MUTATIONS IN THE GENOMES OF SARS-COV-2 FROM CLINICAL SAMPLES OBTAINED IN LATE MARCH-EARLY APRIL FROM PATIENTS IN MOSCOW

2020 ◽  
Author(s):  
V.V. Kaptelova ◽  
◽  
A.S. Speranskaya ◽  
A.E. Samoilov ◽  
A.V. Valdokhina ◽  
...  
Keyword(s):  
Viral Load ◽  
Viral Replication ◽  
Recent Work ◽  
Disease Transmission ◽  
Virus Transmission ◽  
High Viral Load ◽  
Virus Genome ◽  
Clinical Samples ◽  
Whole Genome ◽  
S Protein

Many papers suggested that D614G mutation in the viral spike (S) protein SARS-CoV-2 can influence the ability of virus transmission. In recent work [1], it was shown D614G influences the rate of disease transmission only in combination with the P323L mutation in the viral polymerase. We have sequenced 28 full genomes of SARS-CoV-2, obtained from clinical material from patients of different ages. The analyzed isolates belong to clades B.1 (GH) and B1.1 (GR). Combinations of mutations P323L and D614G were found in all genomes. These differences can be explained by sampling: the samples for the sequencing of the whole genome were selected with high viral load, it can be related to the rate of viral replication in intra-host. That, in turn, can be dependent on the presence of P323L/D614G mutations in the virus genome.

scholarly journals SARS-CoV2 genome analysis of Indian isolates and molecular modelling of D614G mutated spike protein with TMPRSS2 depicted its enhanced interaction and virus infectivity

2020 ◽  
Author(s):  
Sunil Raghav ◽  
Arup Ghosh ◽  
Jyotirmayee Turuk ◽  
Sugandh Kumar ◽  
Atimukta Jha ◽  
...  
Keyword(s):  
Molecular Modelling ◽  
Genome Analysis ◽  
Disease Transmission ◽  
Amplicon Sequencing ◽  
Virus Genome ◽  
Host Cells ◽  
Spike Protein ◽  
Disease Spread ◽  
Clinical Samples ◽  
Virus Infectivity

AbstractCOVID-19 that emerged as a global pandemic is caused by SARS-CoV-2 virus. The virus genome analysis during disease spread reveals about its evolution and transmission. We did whole genome sequencing of 225 clinical strains from the state of Odisha in eastern India using ARTIC protocol-based amplicon sequencing. Phylogenetic analysis identified the presence of all five reported clades 19A, 19B, 20A, 20B and 20C in the population. The analyses revealed two major routes for the introduction of the disease in India i.e. Europe and South-east Asia followed by local transmission. Interestingly, 19B clade was found to be much more prevalent in our sequenced genomes (17%) as compared to other genomes reported so far from India. The haplogroup analysis for clades showed evolution of 19A and 19B in parallel whereas the 20B and 20C appeared to evolve from 20A. Majority of the 19A and 19B clades were present in cases that migrated from Gujarat state in India suggesting it to be one of the major initial points of disease transmission in India during month of March and April. We found that with the time 20A and 20B clades evolved drastically that originated from central Europe. At the same time, it has been observed that 20A and 20B clades depicted selection of four common mutations i.e. 241 C>T (5’UTR), P323L in RdRP, F942F in NSP3 and D614G in the spike protein. We found an increase in the concordance of G614 mutation evolution with the viral load in clinical samples as evident from decreased Ct value of spike and Orf1ab gene in qPCR. Molecular modelling and docking analysis identified that D614G mutation enhanced interaction of spike with TMPRSS2 protease, which could impact the shedding of S1 domain and infectivity of the virus in host cells.

scholarly journals Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 841 ◽  
Author(s):  
Carole Grädel ◽  
Miguel A. Terrazos Miani ◽  
Christian Baumann ◽  
Maria Teresa Barbani ◽  
Stefan Neuenschwander ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Rna Sequencing ◽  
Genome Sequencing ◽  
Illumina Miseq ◽  
High Viral Load ◽  
Clinical Samples ◽  
Whole Genome ◽  
Consensus Sequences ◽  
Positive Stool ◽  
Stool Samples

Enteroviruses are small RNA viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. In routine diagnostic laboratories, enteroviruses are identified by PCR-based methods, often combined with partial sequencing for genotyping. In this proof-of-principle study, we assessed direct RNA sequencing (DRS) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. The approach was complemented by sequencing the corresponding viral cDNA via Illumina MiSeq sequencing. DRS of total RNA extracted from three different enterovirus-positive stool samples produced long RNA fragments, covering between 59% and 99.6% of the most similar reference genome sequences. The identification of the enterovirus sequences in the samples was confirmed by short-read cDNA sequencing. Sequence identity between DRS and Illumina MiSeq enterovirus consensus sequences ranged between 94% and 97%. Here, we show that nanopore DRS can be used to correctly identify enterovirus genotypes from patient stool samples with high viral load and that the approach also provides rich metatranscriptomic information on sample composition for all life domains.

scholarly journals Predictive factors for response to Lamivudine in chronic hepatitis B

2000 ◽  
Vol 42 (4) ◽  
pp. 189-196 ◽  
Author(s):  
Luiz Caetano da SILVA ◽  
Luís Edmundo Pinto da FONSECA ◽  
Flair José CARRILHO ◽  
Venâncio Avancini Ferreira ALVES ◽  
Roberta SITNIK ◽  
...  
Keyword(s):  
Chronic Hepatitis ◽  
Viral Load ◽  
Hepatitis B ◽  
Viral Replication ◽  
Chronic Hepatitis B ◽  
Predictive Factors ◽  
Prospective Trial ◽  
High Sensitivity ◽  
High Viral Load ◽  
Pre Treatment

BACKGROUND: Lamivudine has been shown to be an efficient drug for chronic hepatitis B (CHB) treatment. AIM: To investigate predictive factors of response, using a quantitative method with high sensitivity. METHODS: We carried out a prospective trial of lamivudine in 35 patients with CHB and evidence for viral replication, regardless to their HBeAg status. Lamivudine was given for 12 months at 300 mg daily and 150 mg thereafter. Response was considered when DNA was undetectable by PCR after 6 months of treatment. Viral replication was monitored by end-point dilution PCR. Mutation associated with resistance to lamivudine was detected by DNA sequencing in non-responder patients. RESULTS: Response was observed in 23/35 patients (65.7%) but only in 5/15 (33.3%) HBeAg positive patients. Only three pre-treatment variables were associated to low response: HBeAg (p = 0.006), high viral load (DNA-VHB > 3 x 10(6) copies/ml) (p = 0.004) and liver HBcAg (p = 0.0028). YMDD mutations were detected in 7/11 non-responder patients. CONCLUSIONS: HBeAg positive patients with high viral load show a high risk for developing drug resistance. On the other hand, HBeAg negative patients show a good response to lamivudine even with high viremia.

scholarly journals Characterization of SARS-CoV-2 and host entry factors distribution in a COVID-19 autopsy series

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Xiao-Ming Wang ◽  
Rahul Mannan ◽  
Lanbo Xiao ◽  
Eman Abdulfatah ◽  
Yuanyuan Qiao ◽  
...  
Keyword(s):  
Viral Load ◽  
Viral Replication ◽  
Transcriptional Repression ◽  
High Viral Load ◽  
Lung Epithelial Cells ◽  
Qrt Pcr ◽  
Organ Systems ◽  
Kidney Liver ◽  
The Impact

Abstract Background SARS-CoV-2 is a highly contagious virus that causes the disease COVID-19. We have recently reported that androgens regulate the expression of SARS-CoV-2 host entry factors ACE2 and TMPRSS2, and androgen receptor (AR) in lung epithelial cells. We also demonstrated that the transcriptional repression of the AR enhanceosome inhibited SARS-CoV-2 infection in vitro. Methods To better understand the various sites of SARS-CoV-2 infection, and presence of host entry factors, we extensively characterized the tissue distribution and localization of SARS-CoV-2 virus, viral replication, and host entry factors in various anatomical sites sampled via autopsy. We applied RNA in-situ-hybridization (RNA-ISH), immunohistochemistry (IHC) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) approaches. We also assessed histopathological changes in SARS-CoV-2 infected tissues. Results We detect SARS-CoV-2 virus and viral replication in pulmonary tissues by RNA-ISH and IHC and a variety of non-pulmonary tissues including kidney, heart, liver, spleen, thyroid, lymph node, prostate, uterus, and colon by qRT-PCR. We observe heterogeneity in viral load and viral cytopathic effects among various organ systems, between individuals and within the same patient. In a patient with a history of kidney transplant and under immunosuppressant therapy, we observe an unusually high viral load in lung tissue by RNA-ISH, IHC and qRT-PCR. SARS-CoV-2 virus is also detected in this patent’s kidney, liver and uterus. We find ACE2, TMPRSS2 and AR expression to overlap with the infection sites. Conclusions This study portrays the impact of dispersed SARS-CoV-2 infection in diverse organ systems, thereby facilitating avenues for systematic therapeutic approaches.

scholarly journals Clinical, immunological and genomic characterization of asymptomatic and symptomatic cases with SARS-CoV-2 infection, India

2021 ◽  
Author(s):  
Sanchari Chatterjee ◽  
Ankita Datey ◽  
Soumya Sengupta ◽  
Arup Ghosh ◽  
Atimukta Jha ◽  
...  
Keyword(s):  
Viral Load ◽  
Biochemical Parameters ◽  
Mutational Analysis ◽  
Control Strategies ◽  
High Viral Load ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Serum Samples ◽  
Asymptomatic Patients ◽  
Significant Difference

Background: The current global pandemic of Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 led to the investigation with clinical, biochemical, immunological and genomic information of the patients to understand the pathophysiology of this viral infection. Methods: Samples were collected from six asymptomatic and six symptomatic SARS-CoV-2 confirmed hospitalized patients in Bhubaneswar, Odisha, India. Clinical details, biochemical parameters, treatment regime were collected from hospital, viral load was determined by RT-PCR, levels of cytokines and circulating antibodies in plasma were assessed by Bioplex and isotyping respectively. In addition, the whole genome sequencing of viral strains and mutational analysis were carried out. Findings Analysis of the biochemical parameters highlighted the increased levels of C-Reactive protein (CRP), lactate dehydrogenase (LDH), serum SGPT, serum SGOT and ferritin in symptomatic patients indicating that patients with higher levels of few biochemical parameters might experience severe pathophysiological complications after SARS-CoV-2 infection. This was also observed that symptomatic patients were mostly with one or more comorbidities, especially diabetes (66.6%). Surprisingly the virological estimation revealed that there was no significant difference in viral load of oropharyngeal (OP) samples between the two groups. This suggests that the viral load in OP sample does not correlate with disease severity and both asymptomatic and symptomatic patients are equally capable of transmitting the virus. Whereas, viral load was higher in plasma and serum samples of symptomatic patients suggesting that the development of clinical complications is mostly associated to high viral load in plasma and serum. This also demonstrated that the patients with high viral load in plasma and serum samples were found to develop sufficient amounts of antibodies (IgG, IgM and IgA). Interestingly, the levels of 7 cytokines (IL-6, IL-1@, IP-10, IL-8, IL-10, IFN-@2, IL-15) were found to be highly elevated in symptomatic patients, while three cytokines (soluble CD40L, GRO and MDC) were remarkably higher in asymptomatic patients. Therefore, this data suggest that cytokines and chemokines may serve as predictive indicator of SARS-CoV-2 infection and contribute to understand the pathogenesis of COVID-19. The whole genome sequence analysis revealed that the current isolates were clustered with 19B, 20A and 20B clades, however acquired 11 additional changes in Orf1ab, spike, Orf3a, Orf8 and nucleocapsid proteins. The data also confirmed that the D614G mutation in spike protein is mostly linked with severe SARS-CoV-2 infection as two patients with this mutation passes away. Interpretation This is the first comprehensive study of SARS CoV-2 patients from India. This will contribute to a better understanding of the pathophysiology of SARS-CoV-2 infection and advance in the implementation of effective disease control strategies.

scholarly journals Whole genome sequencing of human enteroviruses from clinical samples by nanopore direct RNA sequencing

2020 ◽  
Author(s):  
C. Grädel ◽  
M.A. Terrazos Miani ◽  
C. Baumann ◽  
MT Barbani ◽  
S. Neuenschwander ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Rna Sequencing ◽  
Genome Sequencing ◽  
Illumina Miseq ◽  
High Viral Load ◽  
Clinical Samples ◽  
Whole Genome ◽  
Consensus Sequences ◽  
Positive Stool ◽  
Stool Samples

AbstractEnteroviruses are small RNA viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. In routine diagnostic laboratories, those viruses are identified by PCR based methods, often combined with partial sequencing for genotyping. In this proof-of-principle study, we assessed direct RNA sequencing (DRS) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. Results of the approach were complemented with those obtained by sequencing the corresponding viral cDNA via Illumina MiSeq sequencing. DRS of total RNA extracted from three different enterovirus-positive stool samples produced long RNA fragments, covering between 59% to 99.6 % of the best reference genomes. The identification of the enterovirus sequences in the sample was confirmed by the short-read cDNA sequencing. Sequence identity between DRS and Illumina MiSeq enterovirus consensus sequences ranged between 94-97%. Here we show that nanopore DRS can be used to correctly identify the genotypes of enteroviruses from patient stool samples with high viral load.

scholarly journals Porcine reproductive and respiratory syndrome virus whole-genome sequencing efficacy with field clinical samples using a poly(A)-tail viral genome purification method

2020 ◽  
pp. 104063872095241
Author(s):  
Carl A. Gagnon ◽  
Christian Lalonde ◽  
Chantale Provost
Keyword(s):  
Viral Load ◽  
Whole Genome Sequencing ◽  
Success Rate ◽  
Genome Sequencing ◽  
Viral Genome ◽  
Clinical Samples ◽  
Respiratory Syndrome Virus ◽  
Whole Genome ◽  
Purification Method ◽  
Entire Viral Genome

The genomic surveillance of porcine reproductive and respiratory syndrome virus (PRRSV) is based on sequencing of the ORF5 gene of the virus, which covers only 4% of the entire viral genome. It is expected that PRRSV whole-genome sequencing (WGS) will improve PRRSV genomic data and allow better understanding of clinical discrepancies observed in the field when using ORF5 sequencing. Our main objective was to implement an efficient method for WGS of PRRSV from clinical samples. The viral genome was purified using a poly(A)-tail viral genome purification method and sequenced using Illumina technology. We tested 149 PRRSV-positive samples: 80 sera, 33 lungs, 33 pools of tissues, 2 oral fluids, and 1 processing fluid (i.e., castration liquid). Overall, WGS of 67.1% of PRRSV-positive cases was successful. The viral load, in particular for tissues, had a major impact on the PRRSV WGS success rate. Serum was the most efficient type of sample to conduct PRRSV WGS poly(A)-tail assays, with a success rate of 76.3%, and this result can be explained by improved sequencing reads dispersion matching throughout the entire viral genome. WGS was unsuccessful for all pools of tissue and lung samples with Cq values > 26.5, whereas it could still be successful with sera at Cq ≤ 34.1. Evaluation of results of highly qualified personnel confirmed that laboratory skills could affect PRRSV WGS efficiency. Oral fluid samples seem very promising and merit further investigation because, with only 2 samples of low viral load (Cq = 28.8, 32.8), PRRSV WGS was successful.

Update in management of hepatitis B in pregnancy and prevention of mother to child hepatitis B transmission

2018 ◽  
Vol 1 (3) ◽  
pp. 1-8
Author(s):  
Naichaya Chamroonkul
Keyword(s):  
Chronic Hepatitis ◽  
Viral Load ◽  
Hepatitis B ◽  
Chronic Hepatitis B ◽  
High Viral Load ◽  
World Population ◽  
Hepatitis B Infection ◽  
Mother To Child Transmission ◽  
Child Transmission ◽  
Mother To Child

Even with two decades of widespread using hepatitis B vaccination, chronic hepatitis B remains a major global health problem. In Thailand, the prevalence of chronic hepatitis B infection was down from 8 - 10% in last decade to 5% recently. Failure to control mother to child transmission is one of the important barriers to the total elimination of hepatitis B infection from world population. In the majority, vertical transmission can be prevented with a universal screening program, immunoprophylaxis by administration of hepatitis B vaccine and hepatitis B immunoglobulin (HBIg) for babies born to mothers with HBV. However, in mothers with a high viral load, the chance of immunoprophylaxis failure remains high. To date, there are standard recommendations by all international liver societies including AASLD, EASL and APASL suggest introducing an antiviral agent during the third trimester to CHB pregnant women with a high viral load. Previous US FDA pregnancy category B agents such as Tenofovir and Telbivudine are allowed through all trimesters of pregnancy and are effective for prevention of mother to child transmission. Breastfeeding for patients who receive antiviral agents can be allowed after a risk-benefit discussion with the patient and family.

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