Phosphoproteomic Analysis Reveals the Importance of Kinase Regulation During Orbivirus Infection.

Bluetongue virus (BTV) causes infections in wild and domesticated ruminants with high morbidity and mortality and is responsible for significant economic losses in both developing and developed countries. BTV serves as a model for the study of other members of the Orbivirus genus. Previously, the importance of casein kinase 2 for BTV replication was demonstrated. To identify intracellular signaling pathways and novel host-cell kinases involved during BTV infection, the phosphoproteome of BTV infected cells was analyzed. Over 1000 phosphosites were identified using mass spectrometry, which were then used to determine the corresponding kinases involved during BTV infection. This analysis yielded protein kinase A (PKA) as a novel kinase activated during BTV infection. Subsequently, the importance of PKA for BTV infection was validated using a PKA inhibitor and activator. Our data confirmed that PKA was essential for efficient viral growth. Further, we showed that PKA is also required for infection of equid cells by African horse sickness virus, another member of the Orbivirus genus. Thus, despite their preference in specific host species, orbiviruses may utilize the same host signaling pathways during their replication

Selection and Characterization of Rupintrivir-Resistant Norwalk Virus Replicon Cells In Vitro.

Human norovirus (HuNoV) is a major cause of nonbacterial gastroenteritis worldwide, yet despite its impact on society, vaccines and antivirals are currently lacking. A HuNoV replicon system has been widely applied to the evaluation of antiviral compounds and has thus accelerated the process of drug discovery against HuNoV infection. Rupintrivir, an irreversible inhibitor of the human rhinovirus 3C protease, has been reported to inhibit the replication of the Norwalk virus replicon via the inhibition of the norovirus protease. Here we report, for the first time, the generation of rupintrivir-resistant human Norwalk virus replicon cells in vitro Sequence analysis revealed that these replicon cells contained amino acid substitutions of alanine 105 to valine (A105V) and isoleucine 109 to valine (I109V) in the viral protease NS6. The application of a cell-based fluorescence resonance energy transfer (FRET) assay for protease activity demonstrated that these substitutions were involved in the enhanced resistance to rupintrivir. Furthermore, we validated the effect of these mutations using reverse genetics in murine norovirus (MNV), demonstrating that a recombinant MNV strain with a single I109V substitution in the protease also showed reduced susceptibility to rupintrivir. In summary, using a combination of different approaches, we have demonstrated that, under the correct conditions, mutations in the norovirus protease that lead to the generation of resistant mutants can rapidly occur

Vesivirus 2117 capsids more closely resemble sapovirus and lagovirus particles than other known vesivirus structures

Vesivirus 2117 is an adventitious agent that in 2009, was identified as a contaminant of CHO cells propagated in bioreactors at a pharmaceutical manufacturing plant belonging to Genzyme. The consequent interruption in supply of Fabrazyme and Cerezyme (drugs used to treat Fabry and Gaucher disease respectively), caused significant economic losses. Vesivirus 2117 is a member of the Caliciviridae; a family of small icosahedral viruses encoding a positive sense RNA genome. We have used cryo-electron microscopy and three dimensional image reconstruction to calculate a structure of vesivirus 2117 virus like particles as well as feline calicivirus and a chimeric sapovirus. We present a structural comparison of several members of the Caliciviridae, showing that the distal P domain of vesivirus 2117 is morphologically distinct from that seen in other known vesivirus structures. Furthermore, at intermediate resolutions we found a high level of structural similarity between vesivirus 2117 and Caliciviridae from other genera, such as sapovirus and rabbit haemorrhagic disease virus. Phylogenetic analysis confirms vesivirus 2117 as a vesivirus closely related to canine vesiviruses. We postulate that morphological differences in virion structure seen between vesivirus clades may reflect differences in receptor usage

UK circulating strains of human parainfluenza 3: an amplicon based next generation sequencing method and phylogenetic analysis.

Background: Human parainfluenza viruses type 3 (HPIV3) are a prominent cause of respiratory infection with a significant impact in both pediatric and transplant patient cohorts.  Currently there is a paucity of whole genome sequence data that would allow for detailed epidemiological and phylogenetic analysis of circulating strains in the UK. Although it is known that HPIV3 peaks annually in the UK, to date there are no whole genome sequences of HPIV3 UK strains available.  Methods: Clinical strains were obtained from HPIV3 positive respiratory patient samples collected between 2011 and 2015.  These were then amplified using an amplicon based method, sequenced on the Illumina platform and assembled using a new robust bioinformatics pipeline. Phylogenetic analysis was carried out in the context of other epidemiological studies and whole genome sequence data currently available with stringent exclusion of significantly culture-adapted strains of HPIV3. Results: In the current paper we have presented twenty full genome sequences of UK circulating strains of HPIV3 and a detailed phylogenetic analysis thereof.  We have analysed the variability along the HPIV3 genome and identified a short hypervariable region in the non-coding segment between the M (matrix) and F (fusion) genes. The epidemiological classifications obtained by using this region and whole genome data were then compared and found to be identical. Conclusions: The majority of HPIV3 strains were observed at different geographical locations and with a wide temporal spread, reflecting the global distribution of HPIV3. Consistent with previous data, a particular subcluster or strain was not identified as specific to the UK, suggesting that a number of genetically diverse strains circulate at any one time. A small hypervariable region in the HPIV3 genome was identified and it was shown that, in the absence of full genome data, this region could be used for epidemiological surveillance of HPIV3.Wellcome Open Grant PHE PhD studentshi

In vitro sensitivity of human parainfluenza 3 clinical isolates to ribavirin, favipiravir and zanamivir.

BACKGROUND: Human parainfluenza type 3 (HPIV3) is an important respiratory pathogen. Although a number of potential therapeutic candidates exist, there is currently no licensed therapy or vaccine. Ribavirin (RBV), favipiravir (FVP) and zanamivir (ZNV) are inhibitors with proven activity against influenza and with potential inhibitory activity against HPIV3 laboratory adapted strains in vitro. OBJECTIVES: To evaluate RBV, FVP and ZNV as inhibitors of minimally passaged UK clinical strains of HPIV3 as well as a laboratory adapted strain MK9 in vitro. STUDY DESIGN: The inhibitory action of RBV, FVP and ZNV was evaluated against nine minimally passaged clinical strains and a laboratory adapted strain MK9 using plaque reduction and growth curve inhibition in a cell culture model. RESULTS: Clinical isolates were found to be at least as susceptible as the laboratory adapted strains to RBV and FVP and significantly more susceptible to ZNV. However the inhibitory concentrations achieved by ZNV against clinical strains remain prohibitively high in vivo. CONCLUSIONS: RBV, FVP and ZNV were found to be effective inhibitors of HPIV3 in vitro. The lack of efficacy of RBV in vivo may be due to inability to reach required therapeutic levels. FVP, on the other hand, is a good potential therapeutic agent against HPIV3. Further studies using wild type clinical strains, as well as better formulation and delivery mechanisms may improve the utility of these three inhibitors.This work was supported by Public Health England (PHE) PhD studentship fund 201

A Cell-based Fluorescence Resonance Energy Transfer (FRET) Sensor Reveals Inter- and Intragenogroup Variations in Norovirus Protease Activity and Polyprotein Cleavage.

The viral protease represents a key drug target for the development of antiviral therapeutics. Because many protease inhibitors mimic protease substrates, differences in substrate recognition between proteases may affect their sensitivity to a given inhibitor. Here we use a cell-based FRET sensor to investigate the activity of different norovirus proteases upon cleavage of various norovirus cleavage sites inserted into a linker region separating cyan fluorescent protein and yellow fluorescent protein. Using this system, we demonstrate that differences in substrate processing exist between proteases from human noroviruses (genogroups I (GI) and II) and the commonly used murine norovirus (MNV, genogroup V) model. These altered the cleavage efficiency of specific cleavage sites both within and between genogroups. The differences observed between these proteases may affect sensitivity to protease inhibitors and the suitability of MNV as a model system for testing such molecules against the human norovirus protease. Finally, we demonstrate that replacement of MNV polyprotein cleavage sites with the GI or GII equivalents, with the exception of the NS6-7 junction, leads to the production of infectious virus when the MNV NS6 protease, but not the GI or GII proteases, are present.This work was funded by a Wellcome Trust Senior Fellowship awarded to IG Ref: WT097997MA) and a Marie Curie Fellowship awarded to TS (Ref: 628373).This is the final version of the article. It was first available from the American Society for Biochemistry and Molecular Biology via http://dx.doi.org/10.1074/jbc.M115.68823

UK circulating strains of human parainfluenza 3: an amplicon based next generation sequencing method and phylogenetic analysis [version 2; referees: 2 approved]

Background: Human parainfluenza viruses type 3 (HPIV3) are a prominent cause of respiratory infection with a significant impact in both pediatric and transplant patient cohorts.  Currently there is a paucity of whole genome sequence data that would allow for detailed epidemiological and phylogenetic analysis of circulating strains in the UK. Although it is known that HPIV3 peaks annually in the UK, to date there are no whole genome sequences of HPIV3 UK strains available.  Methods: Clinical strains were obtained from HPIV3 positive respiratory patient samples collected between 2011 and 2015.  These were then amplified using an amplicon based method, sequenced on the Illumina platform and assembled using a new robust bioinformatics pipeline. Phylogenetic analysis was carried out in the context of other epidemiological studies and whole genome sequence data currently available with stringent exclusion of significantly culture-adapted strains of HPIV3. Results: In the current paper we have presented twenty full genome sequences of UK circulating strains of HPIV3 and a detailed phylogenetic analysis thereof.  We have analysed the variability along the HPIV3 genome and identified a short hypervariable region in the non-coding segment between the M (matrix) and F (fusion) genes. The epidemiological classifications obtained by using this region and whole genome data were then compared and found to be identical. Conclusions: The majority of HPIV3 strains were observed at different geographical locations and with a wide temporal spread, reflecting the global distribution of HPIV3. Consistent with previous data, a particular subcluster or strain was not identified as specific to the UK, suggesting that a number of genetically diverse strains circulate at any one time. A small hypervariable region in the HPIV3 genome was identified and it was shown that, in the absence of full genome data, this region could be used for epidemiological surveillance of HPIV3

On the value of preprints: an early career researcher perspective

Peer-reviewed journal publication is the main means for academic researchers in the life sciences to create a permanent, public record of their work. These publications are also the de facto currency for career progress, with a strong link between journal brand recognition and perceived value. The current peer-review process can lead to long delays between submission and publication, with cycles of rejection, revision and resubmission causing redundant peer review. This situation creates unique challenges for early career researchers (ECRs), who rely heavily on timely publication of their work to gain recognition for their efforts. ECRs face changes in the academic landscape including the increased interdisciplinarity of life sciences research, expansion of the researcher population and consequent shifts in employer and funding demands. The publication of preprints, publicly available scientific manuscripts posted on dedicated preprint servers prior to journal managed peer-review, can play a key role in addressing these ECR challenges. Preprinting benefits include rapid dissemination of academic work, open access, establishing priority or concurrence, receiving feedback and facilitating collaborations. While there is a growing appreciation for and adoption of preprints, a minority of all articles in life sciences and medicine are preprinted. The current low rate of preprint submissions in life sciences and ECR concerns regarding preprinting needs to be addressed. We provide a perspective from an interdisciplinary group of early career researchers on the value of preprints and advocate the wide adoption of preprints to advance knowledge and facilitate career development

IFIT3 and IFIT2/3 promote IFIT1-mediated translation inhibition by enhancing binding to non-self RNA.

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed during the cell-intrinsic immune response to viral infection. IFIT1 inhibits translation by binding directly to the 5' end of foreign RNAs, particularly those with non-self cap structures, precluding the recruitment of the cap-binding eukaryotic translation initiation factor 4F and ribosome recruitment. The presence of IFIT1 imposes a requirement on viruses that replicate in the cytoplasm to maintain mechanisms to avoid its restrictive effects. Interaction of different IFIT family members is well described, but little is known of the molecular basis of IFIT association or its impact on function. Here, we reconstituted different complexes of IFIT1, IFIT2 and IFIT3 in vitro, which enabled us to reveal critical aspects of IFIT complex assembly. IFIT1 and IFIT3 interact via a YxxxL motif present in the C-terminus of each protein. IFIT2 and IFIT3 homodimers dissociate to form a more stable heterodimer that also associates with IFIT1. We show for the first time that IFIT3 stabilizes IFIT1 protein expression, promotes IFIT1 binding to a cap0 Zika virus reporter mRNA and enhances IFIT1 translation inhibition. This work reveals molecular aspects of IFIT interaction and provides an important missing link between IFIT assembly and function.This work was supported by a joint Royal Society/Wellcome Trust Sir Henry Dale Fellowship (202471/Z/16/Z) and a Royal Society Research Grant (RG140708) to TRS. HVM is supported by a University of Cambridge, Department of Pathology PhD studentship. XYL is supported by a King’s Scholarship from the Malaysian government. TJS is supported by a Wellcome Trust PhD studentship (105389/Z/14/Z). RCF and DSM are supported by CAPES Computational Biology (23038.010048/2013-27). DSM is also supported by the Academy of Medical Sciences/UK (NAF004/1005). SCG is a Sir Henry Dale Fellow (098406/Z/12/Z) co-funded by the Wellcome Trust and Royal Society

Evidence for human norovirus infection of dogs in the United kingdom.

Human noroviruses (HuNoVs) are a major cause of viral gastroenteritis, with an estimated 3 million cases per year in the United Kingdom. HuNoVs have recently been isolated from pet dogs in Europe (M. Summa, C.-H. von Bonsdorff, and L. Maunula, J Clin Virol 53:244-247, 2012, http://dx.doi.org/10.1016/j.jcv.2011.12.014), raising concerns about potential zoonotic infections. With 31% of United Kingdom households owning a dog, this could prove to be an important transmission route. To examine this risk, canine tissues were studied for their ability to bind to HuNoV in vitro. In addition, canine stool samples were analyzed for the presence of viral nucleic acid, and canine serum samples were tested for the presence of anti-HuNoV antibodies. The results showed that seven different genotypes of HuNoV virus-like particles (VLPs) can bind to canine gastrointestinal tissue, suggesting that infection is at least theoretically possible. Although HuNoV RNA was not identified in stool samples from 248 dogs, serological evidence of previous exposure to HuNoV was obtained in 43/325 canine serum samples. Remarkably, canine seroprevalence for different HuNoV genotypes mirrored the seroprevalence in the human population. Though entry and replication within cells have not been demonstrated, the canine serological data indicate that dogs produce an immune response to HuNoV, implying productive infection. In conclusion, this study reveals zoonotic implications for HuNoV, and to elucidate the significance of this finding, further epidemiological and molecular investigations will be essential.This collaborative project was facilitated by the Society of Microbiology's President's Fund awarded to S.L.C. and by the Region des Pays de la Loire ARMINA project. This work was supported by a Ph.D. studentship from the Medical Research Council to S.L.C. and a Wellcome Trust Senior Fellowship to I.G. (WT097997MA). I.G. is a Wellcome Senior Fellow.This is the final published version of the article. It was originally published in the Journal of Clinical Microbiology (Caddy S, et al., Journal of Clinical Microbiology, 2015, 53, 1873-1883, doi:10.1128/JCM.02778-14). The final version is available at http://dx.doi.org/10.1128/JCM.02778-1