Bluetongue virus diagnosis of clinical cases by a duplex reverse transcription-PCR: a comparison with conventional methods

Billinis, Charalambos; Koumbati, Maria; Spyrou, Vassiliki; Nomikou, Kyriaki; Mangana, Olga; Panagiotidis, Christos Α.; Papadopoulos, O.

doi:10.1016/s0166-0934(01)00360-3

Cited by 40 publications

(22 citation statements)

References 47 publications

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“…Only a few reports have evaluated real-time PCR for detection of BTV burden in clinical samples [19][20][21]. A few researchers have targeted the NS3 gene for the development of a real-time assay [22,12,23]. In this study, we developed and evaluated a real-time RT-PCR assay using primers specifically for a highly conversed region in segment 10 (NS3 gene) of BTV.…”

Section: Discussionmentioning

confidence: 99%

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Comparison of bluetongue virus detection and quantitation methods in south India

Subhadra

Kumar

Suryanarayana

et al. 2014

J Infect Dev Ctries

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Introduction: Bluetongue (BT), a vector-borne viral disease, primarily affects sheep. Of the 26 serotypes of BTV identified so far, 22 are reported to be circulating in India. Due to an increase in vector population and delays in disease diagnosis, the BT control program heavily relies on rapid and confirmatory diagnosis. Polymerase chain reaction (PCR)-based real-time detection assays may be an ideal method to detect the BTV genome in animal blood at an early stage of infection. Methodology: In this study, a SYBR green-based real-time RT-PCR assay was evaluated, validated, and compared with conventional RT-PCR. The specificity and sensitivity of an assay using BTV-2 RNA extracted from tenfold serially diluted (starting from 1.0 TCID 50 /mL) cell culture virus was also evaluated. Results: While conventional RT-PCR could detect 3.16×10 2 TCID 50 of virus/mL, the real-time PCR test had a detection limit of 3.16×10

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Section: Discussionmentioning

confidence: 99%

“…Total RNA from whole blood was extracted using the procedure described by Billinis et al [12]. Necessary precautions were taken to avoid RNAse contamination during extraction as described by Sambrook et al [13].…”

Section: Extraction Of Viral Rna and Cdna Synthesismentioning

confidence: 99%

Comparison of bluetongue virus detection and quantitation methods in south India

Subhadra

Kumar

Suryanarayana

et al. 2014

J Infect Dev Ctries

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“…(11), Toxoplasma gondii, Neospora caninum (18), Brucella spp. (1), and bovid herpesvirus 1 (26) and by RT-PCR for bluetongue virus (3,8). Conversely, the pestivirus genome was detected with two different RT-PCR protocols (7,24), and the virus was characterized as Hobi-like by a species-specific nested PCR (7) (Fig.…”

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confidence: 99%

Hobi-Like Pestivirus in Aborted Bovine Fetuses

Decaro¹,

Lucente²,

Mari³

et al. 2012

J Clin Microbiol

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An outbreak of abortion affecting multiparous cows was associated with Hobi-like pestivirus infection. Viral RNA and antigens were detected in the tissues of two aborted fetuses. Molecular assays for other common abortogenic agents tested negative. At the genetic level, the Hobi-like pestivirus displayed the closest relatedness to Italian, Australian, and South American viruses, whereas it diverged from the prototype Thai isolate. These findings may have important implications for the pestivirus control/ eradication programs in cattle herds. Bovine viral diarrhea viruses (BVDVs) are members of the genus Pestivirus (family Flaviviridae), responsible for a number of clinical signs, including subclinical infections, immunosuppression, acute diarrhea, respiratory disease, reproductive failures, and mucosal disease in persistently infected calves. Reproductive disorders caused by BVDVs vary according to the fetal age and include embryo death, abortion, mummification, congenital abnormalities, or stillbirths (2). Thus far, two different BVDV species have been recognized, BVDV-1 and BVDV-2, that cocirculate in cattle herds worldwide (20). In 2004, an atypical pestivirus, strain D32/00_Hobi, was isolated from a contaminated batch of fetal calf serum (FCS) (19). The virus was distantly related to BVDV-1/BVDV-2, and it was proposed as a prototype of a new pestivirus species (14, 15). Hobi-like sequences have been repeatedly detected in commercial FCS batches (17,22,23), whereas there are few reports on natural infections (4, 5, 21, 23) and clinical outbreaks (5). The virus has been recently detected in aborted bovine fetuses in Brazil, thus suggesting direct clinical implications (4).Here, we report the isolation and genetic characterization of a Hobi-like strain detected from aborted fetuses in southern Italy. The abortion outbreak occurred in June 2011 in a cattle herd where a Hobi-like pestivirus-associated respiratory disease had been recently described (5). Abortion was observed in eight multiparous cows in a group of 270 lactating Holstein cows and occurred between the fourth and sixth months of pregnancy. The animals neither showed prodromal signs nor presented postabortion complications. Two aborted fetuses (280/11-A, 280/11-B) were sent to our laboratory, and tissue samples were collected from lungs, spleens, livers, kidneys, and placentas for diagnostic investigations. Nucleic acids were purified using the DNeasy tissue kit (Qiagen) and QIAamp RNeasy minikit (Qiagen). Reverse transcription (RT)-PCR and PCR assays were performed using SuperScript one-step RT-PCR for long templates (Life Technologies) and LA PCR kit version 2.1 (TaKaRa Bio Inc.), respectively. Positive and negative controls were processed in parallel to the screened samples. The samples tested negative by PCR for Chlamydophila spp. 3, 8). Conversely, the pestivirus genome was detected with two different 24), and the virus was characterized as Hobi-like by a species-specific nested PCR (7) (Fig. 1A). Viral titers, quantified by a TaqMan-based real...

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“…Edirne and Kirklareli) were perceived (distinguished) because of their high seroprevalence status for BTV-9 and BTV-16. On the basis of geographic features or BT occurrence data from neighboring countries, one might be postulated that BTV-9 is predominately spread in Bulgaria, Greece and Turkey while BTV-16 is the most threaten serotype for Bulgaria and Turkey (1,2,3,4,24). This speculation need to be further investigated by species distribution along the region.…”

Section: Discussionmentioning

confidence: 99%

Seroprevalence of culicoides-borne disease in cattle in European Turkey

ÖZGÜNLÜK¹

2007

Ankara Üniversitesi Veteriner Fakültesi Dergisi

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Summary:In this research, seroprevalence of BTV serotypes (BTV-4, BTV-9, BTV-16), and occurrence of Akabane and Ephemeral fever viruses were investigated in 557 cattle sera collected from 21 locations in European Turkey (Thrace). Virus neutralization test was used to discriminate antibody response against BTV serotypes while commercial blocking-ELISA systems were used to detect antibodies for Akabane and ephemeral fever viruses. The overall seroprevalence of BTV was recorded as 73.54%, comprising individual percentages for BTV-4, BTV-9 and BTV-16 were 69.04%, 71.38% and 80.2%, respectively. The seroprevalence of Ephemeral fever virus infection in Thrace varied between 2.5 and 15.3% on the province basis, whilst antibody percentage for Akabane virus was found as low as 0.14% in the study.Key words: Akabane, bluetongue, ephemeral fever, seroprevalence. Trakya bölgesinde sığırlarda sivrisineklerle nakledilen enfeksiyonların seroprevalansıÖzet: Trakya bölgesinde, 2003 yılında 5 ile bağlı 21 ilçe/köyden toplam 557 sığır, Mavidil, Akabane ve Ephemeral Fever enfeksiyonlarının seroprevalanslarının tespiti amacıyla random sampling metoduyla örneklendi. Serum örnekleri Mavidil virusunun 3 farklı serotipine (BTV-serotip 4, BTV-serotip 9 ve BTV-serotip 16) karşı virus nötralizasyon testine tabi tutuldu. Aynı serum örneklerine Akabane ve Ephemeral Fever virus antikorlarının tespiti için blocking ELISA testi uygulandı. Trakya bölgesinde mavi dil enfeksiyonunun seroprevalansı %73.54 olarak tespit edilirken, BTV serotip-4 %69.04, BTV serotip-9 %71.38, BTV serotip-16 ise %80.2 olarak saptandı. Akabane enfeksiyonunun seroprevalansı Trakya bölgesinde %0.14 olarak belirlenirken, Ephemeral fever enfeksiyonunun seroprevalansı örneklenen iller bazında %2.5-15.3 olarak tespit edildi.

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Bluetongue virus diagnosis of clinical cases by a duplex reverse transcription-PCR: a comparison with conventional methods

Cited by 40 publications

References 47 publications

Comparison of bluetongue virus detection and quantitation methods in south India

Comparison of bluetongue virus detection and quantitation methods in south India

Hobi-Like Pestivirus in Aborted Bovine Fetuses

Seroprevalence of culicoides-borne disease in cattle in European Turkey

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