Dalan Bailey

Norovirus Polymerase Fidelity Contributes to Viral Transmission In Vivo

Virus replication fidelity and hence the intrahost genetic diversity of viral populations are known to be intricately linked to viral pathogenesis and tropism as well as to immune and antiviral escape during infection. In this study, we investigated whether changes in replication fidelity can impact the ability of a virus to transmit between susceptible hosts by the use of a mouse model for norovirus. We show that a variant encoding a high-fidelity polymerase is transmitted less efficiently between mice than the wild-type strain. This constitutes the first experimental demonstration that the polymerase fidelity of viruses can impact transmission of infection in their natural hosts. These results provide further insight into potential reasons for the global emergence of pandemic human noroviruses that display alterations in the replication fidelity of their polymerases compared to nonpandemic strains. ABSTRACT Intrahost genetic diversity and replication error rates are intricately linked to RNA virus pathogenesis, with alterations in viral polymerase fidelity typically leading to attenuation during infections in vivo. We have previously shown that norovirus intrahost genetic diversity also influences viral pathogenesis using the murine norovirus model, as increasing viral mutation frequency using a mutagenic nucleoside resulted in clearance of a persistent infection in mice. Given the role of replication fidelity and genetic diversity in pathogenesis, we have now investigated whether polymerase fidelity can also impact virus transmission between susceptible hosts. We have identified a high-fidelity norovirus RNA-dependent RNA polymerase mutant (I391L) which displays delayed replication kinetics in vivo but not in cell culture. The I391L polymerase mutant also exhibited lower transmission rates between susceptible hosts than the wild-type virus and, most notably, another replication defective mutant that has wild-type levels of polymerase fidelity. These results provide the first experimental evidence that norovirus polymerase fidelity contributes to virus transmission between hosts and that maintaining diversity is important for the establishment of infection. This work supports the hypothesis that the reduced polymerase fidelity of the pandemic GII.4 human norovirus isolates may contribute to their global dominance. IMPORTANCE Virus replication fidelity and hence the intrahost genetic diversity of viral populations are known to be intricately linked to viral pathogenesis and tropism as well as to immune and antiviral escape during infection. In this study, we investigated whether changes in replication fidelity can impact the ability of a virus to transmit between susceptible hosts by the use of a mouse model for norovirus. We show that a variant encoding a high-fidelity polymerase is transmitted less efficiently between mice than the wild-type strain. This constitutes the first experimental demonstration that the polymerase fidelity of viruses can impact transmission of infection in their natural hosts. These results provide further insight into potential reasons for the global emergence of pandemic human noroviruses that display alterations in the replication fidelity of their polymerases compared to nonpandemic strains.

Human liver sinusoidal endothelial cells promote intracellular crawling of lymphocytes during recruitment‐ a new step in migration

The recruitment of lymphocytes via the hepatic sinusoidal channels and positioning within liver tissue is a critical event in the development and persistence of chronic inflammatory liver diseases. The hepatic sinusoid is a unique vascular bed lined by hepatic sinusoidal endothelial cells (HSECs), a functionally and phenotypically distinct subpopulation of endothelial cells. Using flow‐based adhesion assays to study the migration of lymphocytes across primary human HSECs, we found that lymphocytes enter into HSECs, confirmed by electron microscopy demonstrating clear intracellular localization of lymphocytes in vitro and by studies in human liver tissues. Stimulation by interferon‐γ increased intracellular localization of lymphocytes within HSECs. Furthermore, using confocal imaging and time‐lapse recordings, we demonstrated “intracellular crawling” of lymphocytes entering into one endothelial cell from another. This required the expression of intracellular adhesion molecule‐1 and stabilin‐1 and was facilitated by the junctional complexes between HSECs. Conclusion: Lymphocyte migration is facilitated by the unique structure of HSECs. Intracellular crawling may contribute to optimal lymphocyte positioning in liver tissue during chronic hepatitis. (Hepatology 2017;65:294‐309).

Co-circulation of Peste-des-Petits-Ruminants Virus Asian lineage IV with Lineage II in Nigeria

Peste-des-petits-ruminants (PPR), a major small ruminant transboundary animalxa0disease, is endemic in Nigeria. Strains of the causal agent, peste-des-petits-ruminants virus (PPRV), have been differentiated into four genetically distinct lineages based on the partial sequence of the virus nucleoprotein (N) or fusion (F) genes. Peste-des-petits-ruminants virus strains that were identified initially in Africa were grouped into lineages I, II and III and viruses from Asia were classified as lineage IV and referred to as the Asian lineage. Many recent reports indicate that the Asian lineage is now also present in Africa. With this in mind, this study was conducted to reassess the epidemiology of PPRV in Nigeria. A total of 140 clinical samples from 16 sheep and 63 goats with symptoms suggestive of PPR were collected from different states of Nigeria during a four-year period (2010-2013). They were analysed by the amplification of fragments of the N gene. Results for 33 (42%) animals were positive. The phylogenetic analysis of the N gene sequences with those available in GenBank showed that viruses that were detected belong to both lineage II and IV. Based on an analysis of the N gene sequences, the lineage IV isolates grouped into two clades, one being predominant in the north-eastern part of the country and the other found primarily in the southern regions of the country. This study reports the presence of PPRV Asian lineage IV in Nigeria for the first time.

Identifying novel protein interactions: Proteomic methods, optimisation approaches and data analysis pipelines.

The technological revolution in high-throughput nucleic acid and protein analysis in the last 15 years has launched the field of omics and led to great advances in our understanding of cell biology. Consequently the study of the cellular proteome and protein dynamics, in particular interactomics, has been a matter of intense investigation, specifically the determination and description of complex protein interaction networks in the cell, not only with other proteins but also with RNA and DNA. The analysis of these interactions, beginning with their identification and ultimately resulting in structural level examination, is one of the cornerstones of modern biological science underpinning basic research and impacting on applied biology, biomedicine and drug discovery. In this review we summarise a selection of emerging and established techniques currently being applied in this field with a particular focus on affinity-based purification systems and their optimisation, including tandem affinity purification (TAP) tagging, isolation of proteins on nascent DNA (IPOND) and RNA-protein immunoprecipitation in tandem (RIPiT). The recent application of quantitative proteomics to improve stringency and specificity is also discussed, including the use of metabolic labelling by stable isotope labelling by amino acids in cell culture (SILAC), localization of organelle proteins by isotope tagging (LOPIT) and proximity-dependent biotin identification (BioID). Finally, we describe a range of software resources that can be applied to interactomics, both to handle raw data and also to scrutinise its broader biological context. In this section we focus especially on open-access online interactomic databases such as Reactome and IntAct.

The measles virus receptor SLAMF1 can mediate particle endocytosis

ABSTRACT The signaling lymphocyte activation molecule F1 (SLAMF1) is both a microbial sensor and entry receptor for measles virus (MeV). Herein, we describe a new role for SLAMF1 to mediate MeV endocytosis that is in contrast with the alternative, and generally accepted, model that MeV genome enters cells only after fusion at the cell surface. We demonstrated that MeV engagement of SLAMF1 induces dramatic but transient morphological changes, most prominently in the formation of membrane blebs, which were shown to colocalize with incoming viral particles, and rearrangement of the actin cytoskeleton in infected cells. MeV infection was dependent on these dynamic cytoskeletal changes as well as fluid uptake through a macropinocytosis-like pathway as chemical inhibition of these processes inhibited entry. Moreover, we identified a role for the RhoA-ROCK-myosin II signaling axis in this MeV internalization process, highlighting a novel role for this recently characterized pathway in virus entry. Our study shows that MeV can hijack a microbial sensor normally involved in bacterial phagocytosis to drive endocytosis using a complex pathway that shares features with canonical viral macropinocytosis, phagocytosis, and mechanotransduction. This uptake pathway is specific to SLAMF1-positive cells and occurs within 60 min of viral attachment. Measles virus remains a significant cause of mortality in human populations, and this research sheds new light on the very first steps of infection of this important pathogen. IMPORTANCE Measles is a significant disease in humans and is estimated to have killed over 200 million people since records began. According to current World Health Organization statistics, it still kills over 100,000 people a year, mostly children in the developing world. The causative agent, measles virus, is a small enveloped RNA virus that infects a broad range of cells during infection. In particular, immune cells are infected via interactions between glycoproteins found on the surface of the virus and SLAMF1, the immune cell receptor. In this study, we have investigated the steps governing entry of measles virus into SLAMF1-positive cells and identified endocytic uptake of viral particles. This research will impact our understanding of morbillivirus-related immunosuppression as well as the application of measles virus as an oncolytic therapeutic.

Future research to underpin successful peste des petits ruminants virus (PPRV) eradication

Peste des petits ruminants virus (PPRV) is a significant pathogen of small ruminants and is prevalent in much of Africa, the Near and Middle East and Asia. Despite the availability of an efficacious and cheap live-attenuated vaccine, the virus has continued to spread, with its range stretching from Morocco in the west to China and Mongolia in the east. Some of the worlds poorest communities rely on small ruminant farming for subsistence and the continued endemicity of PPRV is a constant threat to their livelihoods. Moreover, PPRVs effects on the worlds population are felt broadly across many economic, agricultural and social situations. This far-reaching impact has prompted the Food and Agriculture Organization of the United Nations (FAO) and the World Organisation for Animal Health (OIE) to develop a global strategy for the eradication of this virus and its disease. PPRV is a morbillivirus and, given the experience of these organizations in eradicating the related rinderpest virus, the eradication of PPRV should be feasible. However, there are many critical areas where basic and applied virological research concerning PPRV is lacking. The purpose of this review is to highlight areas where new research could be performed in order to guide and facilitate the eradication programme. These areas include studies on disease transmission and epidemiology, the existence of wildlife reservoirs and the development of next-generation vaccines and diagnostics. With the support of the international virology community, the successful eradication of PPRV can be achieved.

Serological evidence of camel exposure to peste des petits ruminants virus (PPRV) in Nigeria

Peste des petits ruminants (PPR), a viral disease of sheep and goats, is endemic in Nigeria. There are reports indicating the involvement of peste des petits ruminants virus (PPRV), the causative agent of PPR, in a camel respiratory syndrome in Africa. Considering that camels share the same grazing land and drinking points with other ruminants, this study was undertaken to determine the seroprevalence and extent of PPRV antibodies in Nigerian camels. A total of 1517 camel sera samples were collected from four states (Borno, Kano, Kastina and Sokoto). The seroprevalence was determined by the H-protein-based competitive ELISA. The overall prevalence was 3.36xa0% (51/1517, 95xa0% confidence interval of 2.51–4.39xa0%). There was no significant differences in prevalence between states (pu2009=u20090.8921) and between male and female camels (pu2009=u20090.7424). The prevalence differed significantly (pu2009<u20090.00001) by body condition score; camels with poor body condition score has higher (16.67xa0%) antibody seroprevalence to PPR compared to those with fair and good body condition score. There was a statistically significant difference between camels aged ≤ 5xa0years and those >5xa0years (pu2009=u20090.0042). These results show occasional transient PPRV infection of camels in Nigeria, and there is the need to include camels among species to be studied in elucidating the epidemiology of the disease in sheep and goats.

Detection of protein-protein interactions using tandem affinity purification.

Tandem affinity purification (TAP) is an invaluable technique for identifying interaction partners for an affinity tagged bait protein. The approach relies on the fusion of dual tags to the bait before separate rounds of affinity purification and precipitation. Frequently two specific elution steps are also performed to increase the specificity of the overall technique. In the method detailed here, the two tags used are protein G and a short streptavidin binding peptide; however, many variations can be employed. In our example the tags are separated by a cleavable tobacco etch virus protease target sequence, allowing for specific elution after the first round of affinity purification. Proteins isolated after the final elution step in this process are concentrated before being identified by mass spectrometry. The use of dual affinity tags and specific elution in this technique dramatically increases both the specificity and stringency of the pull-downs, ensuring a low level of background nonspecific interactions.

Serosurvey of peste des petits ruminants virus in small ruminants from different agro-ecological zones of Nigeria

Peste des petits ruminants, caused by the peste des petits ruminants virus (PPRV), is a highly contagious and economically important transboundary viral disease of domestic and wild small ruminants and a major hindrance to small-ruminant production in Nigeria. The seroprevalence and distribution of PPRV antibodies in small ruminants in rural households, farms, live animal markets and slaughter slabs across the six different agro-ecological zones of Nigeria were determined. A total of 4548 serum samples from 3489 goats and 1059 sheep were collected in 12 states. A PPRV competitive enzyme-linked immunosorbent assay was used to test the samples and the data analysed with R statistical software version 3.0.1. The study animals included all ages and both sexes. The overall prevalence estimate of sera positive for PPRV antibodies was 23.16% (n = 1018 positive samples per 4548 total samples, 95% confidence interval: 21.79% – 24.57%). There were significant differences in the seroprevalence between the states (p = 0.001). Taraba State had the highest seroprevalence of 29.51%, whilst the lowest seroprevalence of 14.52% was observed in Cross River State. There were no significant differences in the PPRV seroprevalence between male and female animals (p = 0.571), age (p = 0.323) and between species (p = 0.639). These data indicate the current seroprevalence to PPRV in the small-ruminant population in Nigeria.

WITHDRAWN: Identification of peste-des-petits ruminants virus (PPRV) lineage IV, the Asian lineage, in Nigeria and co-circulation with PPRV lineage II.

This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.