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April 21, 2020  |  

Identification and characterization of chicken circovirus from commercial broiler chickens in China.

Circoviruses are found in many species, including mammals, birds, lower vertebrates and invertebrates. To date, there are no reports of circovirus-induced diseases in chickens. In this study, we identified a new strain of chicken circovirus (CCV) by PacBio third-generation sequencing samples from chickens with acute gastroenteritis in a Shandong commercial broiler farm in China. The complete genome of CCV was verified by inverse PCR. Genomic analysis revealed that CCV codes two inverse open reading frames (ORFs), and a potential stem-loop structure was present at the 5′ end with a structure typical of a circular virus. Phylogenetic tree analysis showed that CCV formed an independent branch between mammalian and avian circovirus, and homology analysis indicated that the homology of CCV with 21 other known circoviruses was less than 40%. Thus, this CCV strain represents a new species in the genus Circovirus. The infection rate of CCV in 12 chickens with diarrhoea was 100%, but no CCV was found in healthy chickens, thereby indicating that the novel CCV strain is highly associated with acute infectious gastroenteritis in chickens. The emergence of a novel CCV in commercial broiler chickens is highly concerning for the broiler industry. © 2019 Blackwell Verlag GmbH.


April 21, 2020  |  

Integrative functional genomics decodes herpes simplex virus 1

Since the genome of herpes simplex virus 1 (HSV-1) was first sequenced more than 30 years ago, its predicted 80 genes have been intensively studied. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identified a total of 201 viral transcripts and 284 open reading frames (ORFs) including all known and 46 novel large ORFs. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of novel viral ORFs as well as N-terminal extensions (NTEs) and truncations thereof. We show that key viral regulators and structural proteins possess NTEs, which initiate from non-canonical start codons and govern subcellular protein localization and packaging. We validated a novel non-canonical large spliced ORF in the ICP0 locus and identified a 93 aa ORF overlapping ICP34.5 that is thus also deleted in the FDA-approved oncolytic virus Imlygic. Finally, we extend the current nomenclature to include all novel viral gene products. Taken together, this work provides a valuable resource for future functional studies, vaccine design and oncolytic therapies.


April 21, 2020  |  

Multiple Long-read Sequencing Survey of Herpes Simplex Virus Lytic Transcriptome

Long-read sequencing (LRS) has become increasingly important in RNA research due to its strength in resolving complex transcriptomic architectures. In this regard, currently two LRS platforms have demonstrated adequate performance: the Single Molecule Real-Time Sequencing by Pacific Biosciences (PacBio) and the nanopore sequencing by Oxford Nanopore Technologies (ONT). Even though these techniques produce lower coverage and are more error prone than short-read sequencing, they continue to be more successful in identifying transcript isoforms including polycistronic and multi-spliced RNA molecules, as well as transcript overlaps. Recent reports have successfully applied LRS for the investigation of the transcriptome of viruses belonging to various families. These studies have substantially increased the number of previously known viral RNA molecules. In this work, we used the Sequel and MinION technique from PacBio and ONT, respectively, to characterize the lytic transcriptome of the herpes simplex virus type 1 (HSV-1). In most samples, we analyzed the poly(A) fraction of the transcriptome, but we also performed random oligonucleotide-based sequencing. Besides cDNA sequencing, we also carried out native RNA sequencing. Our investigations identified more than 160 previously undetected transcripts, including coding and non-coding RNAs, multi-splice transcripts, as well as polycistronic and complex transcripts. Furthermore, we determined previously unsubstantiated transcriptional start sites, polyadenylation sites, and splice sites. A large number of novel transcriptional overlaps were also detected. Random-primed sequencing revealed that each convergent gene pair produces non-polyadenylated read-through RNAs overlapping the partner genes. Furthermore, we identified novel replication-associated transcripts overlapping the HSV-1 replication origins, and novel LAT variants with very long 5’ regions, which are co-terminal with the LAT-0.7kb transcript. Overall, our results demonstrated that the HSV-1 transcripts form an extremely complex pattern of overlaps, and that entire viral genome is transcriptionally active. In most viral genes, if not in all, both DNA strands are expressed.


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