24 research outputs found
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Human Parainfluenza Virus 3: Genetic Diversity, Virulence and Antiviral Susceptibility
Human parainfluenza 3 (HPIV3) is a member of the Paramyxoviridae, a single strain negative-sense non-segmented RNA virus in the order Mononegavirales. It is a respiratory pathogen with a broad spectrum of presentations for which there is currently neither a vaccine nor licensed treatment for HPIV3.
To date most research on HPIV3 has been conducted using significantly culture adapted reference strains. Therefore, minimally adapted clinical strains were grown in two cell culture systems: immortalised and primary. Plaque phenotype, growth kinetics and inflammatory response triggered were evaluated and it was found that there is a range of phenotypes exhibited by clinical strains with potential implications in vivo.
To examine the genetic diversity of circulating strains of HPIV3 in the UK, a new amplicon based sequencing pipeline for whole genome sequencing of HPIV3 was developed and validated. A short hypervariable region in the HPIV3 genome was identified and evaluated as a potential candidate for subsequent phylogenetic analysis compared to whole genome data. This method was then applied to tracking an HPIV3 outbreak that took place on a paediatric oncology ward. It was found to be a point-source outbreak and the clinical impact in this setting, as well as the infection control procedures involved were evaluated.
Finally a robust in vitro model for the evaluation of potential therapeutic candidates for HPIV3, based on a panel of minimally passaged clinical strains as well as a culture-adapted reference strain, was set up. This model was applied to three potential inhibitors of HPIV3: ribavirin, favipiravir and zanamivir. The results showed that clinical strains were at least as susceptible to ribavirin and favipiravir as the laboratory reference strain and significantly more susceptible to zanamivir. This indicates that further work on minimally adapted clinical strains is essential to further the understanding of this important virus.Public Health England (PHE) - this PhD thesis
Wellcome Trust (Prof Ian Goodfellow
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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
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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
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
Unrecognised Outbreak: Human parainfluenza virus infections in a pediatric oncology unit. Â A new diagnostic PCR and virus monitoring system may allow early detection of future outbreaks.
Background: Human parainfluenza viruses (HPIVs) are significant causes of both upper and lower respiratory tract infections with type 3 (HPIV3) causing the most severe disease in the immunocompromised cohorts. The objective of this study was to analyse the epidemiological nature of a cluster of cases of HPIV3 in a pediatric oncology unit of a major teaching hospital. Methods: In order to determine whether the activity observed represented a deviation from the norm, seasonal trends of HPIV3 in the surrounding geographical area as well as on the ward in question were analysed. The genetic link between cases was established by the phylogenetic analysis of the non-coding hypervariable region between the M (Matrix) and F (fusion) genes of HPIV3. The 15 cases involved and 15 unrelated cases were sequenced. Transmission routes were subsequently inferred and visualized using Konstanz Information Miner (KNIME) 3.3.2. Results: Of the 15 cases identified, 14 were attributed to a point source outbreak. Two out of 14 outbreak cases were found to differ by a single mutation A182C. The outbreak strain was also seen in 1 out of 15 unrelated cases, indicating that it was introduced from the community. Transmission modeling was not able to link all the cases and establish a conclusive chain of transmission. No staff were tested during the outbreak period. No deaths occurred as a result of the outbreak. Conclusion: A point source outbreak of HPIV3 was recognized post factum on an oncology pediatric unit in a major teaching hospital. This raised concern about the possibility of a future more serious outbreak. Weaknesses in existing systems were identified and a new dedicated respiratory virus monitoring system introduced. Pediatric oncology units require sophisticated systems for early identification of potentially life-threatening viral outbreaks
SARS-CoV-2 evolution during treatment of chronic infection
The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein1 and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals
SARS-CoV-2 evolution during treatment of chronic infection
SARS-CoV-2 Spike protein is critical for virus infection via engagement of ACE21, and is a major
54 antibody target. Here we report chronic SARS-CoV-2 with reduced sensitivity to neutralising
55 antibodies in an immune suppressed individual treated with convalescent plasma, generating
56 whole genome ultradeep sequences over 23 time points spanning 101 days. Little change was
57 observed in the overall viral population structure following two courses of remdesivir over the
58 first 57 days. However, following convalescent plasma therapy we observed large, dynamic
59 virus population shifts, with the emergence of a dominant viral strain bearing D796H in S2 and
60 H69/V70 in the S1 N-terminal domain NTD of the Spike protein. As passively transferred
61 serum antibodies diminished, viruses with the escape genotype diminished in frequency, before
62 returning during a final, unsuccessful course of convalescent plasma. In vitro, the Spike escape
63 double mutant bearing H69/V70 and D796H conferred modestly decreased sensitivity to
64 convalescent plasma, whilst maintaining infectivity similar to wild type. D796H appeared to be
65 the main contributor to decreased susceptibility but incurred an infectivity defect. The
66 H69/V70 single mutant had two-fold higher infectivity compared to wild type, possibly
67 compensating for the reduced infectivity of D796H. These data reveal strong selection on SARS68
CoV-2 during convalescent plasma therapy associated with emergence of viral variants with
69 evidence of reduced susceptibility to neutralising antibodies.COG-UK is supported by funding from the Medical Research Council (MRC) part of UK Research & Innovation (UKRI), the National Institute of Health Research (NIHR) and Genome Research Limited, operating as the Wellcome Sanger Institute
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Treatment of COVID-19 with remdesivir in the absence of humoral immunity: a case report
Abstract: The response to the coronavirus disease 2019 (COVID-19) pandemic has been hampered by lack of an effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antiviral therapy. Here we report the use of remdesivir in a patient with COVID-19 and the prototypic genetic antibody deficiency X-linked agammaglobulinaemia (XLA). Despite evidence of complement activation and a robust T cell response, the patient developed persistent SARS-CoV-2 pneumonitis, without progressing to multi-organ involvement. This unusual clinical course is consistent with a contribution of antibodies to both viral clearance and progression to severe disease. In the absence of these confounders, we take an experimental medicine approach to examine the in vivo utility of remdesivir. Over two independent courses of treatment, we observe a temporally correlated clinical and virological response, leading to clinical resolution and viral clearance, with no evidence of acquired drug resistance. We therefore provide evidence for the antiviral efficacy of remdesivir in vivo, and its potential benefit in selected patients
Treatment of COVID-19 with remdesivir in the absence of humoral immunity: a case report
Abstract: The response to the coronavirus disease 2019 (COVID-19) pandemic has been hampered by lack of an effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antiviral therapy. Here we report the use of remdesivir in a patient with COVID-19 and the prototypic genetic antibody deficiency X-linked agammaglobulinaemia (XLA). Despite evidence of complement activation and a robust T cell response, the patient developed persistent SARS-CoV-2 pneumonitis, without progressing to multi-organ involvement. This unusual clinical course is consistent with a contribution of antibodies to both viral clearance and progression to severe disease. In the absence of these confounders, we take an experimental medicine approach to examine the in vivo utility of remdesivir. Over two independent courses of treatment, we observe a temporally correlated clinical and virological response, leading to clinical resolution and viral clearance, with no evidence of acquired drug resistance. We therefore provide evidence for the antiviral efficacy of remdesivir in vivo, and its potential benefit in selected patients