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Monkeypox virus (MPXV) belongs to the Orthopoxvirus genus along with smallpox and cowpox. The first human case of MPXV was reported on September 1, 1970, at a hospital in Basankusu, Democratic Republic of the Congo. MPXV is primarily transmitted from wild animals like rodents and primates to humans, causing a rare zoonotic disease known as monkeypox. Human-to-human transmission is limited. Monkeypox symptoms are similar to smallpox but less severe. Initially confined to remote areas in Central and West Africa, MPXV has evolved into two clades: the more virulent Central African (Congo Basin) clade and the less virulent West African clade.
Monkeypox virus is an enveloped double-stranded DNA virus with low dependence on the host cell's DNA and RNA replication machinery, allowing it to replicate in the host cell cytoplasm. Similar to other viruses in the Orthopoxvirus genus, MPXV likely replicates into morphologically distinct intracellular mature virions (IMVs) and extracellular enveloped virions (EEVs). IMVs have a robust physical structure, facilitating transmission between hosts, while the more fragile EEVs, encased in an envelope, evade host immune clearance, making them suitable for intercellular spread. MPXV efficiently infects human primary monocytes, suppressing CD4+ and CD8+ T cell activation, eliminating local T cell responses, and avoiding systemic immunosuppression and immune surveillance.
The best diagnostic samples for monkeypox come from skin lesions, specifically vesicles and pustules, as well as dried scabs. Lesion samples must be stored in a dry, sterile tube (without viral transport media) and kept cold. Clinical diagnosis of monkeypox must consider other rash diseases such as chickenpox, measles, bacterial skin infections, scabies, syphilis, and drug-related allergies. Lymphadenopathy in the early stages may be the only clinical feature distinguishing monkeypox from chickenpox or smallpox.
Under electron microscopy, MPXV appears as a brick-shaped cytoplasmic particle with lateral bodies and a central core measuring approximately 200-300 nm. Since Orthopoxvirus species cannot be morphologically distinguished, this method does not confirm the diagnosis but suggests the virus belongs to the Poxviridae family.
Routine detection of MPXV DNA in clinical samples or MPXV-infected cell cultures can be performed using PCR or real-time PCR, preferably in a biosafety level 3 facility. Real-time PCR targeting the conserved regions of the extracellular enveloped protein gene (B6R), DNA polymerase gene, E9L, DNA-dependent RNA polymerase subunit 18, rpo18, and F3L genes is recommended.
PCR-amplified gene or gene fragment restriction length fragment polymorphism (RFLP) can also be used to detect MPXV DNA, but RFLP is time-consuming and requires virus culture. RFLP of PCR products also requires enzyme digestion followed by gel electrophoresis, making it less suitable in clinical settings where speed, sensitivity, and specificity are critical.
Whole-genome sequencing using NGS remains the gold standard for distinguishing MPXV from other Orthopoxviruses (OPV), but this technique is costly and requires substantial computational resources for downstream data processing. Therefore, NGS may not be suitable for resource-limited countries in sub-Saharan Africa.
Real-time PCR remains the preferred method for routine diagnosis of MPXV, but on-site genome sequencing techniques, such as Oxford Nanopore MinION, are essential for providing real-time viral genomic data, which is crucial for evidence-based epidemiological interventions.
Monkeypox virus immunodetection methods mainly include enzyme-linked immunosorbent assay (ELISA) for detecting IgG and IgM antibodies and immunohistochemistry for detecting viral antigens. Immunochemical analysis using MPXV antibodies can distinguish between Poxvirus infections and herpesvirus.
It has been shown that antiviral antibodies and T cell responses increase before and after the onset of disease, with MPXV IgM and IgG detected in serum approximately 5 days and more than 8 days after the rash onset, respectively. The presence of IgM and IgG antibodies in unvaccinated individuals with a history of rash and severe disease may suggest MPXV infection. IgM-capture ELISA positivity indicates recent exposure to MPXV, while IgG-capture ELISA positivity suggests prior exposure through vaccination or natural infection. Simultaneous presence of IgM and IgG in a sample suggests previous exposure in individuals vaccinated or naturally infected with MPXV. However, Orthopoxviruses have serological cross-reactivity, and antigen and antibody detection methods cannot provide monkeypox-specific confirmation but may be feasible for serological surveillance in MPXV-endemic areas.
References
1. Sarah Keasey, Christine Pugh, Alexander Tikhonov, et al. Proteomic Basis of the Antibody Response to Monkeypox Virus Infection Examined in Cynomolgus Macaques and a Comparison to Human Smallpox Vaccination[J]. Plos one, 2010, 5(12): e15547.
2. Emmanuel Alakunle, Ugo Moens, Godwin Nchind, et al. Monkeypox Virus in Nigeria: Infection Biology, Epidemiology, and Evolution[J]. Viruses, 2020, 12(11):1257.
3. Erika Hammarlund, Anindya Dasgupta, Clemencia Pinilla, et al. Monkeypox virus evades antiviral CD4+ and CD8+ T cell responses by suppressing cognate T cell activation[J]. Proc Natl Acad Sci USA, 2008, 105(38): 14567-72.
