A multiplex method for detection of SARS-CoV-2 variants based on MALDI-TOF mass spectrometry.

The recent outbreak of the coronavirus disease 2019 (COVID-19) pandemic and the continuous evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have highlighted the significance of new detection methods for global monitoring and prevention. Although quantitative reverse transcription PCR (RT-qPCR), the current gold standard for diagnosis, performs excellently in genetic testing, its multiplexing capability is limited because of the signal crosstalk of various fluorophores. Herein, we present a highly efficient platform which combines 17-plex assays with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), enabling the targeting of 14 different mutation sites of the spike gene. Diagnosis using a set of 324 nasopharyngeal swabs or sputum clinical samples with SARS-CoV-2 MS method was identical to that with the RT-qPCR. The detection consistency of mutation sites was 97.9% (47/48) compared to Sanger sequencing without cross-reaction with other respiratory-related pathogens. Therefore, the MS method is highly potent to track and assess SARS-CoV-2 changes in a timely manner, thereby aiding the continuous response to viral variation and prevention of further transmission.

[Epidemic Characteristics of the Novel Coronavirus Delta Variant in Guangzhou and Grid Crowd Management Based on Public Security Forensic Perspective].

ABSTRACT: Abstract： Objective To analyze the first epidemic spread of the novel coronavirus Delta variant in China based on public security forensic perspective, investigate its transmission characteristics, contributing factors, and epidemiologic research experience, and provide a reference for the prevention and control of the epidemic caused by the novel coronavirus variant. Methods Based on the information that public security forensic experts obtained from front-line epidemiologic research, the gender, age, place of residence, transmission route and infectivity of the coronavirus disease 2019 （COVID-19） confirmed cases, asymptomatic infected persons and their close contacts in Guangzhou caused by the novel coronavirus Delta variant were analyzed. The basic reproduction number （R0） during this epidemic in Guangzhou was calculated. Results Among the 153 cases infected with novel coronavirus Delta variant in the epidemic, 63 cases were male and 90 cases were female, their age ranging from 1 to 92 years, with a median age of 49 years. The main route of transmission was close contact, including dining together, co-living, and close contact in the same residential building. There were 31 cases of family clusters, 25 of which were in Liwan District. The epidemic lasted from May 26 to May 29, and the R0 remained above 4.0. After May 30, R0 began to decline and remained below 1.0 from June 7. Conclusion The novel coronavirus Delta variant is highly infectious, the crowd is generally susceptible to infection and family cluster cases are easy to occur. So, it is necessary to precisely prevent and control this strain. Public security forensic experts have both medical literacy and criminal investigation capabilities, they can play a more professional role in epidemic prevention and control.

Epidemic Characteristics of the Novel Coronavirus Delta Variant in Guangzhou and Grid Crowd Management Based on Public Security Forensic Perspective / 法医学杂志

Abstract： Objective To analyze the first epidemic spread of the novel coronavirus Delta variant in China based on public security forensic perspective, investigate its transmission characteristics, contributing factors, and epidemiologic research experience, and provide a reference for the prevention and control of the epidemic caused by the novel coronavirus variant. Methods Based on the information that public security forensic experts obtained from front-line epidemiologic research, the gender, age, place of residence, transmission route and infectivity of the coronavirus disease 2019 （COVID-19） confirmed cases, asymptomatic infected persons and their close contacts in Guangzhou caused by the novel coronavirus Delta variant were analyzed. The basic reproduction number （R0） during this epidemic in Guangzhou was calculated. Results Among the 153 cases infected with novel coronavirus Delta variant in the epidemic, 63 cases were male and 90 cases were female, their age ranging from 1 to 92 years, with a median age of 49 years. The main route of transmission was close contact, including dining together, co-living, and close contact in the same residential building. There were 31 cases of family clusters, 25 of which were in Liwan District. The epidemic lasted from May 26 to May 29, and the R0 remained above 4.0. After May 30, R0 began to decline and remained below 1.0 from June 7. Conclusion The novel coronavirus Delta variant is highly infectious, the crowd is generally susceptible to infection and family cluster cases are easy to occur. So, it is necessary to precisely prevent and control this strain. Public security forensic experts have both medical literacy and criminal investigation capabilities, they can play a more professional role in epidemic prevention and control.

Analysis of sequence diversity and selection pressure in HIV-1 clade C gp41 from India.

Evaluation of viral diversity is critical for the rational design of treatment modalities against Human immunodeficiency virus (HIV). Predominated by HIV-1 clade C (HIV-1C), the epidemic in India represents the third largest population infected with HIV-1 globally. Glycoprotein 41 (gp41) is critical for viral replication and is a target for the design of therapeutic strategies. However, documentation of viral diversity of gp41 gene in infected individuals from India remains limited. Present study employed high throughput sequencing to examine variation in gp41 amplicons generated from blood derived viruses in 24 HIV-1C infected individuals from Mumbai, India. Sequence diversity profiles were documented in different functional domains of gp41. Furthermore, through a meta-analysis approach, all reported gp41 sequences from India (N = 70) were compared with those from South Africa (N = 126), country with the largest HIV epidemic globally, also predominated by HIV-1C. A total of 44 positions displayed statistically significant differential (p < 0.05) Shannon entropy in the two regions. This comparison also identified 11 codon sites undergoing distinct selection, 8 of which remained differentially selected in an extended comparison of data from Asia (N = 137) and Africa(N = 383). Assessment of correlated mutation networks associated with differentially selected residues revealed common as well as distinct interaction networks. Furthermore, codon usage analysis revealed 17 differentially selected codons (Mann-Whitney test, p < 0.001) in Asia and Africa. Dissimilar trends in GC content across codon positions were also observed. In depth understanding of these divergent evolutionary signatures through extended analysis with larger data-sets would assist development of effective interventions being considered for HIV-1C.

Viral Enrichment Methods Affect the Detection but Not Sequence Variation of West Nile Virus in Equine Brain Tissue.

West Nile virus (WNV), a small, positive sense, single stranded RNA virus continues to encroach into new locales with emergence of new viral variants. Neurological disease in the equine can be moderate to severe in the face of low to undetectable virus loads. Physical methods of virus enrichment may increase sensitivity of virus detection and enhance analysis of viral diversity, especially for deep sequencing studies. However, the use of these techniques is limited mainly to non-neural tissues. We investigated the hypothesis that elimination of equine brain RNA enhances viral detection without limiting viral variation. Eight different WNV viral RNA enrichment and host RNA separation methods were evaluated to determine if elimination of host RNA enhanced detection of WNV and increase the repertoire of virus variants for sequencing. Archived brain tissue from 21 different horses was inoculated with WNV, homogenized, before enrichment and separation. The protocols utilized combinations of low-speed centrifugation, syringe filtration, and nuclease treatment. Viral and host RNA were analyzed using real-time PCR targeting the WNV Envelope (E) protein and equine G3PDH to determine relative sensitivity for WNV and host depletion, respectively. To determine the effect of these methods on viral variation, deep sequencing of the E protein was performed. Our results demonstrate that additional separation and enrichment methods resulted in loss of virus in the face of host RNA depletion. DNA sequencing showed no significant difference in total sequence variation between the RNA enrichment protocols. For equine brain infected with WNV, direct RNA extraction followed by host RNA depletion was most suitable. This study highlights the importance of evaluating viral enrichment and separation methods according to tissue type before embarking on studies where quantification of virus and viral variants is essential to the outcome of the study.