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We have developed a high-throughput sequencing (HTS) workflow for investigating paramyxovirus transcription and replication. We show that sequencing of oligo-dT selected polyadenylated mRNAs, without considering the orientation of the RNAs from which they had been generated, cannot accurately be used to analyse the abundance of viral mRNAs because genomic RNA co-purifies with the viral mRNAs. The best method is directional sequencing of infected cell RNA that has physically been depletion of ribosomal and mitochondrial RNA followed by bioinformatic steps to differentiate data originating from genomes from viral mRNAs and antigenomes. This approach has the advantage that the abundance of viral mRNA (and antigenomes) and genomes can be analysed and quantified from the same data. We investigated the kinetics of viral transcription and replication during infection of A549 cells with parainfluenza virus type 2 (PIV2), PIV3, PIV5 or mumps virus, and determined the abundance of individual viral mRNAs and readthrough mRNAs. We found that the mRNA abundance gradients differed significantly between all four viruses, but that for each virus the pattern remained relatively stable throughout infection. We suggest that rapid degradation of nonpolyadenylated mRNAs may be primarily responsible for the shape of the mRNA abundance gradient in parainfluenza virus 3, whereas a combination of this factor and disengagement of RNA polymerase at intergenic sequences, particularly those at the NP:P and P:M gene boundaries, may be responsible in the other viruses. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) genotype 3 (GT-3) represents 22–30% of all infections and is the second most common genotype among all HCV genotypes. It has two main subtypes, GT-3a and GT-3b, that present epidemiological differences in transmission groups. This report generated 56 GT-3a and 64 GT-3b whole-genome sequences to conduct an evolutionary kinetics and selective force analysis with reference sequences from various countries. Evolutionary analysis showed that HCV GT-3a worldwide might have been transmitted from the Indian subcontinent to South Asia, Europe, North America and then become endemic in China. In China, GT-3a may have been transmitted by intravenous drug users (IDUs) and become endemic in the general population, while GT-3b may have originated from IDUs and then underwent mutual transmission between blood donors (BDs) and IDUs, ultimately becoming independently endemic in IDUs. Furthermore, the spread of GT-3a and GT-3b sequences from BD and IDU populations exhibit different selective pressures: the proportion of positively selected sites (PPSs) in E1 and E2 from IDUs was higher than in BDs. The number of positive selection sites was higher in GT-3b and IDUs. These results indicate that different selective constraints act along with the GT-3a and GT-3b genomes from IDUs and BDs. In addition, GT-3a and GT-3b have different transmission routes in China, which allows us to formulate specific HCV prevention and control strategies in China. [ABSTRACT FROM AUTHOR]

• Hepatitis E Virus (HEV) is emerging as a public health concern across Europe. • Deep sequencing would shed light on the epidemiology and transmission of HEV by offering whole genome sequences. • New protocol for HEV sequencing using target enrichment approach would replace limited traditional method. • The new method is validated for Genotype 1 and 3 and needs further validation using for genotypes 2 and 4. Hepatitis E Virus (HEV) is emerging as a public health concern across Europe and tools for complete genome data to aid epidemiological and virulence analysis are needed. The high sequence heterogeneity observed amongst HEV genotypes has restricted most analyses to subgenomic regions using PCR-based methods, which can be unreliable due to poor primer homology. We designed a panel of custom-designed RNA probes complementary to all published HEV full genome NCBI sequences. A target enrichment protocol was performed according to the NimbleGen® standard protocol for Illumina® library preparation. Optimisation of this protocol was performed using 40 HEV RNA-positive serum samples and the World Health Organization International Reference Panel for Hepatitis E Virus RNA Genotypes for Nucleic Acid Amplification Technique (NAT)-Based Assays and related reference materials. Deep sequencing using this target enrichment protocol resulted in whole genome consensus sequences from samples with a viral load range of 1.25 × 104-1.17 × 107 IU/mL. Phylogenetic analysis of these sequences recapitulated and extended the partial genome results obtained from genotyping by Sanger sequencing (genotype 1, ten samples and genotype 3, 30 samples). The protocol is highly adaptable to automation and could be used to sequence full genomes of large sample numbers. A more comprehensive understanding of hepatitis E virus transmission, epidemiology and viral phenotype prediction supported by an efficient method of sequencing the whole viral genome will facilitate public health initiatives to reduce the prevalence and mitigate the harm of HEV infection in Europe. [ABSTRACT FROM AUTHOR]

Canine distemper virus (CDV) has recently emerged as an extinction threat for the endangered Amur tiger (Panthera tigris altaica). CDV is vaccine-preventable, and control strategies could require vaccination of domestic dogs and/or wildlife populations. However, vaccination of endangered wildlife remains controversial, which has led to a focus on interventions in domestic dogs, often assumed to be the source of infection. Effective decision making requires an understanding of the true reservoir dynamics, which poses substantial challenges in remote areas with diverse host communities. We carried out serological, demographic, and phylogenetic studies of dog and wildlife populations in the Russian Far East to show that a number of wildlife species are more important than dogs, both in maintaining CDV and as sources of infection for tigers. Critically, therefore, because CDV circulates among multiple wildlife sources, dog vaccination alone would not be effective at protecting tigers. We show, however, that low-coverage vaccination of tigers themselves is feasible and would produce substantive reductions in extinction risks. Vaccination of endangered wildlife provides a valuable component of conservation strategies for endangered species. [ABSTRACT FROM AUTHOR]

The genomic characteristics of human cytomegalovirus (HCMV) strains sequenced directly from clinical pathology samples were investigated, focusing on variation, multiple-strain infection, recombination, and gene loss. A total of 207 datasets generated in this and previous studies using target enrichment and high-throughput sequencing were analyzed, in the process enabling the determination of genome sequences for 91 strains. Key findings were that (i) it is important to monitor the quality of sequencing libraries in investigating variation; (ii) many recombinant strains have been transmitted during HCMV evolution, and some have apparently survived for thousands of years without further recombination; (iii) mutants with nonfunctional genes (pseudogenes) have been circulating and recombining for long periods and can cause congenital infection and resulting clinical sequelae; and (iv) intrahost variation in single-strain infections is much less than that in multiple-strain infections. Future population-based studies are likely to continue illuminating the evolution, epidemiology, and pathogenesis of HCMV. [ABSTRACT FROM AUTHOR]