAM), Teramo, Italy 3

Abstract In this rapid communication, a novel atypical bluetongue virus (BTV) strain detected in goats in the Piedmont region (north-western Italy) is described. This strain, BTV-Z ITA2017, is most related in Seg-2/VP-2 (83.8% nt/82.7% aa) to strain TOV of BTV25. Reactive antisera of goats positive by cELISA for BTV antibodies failed to neutralize a chimeric virus expressing the outermost protein of TOV. Infected animals displayed low levels of RNAemia and absence of clinical signs consistent with

Istituto Zooprofilattico del Piemonte, Liguria e Valle d’Aosta (IZSTO), Torino, Italy

bluetongue infection, a scenario described in animals infected with atypical BTV

4

strains.

Department of Virology, Wageningen Bioveterinary Research, Lelystad, The Netherlands 5

Department of Biochemistry, Centre for Human Metabolomics, North-West University, South Africa

KEYWORDS

arboviruses, diagnostics, emerging diseases, virus

Correspondence Alessio Lorusso, OIE Reference Laboratory for Bluetongue, Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise (IZSAM), Teramo, Italy. Email: [email protected] Funding information Italian Ministry of Health, Grant/Award Number: IZS AM 01/14 RC

1 | INTRODUCTION

medicine. The need to improve the design, conduct and analysis in a fast and efficient manner is also gathering momentum, with greater

Infectious diseases constitute one of the main challenges to medical

emphasis from the scientific community for emerging and potentially

science in the coming century. The impressive development of

catastrophic human infectious diseases. Prompt and accurate diagno-

molecular technologies and of bioinformatics has greatly increased

sis is also demanding for infectious diseases of domestic and wild

our knowledge of the evolution, transmission and pathogenicity of

animals which have a significant impact on livestock trade, animal

infectious diseases. Likewise, the application of molecular technology

production or clinical practice. Bluetongue (BT), a Culicoides-borne

in clinical diagnosis is a rapidly developing area and is predicted to

infectious disease caused by bluetongue viruses (BTV), is certainly

greatly improve the speed, efficiency and accuracy of diagnostic

included within this group of diseases. BT has indeed severe

Transbound Emerg Dis. 2018;1–5.

wileyonlinelibrary.com/journal/tbed

© 2018 Blackwell Verlag GmbH

|

1

2

|

MARCACCI

BTV-X ITL2015

ET AL.

economic repercussions for the livestock industry (Velthuis, Saat-

ITL2015 (BTV-6VP2/VP5

kamp, Mourits, de Koeijer, & Elbers, 2010) due to direct losses

positive serum samples. BTV-6VP2/VP5

caused by the infection but also due to indirect losses because of

was obtained by reverse genetics as previously described (van Rijn,

restrictions on animal trade. For decades, only 24 serotypes of BTV

Sandra, et al., 2016). Molecular and serological screening by RT-

were recognized (Maan et al., 2008). The existence of multiple sero-

qPCRNS3 and cELISA, respectively, were repeated in September

types had, and still has, profound consequences for prophylactic

2017. Isolation on cell cultures and eggs was also attempted fol-

countermeasures. Indeed, these serotypes exist in a complex net-

lowing procedures described in our previous studies (Lorusso et al.,

) was performed on cELISA BTV-X ITL2015

chimeric virus

work of serological cross-relationships, varying from partial to no

2013, 2014, 2016). Serotyping was attempted on qRT-PCRNS3-posi-

protection between heterologous strains. Serotype specificity is

tive samples using the TaqVet European BTV (1-2-4-6-8-9-16)

determined by variation in the outer capsid proteins of the virion,

Typing kit (BTVEuropean

especially VP-2 (Huisman & Erasmus, 1981) and, for a less degree,

[LSI-Thermo Fisher Scientific]. RNA was further purified (Savini

VP-5 (Shaw et al., 2013). As for the development of ground-breaking

et al., 2017) from one blood sample (sample 12, Table 1) showing

molecular technologies or innovative metagenomic protocols, in the

the lowest CT cycle by RT-qPCRNS3 for the sequence-independent

last years novel BTV serotypes including BTV-25 (TOV strain) from

single-primer amplification (SISPA)/next-generation sequencing pro-

Switzerland, BTV-26 from Kuwait, BTV-27 (variants 01, 02 and 03)

tocol and bioinformatic analyses routinely used at IZSAM (Marcacci

viruses from Corsica (France), BTV-XJ1407 from China, a BTV strain

et al., 2016; Savini et al., 2017). Multiple sequence alignment for

isolated from a sheep pox vaccine (SP vaccine-derived BTV) and

Seg-2 with the homologues sequences of extant serotypes (includ-

BTV-X ITL2015 from Sardinia (Italy) have been described by

ing atypical serotypes) retrieved from GenBank was conducted

typing,

LSI VetMAXTM European BTV Typing

researchers within the field (Bumbarov, Golender, Erster, & Khinich, 2016; Hofmann et al., 2008; Jenckel et al., 2015; Maan, Maan, Nomikou, Batten, et al., 2011; Maan, Maan, Nomikou, Veronesi, et al., 2011; Savini et al., 2017; Schulz et al., 2016; Sun et al., 2016;

T A B L E 1 BTV RT-qPCRNS3 and cELISA performed on purified RNA from blood and serum samples, respectively, of goats sampled in July and September 2017

Zientara et al., 2014). In addition, a novel BTV strain partially related

Animal

RT-qPCRNS3 July 2017 CT

RT-qPCRNS3 September 2017 CT

cELISA July

cELISA September

1

35

neg

+

+

2

35

neg

+

+

3

34

neg

+

+

4

neg

neg

5

34

neg

+

+

ard et al., have been demonstrated (Batten et al., 2013, 2014; Bre

6

35

neg

+

+

2017; Chaignat et al., 2009, 2010; Planzer et al., 2011; Savini et al.,

7

39

neg

+

+

2017).

8

39

43

+

+

9

neg

neg

10

40

neg

to BTV-26 and to the SP vaccine-derived BTV has been very recently identified in sheep imported from Libya to Tunisia (Lorusso et al., 2018). The rate of discovery of these additional BTVs is becoming exponential. This certainly raises questions regarding the origin and the role of these viruses in the ecobiology of BTVs also considering that, at least for some of them, new biological characteristics with respect to classical BTV serotypes, including horizontal transmission capabilities and the inability to grow in cell cultures,

2 | MATERIALS AND METHODS

+

+ +

+ +

11

neg

neg

12a

32a

39

+

+

On 4 July 2017, whole blood samples were collected from 24

13

37

35

+

+

goats located in a farm in the municipality of Pieve Vergonte, pro-

14

36

38

+

+

vince of Verbano-Cusio-Ossola, Piedmont region, north-western

15

34

38

+

+

16

41

43

+

+

17

39

43

+

+

18

35

44

+

+

19

35

neg

+

+

20

35

40

+

+

qPCR1–24 (Polci et al., 2007). Serum samples were also collected

21

neg

neg

and screened by cELISA for BTV antibodies (Lelli et al., 2003).

22

35

36

+

+

Serum-neutralization (SN, Savini et al., 2004) for serotypes 1–24,

23

37

neg

+

+

24

35

neg

+

+

Italy, for BT surveillance. The animals did not show signs of BTV infection. Blood samples, after RNA purification, were first tested by a generic quantitative real-time RT-PCR (RT-qPCR) assay which detects all existing BTV serotypes (RT-qPCRNS3, Hofmann et al., 2008). Positive samples by RT-qPCRNS3 were screened by the RTqPCR assay which detects BTV strains of serotypes 1–24, RT-

VP2/VP5 BTV 25

25 (BTV-6

, van Rijn, van Water, Maris-Veldhuis, &

van Gennip, 2016), BTV-26, BTV-27 v01-03 (kindly donated by Stephan Zientara and Emmanuel Breard, Anses, France) and BTV-X

Neg, negative. Sample used for NGS analysis.

a

+

|

3

69.4% nt/67.9% aa

using MAFFT (version 7.017; Katoh & Standley, 2013). Seg-2 sequences were used for phylogenetic analysis using the maximumlikelihood (ML) method implemented in MEGA version 6 (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013). To assess the robustness of individual nodes on the phylogenetic trees, a bootstrap

65.9% nt/67.5% aa

resampling analysis (500 replications) was performed.

3 | RESULTS AND DISCUSSION Results of both qRT-PCRNS3 screening are summarized in Table 1.

67.4% nt/69.4% aa

69.9% nt/76.8% aa

68.8% nt/76.3% aa 71.7% nt/76.3% aa na

73.1% nt/82.1% aa 77.6% nt/91.3% aa

68.9% nt/74.9% aa na

72.3% nt/81.4% aa

63.3% nt/60.9% aa

76.5% nt/88.5% aa 80.0% nt/91.3% aa na

75.5% nt/87.4% aa 77.2% nt/87.9% aa

71.6% nt/74.0% aa na

75.2% nt/87.5% aa

BTV SP vacc

BTV-X ITL2015

BTV-Y TUN 2017

ET AL.

In July, 20 of 24 animals were positive for BTV RNA by qRTPCRNS3. In September, the number of RNAemic animals dropped to 10. CT values of BTV RNA in these animals were lower (range 36–44) to those obtained by the same molecular screening per-

71.5% nt/79.8% aa

78.1% nt/81.8%

70.2% nt/76.9% aa

79.7% nt/81.8% aa

be RNAemic in July, were demonstrated to be negative for BTV RNA in September. All positive RT-qPCRNS3 turned negative by RT-qPCR1–24 and BTVEuropean

typing.

Twenty of 24 animals were

demonstrated to be positive for BTV antibodies by cELISA in July. Conversely, all serum samples were positive by cELISA in September, demonstrating the seroconversion of all sampled animals. However, cELISA -positive serum samples of goats failed to neutralize all available reference and atypical (including chimeric strains) BTV serotypes by SN (data not shown). Nearly the full-length genome sequences for seven (including Seg-1, Seg-2, Seg-3, Seg-4, Seg-5, Seg-6 and Seg-8, Table 2) of ten segments were obtained

77.3% nt/81.8% aa

70.1% nt/76.2% aa 70.1% nt/74.9% aa

70.0% nt/76.3% aa

78.4% nt/92.5% aa 78.8% nt/90.3% aa

70.2% nt/74.3% aa

83.8% nt/95.3% aa 84.1% nt/95.6% aa

79.1% nt/90.1% aa

71.9% nt/74.0% aa 72.6% nt/73.1% aa

84.4% nt/95.1% aa

81.6% nt/91.6% aa 83.2% nt/92.1% aa 83.4% nt/9292.4% aa

73.6% nt/74.0% aa

BTV-28 CHINA BTV-27 FRA v03 BTV-27 FRA v02

formed in July (range 32–40). Ten animals, which were shown to

by NGS from sample no. 12 (CT 32), with coverage ranging from 17X to 61X. This virus has been identified as BTV-Z ITA2017, and genome sequences have been deposited with the GenBank database (acc. nos. MF673720–MF673726). BLAST revealed the high-

84.4% nt/95.3% aa

78.7% nt/90.5% aa

69.7% nt/74.9% aa

77.4% nt/85.5% aa

79.4% nt/82.8% aa

76.3% nt/88.5% aa

73.4% nt/81.3% aa

69.3% nt/76.2% aa

68.1% nt/74.0% aa

67.1% nt/69.4% aa

82.8% nt/93.7% aa

89.6% nt/95.4% aa

76.5% nt/80.5% aa

83.2% nt/91.3% aa

97.1% nt/97.1% aa

newly described BTV serotypes, particularly with strain TOV of BTV-25 as illustrated in Table 2. Phylogenetically, BTV-Z ITA2017 clusters with TOV within the same clade of the atypical BTV serotypes (Figure 1). Percentage of nt identity of the genome segments of BTV-Z ITA2017 with homologous segments of BTV-25 strain TOV ranges from 76.5% (Seg-5) to 97.1% (Seg-8). Seg-2 nt identity with TOV is 83.8%/82.7% aa, whereas with extant atypical BTV serotypes ranges from 62.6 nt/60.6 aa (BTV-26) to 72.6 nt/74.1 aa (BTV-27 v01). It has been established that different isolates of a single BTV serotype show >68.4%/72.6% nt/aa identity in Seg2/VP-2, with 40.5%/22.1% nt/aa to 71.5%/77.8% nt/aa identity between different serotypes (Maan, Maan, Nomikou, Veronesi, et al., 2011). Accordingly, BTV-Z ITA2017 should belong to the 25th serotype together with TOV; however, BTV-positive serum samples of goats failed to neutralize BTV-6VP2/VP5

BTV 25

(data not

na, not available.

Seg-8 691nt/230aa

Seg-6 521nt/173aa

shown). Moreover, it was not possible to test by SN reactive antisSeg-5 994nt/331aa

Seg-4 1775nt/591aa

72.6% nt/74.1% aa 62.6% nt/60.6% aa 83.8% nt/82.7% aa

Seg-3 1739nt/579aa

83.2% nt/92.2% aa 75.4% nt/88.0% aa 83.7% nt/93.4% aa

Seg-2 2290nt/763aa

Seg-1 3478nt/1159aa

BTV-27 FRA v01 BTV-26 BTV-25 TOV

est nucleotide (nt) sequences identity of BTV-Z ITA2017 with the

BTV-Z ITA2017

T A B L E 2 Percentage of nt and aa identities of seven segments of BTV-Z ITA2017 with BTV-25, BTV-26, the three variants of BTV-27 (BTV-27/FRA2014/v01-v03), BTV XJ1407 from China, SP vaccine-derived BTV, BTV-X ITL2015 and BTV-Y TUN2017

MARCACCI

era for TOV with BTV-Z ITA2017 as all isolation procedures for BTV-Z ITA2017 failed. Therefore, based upon the current data, it is difficult to determine whether BTV-Z ITA2017 represents a novel BTV serotype or a novel strain belonging to the 25th serotype. Further serological experiments are warranted as well as

4

|

MARCACCI

ET AL.

F I G U R E 1 Maximum-likelihood unrooted phylogenetic relationships of BTV-Z ITA2017 (black circle) strain based on segment 2. The phylogenetic tree was constructed with MEGA 6 (Tamura et al., 2013) using 500 bootstrap replicates and the “best fitted model”. Bar indicates the estimated numbers of nt substitutions per site. Bootstrap values ≥70 are indicated novel genomic criteria for definition of BTV serotypes. Overall, in

and does not imply recommendation or endorsement by the OIE

this short communication, the presence of a novel atypical BTV

Reference Laboratory for Bluetongue of Teramo.

strain in goats in Piedmont region, north-western Italy, has been revealed. This strain is most related in Seg-2/VP-2 to strain TOV of BTV-25. Infected animals displayed low levels of RNAemia and clinical signs of BT infection, like those induced by virulent strains of BTV 1-24 serotypes, were absent. This scenario has been

ORCID Alessio Lorusso

http://orcid.org/0000-0001-6156-8212

already described in animals infected with other atypical BTV strains. Additional experiments are currently ongoing to obtain the

REFERENCES

whole genome sequence and a viable isolate to use for serological purposes.

ACKNOWLEDGEMENTS Funding was provided by the Italian Ministry of Health (IZS AM 01/ 14 RC, Ricerca Corrente 2014 “Approccio metagenomico per una diagnosi rapida e accurata di alcune infezioni batteriche e virali”, recipient Giovanni Savini). Mention of trade names or commercial products in this article is solely for the purpose of providing specific information

Batten, C., Darpel, K., Henstock, M., Fay, P., Veronesi, E., Gubbins, S., & Oura, C. (2014). Evidence for transmission of bluetongue virus serotype 26 through direct contact. PLoS ONE, 9(5), e96049. Batten, C., Henstock, M. R., Steedman, H. M., Waddington, S., Edwards, L., & Oura, C. (2013). Bluetongue virus serotype 26: Infection kinetics, pathogenesis and possible contact transmission in goats. Veterinary Microbiology, 162(1), 62–67. https://doi.org/10.1016/j.vetmic. 2012.08.014 ard, E., Schulz, C., Sailleau, C., Bernelin-Cottet, C., Viarouge, C., Vitour, Bre D., . . . Beer, M. (2017). Bluetongue virus serotype 27: Experimental infection of goats, sheep and cattle with three BTV-27 variants

MARCACCI

|

ET AL.

reveal atypical characteristics and likely direct contact transmission BTV-27 between goats. Transboundary and Emerging Diseases, [Epub ahead of print]. https://doi.org/10.1111/tbed.12780. Bumbarov, V., Golender, N., Erster, O., & Khinich, Y. (2016). Detection and isolation of Bluetongue virus from commercial vaccine batches. Vaccine, 34(28), 3317–3323. https://doi.org/10.1016/j.vaccine.2016.03.097 Chaignat, V., Schwermer, H., Casati, S., Planzer, J., Worwa, G., Vanzetti, €r, B. (2010). Occurrence and spatial distribution of ToggenT., . . . Thu burg Orbivirus in Switzerland. Small Ruminant Research, 93, 157–164. https://doi.org/10.1016/j.smallrumres.2010.05.016 Chaignat, V., Worwa, G., Scherrer, N., Hilbe, M., Ehrensperger, F., Batten, C., . . . Thuer, B. (2009). Toggenburg Orbivirus, a new bluetongue virus: Initial detection, first observations in field and experimental infection of goats and sheep. Veterinary Microbiology, 138, 11–19. https://doi.org/10.1016/j.vetmic.2009.02.003 Hofmann, M.A., Renzullo, S., Mader, M., Chaignat, V., Worwa, G., & Thuer, B. (2008). Genetic characterization of Toggenburg orbivirus, a new bluetongue virus, from goats, Switzerland. Emerging infectious diseases, 14(12), 1855–1861. https://doi.org/10.3201/eid1412. 080818 https://doi.org/10.3201/eid1412.080818 Huisman, H., & Erasmus, B. J. (1981). Identification of the serotype-specific and group-specific antigens of bluetongue virus. Onderstepoort Journal of Veterinary Research, 48(2), 51–58. ard, E., Schulz, C., Sailleau, C., Viarouge, C., Hoffmann, B., Jenckel, M., Bre . . . Zientara, S. (2015). Complete coding genome sequence of putative novel bluetongue virus serotype 27. Genome Announcements, 3 (2), pii: e00016–15.https://doi.org/10.1128/genomea.00016-15 Katoh, K., & Standley, D. M. (2013). MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution, 30, 772–780. https://doi.org/10. 1093/molbev/mst010 Lelli, R., Portanti, O., Langella, V., Luciani, M., Di Emidio, B., & Conte, A. M. (2003). Production of a competitive ELISA kit for the serological diagnosis of bluetongue disease. Veterinaria Italiana, 47, 5–12. Lorusso, A., Baba, D., Spedicato, M., Teodori, L., Bonfini, B., Marcacci, M., . . . Savini, G. (2016). Bluetongue virus surveillance in the Islamic Republic of Mauritania: Is serotype 26 circulating among cattle and dromedaries? Infection, Genetics and Evolution, 40, 109–112. https://d oi.org/10.1016/j.meegid.2016.02.036 Lorusso, A., Sghaier, S., Ancora, M., Marcacci, M., Di Gennaro, A., Portanti, O., . . . Savini, G. (2014). Molecular epidemiology of bluetongue virus serotype 1 circulating in Italy and its connection with northern Africa. Infection, Genetics and Evolution, 28, 144–149. https://doi.org/ 10.1016/j.meegid.2014.09.014 Lorusso, A., Sghaier, S., Carvelli, A., Di Gennaro, A., Leone, A., Marini, V., . . . Savini, G. (2013). Bluetongue virus serotypes 1 and 4 in Sardinia during autumn 2012: New incursions or re-infection with old strains? Infection, Genetics and Evolution, 19, 81–87. https://doi.org/10.1016/ j.meegid.2013.06.028 Lorusso, A., Sghaier, S., Di Domenico, M., Barbria, M. E., Zaccaria, G., Portanti, O., . . . Savini, G. (2018). Analysis of Bluetongue serotype 3 spread in Tunisia and discovery of a novel strain related to the Bluetongue virus isolated from a commercial sheep pox vaccine. Infection, Genetics and Evolution, submitted manuscript. Maan, S., Maan, N. S., Nomikou, K., Batten, C., Antony, F., Belaganahalli, M. N., & Mertens, P. P. C. (2011). Novel bluetongue virus serotype from Kuwait. Emerging Infectious Diseases Journal, 17(5), 886–889. https://doi.org/10.3201/eid1705.101742 Maan, S., Maan, N. S., Nomikou, K., Veronesi, E., Bachanek-Bankowska, K., Belaganahalli, M. N., . . . Mertens, P. P. C. (2011). Complete genome characterisation of a novel 26th bluetongue virus serotype from Kuwait. PLoS ONE, 6(10), e26147. https://doi.org/10.1371/journal.pone.0026147 Maan, S., Maan, N. S., Ross-smith, N., Batten, C. A., Shaw, A. E., Anthony, S. J., & Mertens, P. P. C. (2008). Sequence analysis of bluetongue virus serotype 8 from the Netherlands 2006 and comparison to other

5

European strains. Virology, 377(2), 308–318. https://doi.org/10.1016/ j.virol.2008.04.028 Marcacci, M., De Luca, E., Zaccaria, G., Di Tommaso, M., Mangone, I., Aste, G., & Lorusso, A. (2016). Genome characterization of feline morbillivirus from Italy. Journal of Virological Methods, 234, 160–163. https://doi.org/10.1016/j.jviromet.2016.05.002 Planzer, J., Kaufmann, C., Worwa, G., Gavier-Widen, D., Hofmann, M. A., Chaignat, V., & Thur, B. (2011). In vivo and in vitro propagation and transmission of Toggenburg orbivirus. Research in Veterinary Science, 91, 163–168. https://doi.org/10.1016/j.rvsc.2011.03.007 Polci, A., Camma, C., Serini, S., Di Gialleonardo, L., Monaco, F., & Savini, G. (2007). Real-time polymerase chain reaction to detect bluetongue virus in blood samples. Veterinaria Italiana, 43(1), 77–88. , van Rijn, P. A., Sandra, G. P., van de Water, S. G. P., Feenstra, F., Rene G. P., & van Gennip, RG. (2016). Requirements for reverse genetics of bluetongue virus (BTV) and African horse sickness virus (AHSV). Virology Journal, 13, 119, 1-9. https://doi.org/rdcu.be/nuox van Rijn, P. A., van Water, S. G. P., Maris-Veldhuis, M., & van Gennip, H. G. P. (2016). Experimental infection of small ruminants with bluetongue virus expressing Toggenburg Orbivirus proteins. Veterinary Microbiology, 192, 145–151. https://doi.org/10.1016/j.vetmic.2016.07.013 Savini, G., Monaco, F., Migliaccio, P., Casaccia, C., Salucci, S., & Di Ventura, M. (2004). Virological and serological responses in sheep following field vaccination with bivalent live modified vaccine against bluetongue virus serotypes 2 and 9. Veterinaria Italiana, 40, 631–634. Savini, G., Puggioni, G., Meloni, G., Marcacci, M., Di Domenico, M., Rocchigiani, A. M., . . . Lorusso, A. (2017). Novel putative Bluetongue virus in healthy goats from Sardinia, Italy. Infection, Genetics and Evolution, 21 (51), 108–117. https://doi.org/10.1016/j.meegid.2017.03.021 ard, E., Sailleau, C., Jenckel, M., Viarouge, C., Vitour, D., . . . Schulz, C., Bre Zientara, S. J. (2016). Bluetongue virus serotype 27: Detection and characterization of two novel variants in Corsica, France. Journal of General Virology, 97(9), 2073–2083. Shaw, A. E., Ratinier, M., Nunes, S. F., Nomikou, K., Caporale, M., Golder, M., . . . Palmarini, M. (2013). Reassortment between two serologically unrelated bluetongue virus strains is flexible and can involve any genome segment. Journal of Virology, 87, 543–557. https://doi.org/10. 1128/JVI.02266-12 Sun, E. C., Huang, L. P., Xu, Q. Y., Wang, H. X., Xue, X. M., Lu, P., . . . Wu, D. L. (2016). Emergence of a novel bluetongue virus serotype, China 2014. Transboundary and Emerging Diseases, 63, 585–589. https://doi.org/10.1111/tbed.12560 Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30, 2725–2729. https://doi.org/10. 1093/molbev/mst197 Velthuis, A. G., Saatkamp, H. W., Mourits, M. C., de Koeijer, A. A., & Elbers, A. R. (2010). Financial consequences of the Dutch bluetongue serotype 8 epidemics of 2006 and 2007. Preventive Veterinary Medicine, 93(4), 294–304. https://doi.org/10.1016/j.prevetmed.2009.11.007 € per, D., Beer, M., Jenckel, M., . . . Zientara, S., Sailleau, C., Viarouge, C., Ho Vitour, D. (2014). Novel bluetongue virus in goats, Corsica, France, 2014. Emerging Infectious Diseases Journal, 20(12), 2123–2125. https://doi.org/10.3201/eid2012.140924

How to cite this article: Marcacci M, Sant S, Mangone I, et al. One after the other: A novel Bluetongue virus strain related to Toggenburg virus detected in the Piedmont region (North-western Italy), extends the panel of novel atypical BTV strains. Transbound Emerg Dis. 2018;00:1–5. https://doi.org/10.1111/tbed.12822