ELISA for Monkeypox

What Is Monkeypox

Monkeypox virus is a zoonotic virus endemic to Central Africa. Although active disease surveillance has assessed monkeypox disease prevalence and geographic range, information about virus diversity is lacking. We therefore assessed genome diversity of viruses in 60 samples obtained from humans with primary and secondary cases of infection from 2005 through 2007.

We detected 4 distinct lineages and a deletion that resulted in gene loss in 10 (16.7%) samples and that seemed to correlate with human-to-human transmission (p = 0.0544). The data suggest a high frequency of spillover events from the pool of viruses in nonhuman animals, active selection through genomic destabilization and gene loss, and increased disease transmissibility and severity. The potential for accelerated adaptation to humans should be monitored through improved surveillance.

ELISA For Monkeypox

Although smallpox has been eradicated, other diseases caused by virulent orthopoxviruses such as monkeypox virus (MPV) remain endemic in remote areas of western and central sub-Saharan Africa, and represent a potential biothreat due to international travel and/or inadvertent exposure. Unfortunately, extensive antigenic cross-reactivity among orthopoxviruses presents a challenge to serological diagnosis.

We previously reported a 20mer peptide-based ELISA that identified recent MPV infection with >90% sensitivity and >90% specificity. However, the sensitivity of this approach was not determined with samples obtained at later time points after antibody titers had declined from their peak levels. To improve assay sensitivity for detecting MPV-specific antibodies at later time points, we compared diagnostic 20mer peptides to 30mer peptides.

In addition, optimal 30mer peptides were tested in combination or after conjugating selected peptides to a carrier protein (bovine serum albumin) to further improve assay performance. An optimized combination of four unconjugated 30mer peptides provided 100% sensitivity for detecting MPV infection at 2-6 months post-infection, 45% sensitivity for detecting MPV infection at >2 years post-infection, and 99% specificity. However, an optimized combination of two peptide conjugates provided 100% sensitivity for detecting MPV infection at 2-6 months post-infection, 90% sensitivity for detecting MPV infection at >2 years post-infection, and 97% specificity.

Peptide-based ELISA tests provide a relatively simple approach for serological detection of MPV infection. Moreover, the systematic approach used here to optimize diagnostic peptide reagents is applicable to developing improved diagnostics to a broad range of other viruses, and may be particularly useful for distinguishing between closely-related viruses within the same genus or family.

Pathogenesis Of Fulminant Monkeypox.

The pathogenesis of severe human monkeypox, which causes systemic and fulminant infections, is not clear. This study presents a case repot of fulminant monkeypox with bacterial sepsis after experimental infection with monkeypox virus in a cynomolgus monkey (Macaca fascicularis). In our previous study (Saijo et al., 2009, J Gen Virol), two cynomolgus monkeys became moribund after experimental infection with monkeypox virus Liberia strain, West African strain.

One exhibited typical monkeypox-related papulovesicular lesions. The other monkey presented fulminant clinical symptoms with a characteristic flat red rash similar to that found in smallpox, which is associated with extremely high fatality rates. In this study, we found that the monkey with flat red rash had high levels of viremia and neutropenia, as well as high plasma levels of pro-inflammatory cytokines and chemokines compared with the other monkey. Monkeypox virus replicates in epithelial cells and macrophages in various organs. Sepsis due to Gram-positive cocci was confirmed histopathologically in the monkey with flat red rash. The lack of inflammatory response in the lesion suggested that the monkey with sepsis experienced strong immune suppression during the viral infection. The neutropenia and excessive inflammatory cytokine responses indicate that neutrophils play key roles in the pathogenesis of systemic and fulminant human monkeypox virus infections with sepsis.

Human monkeypox, i.e., monkeypox virus infection of humans, is the most important orthopoxvirus infection at present now that smallpox, which is caused by Variola virus, has been eradicated. Monkeypox virus is a zoonotic agent in rodents, nonhuman primate species, and humans. Outbreaks of human monkeypox occur occasionally in remote villages in central and western Africa near tropical rainforests . In 2003, an outbreak of human monkeypox with fever and skin eruptions occurred in Midwestern USA. The patients were found to be infected with a West African strain of monkeypox virus caused by close contact with sick pet prairie dogs (Cynomys spp.), which may have been in contact with wild rodents imported from Ghana . There were no deaths during the USA outbreak, but some pediatric patients developed serious complications that could have resulted in death . The disease course is often milder than that of smallpox. However, the mortality rate with monkeypox infection has been reported as 1.5-17% in Africa .

The clinical features of classical smallpox with a high mortality rate, i.e., Variola major, are classified as ordinary type, modified type, Variola sine eruptione, hemorrhagic type, and flat type . The hemorrhagic type, with widespread hemorrhages in the skin and mucous membranes, and the flat type, where the pustules remain flat, were usually fatal. The pathophysiological processes of these fulminant types of smallpox are not well understood. Cynomolgus (Macaca fascicularis) and rhesus macaques have been used as nonhuman primate models of smallpox and orthopoxvirus infections .

The virulence and pathophysiology of monkeypox two strains, Zr-599 and Liberia (Congo Basin and West African monkeypox virus strains, respectively), were evaluated in cynomolgus monkeys , which showed that Zr-599 was more virulent than Liberia. The clinically advanced stage was characterized by poxviral papulovesicular rashes on days 7-9 post-inoculation (p.i.). Three Zr-599-inoculated monkeys and one Liberia-inoculated monkey were moribund during days 13-18 p.i. The other monkeys recovered and survived for the observational period of 22 days p.i. However, one Liberia-inoculated monkey did not exhibit typical rashes, although it was debilitated. A postmortem pathological examination on day 10 p.i. showed that the monkey had sepsis with Gram-positive cocci. The present study investigated the clinical, immunological, and histopathological features of the monkey with fulminant monkeypox to elucidate the pathogenesis and risk factors related to this severe orthopoxvirus infection with sepsis.

Monkeypox Symptoms.

In contrast, varicella is a febrile rash illness characterized by the absence of a significant febrile prodrome and by lesions at different stages, with bumps, blisters, and scabbed lesions existing at the same time . Regarding operational perspectives, diagnosis can be challenging because MPX atypical forms cannot always be easily distinguished from severe varicella or from smallpox.