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
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1
2
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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
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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
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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
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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
