Distemper, extinction, and vaccination of the Amur tiger.

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.

Development of a Reverse Genetics System for Toscana Virus (Lineage A).

Toscana virus (TOSV) is a Phlebovirus in the Phenuiviridae family, order Bunyavirales, found in the countries surrounding the Mediterranean. TOSV is an important cause of seasonal acute meningitis and encephalitis within its range. Here, we determined the full sequence of the TOSV strain 1500590, a lineage A virus obtained from an infected patient (Marseille, 2007) and used this in combination with other sequence information to construct functional cDNA plasmids encoding the viral L, M, and S antigenomic sequences under the control of the T7 RNA promoter to recover recombinant viruses. Importantly, resequencing identified two single nucleotide changes to a TOSV reference genome, which, when corrected, restored functionality to the polymerase L and made it possible to recover infectious recombinant TOSV (rTOSV) from cDNA, as well as establish a minigenome system. Using reverse genetics, we produced an NSs-deletant rTOSV and also obtained viruses expressing reporter genes instead of NSs. The availability of such a system assists investigating questions that require genetic manipulation of the viral genome, such as investigations into replication and tropism, and beyond these fundamental aspects, also the development of novel vaccine design strategies.

Alcelaphine herpesvirus 1 genes A7 and A8 regulate viral spread and are essential for malignant catarrhal fever.

Alcelaphine herpesvirus 1 (AlHV-1) is a gammaherpesvirus that is carried asymptomatically by wildebeest. Upon cross-species transmission to other ruminants, including domestic cattle, AlHV-1 induces malignant catarrhal fever (MCF), which is a fatal lymphoproliferative disease resulting from proliferation and uncontrolled activation of latently infected CD8+ T cells. Two laboratory strains of AlHV-1 are used commonly in research: C500, which is pathogenic, and WC11, which has been attenuated by long-term maintenance in cell culture. The published genome sequence of a WC11 seed stock from a German laboratory revealed the deletion of two major regions. The sequence of a WC11 seed stock used in our laboratory also bears these deletions and, in addition, the duplication of an internal sequence in the terminal region. The larger of the two deletions has resulted in the absence of gene A7 and a large portion of gene A8. These genes are positional orthologs of the Epstein-Barr virus genes encoding envelope glycoproteins gp42 and gp350, respectively, which are involved in viral propagation and switching of cell tropism. To investigate the degree to which the absence of A7 and A8 participates in WC11 attenuation, recombinant viruses lacking these individual functions were generated in C500. Using bovine nasal turbinate and embryonic lung cell lines, increased cell-free viral propagation and impaired syncytia formation were observed in the absence of A7, whereas cell-free viral spread was inhibited in the absence of A8. Therefore, A7 appears to be involved in cell-to-cell viral spread, and A8 in viral cell-free propagation. Finally, infection of rabbits with either mutant did not induce the signs of MCF or the expansion of infected CD8+ T cells. These results demonstrate that A7 and A8 are both essential for regulating viral spread and suggest that AlHV-1 requires both genes to efficiently spread in vivo and reach CD8+ T lymphocytes and induce MCF.

Metaviromics Reveals Unknown Viral Diversity in the Biting Midge Culicoides impunctatus.

Biting midges (Culicoides species) are vectors of arboviruses and were responsible for the emergence and spread of Schmallenberg virus (SBV) in Europe in 2011 and are likely to be involved in the emergence of other arboviruses in Europe. Improved surveillance and better understanding of risks require a better understanding of the circulating viral diversity in these biting insects. In this study, we expand the sequence space of RNA viruses by identifying a number of novel RNA viruses from Culicoides impunctatus (biting midge) using a meta-transcriptomic approach. A novel metaviromic pipeline called MetaViC was developed specifically to identify novel virus sequence signatures from high throughput sequencing (HTS) datasets in the absence of a known host genome. MetaViC is a protein centric pipeline that looks for specific protein signatures in the reads and contigs generated as part of the pipeline. Several novel viruses, including an alphanodavirus with both segments, a novel relative of the Hubei sobemo-like virus 49, two rhabdo-like viruses and a chuvirus, were identified in the Scottish midge samples. The newly identified viruses were found to be phylogenetically distinct to those previous known. These findings expand our current knowledge of viral diversity in arthropods and especially in these understudied disease vectors.

Human Cytomegalovirus Genomes Sequenced Directly From Clinical Material: Variation, Multiple-Strain Infection, Recombination, and Gene Loss.

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.

Genome Sequences of Five African Swine Fever Virus Genotype IX Isolates from Domestic Pigs in Uganda.

Complete genome sequences of five African swine fever virus isolates were determined directly from clinical material obtained from domestic pigs in Uganda. Four sequences were essentially identical to each other, and all were closely related to the only known genome sequence of p72 genotype IX.

Development of reverse genetics systems and investigation of host response antagonism and reassortment potential for Cache Valley and Kairi viruses, two emerging orthobunyaviruses of the Americas.

Orthobunyaviruses such as Cache Valley virus (CVV) and Kairi virus (KRIV) are important animal pathogens. Periodic outbreaks of CVV have resulted in the significant loss of lambs on North American farms, whilst KRIV has mainly been detected in South and Central America with little overlap in geographical range. Vaccines or treatments for these viruses are unavailable. One approach to develop novel vaccine candidates is based on the use of reverse genetics to produce attenuated viruses that elicit immune responses but cannot revert to full virulence. The full genomes of both viruses were sequenced to obtain up to date genome sequence information. Following sequencing, minigenome systems and reverse genetics systems for both CVV and KRIV were developed. Both CVV and KRIV showed a wide in vitro cell host range, with BHK-21 cells a suitable host cell line for virus propagation and titration. To develop attenuated viruses, the open reading frames of the NSs proteins were disrupted. The recombinant viruses with no NSs protein expression induced the production of type I interferon (IFN), indicating that for both viruses NSs functions as an IFN antagonist and that such attenuated viruses could form the basis for attenuated viral vaccines. To assess the potential for reassortment between CVV and KRIV, which could be relevant during vaccination campaigns in areas of overlap, we attempted to produce M segment reassortants by reverse genetics. We were unable to obtain such viruses, suggesting that it is an unlikely event.

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.

Suppression of costimulation by human cytomegalovirus promotes evasion of cellular immune defenses.

CD58 is an adhesion molecule that is known to play a critical role in costimulation of effector cells and is intrinsic to immune synapse structure. Herein, we describe a virally encoded gene that inhibits CD58 surface expression. Human cytomegalovirus (HCMV) UL148 was necessary and sufficient to promote intracellular retention of CD58 during HCMV infection. Blocking studies with antagonistic anti-CD58 mAb and an HCMV UL148 deletion mutant (HCMV∆UL148) with restored CD58 expression demonstrated that the CD2/CD58 axis was essential for the recognition of HCMV-infected targets by CD8+ HCMV-specific cytotoxic T lymphocytes (CTLs). Further, challenge of peripheral blood mononuclear cells ex vivo with HCMV∆UL148 increased both CTL and natural killer (NK) cell degranulation against HCMV-infected cells, including NK-driven antibody-dependent cellular cytotoxicity, showing that UL148 is a modulator of the function of multiple effector cell subsets. Our data stress the effect of HCMV immune evasion functions on shaping the immune response, highlighting the capacity for their potential use in modulating immunity during the development of anti-HCMV vaccines and HCMV-based vaccine vectors.

Integrating patient and whole-genome sequencing data to provide insights into the epidemiology of seasonal influenza A(H3N2) viruses.

Genetic surveillance of seasonal influenza is largely focused on sequencing of the haemagglutinin gene. Consequently, our understanding of the contribution of the remaining seven gene segments to the evolution and epidemiological dynamics of seasonal influenza is relatively limited. The increased availability of next-generation sequencing technologies allows rapid and economic whole-genome sequencing (WGS) of influenza virus. Here, 150 influenza A(H3N2) positive clinical specimens with linked epidemiological data, from the 2014/15 season in Scotland, were sequenced directly using both Sanger sequencing of the HA1 region and WGS using the Illumina MiSeq platform. Sequences generated by the two methods were highly correlated, and WGS provided on average >90 % whole genome coverage. As reported in other European countries during 2014/15, all strains belonged to genetic group 3C, with subgroup 3C.2a predominating. Multiple inter-subgroup reassortants were identified, including three 3C.3 viruses descended from a single reassortment event, which had persisted in the population. Cases of severe acute respiratory illness were significantly clustered on phylogenies of multiple gene segments indicating potential genetic factors warranting further investigation. Severe cases were also more likely to be associated with reassortant viruses and to occur later in the season. These results suggest that WGS provides an opportunity to develop our understanding of the relationship between the influenza genome and disease severity and the epidemiological consequences of within-subtype reassortment. Therefore, increased levels of WGS, linked to clinical and epidemiological data, could improve influenza surveillance.

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-ß treatment - suggesting a virus-specific signature - and we identified a group of ISGs that were highly up-regulated following IFN-ß treatment. Moreover, a high rate of down-regulation was observed for a wide panel of pro-inflammatory cytokines upon IFN-ß 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.

Proteomic and Functional Analyses of the Virion Transmembrane Proteome of Cyprinid Herpesvirus 3.

Virion transmembrane proteins (VTPs) mediate key functions in the herpesvirus infectious cycle. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses. The present study was devoted to CyHV-3 VTPs. Using mass spectrometry approaches, we identified 16 VTPs of the CyHV-3 FL strain. Mutagenesis experiments demonstrated that eight of these proteins are essential for viral growth in vitro (open reading frame 32 [ORF32], ORF59, ORF81, ORF83, ORF99, ORF106, ORF115, and ORF131), and eight are nonessential (ORF25, ORF64, ORF65, ORF108, ORF132, ORF136, ORF148, and ORF149). Among the nonessential proteins, deletion of ORF25, ORF132, ORF136, ORF148, or ORF149 affects viral replication in vitro, and deletion of ORF25, ORF64, ORF108, ORF132, or ORF149 impacts plaque size. Lack of ORF148 or ORF25 causes attenuation in vivo to a minor or major extent, respectively. The safety and efficacy of a virus lacking ORF25 were compared to those of a previously described vaccine candidate deleted for ORF56 and ORF57 (Δ56-57). Using quantitative PCR, we demonstrated that the ORF25 deleted virus infects fish through skin infection and then spreads to internal organs as reported previously for the wild-type parental virus and the Δ56-57 virus. However, compared to the parental wild-type virus, the replication of the ORF25-deleted virus was reduced in intensity and duration to levels similar to those observed for the Δ56-57 virus. Vaccination of fish with a virus lacking ORF25 was safe but had low efficacy at the doses tested. This characterization of the virion transmembrane proteome of CyHV-3 provides a firm basis for further research on alloherpesvirus VTPs.IMPORTANCE Virion transmembrane proteins play key roles in the biology of herpesviruses. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses and the causative agent of major economic losses in common and koi carp worldwide. In this study of the virion transmembrane proteome of CyHV-3, the major findings were: (i) the FL strain encodes 16 virion transmembrane proteins; (ii) eight of these proteins are essential for viral growth in vitro; (iii) seven of the nonessential proteins affect viral growth in vitro, and two affect virulence in vivo; and (iv) a mutant lacking ORF25 is highly attenuated but induces moderate immune protection. This study represents a major breakthrough in understanding the biology of CyHV-3 and will contribute to the development of prophylactic methods. It also provides a firm basis for the further research on alloherpesvirus virion transmembrane proteins.

Inherited Chromosomally Integrated Human Herpesvirus 6 Genomes Are Ancient, Intact, and Potentially Able To Reactivate from Telomeres.

The genomes of human herpesvirus 6A (HHV-6A) and HHV-6B have the capacity to integrate into telomeres, the essential capping structures of chromosomes that play roles in cancer and ageing. About 1% of people worldwide are carriers of chromosomally integrated HHV-6 (ciHHV-6), which is inherited as a genetic trait. Understanding the consequences of integration for the evolution of the viral genome, for the telomere, and for the risk of disease associated with carrier status is hampered by a lack of knowledge about ciHHV-6 genomes. Here, we report an analysis of 28 ciHHV-6 genomes and show that they are significantly divergent from the few modern nonintegrated HHV-6 strains for which complete sequences are currently available. In addition, ciHHV-6B genomes in Europeans are more closely related to each other than to ciHHV-6B genomes from China and Pakistan, suggesting regional variation of the trait. Remarkably, at least one group of European ciHHV-6B carriers has inherited the same ciHHV-6B genome, integrated in the same telomere allele, from a common ancestor estimated to have existed 24,500 ± 10,600 years ago. Despite the antiquity of some, and possibly most, germ line HHV-6 integrations, the majority of ciHHV-6B (95%) and ciHHV-6A (72%) genomes contain a full set of intact viral genes and therefore appear to have the capacity for viral gene expression and full reactivation.IMPORTANCE Inheritance of HHV-6A or HHV-6B integrated into a telomere occurs at a low frequency in most populations studied to date, but its characteristics are poorly understood. However, stratification of ciHHV-6 carriers in modern populations due to common ancestry is an important consideration for genome-wide association studies that aim to identify disease risks for these people. Here, we present full sequence analysis of 28 ciHHV-6 genomes and show that ciHHV-6B in many carriers with European ancestry most likely originated from ancient integration events in a small number of ancestors. We propose that ancient ancestral origins for ciHHV-6A and ciHHV-6B are also likely in other populations. Moreover, despite their antiquity, all of the ciHHV-6 genomes appear to retain the capacity to express viral genes, and most are predicted to be capable of full viral reactivation. These discoveries represent potentially important considerations in immunocompromised patients, in particular in organ transplantation and in stem cell therapy.

Characterization of Human Cytomegalovirus Genome Diversity in Immunocompromised Hosts by Whole-Genome Sequencing Directly From Clinical Specimens.

Background: Advances in next-generation sequencing (NGS) technologies allow comprehensive studies of genetic diversity over the entire genome of human cytomegalovirus (HCMV), a significant pathogen for immunocompromised individuals. Methods: Next-generation sequencing was performed on target enriched sequence libraries prepared directly from a variety of clinical specimens (blood, urine, breast milk, respiratory samples, biopsies, and vitreous humor) obtained longitudinally or from different anatomical compartments from 20 HCMV-infected patients (renal transplant recipients, stem cell transplant recipients, and congenitally infected children). Results: De novo-assembled HCMV genome sequences were obtained for 57 of 68 sequenced samples. Analysis of longitudinal or compartmental HCMV diversity revealed various patterns: no major differences were detected among longitudinal, intraindividual blood samples from 9 of 15 patients and in most of the patients with compartmental samples, whereas a switch of the major HCMV population was observed in 6 individuals with sequential blood samples and upon compartmental analysis of 1 patient with HCMV retinitis. Variant analysis revealed additional aspects of minor virus population dynamics and antiviral-resistance mutations. Conclusions: In immunosuppressed patients, HCMV can remain relatively stable or undergo drastic genomic changes that are suggestive of the emergence of minor resident strains or de novo infection.

Control of immune ligands by members of a cytomegalovirus gene expansion suppresses natural killer cell activation.

The human cytomegalovirus (HCMV) US12 family consists of ten sequentially arranged genes (US12-21) with poorly characterized function. We now identify novel natural killer (NK) cell evasion functions for four members: US12, US14, US18 and US20. Using a systematic multiplexed proteomics approach to quantify ~1300 cell surface and ~7200 whole cell proteins, we demonstrate that the US12 family selectively targets plasma membrane proteins and plays key roles in regulating NK ligands, adhesion molecules and cytokine receptors. US18 and US20 work in concert to suppress cell surface expression of the critical NKp30 ligand B7-H6 thus inhibiting NK cell activation. The US12 family is therefore identified as a major new hub of immune regulation.

Identification of compounds with anti-human cytomegalovirus activity that inhibit production of IE2 proteins.

Using a high throughput screening methodology we surveyed a collection of largely uncharacterized validated or suspected kinase inhibitors for anti-human cytomegalovirus (HCMV) activity. From this screen we identified three structurally related 5-aminopyrazine compounds (XMD7-1, -2 and -27) that inhibited HCMV replication in virus yield reduction assays at low micromolar concentrations. Kinase selectivity assays indicated that each compound was a kinase inhibitor capable of inhibiting a range of cellular protein kinases. Western blotting and RNA sequencing demonstrated that treatment of infected cells with XMD7 compounds resulted in a defect in the production of the major HCMV transcriptional transactivator IE2 proteins (IE2-86, IE2-60 and IE2-40) and an overall reduction in transcription from the viral genome. However, production of certain viral proteins was not compromised by treatment with XMD7 compounds. Thus, these novel anti-HCMV compounds likely inhibited transcription from the viral genome and suppressed production of a subset of viral proteins by inhibiting IE2 protein production.

Full Genome Sequence and sfRNA Interferon Antagonist Activity of Zika Virus from Recife, Brazil.

BACKGROUND: The outbreak of Zika virus (ZIKV) in the Americas has transformed a previously obscure mosquito-transmitted arbovirus of the Flaviviridae family into a major public health concern. Little is currently known about the evolution and biology of ZIKV and the factors that contribute to the associated pathogenesis. Determining genomic sequences of clinical viral isolates and characterization of elements within these are an important prerequisite to advance our understanding of viral replicative processes and virus-host interactions. METHODOLOGY/PRINCIPAL FINDINGS: We obtained a ZIKV isolate from a patient who presented with classical ZIKV-associated symptoms, and used high throughput sequencing and other molecular biology approaches to determine its full genome sequence, including non-coding regions. Genome regions were characterized and compared to the sequences of other isolates where available. Furthermore, we identified a subgenomic flavivirus RNA (sfRNA) in ZIKV-infected cells that has antagonist activity against RIG-I induced type I interferon induction, with a lesser effect on MDA-5 mediated action. CONCLUSIONS/SIGNIFICANCE: The full-length genome sequence including non-coding regions of a South American ZIKV isolate from a patient with classical symptoms will support efforts to develop genetic tools for this virus. Detection of sfRNA that counteracts interferon responses is likely to be important for further understanding of pathogenesis and virus-host interactions.

Investigation of Amphibian Mortality Events in Wildlife Reveals an On-Going Ranavirus Epidemic in the North of the Netherlands.

In the four years following the first detection of ranavirus (genus Ranavirus, family Iridoviridae) infection in Dutch wildlife in 2010, amphibian mortality events were investigated nationwide to detect, characterize and map ranaviruses in amphibians over time, and to establish the affected host species and the clinico-pathological presentation of the disease in these hosts. The ultimate goal was to obtain more insight into ranavirus disease emergence and ecological risk. In total 155 dead amphibians from 52 sites were submitted between 2011 and 2014, and examined using histopathology, immunohistochemistry, virus isolation and molecular genetic characterization. Ranavirus-associated amphibian mortality events occurred at 18 sites (35%), initially only in proximity of the 2010 index site. Specimens belonging to approximately half of the native amphibian species were infected, including the threatened Pelobates fuscus (spadefoot toad). Clustered massive outbreaks involving dead adult specimens and ranavirus genomic identity indicated that one common midwife toad virus (CMTV)-like ranavirus strain is emerging in provinces in the north of the Netherlands. Modelling based on the spatiotemporal pattern of spread showed a high probability that this emerging virus will continue to be detected at new sites (the discrete reproductive power of this outbreak is 0.35). Phylogenetically distinct CMTV-like ranaviruses were found in the south of the Netherlands more recently. In addition to showing that CMTV-like ranaviruses threaten wild amphibian populations not only in Spain but also in the Netherlands, the current spread and risk of establishment reiterate that understanding the underlying causes of CMTV-like ranavirus emergence requires international attention.

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.