Identification and genetic characterization of bovine enterovirus by combination of two next generation sequencing platforms

Beato, Maria Serena; Marcacci, Maurilia; Schiavon, Eliana; Bertocchi, L.; Domenico, Marco Di; Peserico, Alessia; Mion, Monica; Zaccaria, Guendalina; Cavicchio, Lara; Mangone, Iolanda; Soranzo, E.; Patavino, Claudio; Cammà, Cesare; Lorusso, Alessio

doi:10.1016/j.jviromet.2018.07.002

Cited by 15 publications

(13 citation statements)

References 19 publications

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“…Two hundred µL of the brain homogenate were enrolled for nucleic acid purification through the High Pure Viral Nucleic Acid Kit (Roche, Basel, Switzerland) and used for metagenomic analysis. Nucleic acid elution was divided into two aliquots to perform RNA and DNA sequencing, as previously described by our group [27,30]. After Turbo DNAse incubation, RNA was processed by means of the Sequence Independent Single Primer Amplification (SISPA) method to obtain cDNA [31,32].…”

Section: Shotgun Metagenomics By Minionmentioning

confidence: 99%

Detection of Astrovirus in a Cow with Neurological Signs by Nanopore Technology, Italy

Zaccaria

Lorusso

Hierweger

et al. 2020

Viruses

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In this study, starting from nucleic acids purified from the brain tissue, Nanopore technology was used to identify the etiological agent of severe neurological signs observed in a cow which was immediately slaughtered. Histological examination revealed acute non-suppurative encephalomyelitis affecting the brainstem, cerebrum, cerebellum, and medulla oblongata, while by using PCR-based assays, the nucleic acids of major agents for neurological signs were not detected. By using Nanopore technology, 151 sequence reads were assigned to Bovine Astrovirus (BoAstV). Real-time RT-PCR and in situ hybridization (ISH) confirmed the presence of viral RNA in the brain. Moreover, using the combination of fluorescent ISH and immunofluorescence (IF) techniques, it was possible to detect BoAstV RNA and antigens in the same cells, suggesting the active replication of the virus in infected neurons. The nearly whole genome of the occurring strain (BoAstV PE3373/2019/Italy), obtained by Illumina NextSeq 500, showed the highest nucleotide sequence identity (94.11%) with BoAstV CH13/NeuroS1 26,730 strain, an encephalitis-associated bovine astrovirus. Here, we provide further evidence of the role of AstV as a neurotropic agent. Considering that in a high proportion of non-suppurative encephalitis cases, which are mostly indicative of a viral infection, the etiologic agent remains unknown, our result underscores the value and versatility of Nanopore technology for a rapid diagnosis when the PCR-based algorithm gives negative results.

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Section: Shotgun Metagenomics By Minionmentioning

confidence: 99%

Detection of Astrovirus in a Cow with Neurological Signs by Nanopore Technology, Italy

Zaccaria

Lorusso

Hierweger

et al. 2020

Viruses

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“…This showed high levels of identity between the BTV TUN2016 and the BTV‐3 SAR2018 strains confirming that NGS is becoming a central and crucial diagnostic technique enabling the identification and characterization of a given BTV strain (and thus its potential origin) in a rapid time period as described in previous reports (Marcacci et al., ; Savini et al., ). This aspect, combined with the availability of portable third generation sequencers (Beato et al., ; Peserico et al., ) would make possible the molecular diagnosis of BTV also in field conditions. BTV‐3w circulation was detected in sentinel animals before the onset of the disease in other susceptible animals.…”

mentioning

confidence: 99%

Western Bluetongue virus serotype 3 in Sardinia, diagnosis and characterization

Cappai

Rolesu

Loi

et al. 2019

Transbound Emerg Dis

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Over the last 20 years, Italy has experienced multiple incursions of different serotypes of Bluetongue virus (BTV), a Culicoides‐borne arbovirus, the causative agent of bluetongue (BT), a major disease of ruminants. The majority of these incursions originated from Northern Africa, likely because of wind‐blown dissemination of infected midges. Here, we report the first identification of BTV‐3 in Sardinia, Italy. BTV‐3 circulation was evidenced in sentinel animals located in the province of Sud Sardegna on September 19, 2018. Prototype strain BTV‐3 SAR2018 was isolated on cell culture. BTV‐3 SAR2018 sequence and partial sequences obtained by next‐generation sequencing from nucleic acids purified from the isolate and blood samples, respectively, were demonstrated to be almost identical (99–100% of nucleotide identity) to BTV‐3 TUN2016 identified in Tunisia in 2016 and 2017, a scenario already observed in past incursions of other BTV serotypes originating from Northern Africa.

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“…41 Deep sequencing-based approaches using viral nucleic acid enrichment methods have been described to address this issue, including targeted and untargeted library preparations, like SISPA. 21,22 The methodology in this study demonstrates the application of culture-based viral enrichment followed by random, strand-switching MinION sequencing for accurately detecting and characterizing RNA viruses. RNA viruses with varying genome compositions (single stranded [positive- and negative-sense], double stranded, and segmented) were used to demonstrate the ability of untargeted strand-switching to obtain complete CDS of genotyping regions and whole genomes with viral culture enrichment methods.…”

Section: Discussionmentioning

confidence: 99%

“…19,20 Other methods such as sequence independent primer amplification (SISPA) have been used with MinION to obtain whole genome sequences for bovine enterovirus from culture and canine distemper virus from the brain of an affected dog. 21,22 While virus targeting can be accomplished with the previously mentioned rRNA depletion or sequence targeting, it is also possible to couple this newest sequencing technology with classical virus culture for viral enrichment. The aim of this study was to develop a method to simultaneously detect and characterize various RNA viruses from culture by using a randomly primed, strand-switching approach and sequencing on MinION.…”

Section: Introductionmentioning

confidence: 99%

Randomly primed, strand-switching MinION-based sequencing for the detection and characterization of cultured RNA viruses

Young

Lahmers

Sellers

et al. 2019

Preprint

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RNA viruses rapidly mutate, which can result in increased virulence, increased escape 2 0 from vaccine protection, and false negative detection results. Targeted detection methods have a 2 1 limited ability to detect unknown viruses and often provide insufficient data to detect 2 2 coinfections or identify antigenic variants. Random, deep sequencing is a method that can more 2 3 fully detect and characterize RNA viruses and is often coupled with molecular techniques or 2 4 culture methods for viral enrichment. Viral culture coupled with third-generation sequencing 2 5 were tested for the ability to detect and characterize RNA viruses. Cultures of bovine viral 2 6 diarrhea virus, canine distemper virus, epizootic hemorrhagic disease virus, infectious bronchitis 2 7 virus, two influenza A viruses, and porcine respiratory and reproductive syndrome virus were 2 8 sequenced on the MinION platform using a random, reverse primer in a strand-switching 2 9 reaction, coupled with PCR-based barcoding. Reads were taxonomically classified and used for 3 0 reference-based sequence building using a stock personal computer. This method accurately 3 1 detected and identified complete coding sequence genomes with a minimum of 20× coverage 3 2 depth for all seven viruses, including a sample containing two viruses. Each lineage-typing 3 3 region had at least 26× coverage depth for all viruses. Furthermore, analyzing the canine 3 4 distemper virus sample through a pipeline devoid of canine distemper virus reference sequences 3 5 modeled the ability of this protocol to detect unknown viruses. These results show the ability of 3 6 this technique to detect and characterize dsRNA, negative-and positive-sense ssRNA, 3 7nonsegmented, and segmented RNA viruses. 3 8 3 9

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Identification and genetic characterization of bovine enterovirus by combination of two next generation sequencing platforms

Cited by 15 publications

References 19 publications

Detection of Astrovirus in a Cow with Neurological Signs by Nanopore Technology, Italy

Detection of Astrovirus in a Cow with Neurological Signs by Nanopore Technology, Italy

Western Bluetongue virus serotype 3 in Sardinia, diagnosis and characterization

Randomly primed, strand-switching MinION-based sequencing for the detection and characterization of cultured RNA viruses

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