Arch Virol DOI 10.1007/s00705-014-1977-7

ORIGINAL ARTICLE

Detection of bovine torovirus in fecal specimens from calves with diarrhea in Turkey I˙rem Gu¨lac¸tı • Hakan Is¸ ıdan • I˙brahim So¨zdutmaz

Received: 8 October 2013 / Accepted: 31 December 2013 Ó Springer-Verlag Wien 2014

Abstract Bovine torovirus (BToV), a member of the family Coronaviridae, is an established gastrointestinal infectious agent in cattle. In this study, we performed a survey to detect BToV in Turkey between 2009 and 2011 using 235 fecal samples from neonatal calves with diarrhea that were analyzed by the nested reverse transcription (RT) PCR method using primers located in the consensus sequences of the BToV membrane (M) gene. The BToV M gene was detected in 4.7 % (11/235) of the samples using the nested RT-PCR method. The nucleotide sequences of partial M fragments from the BToV isolates, including the newly identified Turkish isolates, showed more than 96 % identity. The result indicates that BToV is one of the pathogens that contribute to neonatal calf diarrhea cases in Turkey.

Introduction Torovirus is a genus of the family Coronaviridae, order Nidovirales, and the members of this genus are enveloped, single-stranded, positive-sense RNA viruses [14, 28, 31]. Toroviruses cause gastroenteritis in mammals and have been detected in humans, horses, cattle, and swine

worldwide [1–3, 6, 9, 13, 16]. BToV was first detected in the USA during an outbreak of diarrhea in cattle in 1979 [32]. Epidemiological studies of BToV have shown that it is widespread throughout the world [7, 10–13, 15, 17, 22– 26, 30, 32, 34]. However, there have not been any reports of BToV infections in Turkey. Human and bovine toroviruses are difficult to grow in cell culture. However there has been one report on a BToV (Aichi/2004) that was isolated in a human rectal adenocarcinoma cell line (HRT-18) from the ileum content of a calf with diarrhea [20]. Since growth of BToV in cell culture was established just a few years ago, there are only a limited number of sequences of BToV available in the GenBank database [4, 5, 8–13, 15, 25, 27]. The aim of this study was to determine the presence of bovine toroviruses in diarrheic feces from calves in Turkey. This report also describes the first detection of BToV in Turkey, using the RT-PCR technique, along with the genetic diversity of the BToV strains based on partial BToV membrane (M) gene sequences.

Materials and methods Specimen collection

˙I. Gu¨lac¸tı The Pendik Veterinary Control and Research Institute, 34890 I˙stanbul, Turkey H. Is¸ ıdan (&) Department of Virology, Cumhuriyet University Faculty of Veterinary Medicine, 58140 Sivas, Turkey e-mail: [email protected] I˙. So¨zdutmaz Department of Virology, Erciyes University Faculty of Veterinary Medicine, 38039 Kayseri, Turkey

A total of 235 diarrheic fecal specimens were collected from calves with diarrhea from 28 provinces of Turkey between 2009 and 2011. Figure 1 indicates the locations where samples were taken and where they were found to be positive for BToV. RNA extraction The fecal samples were diluted in a 109 volume of 0.01 M phosphate-buffered saline (0.138 M NaCl, 0.0027 M KCl,

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Fig. 1 Distribution of BToV-positive samples in Turkey. *Black dots indicate individual samples, and the locations of the positive samples are indicated by arrows

Table 1 Oligonucleotide primer pairs used for nested RT-PCR [24] Target gene

Primer usage

Polarity

Primers

Amplicon length

BoTV

First-step PCR

Forward

50 -TTCTTACTACACTTTTTGGA-30

603 bp

Reverse

50 -ACTCAAACTTAACACTAGAC-30

Forward

50 - TATGTACTATGTTTCCAGCT-30

Reverse

50 -CCAACACAAATCCGCAACGC-30

M gene Nested PCR

409 bp

pH 7.5) and centrifuged at 1000 9 g for 1 min at room temperature. The supernatant was transferred to a new tube, and a second centrifugation was done at 8000 9 g for 5 minutes at room temperature. The RNA was extracted from the supernatant using an RNeasy Kit (QIAGEN GmbH, Hilden, Germany) in accordance with the manufacturer’s instructions. For each extraction period, ddH2O was used as a negative control.

DNA sequencing

RT-PCR

Molecular analysis

A previously described nested RT-PCR for BToV detection was used with primers targeting the BToV M gene (Table 1) [24]. For each reaction, distilled water was used as a negative control. The expected size of the amplicon produced for the first round of PCR was 603 bp, and for the nested PCR, it was 409 bp. The amplification products were analyzed using 1.5 % agarose gel electrophoresis and visualized by UV irradiation of ethidium-bromide-stained gels.

Phylogenetic and bootstrap (1000 replicates) analyses based on nt and aa sequence alignments were done using the neighbour-joining method and the unweighted-pair group method with the average linkages in Molecular Evolutionary Genetics Analysis (MEGA, Version 5.2), with pairwise distance analysis [18, 19, 29]. A sequence similarity search was carried out for partial M gene sequences of bovine torovirus using the LALIGN Query program of the GENESTREAM network server at the

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The nested RT-PCR products corresponding to the BToV M open reading frame (ORF) were sequenced directly by the Pendik Veterinary Control and Research Institute (Department of Genetics) using an ABI Prism BigDye Terminator version 3.1 cycle sequencing kit and an Applied Biosystems 310 DNA analyzer (Applied Biosystems Inc., CA, USA).

Bovine torovirus in Turkey Table 2 GenBank accession numbers of the reference strains of bovine toroviruses used in nucleotide and amino acid sequence comparisons and phylogenetic analysis Designation

Country

Year

Accession number

References

Breda 1

USA

1982

AY427798

[5, 33]

B6

Italy

1990

AJ575374

[27]

B145

The Netherlands

1998

AJ575375

[27]

B150

The Netherlands

1998

AJ575376

[27]

B155

The Netherlands

1998

AJ575377

[27]

Aichi/2004

Japan

2004

AB285126

[15]

K-567

Japan

2004

AB270905

[15]

K-637

Japan

2004

AB270908

[15]

K-645

Japan

2005

AB270911

[15]

K-674

Japan

2005

AB270913

[15]

K-676

Japan

2005

AB270915

[15]

K-683

Japan

2005

AB270917

[15]

K34

South Korea

2004

DQ778041

[25]

K49 K96

South Korea South Korea

2004 2004

DQ778042 DQ778043

[25] [25]

K110

South Korea

2004

DQ778044

[25]

K119

South Korea

2004

DQ778045

[25]

K375

South Korea

2004

DQ778046

[25]

K490

South Korea

2005

DQ778047

[25]

K501

South Korea

2005

DQ778048

[25]

K536

South Korea

2005

DQ778049

[25]

K537

South Korea

2005

DQ778050

[25]

K540

South Korea

2005

DQ778051

[25]

K546

South Korea

2005

DQ778052

[25]

K577

South Korea

2005

DQ778053

[25]

T1

Turkey

2009

KF188708

This study

T2

Turkey

2009

KF188709

This study

T3

Turkey

2010

KF188710

This study

T4 T5

Turkey Turkey

2010 2010

KF188711 KF188712

This study This study

T6

Turkey

2010

KF188713

This study

T7

Turkey

2011

KF188714

This study

T8

Turkey

2011

KF188715

This study

T9

Turkey

2011

KF188716

This study

T10

Turkey

2011

KF188717

This study

T11

Turkey

2011

KF188718

This study

Institut de Ge´ne´tique Humaine, Montpellier, France (http:// xylian.igh.cnrs.fr/bin/lalign-guess.cgi). The GenBank accession numbers are listed in Table 2. Results and discussion BToV gene products corresponding to the M gene were detected in 4.7 % (11/235) of the samples. All of the positive samples were collected from three of the 28 cities

Fig. 2 A phylogenetic bootstrap consensus tree of the partial 387-bp (152 to 538) nucleotide sequence of the M gene of the bovine torovirus strains was made using the neighbor-joining method of Molecular Evolutionary Genetics Analysis [18, 19, 29]. The names of the viruses used are listed in Table 2

in Turkey (Figs. 1, 2). This result suggests that BToV infections are sporadic in diarrheic calves in Turkey, which by way of analogy, was similar to previous findings in reports from South Korea [25]. The nucleotide sequence analysis indicates that some of the Turkish strains were identical for the sequenced fragment of the M gene, and also the location of identical samples supports this identity. Eleven nested PCR products were sequenced, and the results, together with other BToV M gene sequences (see Table 2) from GenBank, were analysed using the LALIGN query program for nucleotide and amino acid similarities [21]. Among the eleven Turkish BToVs, identical sequences were put together in three groups, as shown in Tables 2, 3. Group 1 consisted of strains T1 and T2, group 2 consisted of strains T4 and T7, and group 3 consisted of strains T8, T9, T10 and T11.

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I˙. Gu¨lac¸tı et al. Table 3 Nucleotide (below the diagonal) and amino acid (above the diagonal) sequence identities of the partial (152 to 538) 387 bp M gene sequences

Group 1 includes T1 and T2; group 2 includes T4 and T7; group 3 includes T8 through T11; group 4 includes B150 and B155; group 5 includes K110 through K577 and K49. Isolates included within a group have identical sequences

From these results it was established that Turkish BToVs were closely related to the Italian BToV strain B6, which has more than 97.9 % nucleotide (nt) sequence similarity, and the Dutch BToVs, which have more than 97.4 % nucleotide sequence similarity. However, the sequences of the Turkish strains differed more extensively from the North American strain Breda 1, which had less than 96.4 % nucleotide sequence similarity. Further similarities were also deduced between Turkish BToVs. The North American strain (Breda1) shared 96.4 % nucleotide sequence similarity with the Italian one, which, together with the Dutch BToVs, were 96.6 % similar. Also, the North American strain and the Japanese BToVs shared nucleotide sequence similarities between 95.3 and 96.4 %. Furthermore, the Korean strains and the Turkish BToVs shared nucleotide sequence similarities between 96.1 and 96.6 %, and the Turkish ones, between 95.3 to 96.4 %. These results indicate that the North American strain is the most divergent one (less than 96.6 % sequence similarity) in comparison with the others. The phylogenetic tree (see Fig. 1) indicates that Turkish BToVs were more similar to European, Japanese and Korean strains. This could be due to Turkey’s geographical location, which is more proximate, central and connected with Europe, Asia and Africa. Therefore, further studies will be needed to achieve a more accurate characterization of the BToV. In conclusion, BToV infections are very rare and sporadic in diarrheic calves in Turkey. Based on the partial M

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gene sequence, Turkish BToVs were found to be more closely related to European BToVs than the others. Acknowledgments We thank to Dr. Eray ATIL from the Pendik Veterinary Control and Research Institute (I˙stanbul, Turkey) for sequencing the PCR products. The authors declare that they have no conflict of interest.

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Detection of bovine torovirus in fecal specimens from calves with diarrhea in Turkey.

Bovine torovirus (BToV), a member of the family Coronaviridae, is an established gastrointestinal infectious agent in cattle. In this study, we perfor...
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