Patients with signs of COVID-19 were tested through diagnostic RT-PCR for SARS-CoV-2 using RNA extracted from the nasopharyngeal/nasal swabs.To determine the variants of SARS-CoV-2 circulating in the state of Nevada,s...Patients with signs of COVID-19 were tested through diagnostic RT-PCR for SARS-CoV-2 using RNA extracted from the nasopharyngeal/nasal swabs.To determine the variants of SARS-CoV-2 circulating in the state of Nevada,specimens from 200 COVID-19 patients were sequenced through our robust sequencing platform,which enabled sequencing of SARS-CoV-2 from specimens with even very low viral loads,without the need of culture-based amplification.High genome coverage allowed the identification of single and multi-nucleotide variants in SARS-CoV-2 in the community and their phylogenetic relationships with other variants present during the same period of the outbreak.We report the occurrence of a novel mutation at 323aa (314aa of orf1b) of nsp12 (RNA-dependent RNA polymerase) changed to phenylalanine(F) from proline (P),in the first reported isolate of SARS-CoV-2,Wuhan-Hu-1.This 323F variant was present at a very high frequency in Northern Nevada.Structural modeling determined this mutation in the interface domain,which is important for the association of accessory proteins required for the polymerase.In conclusion,we report the introduction of specific SARS-CoV-2 variants at very high frequency in distinct geographic locations,which is important for understanding the evolution and circulation of SARS-CoV-2variants of public health importance,while it circulates in humans.展开更多
We sought to determine the characteristics of viral specimens associated with fatal cases,asymptomatic cases and non-fatal symptomatic cases of COVID-19.This included the analysis of 1264 specimens found reactive for ...We sought to determine the characteristics of viral specimens associated with fatal cases,asymptomatic cases and non-fatal symptomatic cases of COVID-19.This included the analysis of 1264 specimens found reactive for at least two SARS-CoV-2 specific loci from people screened for infection in Northern Nevada in March-May of 2020.Of these,30 were specimens from fatal cases,while 23 were from positive,asymptomatic cases.We assessed the relative amounts of SARS-CoV-2 RNA from sample swabs by real-time PCR and use of the threshold crossing value(Ct).Moreover,we compared the amount of human RNase P found on the same swabs.A considerably higher viral load was found to be associated with swabs from cases involving fatality and the difference was found to be strongly statistically significant.Noting this difference,we sought to assess whether any genetic correlation could be found in association with virus from fatal cases using whole genome sequencing.While no common genetic elements were discerned,one branch of epidemiologically linked fatal cases did have two point mutations,which no other of 156 sequenced cases from northern Nevada had.The mutations caused amino acid changes in the 3′-5′exonuclease protein,and the product of the gene,orf8.展开更多
基金supported by University of Nevada, RenoDepartment of Microbiology & Immunology, UNR School of MedicineNevada IDeA Network of Biomedical Research Excellence (INBRE) from the National Institute of General Medical Sciences (GM 103440 and GM 104944) and from the National Institutes of Health。
文摘Patients with signs of COVID-19 were tested through diagnostic RT-PCR for SARS-CoV-2 using RNA extracted from the nasopharyngeal/nasal swabs.To determine the variants of SARS-CoV-2 circulating in the state of Nevada,specimens from 200 COVID-19 patients were sequenced through our robust sequencing platform,which enabled sequencing of SARS-CoV-2 from specimens with even very low viral loads,without the need of culture-based amplification.High genome coverage allowed the identification of single and multi-nucleotide variants in SARS-CoV-2 in the community and their phylogenetic relationships with other variants present during the same period of the outbreak.We report the occurrence of a novel mutation at 323aa (314aa of orf1b) of nsp12 (RNA-dependent RNA polymerase) changed to phenylalanine(F) from proline (P),in the first reported isolate of SARS-CoV-2,Wuhan-Hu-1.This 323F variant was present at a very high frequency in Northern Nevada.Structural modeling determined this mutation in the interface domain,which is important for the association of accessory proteins required for the polymerase.In conclusion,we report the introduction of specific SARS-CoV-2 variants at very high frequency in distinct geographic locations,which is important for understanding the evolution and circulation of SARS-CoV-2variants of public health importance,while it circulates in humans.
文摘We sought to determine the characteristics of viral specimens associated with fatal cases,asymptomatic cases and non-fatal symptomatic cases of COVID-19.This included the analysis of 1264 specimens found reactive for at least two SARS-CoV-2 specific loci from people screened for infection in Northern Nevada in March-May of 2020.Of these,30 were specimens from fatal cases,while 23 were from positive,asymptomatic cases.We assessed the relative amounts of SARS-CoV-2 RNA from sample swabs by real-time PCR and use of the threshold crossing value(Ct).Moreover,we compared the amount of human RNase P found on the same swabs.A considerably higher viral load was found to be associated with swabs from cases involving fatality and the difference was found to be strongly statistically significant.Noting this difference,we sought to assess whether any genetic correlation could be found in association with virus from fatal cases using whole genome sequencing.While no common genetic elements were discerned,one branch of epidemiologically linked fatal cases did have two point mutations,which no other of 156 sequenced cases from northern Nevada had.The mutations caused amino acid changes in the 3′-5′exonuclease protein,and the product of the gene,orf8.
