Development of a Real-Time, TaqMan Reverse Transcription-PCR Assay for Detection and Differentiation of Lyssavirus Genotypes 1, 5, and 6

Wakeley, Philip R.; Johnson, Nicholas; McElhinney, L. M.; Marston, Denise A.; Sawyer, Jason; Fooks, Anthony R.

doi:10.1128/jcm.43.6.2786-2792.2005

Cited by 133 publications

(162 citation statements)

References 24 publications

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“…Amplification and detection of viral RNA were undertaken using a hemi-nested RT-PCR (Heaton et al, 1997). Oral swabs were also tested by real-time PCR (Wakeley et al, 2005). The viral load was estimated from RNA samples extracted from the organs of three bats (IDs 34629, 34841 and 34620) from the s.c. group.…”

Section: Methodsmentioning

confidence: 99%

“…The viral load was estimated from RNA samples extracted from the organs of three bats (IDs 34629, 34841 and 34620) from the s.c. group. A standard curve was generated using purified amplicons generated with primers Jw12 and N165 (Wakeley et al, 2005) and this was used to calculate the absolute number of target copies in a particular sample, as described previously (Johnson et al, 2006a). Target samples and standards were amplified using primers Jw12 and N165, and detected using probe GT5 (Wakeley et al, 2005).…”

Section: Methodsmentioning

confidence: 99%

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Experimental infection of serotine bats (Eptesicus serotinus) with European bat lyssavirus type 1a

Freuling

Vos

Johnson³

et al. 2009

Journal of General Virology

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The serotine bat (Eptesicus serotinus) accounts for the vast majority of bat rabies cases in Europe and is considered the main reservoir for European bat lyssavirus type 1 (EBLV-1, genotype 5). However, so far the disease has not been investigated in its native host under experimental conditions. To assess viral virulence, dissemination and probable means of transmission, captive bats were infected experimentally with an EBLV-1a virus isolated from a naturally infected conspecific from Germany. Twenty-nine wild caught bats were divided into five groups and inoculated by intracranial (i.c.), intramuscular (i.m.) or subcutaneous (s.c.) injection or by intranasal (i.n.) inoculation to mimic the various potential routes of infection. One group of bats was maintained as uninfected controls. Mortality was highest in the i.c.-infected animals, followed by the s.c. and i.m. groups. Incubation periods varied from 7 to 26 days depending on the route of infection. Rabies did not develop in the i.n. group or in the negative-control group. None of the infected bats seroconverted. Viral antigen was detected in more than 50 % of the taste buds of an i.c.-infected animal. Shedding of viable virus was measured by virus isolation in cell culture for one bat from the s.c. group at 13 and 14 days post-inoculation, i.e. 7 days before death. In conclusion, it is postulated that s.c. inoculation, in nature caused by bites, may be an efficient way of transmitting EBLV-1 among free-living serotine bats.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Experimental infection of serotine bats (Eptesicus serotinus) with European bat lyssavirus type 1a

Freuling

Vos

Johnson³

et al. 2009

Journal of General Virology

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“…In Experiment 2, three different RT-PCR tests were used for the detection of viral RNA: a single-round PCR assay (Vos et al, 2004a), a nested PCR assay (Heaton et al, 1997) and a quantitative real-time PCR (Wakeley et al, 2005). Amplicons were sequenced using standard protocols to confirm virus identity.…”

Section: Rt-pcr Hemi-nested Rt-pcr and Quantitative Real-time Pcrmentioning

confidence: 99%

Susceptibility of North American big brown bats (Eptesicus fuscus) to infection with European bat lyssavirus type 1

Franka

Johnson

Müller

et al. 2008

Journal of General Virology

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The aim of this study was to determine the susceptibility of insectivorous bats (using the big brown bat as a model) to infection with European bat lyssavirus type 1a , to assess the dynamics of host immune responses and to evaluate the opportunity for horizontal viral transmission within colonies. Two isolates of EBLV-1a, originating from Slovakia (EBLV-1aSK) and Germany (EBLV-1aGE), were tested. According to EUROBATS, an organization dedicated to the conservation of European bat populations, 45 species of bat are geographically distributed throughout Europe (Eurobats, 2004). So far, EBLV-1 has been found predominantly in specific association with the serotine

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“…Virüs mermi şeklinde, tek iplikcikli negatif polaritede RNA taşımaktadır [1] . Karnivorlar ve yarasalar virüsün temel konakçılarıdır.…”

Section: Introductionunclassified

Doğal Olarak Enfekte Olmuş Farklı Tür Hayvanlardan, Post-Mortem Olarak Elde Edilen Beyin Numunelerinde, Klasik Kuduzun Rutin Laboratuvar Teşhisinde Real Time RT-PCR Testinin Değerlendirilmesi

Gürçay¹,

Parmaksiz²,

Kiliç³

2014

Kafkas Univ Vet Fak Derg

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Bu çalışmada, doğal olarak enfekte olmuş farklı hayvan türlerine ait beyin numunelerinde klasik kuduzun teşhisi için real-time RT-PCR testi değerlendirildi. Çalışma materyalini Elazığ Veteriner Kontrol Enstitüsü'ne 2011 yılı boyunca Türkiye'nin Doğu ve Güneydoğu Anadolu Bölgelerinde bulunan Elazığ, Malatya, Diyarbakır, Mardin, Tunceli, Muş, Bingöl, Batman, Şırnak, Hakkâri ve Bitlis illerinden getirilen 36 köpek, 13 sığır, 6 koyun, 6 eşek, 4 at, 4 tilki, 3 kurt, 2 kedi ve 1 keçiye ait toplam 75 adet kuduz hastalığı şüpheli beyin numunesi oluşturmuştur. Farklı hayvan türlerine ait beyin numunelerinden kuduz virüsü genomunu tespit etmek amacıyla klasik kuduz virüsüne özgü primer ve TaqMan prob seti kullanıldı. Bu primer ve prob seti, beyin numunelerine real-time RT-PCR testi optimize edilerek uygulandı. Laboratuvara ulaşan beyin numunelerine floresan antikor tekniği (FAT) ve deneme hayvanı inokulasyonu (MIT) testleri ile birlikte real time RT-PCR testi de yapıldı. Real-time RT-PCR testi ile 75 beyin numunesinin 53'ünde kuduz virusuna ait spesifik viral RNA tespit edildi. Otoliz olmuş 3 adet köpek beyni numunesine FAT ve MIT uygulanamazken, real-time RT-PCR testi sonucunda bu numunelerin birinde kuduz virusuna ait spesifik viral RNA tespit edildi. Taze (n=72) beyin dokularından etçillerde C. ammonis, otçullarda Cerebellum'dan elde edilen homojenatlara direkt FAT testi uygulandı, 72 beyin numunesinin 51'inde viral antijenler tespit edildi. Aynı homojenatlardan MIT yöntemiyle 72 beyin numunesinin 52'inde virüs izolasyonu sağlandı. Taze beyin numunelerinden elde edilen MIT testi sonuçlarının, real-time RT-PCR testi sonuçları ile tam uyum gösterdiği (%100) görüldü. Sonuç olarak, klasik kuduz teşhisinde direkt beyin dokusuna uygulanan real-time RT-PCR testinin, hem FAT ve MIT uygulanamayan otoliz olmuş numunelerde güvenle kullanılabileceği ve hem de alt yapısı sağlanmış laboratuvarlarda invitro şartlarda FAT için doğrulayıcı test olarak da kullanılabileceği tespit edilmiştir. Anahtar sözcükler: Kuduz Teşhisi, Klasik Kuduz Virüsü, Real-Time RT-PCR Evaluation of Real-Time RT-PCR Assay for Routine Laboratory Diagnosis of Classical Rabies in Post-Mortem Brain Samples from Naturally Infected Different Species of Animals SummaryIn this study; the real-time RT-PCR assay was evaluated for laboratory diagnosis of classical rabies in post mortem brain samples from naturally infected different animals species. The material consisted of 75 suspected brain samples brought from 11 different provinces, belonging Eastern and Southeastern Anatolia region, including Elazig, Malatya, Diyarbakir, Mardin, Sirnak, Batman, Tunceli, Muş, Bingol, Hakkari and Bitlis in year of 2011. These samples were from 36 dogs (33 normal, 3 autolytic), 13 cattle, 6 sheep, 6 donkeys, 4 horses, foxes, 3 wolves, 2 cats , and one goat. The samples were autolytic in 3 of 75 (4%), whereas 72 (96%) of 75 samples were freshly arrived to the laboratory. Due to badly autolysis of 3 samples, they could not be examined by fluorescent antibody test (FAT) and mouse inocula...

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Development of a Real-Time, TaqMan Reverse Transcription-PCR Assay for Detection and Differentiation of Lyssavirus Genotypes 1, 5, and 6

Cited by 133 publications

References 24 publications

Experimental infection of serotine bats (Eptesicus serotinus) with European bat lyssavirus type 1a

Experimental infection of serotine bats (Eptesicus serotinus) with European bat lyssavirus type 1a

Susceptibility of North American big brown bats (Eptesicus fuscus) to infection with European bat lyssavirus type 1

Doğal Olarak Enfekte Olmuş Farklı Tür Hayvanlardan, Post-Mortem Olarak Elde Edilen Beyin Numunelerinde, Klasik Kuduzun Rutin Laboratuvar Teşhisinde Real Time RT-PCR Testinin Değerlendirilmesi

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