Corrigendum: Analysis of tick-borne encephalitis virus-induced host responses in human cells of neuronal origin and interferon-mediated protection

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Analysis of tick-borne encephalitis virus-induced host responses in human cells of neuronal origin and interferon-mediated protection

Tick-borne encephalitis virus (TBEV) is a member of the genus Flavivirus. It can cause serious infections in humans that may result in encephalitis/meningoencephalitis. Although several studies have described the involvement of specific genes in the host response to TBEV infection in the central nervous system (CNS), the overall network remains poorly characterized. Therefore, we investigated the response of DAOY cells (human medulloblastoma cells derived from cerebellar neurons) to TBEV (Neudoerfl strain, Western subtype) infection to characterize differentially expressed genes by transcriptome analysis. Our results revealed a wide panel of interferon-stimulated genes (ISGs) and pro-inflammatory cytokines, including type III but not type I (or II) interferons (IFNs), which are activated upon TBEV infection, as well as a number of non-coding RNAs, including long non-coding RNAs. To obtain a broader view of the pathways responsible for eliciting an antiviral state in DAOY cells we examined the effect of type I and III IFNs and found that only type I IFN pre-treatment inhibited TBEV production. The cellular response to TBEV showed only partial overlap with gene expression changes induced by IFN-β treatment – suggesting a virus-specific signature – and we identified a group of ISGs that were highly up-regulated following IFN-β treatment. Moreover, a high rate of down-regulation was observed for a wide panel of pro-inflammatory cytokines upon IFN-β treatment. These data can serve as the basis for further studies of host–TBEV interactions and the identification of ISGs and/or lncRNAs with potent antiviral effects in cases of TBEV infection in human neuronal cells

Genome sequence of canine herpesvirus

Canine herpesvirus is a widespread alphaherpesvirus that causes a fatal haemorrhagic disease of neonatal puppies. We have used high-throughput methods to determine the genome sequences of three viral strains (0194, V777 and V1154) isolated in the United Kingdom between 1985 and 2000. The sequences are very closely related to each other. The canine herpesvirus genome is estimated to be 125 kbp in size and consists of a unique long sequence (97.5 kbp) and a unique short sequence (7.7 kbp) that are each flanked by terminal and internal inverted repeats (38 bp and 10.0 kbp, respectively). The overall nucleotide composition is 31.6% G+C, which is the lowest among the completely sequenced alphaherpesviruses. The genome contains 76 open reading frames predicted to encode functional proteins, all of which have counterparts in other alphaherpesviruses. The availability of the sequences will facilitate future research on the diagnosis and treatment of canine herpesvirus-associated disease

Genome sequences of two pseudorabies virus strains isolated in Greece

Pseudorabies virus (species Suid herpesvirus 1) belongs to the genus Varicellovirus, subfamily Alphaherpesvirinae, family Herpesviridae, and is the causative agent of an acute and frequently fatal disease that affects mainly pigs. Here, we report the genome sequences of two strains of this virus isolated in Greece in 2010

Human cytomegalovirus: taking the strain

In celebrating the 60th anniversary of the first isolation of human cytomegalovirus (HCMV), we reflect on the merits and limitations of the viral strains currently being used to develop urgently needed treatments. HCMV research has been dependent for decades on the high-passage strains AD169 and Towne, heavily exploiting their capacity to replicate efficiently in fibroblasts. However, the genetic integrity of these strains is so severely compromised that great caution needs to be exercised when considering their past and future use. It is now evident that wild-type HCMV strains are not readily propagated in vitro. HCMV mutants are rapidly selected during isolation in fibroblasts, reproducibly affecting gene RL13, the UL128 locus (which includes genes UL128, UL130 and UL131A) and often the UL/b′ region. As a result, the virus becomes less cell associated, altered in tropism and less pathogenic. This problem is not restricted to high-passage strains, as even low-passage strains can harbour biologically significant mutations. Cloning and manipulation of the HCMV genome as a bacterial artificial chromosome (BAC) offers a means of working with stable, genetically defined strains. To this end, the low-passage strain Merlin genome was cloned as a BAC and sequentially repaired to match the viral sequence in the original clinical sample from which Merlin was derived. Restoration of UL128L to wild type was detrimental to growth in fibroblasts, whereas restoration of RL13 impaired growth in all cell types tested. Stable propagation of phenotypically wild-type virus could be achieved only by placing both regions under conditional expression. In addition to the development of these tools, the Merlin transcriptome and proteome have been characterized in unparalleled detail. Although Merlin may be representative of the clinical agent, high-throughput whole-genome deep sequencing studies have highlighted the remarkable high level of interstrain variation present in circulating virus. There is a need to develop systems capable of addressing the significance of this diversity, free from the confounding effects of genetic changes associated with in vitro adaptation. The generation of a set of BAC clones, each containing the genome of a different HCMV strain repaired to match the sequence in the clinical sample, would provide a pathway to address the biological and clinical effects of natural variation in wild-type HCMV

Purification of nuclei and preparation of nuclear envelopes from skeletal muscle

The nuclear envelope is a complex membrane-protein system that is notoriously difficult to purify because it has many connections to both nuclear and cytoplasmic components. This difficulty is compounded by the fact that the nature of these connections vary in different cell types, and so methods must be significantly adapted according to the cell type from which nuclear envelopes are being purified. Here we present a detailed method for purification of nuclear envelopes from one of the most intransigent tissues: skeletal muscle. We further note in the procedure how this method differs from that for other tissues. Identification of nuclear envelope-specific proteins is principally encumbered by endoplasmic reticulum contamination; therefore, we also present a method to purify sarcoplasmic reticulum from muscle

Guilt by association: the nuclear envelope proteome and disease

The discovery that many inherited diseases are linked to interacting nuclear envelope proteins has raised the possibility that human genetic studies could be assisted by a fusion with proteomics. Two principles could be applied. In the first, the proteome of an organelle associated with a genetically variable disease is determined. The chromosomal locations of the genes encoding the organellar proteins are then determined. If a related disease is linked to a large chromosomal region that includes a gene identified in the organelle, then that gene has an increased likelihood of causing the disease. Directly sequencing this allele from patient samples might speed identification compared with further genetic linkage studies as has been demonstrated for multiple diseases associated with the nuclear envelope. The second principle is that if an organelle has been implicated in the pathology of a particular disorder, then comparison of the organelle proteome from control and patient cells might highlight differences that could indicate the causative protein. The distinct, tissue-specific pathologies associated with nuclear envelope diseases suggest that many tissues will have a set of disorders linked to this organelle, and there are numerous as yet unmapped or partially mapped syndromes that could benefit from such an approach

Virus and host factors affecting the clinical outcome of bluetongue virus infection

Bluetongue is a major infectious disease of ruminants caused by bluetongue virus (BTV), an arbovirus transmitted by Culicoides. Here, we assessed virus and host factors influencing the clinical outcome of BTV infection using a single experimental framework. We investigated how mammalian host species, breed, age, BTV serotypes, and strains within a serotype, affect the clinical course of bluetongue. Results obtained indicate that in small ruminants there is a marked difference in the susceptibility to clinical disease induced by BTV at the host species level, but less so at the breed level. No major differences in virulence were found between divergent serotypes (BTV-8 and BTV-2). However, we observed striking differences in virulence between closely related strains of the same serotype collected towards the beginning and the end of the European BTV-8 outbreak. As observed previously, differences in disease severity were also observed when animals were infected with either blood from a BTV-infected animal or from the same virus isolated in cell culture. Interestingly, with the exception of two silent mutations, full viral genome sequencing showed identical consensus sequences of the virus before and after cell culture isolation. However, deep sequencing analysis revealed a marked decrease in the genetic diversity of the viral population after passaging in mammalian cells. In contrast, passaging in Culicoides cells increased the overall number of low frequency variants compared to virus never passaged in cell culture. Thus, Culicoides might be a source of new viral variants and viral population diversity can be another factor influencing BTV virulence

Genomic and biologic comparisons of cyprinid herpesvirus 3 strains

Cyprinid herpesvirus 3 (CyHV-3) is the archetypal fish alloherpesvirus and the etiologic agent of a lethal disease in common and koi carp. To date, the genome sequences of only four CyHV-3 isolates have been published, but no comparisons of the biologic properties of these strains have been reported. We have sequenced the genomes of a further seven strains from various geographical sources, and have compared their growth in vitro and virulence in vivo. The major findings were: (i) the existence of the two genetic lineages previously described as European and Asian was confirmed, but inconsistencies between the geographic origin and genotype of some strains were revealed; (ii) potential inter-lineage recombination was detected in one strain, which also suggested the existence of a third, as yet unidentified lineage; (iii) analysis of genetic disruptions led to the identification of non-essential genes and their potential role in virulence; (iv) comparison of the in vitro and in vivo properties of strains belonging to the two lineages revealed that inter-lineage polymorphisms do not contribute to the differences in viral fitness observed; and (v) a negative correlation was observed among strains between viral growth in vitro and virulence in vivo. This study illustrates the importance of coupling genomic and biologic comparisons of viral strains in order to enhance understanding of viral evolution and pathogenesis