- Volume 1, Issue 1A, 2019

Influenza A Virus (IAV) is a zoonotic pathogen which causes seasonal epidemics worldwide, resulting in significant morbidity and mortality. Due to rapid virus evolution, available vaccines/antiviral drugs must be re-formulated and re-administered in most years. There is therefore an unmet need for a ‘universal’ vaccine to provide long-lasting, adaptive immunity to multiple strains of IAV. The aim of this study was to assess outer membrane vesicles (OMVs) produced by Bacteroides thetaiotaomicron (Bt) containing IAV antigens as a candidate universal mucosal vaccine. First, using intranasally-delivered fluorescently-labelled OMVs and FACS we demonstrated good uptake of OMVs by dendritic cells in both secondary and tertiary lymphoid tissue, showing OMVs are efficiently trafficked from the mucosa to the lymphatic system. Immunohistology confirmed this and showed expansion of lymphoid tissues and lymphoid follicles after OMV delivery. The stem/stalk of IAV HA is less variable and will generate antibodies that are cross-protective. Thus, we produced OMVs (termed H5F) that contained the stalk region of HA from strain A/VietNam/1203/04 (H5N1). We inoculated mice intranasally with H5F expressing OMVs or wild type OMVs and assessed antibody production and protection from challenge using a lethal dose of a different strain of influenza (PR8; H1N1). OMV-H5F-inoculted mice produced IgG and IgA in the respiratory tract that recognised both H5 HA and H1 HA. OMV-H5F inoculated mice were also protected from lethal challenge and supported lower virus titres. Our results indicate a strong potential for this approach to generate a universal IAV vaccine.

The vast majority of infectious agents enter the body via mucosal sites yet there are very few licensed mucosal vaccines able to generate protective immunity at the sites of pathogen entry. A major obstacle in developing mucosal vaccine is delivering biologically active vaccine antigens (Ag) to mucosa-associated lymphoid tissues to prime protective immune responses. To address these issues we have developed a drug delivery technology platform to deliver intact antigens to the respiratory and gastrointestinal tract using outer membrane vesicles (OMV) naturally produced by Bacteroides thetaiotaomicron (Bt), a non-pathogenic human commensal gut bacterium. We have developed the capability of engineering Bt to express antigens of interest in their OMVs which we have shown are stable for long periods of time across a wide temperatures range. They also have inherent adjuvanticity as shown by the ability of native OMVs to elicit the formation of organised lymphoid follicles comprising dendritic cells and T and B cells in both the upper and lower respiratory tract after intranasal administration. The intransal administration of Bt OMVs expressing the pre-fusion headless hemagglutinin mini-stem protein of influenza type A virus (IAV) subtype, H5N1, induced high titre antigen-specific antibodies in the respiratory mucosa (IgA) and serum (IgG) that conferred heterotypic protection to infection by a H1N1 IAV. Collectively, our data demonstrates the feasibility of using Bt OMVs in mucosal vaccine formulations for respiratory infections.

Chikungunya (CHIKV), an arbovirus that belongs to the Alphavirus genus of the Togaviridae family, causes a disease characterized by acute onset of fever accompanied by arthralgia. CHIKV also has been associated with cases of meningoencephalitis (primarily in neonates), Guillain–Barré syndrome and hemorrhagic disease. The clinical similarities, cross-reactivity and cocirculation of arboviruses in Brazil have complicated their differentiation, highlighting the need for new diagnostic tools. A serologic test can be useful for acute detections as well as for surveillance and epidemiological studies. Point of care tests currently available to detect CHIKV infections have been associated with low accuracy. The aim of this work was to design a synthetic gene and generate a recombinant protein to be used as antigens in diagnostic assays. Computational methods were used to predict its structure and antigenic potential. To confirm predictions, the gene coding for the recombinant CHIKV protein (rCHIKV) was synthetized and the protein was purified by affinity chromatography. Antigenicity of the protein was initially confirmed by western-blot using sera from CHIKV infected mice. Additionally, the seroreactivity of r-CHIKV protein was evaluated using a panel of sera samples from human patients, CHIKV seropositive or not, by indirect IgG ELISA. The r-CHIKV protein showed sensitivity of 95 % and specificity of 96 %. No cross-reactivity was found against sera of Zika and Dengue positive patients. These results indicate that this proteins maybe useful antigen to detect CHIKV infections in ELISA assays. Our next step includes the development of a rapid test kit.

Identifying transient low-level hepatitis B (HBV) surface antigen (sAg) post-vaccination is a well-established phenomenon in haemodialysis patients. These results create a clinical conundrum; should patients be deemed potentially infectious, requiring further testing and heightened infection control precautions, or should negligible risk be assumed? National guidance covering this scenario is lacking; therefore we conducted a survey of the clinical practice of UK virology departments. Thirty-six laboratories were contacted and 17 responses received (47 %). Eleven responders had observed sAg positivity post-vaccination, for a maximum duration of 3 weeks. Clinical management was highly variable. Only 3 hospitals had specific written policies, with others following an internal consensus or a standard protocol for sAg positive patients without recent vaccine. Further testing consisted of HBV serology (3/11), HBV PCR (1/11) or PCR and serology (7/11). Three departments were highly risk averse, recommending universal isolation with dedicated dialysis machines. Two departments did not advise additional precautions and 5 based decisions on risk factors e.g. foreign travel. Heightened precautions were stopped on the basis of negative serology (1/11), PCR (5/11) or serology and PCR (2/11). To summarise, vaccine-derived sAg positivity is commonly encountered on haemodialysis units. Recommendations on the interval between vaccination and testing would alleviate the issue, with this study suggesting a minimum of 3 weeks. Infection control precautions varied considerably. In most cases, patients are risk assessed for isolation as a minimum; however this may depend on local expertise and the availability of isolation facilities. It is clear that national guidance would standardise patient care.

Molecular diagnostics have superseded cell culture and neutralisation as the gold standard for enterovirus (EV) diagnosis and serotyping in resource rich countries. EV positive isolates from Nottingham University Hospitals NHS Trust are referred to the Enteric Virus Unit (EVU) for typing; performed through amplification and sequencing of viral capsid protein-1 (VP1). An audit of local EVU typing data (2013–2017 showed a significant (P<0.005) increase in untypable EV strains isolated from clinical specimens. Following a literature review of published EV typing assays and in silico analyses of 2201 EV genomes, an end point RT-snPCR VP1-typing assay was derived from a widely cited protocol and successfully employed to produce a simple, reliable and cost-effective typing assay. Twenty-three previously untypable isolates were identified, with phylogenetic analyses of the VP1 region showing broad distribution across EV-A and B species. This assay detected serotypes across EV species A-D, including EV-D68, responsible for severe neurological and respiratory disease. Following an increase in severe EV infections, including six cases of suspected acute flaccid myelitis, this RT-snPCR was employed locally as a rapid typing assay. In combination with 5’UTR PCR and an EV-D68 specific PCR, designed to amplify the complete VP1 region, EV-D68 was detected in respiratory and neurological specimens, including the CSF of a patient with brainstem encephalitis.

Nottingham University Hospitals Trust receives circa 13 000 samples for diagnosis of respiratory and neurological viruses per annum, however positive results are achieved in approximately 50 % of respiratory and 20 % of neurological investigations. We therefore aimed to retrospectively extend the diagnostic spectrum for these samples by applying a battery of degenerate PCR assays to surplus diagnostic nucleic acids.218 previously negative respiratory specimens collected in 2016 from children under 5 years old, identified positivity of 11 % for Human Bocavirus, 5 % for Human Enteroviruses (including 3 cases of Enterovirus D68), 4 % for Human Coronaviruses and one individual positive for Trichodysplasia Spinulosa Polyomavirus. Complementary investigation of 1730 previously negative specimens from children and adults with neurological symptoms yielded positive results for Hepatitis E, BK Polyomavirus and Astroviruses in addition to Entero- and Parechoviruses apparently missed by standard diagnostic assays. Our extensive archive further allowed us to investigate relatively rare viral infections in significant numbers. Therefore we also studied the genetic and clinical epidemiology of the human Rubulavirus pathogens Parainfluenza 2 and 4 in 121 and 237 patients respectively between 2013 and 2017. This indicated co-circulation of three clusters of Parainfluenza 4 in Nottingham with greater presentation of subtype 4b than 4a and 2 clades of Parainfluenza 2. 5 fatalities were recorded in the Parainfluenza 2 cohort. In summary, surplus nucleic acid from viral diagnostic laboratories represents a valuable resource for both service development and clinical research. Coronavirus, Bocavirus and Enterovirus testing have since been implemented in the routine diagnostic panel for respiratory investigations.

Herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) infections may be life-threatening in immunocompromised patients. Antiviral treatment is available but resistance can develop. The gold standard for resistance testing is cell culture-based phenotyping which is slow (3–4 weeks) and has a high (50 %) failure rate. Faster (<10 working days) genotypic testing of viral thymidine kinase (TK) and DNA polymerase (DNApol) can be used with >95 % success rate. However, a reference database for interpreting drug susceptibility from genetic data is lacking. We developed an HSV genotypic test and genotype-to-phenotype drug resistance database. We evaluated the service using 325 clinical samples, previously characterised by phenotypic susceptibility testing, from 248 treatment-experienced patients. The median age was 42.5 years [IQR30.0–51.0] and 50.8 % (n=126) were female. Clinical details were as follows: 42.3 % (n=105) haemato-oncology patients; 12.1 % (n=30) HIV-infected; 4.4 % (n=11) unspecified immunosuppression; 2.4 % (n=6) congenital infection; 1.2 % (n=3) solid organ transplant; 37.5 % (n=93) unknown. HSV-1 was identified in 58.2 % (n=189) samples. Phenotypic testing identified resistance in 63.7 % (n=207) samples. Genotypic testing identified a resistance-associated mutation (RAM) in TK and/or DNApol in 200/207 samples, a positive percent agreement of 96.6 %, whereas a RAM was detected in 1/118 susceptible samples, a negative percent agreement of 99.2 %. Most RAM occurred in TK (n=195; 97.0 %) with few in DNApol (n=32; 15.9 %). In summary, through herPHEgen we have developed a robust HSV resistance testing service, providing clinicians with timely and accurate results. This will improve clinical decision-making, optimising treatment efficacy and minimising toxicity in immunocompromised patients with HSV.

Current guidelines indicate that baseline INI resistance testing is not required; however, increased prescribing may lead to increased resistance mutation prevalence. NGS allows the detection of minority variants (MVs) not be detected by conventional methods. Barts Health NHS Trust patient records were reviewed to identify INI naïve patients prescribed INI between March 2014 and March 2017. Baseline plasma samples were extracted for nucleic acid and amplified in a single RT-PCR. Libraries were prepared using the Nextera XT library preparation kit and sequencing performed on an Illumina Miseq. Sequencing reads were assembled into consensus sequences and resistances determined using the Stanford HIV resistance database (HIVdb). MVs were variants <20 % of the overall population. Of 248 patients prescribed INI, 128 patients were excluded from the study due to an undetectable viral load or lack of sample. Of the remaining 121 patients, integrase sequences were successfully obtained from 91. No major INI mutations were detected. Six patients were identified with an HIVdb INI score ≥10: E157Q (4 patients) and T97A (2 patients). Only 1 MV accessory mutations was observed (A128T). Of patients with resistance mutations, 5 received raltegravir- and 2 received dolutegravir-based regimes. No treatment failures were observed. Prevalence of baseline INI mutations increased in recent years: 0/12 (0 %) patients in 2014–2015, 2/32 (6.3 %) patients in 2015–2016 and 5/47 (10.6 %) patients in 2016–2017. Resistance MVs to INI were extremely rare. Although this study supports current baseline INI resistance testing guidelines, increasing mutation prevalence’s may require future revision of guidelines.

Routine clinical diagnosis of patients with a suspected viral infection involves screening with multiple assays, often limited to only a single genus or species. This approach, however, may fail to detect novel species, atypically presenting viruses (i.e a ‘respiratory’ virus causing neurological symptoms) and viruses that are imported from other countries; often due to clinicians focusing on the ‘likeliest’ candidates. High-throughput sequencing (HTS) allows for the identification viruses present within a sample, by sequencing all viral genomes present. Without the inherent bias of limiting screening targeted assays, these unusual viruses are more likely to be detected. Samples taken from patients with an illness of unknown etiology, were grouped into 5 pools; 2 Respiratory, 2 CSF and an EDTA blood pool. The CSF Pools each contained 200 samples, the Respiratory pools 100 and the EDTA blood pool 80. HTS libraries were created from each of these pools and an additional CSF sample from a single patient with encephalitis and were then sequenced using an Illumina HiSeq platform. Human Pegivirus was detected in both CSF Pools, the EDTA pool and a single respiratory pool. Picobirnavirus was detected in a respiratory pool. RT-PCR was used to screen individual samples compromising these pools. BK Polyomavirus and Mastadenovirus C were detected in the CSF of a patient who had presented with encephalitis. Coinfection of these viruses typically cause neurological symptoms only in immune-compromised patients, so this exemplifies the advantage of using HTS for the detection of atypically presenting viruses.

To date, models of human cytomegalovirus (HCMV) latency and reactivation have depended on the use of primary myeloid cells, which have limited availability, are difficult to culture and are challenging to genetically modify. We now show that induced pluripotent stem cells (iPSCs) derived from circulating late outgrowth endothelial progenitors (EPC) can be differentiated down the myeloid lineage, where HCMV latent carriage and reactivation is known to occur in vivo, and act as a model to allow the interrogation of viral and cellular factors involved in latency and reactivation of this persistent human pathogen. In contrast, monocytes generated from iPSCs derived from de-differentiated fibroblasts failed to support HCMV latent carriage. These iPSCs derived from EPCs may also be suitable for in depth genetic interrogation of other viruses which also infect cells of the myeloid lineage, such as HIV and Zika.

As an obligate intracellular parasite, human cytomegalovirus (HCMV) completely relies on host machinery to replicate. Understanding which host factors are required for virus replication contributes to our understanding of virus biology and cell biology, identification of potential targets for antiviral therapy. High-throughput small interfering RNA (siRNA) screens are a powerful approach to identify novel host-virus interactions. Conventional screens often use reporter genes as a proxy for virus replication, rather than measuring production of infectious virus. We developed a two-step siRNA screen that independently measured primary replication and virus production. Screening with a library targeting almost 7000 genes, we identified 37 genes involved in early stages of HCMV replication and 15 genes specifically involved in later aspects, such as late gene expression, assembly and egress. These include factors in ubiquitin-dependent protein degradation pathway, and components of the mediator complex. Furthermore, we showed that the induction of SIN3A, a transcriptional regulator that forms a repressor complex with histone deacetylase 1 and 2, is essential for late gene expression and virus production. This study demonstrates a powerful two-step high throughput approach which identifies key host factors underpinning HCMV replication and informs our understanding of how the virus interacts with its host.

Genome segmentation offers certain evolutionary benefits to a number of pathogenic RNA viruses, including rotaviruses and influenza viruses. However, as the number of RNA segments per virion increases, the task of a non-random selection of a full set of distinct genomic RNAs poses a formidable challenge to maintaining the integrity of segmented genomes. Recently we have identified sequence-specific inter-segment interactions between rotavirus (+)ssRNA genome segment precursors. We have shown that binding of the rotavirus-encoded non-structural protein NSP2 to viral ssRNAs results in the remodeling of RNA, which is conducive to formation of inter-segment contacts. These protein-RNA interactions result in the stabilisation of extended intermolecular RNA-RNA contacts, potentially underpinning transient inter-segment interactions prior to genome encapsidation and replication. Using this approach, we have identified a number of RNA-RNA interaction sites in the rotavirus genome, which are likely to be involved in genome segment assortment process. Having established the role of NSP2 in promoting inter-segment RNA-RNA contacts, we have developed multiplexed imaging tools for directvisualization of the RNA assortment process in rotavirus-infected cells by employing single-molecule RNA FISH. To unravel the mechanisms, by which NSP2 controls the formation of inter-molecular RNA helices, we have applied RNA structure probing methods that allowed us to monitor conformational rearrangements, which are prerequisite for theformation of the RNA assortment complex. Our findings open up unique avenues for understanding the challenges for further improvement of the recently developed fully plasmid-based reverse genetics systems for rotaviruses.

Infectious bronchitis virus (IBV), a gammacoronavirus, causes the economically important poultry disease, infectious bronchitis, resulting in reduced weight gain and egg quality. As observed for many viruses, during replication, IBV shuts off translation of host proteins, preventing synthesis of important products of the innate immunity, which are pivotal in fighting viral infection. This work investigates the role of stress granules in IBV translational control. Stress granules are membranes-less aggregations of stalled translation initiation complexes comprising translation initiation factors, 40S ribosome and RNA binding proteins. These structures serve as sites of storage and sequestration of translational machinery and cellular mRNA while simultaneously enabling intracellular signalling and antiviral responses. It is shown here by immunofluorescence that IBV induces stress granules in only a proportion of infected cells. These stress granules occur late in the virus life cycle and appear canonical, containing multiple stress granule markers and showing mRNA exchange with ribosomes. In addition, stress granule markers are not diverted to sites of virus replication, as seen during replication of some other viruses. Interestingly, IBV infection results in resistance to chemicals that induce stress granules via eukaryotic initiation factor 2α (eIF2α). Consistent with this, eIF2α is not phosphorylated at any time during IBV infection. This also indicates a non-canonical signalling pathway for IBV-induced stress granules. Significantly, stress granule formation is uncoupled from translational arrest as visualised using ribopuromycylation. Therefore, IBV replication both induces and inhibits cellular stress granule signalling in a process that is uncoupled from shut off of host translation.

Chikungunya virus (CHIKV) causes fever and debilitating joint pain, with frequent long-term health implications and cumulating fatalities worldwide. There are no specific antivirals and vaccines, therefore understanding CHIKV replication is essential to establish treatments and preventative measures. Cellular ion channels are druggable targets and are known to facilitate replication of RNA viruses. To determine if the activities of cellular chloride channels (Cl--channels) are required during CHIKV replication, we applied broad-ranging inhibitors and siRNA to mammalian and invertebrate cells. The Cl--channel inhibitors DIDS, 9-ACA and NPPB significantly reduced the titre of released CHIKV progeny at 12 h post-infection in a dose-dependent manner suggesting that Cl--channels are pro-viral factors. Analysis of viral protein expression and time-of-inhibitor-addition studies indicated that CHIKV requires Cl--channels at post-entry and pre-egress stages. Replication of a sub-genomic replicon was restricted and genome copy numbers reduced by Cl--channel inhibition, implying that Cl--channels are involved in genome replication. siRNA knock-down identified the chloride intracellular channels (CLIC) 1 and 4 to be required for the CHIKV infectious cycle with CLIC1 interacting with the viral protein nsP3. We hypothesise that the channels play a role in formation or maintenance of the membranous, viral replication-complexes and that this important role is conservt amongst the mammalian and invertebrate hosts. These findings advance our understanding of CHIKV replication in the two host environments and help to identify drugs/druggable targets for treatment and prevention of CHIKV disease.

Influenza virus encodes a heterotrimeric RNA-dependent RNA polymerase (RdRP), composed of subunits PB1, PB2 and PA, that carries out both transcription and replication of the viral RNA genome segments in the context of ribonucleoproteins. Replication of negative-sense viral RNA (vRNA) is a two-step process, progressing via a positive-sense complementary RNA (cRNA) intermediate. The mechanism of viral genome replication is mostly unknown, though there are multiple reports indicating RdRP dimerisation may be central for the process. Purified RdRPs from human and avian influenza A viruses both form dimers of heterotrimers in solution. Using a combination of X-ray crystallography, SAXS and cryo-EM, we identify the interface involved in RdRP dimerization, which is primarily located on the PA C-terminal domain. We use bimolecular fluorescence complementation (BiFC) to show that influenza RdRP forms dimers in mammalian cells through the interface identified in solution. Using a combination of cell-based and in vitro assays, we show that influenza RdRP dimerisation via the PA-C terminal domain is necessary for copying cRNA back into vRNA during viral genome replication. In addition, we show that a nanobody (a small-domain antibody) that interferes with dimerisation attenuates influenza A virus growth in cell culture. These data provide insight into the mechanism of influenza viral genome replication, and identify a potential novel drug target against influenza A virus.

To elucidate linkage between replication and encapsidation in Picornavirales, we have taken advantage of the bipartite nature of the plant-infecting member of the order, cowpea mosaic virus (CPMV), to decouple the two processes. RNA-free virus-like particles (eVLPs) can be generated by transiently co-expressing the RNA-2-encoded coat protein precursor (VP60) with the RNA-1-encoded 24K protease, in the absence of the replication machinery (Saunders et al., 2009). We have made use of the ability to produce assembled capsids of CPMV in the absence of replication to examine the putative linkage between RNA replication and packaging in the Picornavirales. We show here that the remarkable specificity of packaging observed in CPMV is due to a functional linking between the two processes of viral replication and encapsidation. We have created a series of mutant RNA-1 and RNA-2 molecules and have assessed the effect of the mutations on both the replication and packaging of the viral RNAs. We demonstrate that mutations that affect replication have a concomitant impact on encapsidation, and that RNA-1-mediated replication is required for encapsidation of both RNA-1 and RNA-2. This close coupling between replication and encapsidation provides a means for the specific packaging of viral RNAs. Moreover, we demonstrate that this feature of CPMV can be used to specifically encapsidate custom RNA by placing a sequence of choice between the RNA-2 sequences required for replication, which opens the door to novel research and therapeutic applications in the field of custom RNA packaging and delivery technologies.

Infectious bronchitis virus (IBV), an avian gammacoronavirus, is an important pathogen causing significant animal welfare problems and economic losses to the global poultry industry. Positive-strand RNA viruses, including coronaviruses, induce cellular membrane rearrangements during replication forming replication organelles, which are thought to support efficient viral RNA synthesis. IBV replication has been shown to induce the formation of double membrane vesicles (DMVs), zippered ER and tethered vesicles, known as spherules. Although these are proposed to be the site of viral RNA synthesis, this is as yet unconfirmed and is therefore the focus of these studies. Historically, dsRNA has been used as a marker for sites of coronavirus RNA synthesis, however IBV-associated dsRNA and nsp12 (the viral RNA-dependent RNA polymerase) do not colocalise in infected cells. We have determined the cellular location of the viral genome using Fluorescence In Situ Hybridisation (FISH). By comparing the immunofluorescence labelling of cells permeabilised with different detergents, we have demonstrated that dsRNA can be found within membrane-protected compartments, while nsp12 is not, indicating that the virus could be isolating the dsRNA in DMVs or spherules, affording protection from the host immune response. By incorporating uridine analogues over the course of infection with IBV, we have visualised sites of nascent viral RNA synthesis using super-resolution microscopy. This has shown that dsRNA appears to colocalise more strongly with nascent RNA than nsp12. Using these methods as well as looking at the ultrastructural level we are able to begin to discover the location of sites of IBV RNA synthesis.