Increasing Clinical Severity during a Dengue Virus Type 3 Cuban Epidemic: Deep Sequencing of Evolving Viral Populations

ABSTRACTDuring the dengue virus type 3 (DENV-3) epidemic that occurred in Havana in 2001 to 2002, severe disease was associated with the infection sequence DENV-1 followed by DENV-3 (DENV-1/DENV-3), while the sequence DENV-2/DENV-3 was associated with mild/asymptomatic infections. To determine the role of the virus in the increasing severity demonstrated during the epidemic, serum samples collected at different time points were studied. A total of 22 full-length sequences were obtained using a deep-sequencing approach. Bayesian phylogenetic analysis of consensus sequences revealed that two DENV-3 lineages were circulating in Havana at that time, both grouped within genotype III. The predominant lineage is closely related to Peruvian and Ecuadorian strains, while the minor lineage is related to Venezuelan strains. According to consensus sequences, relatively few nonsynonymous mutations were observed; only one was fixed during the epidemic at position 4380 in the NS2B gene. Intrahost genetic analysis indicated that a significant minor population was selected and became predominant toward the end of the epidemic. In conclusion, greater variability was detected during the epidemic's progression in terms of significant minority variants, particularly in the nonstructural genes. An increasing trend of genetic diversity toward the end of the epidemic was observed only for synonymous variant allele rates, with higher variability in secondary cases. Remarkably, significant intrahost genetic variation was demonstrated within the same patient during the course of secondary infection with DENV-1/DENV-3, including changes in the structural proteins premembrane (PrM) and envelope (E). Therefore, the dynamic of evolving viral populations in the context of heterotypic antibodies could be related to the increasing clinical severity observed during the epidemic.IMPORTANCEBased on the evidence that DENV fitness is context dependent, our research has focused on the study of viral factors associated with intraepidemic increasing severity in a unique epidemiological setting. Here, we investigated the intrahost genetic diversity in acute human samples collected at different time points during the DENV-3 epidemic that occurred in Cuba in 2001 to 2002 using a deep-sequencing approach. We concluded that greater variability in significant minor populations occurred as the epidemic progressed, particularly in the nonstructural genes, with higher variability observed in secondary infection cases. Remarkably, for the first time significant intrahost genetic variation was demonstrated within the same patient during the course of secondary infection with DENV-1/DENV-3, including changes in structural proteins. These findings indicate that high-resolution approaches are needed to unravel molecular mechanisms involved in dengue pathogenesis.

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Nationwide Distribution of Dengue Virus Type 3 (DENV-3) Genotype I and Emergence of DENV-3 Genotype III during the 2019 Outbreak in Bangladesh

Tropical Medicine and Infectious Disease ◽

10.3390/tropicalmed6020058 ◽

2021 ◽

Vol 6 (2) ◽

pp. 58

Author(s):

Snigdha Rahman Titir ◽

Shyamal Kumar Paul ◽

Salma Ahmed ◽

Nazia Haque ◽

Syeda Anjuman Nasreen ◽

...

Keyword(s):

Dengue Virus ◽

Secondary Infection ◽

Capital City ◽

Endemic Region ◽

Genotype I ◽

Genotype Iii ◽

Two Samples ◽

Dengue Virus Type 3 ◽

Type 3 ◽

Epidemiological Characteristics

Bangladesh is an endemic region of dengue fever and experienced an unprecedented large outbreak with more than 100,000 confirmed cases in 2019. To understand the prevalence of dengue antibody in patients and molecular epidemiological characteristics of dengue virus (DENV) in this outbreak, a total of 179 blood samples were collected from patients in 10 districts (seven divisions) covering nearly the whole country from August to December 2019. DENV NS-1 was detected in 162 samples, among which DENV-specific IgM was positive in 119 samples (73.5%), including 60.5% samples also positive for DENV-specific IgG. Sequencing of the partial C-prM gene and its phylogenetic analysis revealed predominance of DENV type 3 genotype I, accounting for 93% of samples examined. DENV-3 genotype III was identified in two samples from separate districts, and only one DENV-2 cosmopolitan genotype was found in the capital city, Dhaka. These findings suggest the predominance of DENV-3 genotype I and occurrence of DENV-3 genotype III, associated with increased incidence of recent secondary infection in Bangladesh in 2019.

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SARS-CoV-2 genomic and quasispecies analyses in cancer patients reveal relaxed intrahost virus evolution

10.1101/2020.08.26.267831 ◽

2020 ◽

Cited By ~ 2

Author(s):

Juliana D. Siqueira ◽

Livia R. Goes ◽

Brunna M. Alves ◽

Pedro S. de Carvalho ◽

Claudia Cicala ◽

...

Keyword(s):

Genetic Diversity ◽

Phylogenetic Analysis ◽

Cancer Patients ◽

Disease Severity ◽

Deep Sequencing ◽

Healthcare Workers ◽

Adverse Outcomes ◽

Genomic Diversity ◽

Consensus Sequences ◽

The Impact

AbstractNumerous factors have been identified to influence susceptibility to SARS-CoV-2 infection and disease severity. Cancer patients are more prone to clinically evolve to more severe COVID-19 conditions, but the determinants of such a more severe outcome remain largely unknown. We have determined the full-length SARS-CoV-2 genomic sequences of cancer patients and healthcare workers (HCW; non-cancer controls) by deep sequencing and investigated the within-host viral quasispecies of each infection, quantifying intrahost genetic diversity. Naso- and oropharyngeal SARS-CoV-2+ swabs from 57 cancer patients and 14 healthcare workers (HCW) from the Brazilian Cancer Institute were collected in April–May 2020. Complete genome amplification using ARTIC network V3 multiplex primers was performed followed by next-generation sequencing. Assemblies were conducted in Geneious R11, where consensus sequences were extracted and intrahost single nucleotide variants (iSNVs) were identified. Maximum likelihood phylogenetic analysis was performed using PhyMLv.3.0 and lineages were classified using Pangolin and CoV-GLUE. Phylogenetic analysis showed that all but one strain belonged to clade B1.1. Four genetically linked mutations known as the globally dominant SARS-CoV-2 haplotype (C241T, C3037T, C14408T and A23403G) were found in the majority of consensus sequences. SNV signatures of previously characterized Brazilian genomes were also observed in most samples. Another 85 SNVs were found at a lower frequency (1.4-19.7%). Cancer patients displayed a significantly higher intrahost viral genetic diversity compared to HCW (p = 0.009). Intrahost genetic diversity in cancer patients was independent of SARS-CoV-2 Ct values, and was not associated with disease severity, use of corticosteroids, or use of antivirals, characteristics that could influence viral diversity. Such a feature may explain, at least in part, the more adverse outcomes to which cancer/COVID-19 patients experience.Author SummaryCancer patients are more prone to clinically evolve to more severe COVID-19 conditions, but the determinants of such a more severe outcome remain largely unknown. In this study, phylogenetic and variation analysis of SARS-CoV-2 genomes from cancer patients and non-cancer healthcare workers at the Brazilian National Cancer Institute were characterized by deep sequencing. Viral genomes showed signatures characteristic of Brazilian viruses, consistent with the hypothesis of local, community transmission rather than virus importation from abroad. Despite most genomes in patients and healthcare workers belonging to the same lineage, intrahost variability was higher in cancer patients when compared to non-cancer counterparts. The intrahost genomic diversity analysis presented in our study highlights the relaxed evolution of SARS-CoV-2 in a vulnerable population of cancer patients. The high number of minor variations can result in the selection of immune escape variants, resistance to potential drugs, and/or increased pathogenicity. The impact of this higher intrahost variability over time warrants further investigation.

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Ultra-deep sequencing approach to analyze viral thymidine kinase genetic diversity within a new herpes simplex type 2 variant (HSV-2v)

Journal of Clinical Virology ◽

10.1016/j.jcv.2015.07.256 ◽

2015 ◽

Vol 70 ◽

pp. S110-S111

Author(s):

J. Marlet ◽

D. Boutolleau ◽

N. Désiré ◽

M. Darty ◽

C. Rodriguez ◽

...

Keyword(s):

Genetic Diversity ◽

Herpes Simplex ◽

Thymidine Kinase ◽

Deep Sequencing ◽

Herpes Simplex Type ◽

Deep Sequencing Approach

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Dengue Virus Type 3 Adaptive Changes during Epidemics in São Jose de Rio Preto, Brazil, 2006–2007

PLoS ONE ◽

10.1371/journal.pone.0063496 ◽

2013 ◽

Vol 8 (5) ◽

pp. e63496 ◽

Cited By ~ 9

Author(s):

Christian Julian Villabona-Arenas ◽

Adriano Mondini ◽

Irene Bosch ◽

Diane Schimitt ◽

Carlos E. Calzavara-Silva ◽

...

Keyword(s):

Dengue Virus ◽

Virus Type ◽

Adaptive Changes ◽

Dengue Virus Type 3 ◽

Type 3

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SARS-CoV-2 genomic analyses in cancer patients reveal elevated intrahost genetic diversity

Virus Evolution ◽

10.1093/ve/veab013 ◽

2021 ◽

Author(s):

Juliana D Siqueira ◽

Livia R Goes ◽

Brunna M Alves ◽

Pedro S de Carvalho ◽

Claudia Cicala ◽

...

Keyword(s):

Genetic Diversity ◽

Phylogenetic Analysis ◽

Cancer Patients ◽

Disease Severity ◽

Healthcare Workers ◽

Viral Population ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Consensus Sequences ◽

Vulnerable Patients

Abstract Numerous factors have been identified to influence susceptibility to SARS-CoV-2 infection and disease severity. Cancer patients are more prone to clinically evolve to more severe COVID-19 conditions, but the determinants of such a more severe outcome remain largely unknown. We have determined the full-length SARS-CoV-2 genomic sequences of cancer patients and healthcare workers (non-cancer controls) by deep sequencing and investigated the within-host viral population of each infection, quantifying intrahost genetic diversity. Naso- and oropharyngeal SARS-CoV-2+ swabs from 57 cancer patients and 14 healthcare workers from the Brazilian National Cancer Institute were collected in April–May 2020. Complete genome amplification using ARTIC network V3 multiplex primers was performed followed by next-generation sequencing. Assemblies were conducted in Geneious R11, where consensus sequences were extracted and intrahost single nucleotide variants were identified. Maximum likelihood phylogenetic analysis was performed using PhyMLv.3.0 and lineages were classified using Pangolin and CoV-GLUE. Phylogenetic analysis showed that all but one strain belonged to clade B1.1. Four genetically linked mutations known as the globally dominant SARS-CoV-2 haplotype (C241T, C3037T, C14408T and A23403G) were found in the majority of consensus sequences. SNV signatures of previously characterized Brazilian genomes were also observed in most samples. Another 85 SNVs were found at a lower frequency (1.4-19.7%) among the consensus sequences. Cancer patients displayed a significantly higher intrahost viral genetic diversity compared to healthcare workers. This difference was independent of SARS-CoV-2 Ct values obtained at the diagnostic tests, which did not differ between the two groups. The most common nucleotide changes of intrahost SNVs in both groups were consistent with APOBEC and ADAR activities. Intrahost genetic diversity in cancer patients was not associated with disease severity, use of corticosteroids, or use of antivirals, characteristics that could influence viral diversity. Moreover, the presence of metastasis, either in general or specifically in the lung, was not associated with intrahost diversity among cancer patients. Cancer patients carried significantly higher numbers of minor variants compared to non-cancer counterparts. Further studies on SARS-CoV-2 diversity in especially vulnerable patients will shed light onto the understanding of the basis of COVID-19 different outcomes in humans.

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