A visual assay panel for the identification of monkeypox virus DNA belonging to the clades I and II.
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ABSTRACT:
SUBMITTER: Huang P
PROVIDER: S-EPMC10241493 | biostudies-literature | 2023 Aug
REPOSITORIES: biostudies-literature
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A visual assay panel for the identification of monkeypox virus DNA belonging to the clades I and II.
Huang Pei P Huang Zanheng Z Liu Meihui M Bai Yujie Y Jin Hongli H Huang Jingbo J Liu Xingqi X Guan Zhenhong Z Duan Ming M Zhang Haili H Li Yuanyuan Y Chiu Sandra S Wang Hualei H
Virologica Sinica 20230605 4
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Project description:IntroductionMonkeypox virus (MPXV) hosts are of multiple species, with a risk of cross-species transmission. This phenomenon poses a threat to unreported affected domestic animals and increases the risk to human public health. Clinical diagnostics continue to face challenges regarding specificity among poxviruses. The need for a rapid and precise assay to differentiate between MPXV clades I and II, as well as goatpox virus (GTPV) is essential for enhancing our capacity for disease prevention, control, and epidemiological investigation.MethodsTo address this need, we have successfully developed a multiplex real-time PCR assay targeting MPXV D14L gene for clade I, MPXV D18L gene for clade II, and GTPV RPO30 gene, which can simultaneously detect MPXV clades I and II as well as GTPV.ResultsThe developed assay demonstrated high sensitivity, with limits of detection at 207.83 copies/reaction for MPXV clade I, 252.07 copies/reaction for MPXV clade II, and 208.72 copies/reaction for GTPV. Importantly, there was no cross-reactivity with other non-pox viruses which infect goats. The assay exhibited excellent repeatability, with coefficients of variation (CV%) for intra-assay and inter-assay ranging from 0.17% to 0.89% and 0.58% to 1.09%, respectively.DiscussionThis assay can serve as a vital resource to safeguard against the MPXV epidemic posing a threat to the life safety of goats, to mitigate potential risks to the sheep farming industry, and to prevent the transmission of MPXV to humans through sheep, which could act as a potential transmission vector for infection.
| S-EPMC11603957 | biostudies-literature
Project description:In endemic regions (West Africa and the Congo Basin), the genetic diversity of monkeypox virus (MPXV) is geographically structured into two major clades (Clades I and II) that differ in virulence and host associations. Clade IIb is closely related to the B.1 lineage, which is dominating a worldwide outbreak initiated in 2022. Lineage B.1 has however accumulated mutations of unknown significance that most likely result from apolipoprotein B mRNA editing catalytic polypeptide-like 3 (APOBEC3) editing. We applied a population genetics-phylogenetics approach to investigate the evolution of MPXV during historical viral spread in Africa and to infer the distribution of fitness effects. We observed a high preponderance of codons evolving under strong purifying selection, particularly in viral genes involved in morphogenesis and replication or transcription. However, signals of positive selection were also detected and were enriched in genes involved in immunomodulation and/or virulence. In particular, several genes showing evidence of positive selection were found to hijack different steps of the cellular pathway that senses cytosolic DNA. Also, a few selected sites in genes that are not directly involved in immunomodulation are suggestive of antibody escape or other immune-mediated pressures. Because orthopoxvirus host range is primarily determined by the interaction with the host immune system, we suggest that the positive selection signals represent signatures of host adaptation and contribute to the different virulence of Clade I and II MPXVs. We also used the calculated selection coefficients to infer the effects of mutations that define the predominant human MPXV1 (hMPXV1) lineage B.1, as well as the changes that have been accumulating during the worldwide outbreak. Results indicated that a proportion of deleterious mutations were purged from the predominant outbreak lineage, whose spread was not driven by the presence of beneficial changes. Polymorphic mutations with a predicted beneficial effect on fitness are few and have a low frequency. It remains to be determined whether they have any significance for ongoing virus evolution.
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Project description:In this study, a rapid method for the detection of Central and West Africa clades of Monkeypox virus (MPXV) using recombinase polymerase amplification (RPA) assay targeting the G2R gene was developed. MPXV, an Orthopoxvirus, is a zoonotic dsDNA virus, which is listed as a biothreat agent. RPA was operated at a single constant temperature of 42°C and produced results within 3 to 10 minutes. The MPXV-RPA-assay was highly sensitive with a limit of detection of 16 DNA molecules/μl. The clinical performance of the MPXV-RPA-assay was tested using 47 sera and whole blood samples from humans collected during the recent MPXV outbreak in Nigeria as well as 48 plasma samples from monkeys some of which were experimentally infected with MPXV. The specificity of the MPXV-RPA-assay was 100% (50/50), while the sensitivity was 95% (43/45). This new MPXV-RPA-assay is fast and can be easily utilised at low resource settings using a solar powered mobile suitcase laboratory.
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Project description:Mpox, the disease caused by monkeypox virus (MPXV) is increasing in Africa and in 2022 spread to more than 100 countries sickening more than 100,000 individuals. Four clades of MPXV have been recognized with differences in severity of disease and extent of human-to-human transmission. Determination of the genetic basis for these differences could help to develop improved therapeutics and vaccines. The Castaneous (CAST) mouse is highly susceptible to MPXV and virulence differences of MPXV clades are statistically significant mimicking their relative severities in humans. The present study was intended to evaluate the CAST mouse as a model for investigating genomic differences by replacing genes of Zaire-1979 005 (Z-79) clade Ia MPXV with homologous gene sequences of the less virulent and less transmissible USA-2003 clade IIa MPXV. The expectation was that some gene replacements would reduce the virulence of Z-79 chimera. Recombinant viruses expressing firefly luciferase were constructed in which partially overlapping Z-79 DNA segments of 5,000 to 13,000 bp containing ∼ 40 genes in total from the two ends of MPXV-79 were replaced with corresponding segments of USA-2003. Virulence was determined by live animal imaging in addition to weight loss and survival. Although there were statistical differences in survival and viral luminescence between the clade Ia and clade IIa MPXVs, no significant difference was found by replacing individual or multiple genes of clade Ia Z-79 with corresponding genes of clade IIa USA-2003. The absence of a significant reduction in virulence can have several explanations that would inform future experiments.
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Project description: Not available
| S-EPMC9574605 | biostudies-literature
Project description:BackgroundThe vaccinia virus (VACV) isolates, Guarani P1 virus (GP1V) and Passatempo virus (PSTV), were isolated during zoonotic outbreaks in Brazil. Each one of them belongs to two different VACV clades, defined by biological aspects that include virulence in mice and phylogenetic analysis. Considering that information about how vaccinia viruses from different groups elicit immune responses in animals is scarce, we investigated such responses in mice infected either by GP1V (group 2) or PSTV (group 1), using VACV Western Reserve strain (VACV-WR) as control.MethodsThe severity of the infections was evaluated in BALB/c mice considering diverse clinical signs and defined scores, and the immune responses triggered by GP1V and PSTV infections were analysed by immune cell phenotyping and intra-cytoplasmic cytokines detection.ResultsWe detected a reduction in total lymphocytes (CD3 +), macrophages (CD14 +), and NK cells (CD3-CD49 +) in animals infected with VACV-WR or GP1V. The VACV-WR and GP1V viruses, belonging to the most virulent group in a murine model, were able to down-modulate the cell immune responses upon mice infection. In contrast, PSTV, a virus considered less virulent in a murine model, showed little ability to down-modulate the mice immune responses. Mice infected with VACV-WR and GP1V viruses presented significant weight loss and developed lesions in their spleens, as well as damage to liver and lungs whereas mice infected with PSTV developed only moderate clinical signs.ConclusionsOur results suggest that VACV immunomodulation in vivo is clade-related and is proportional to the strain's virulence upon infection. Our data corroborate the classification of the different Brazilian VACV isolates into clades 1 and 2, taking into account not only phylogenetic criteria, but also clinical and immunological data.
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