Pathogen reduction of monkeypox virus in plasma and whole blood using riboflavin and UV light.

BACKGROUND: Monkeypox virus has recently emerged from endemic foci in Africa and, since October 20, 2022, more than 73,000 human infections have been reported by the CDC from over 100 countries that historically have not reported monkeypox cases. The detection of virus in skin lesions, blood, semen, and saliva of infected patients with monkeypox infections raises the potential for disease transmission via routes that have not been previously documented, including by blood and plasma transfusions. Methods for protecting the blood supply against the threats of newly emerging disease agents exist and include Pathogen Reduction Technologies (PRT) which utilize photochemical treatment processes to inactivate pathogens in blood while preserving the integrity of plasma and cellular components. Such methods have been employed broadly for over 15 years, but effectiveness of these methods under routine use conditions against monkeypox virus has not been reported. STUDY DESIGN AND METHODS: Monkeypox virus (strain USA_2003) was used to inoculate plasma and whole blood units that were then treated with riboflavin and UV light (Mirasol Pathogen Reduction Technology System, Terumo BCT, Lakewood, CO). The infectious titers of monkeypox virus in the samples before and after riboflavin + UV treatment were determined by plaque assay on Vero cells. RESULTS: The levels of spiked virus present in whole blood and plasma samples exceeded 103 infectious particles per dose, corresponding to greater than 105 DNA copies per mL. Treatment of whole blood and plasma units under standard operating procedures for the Mirasol PRT System resulted in complete inactivation of infectivity to the limits of detection. This is equivalent to a reduction of ≥ 2.86 +/- 0.73 log10 pfu/mL of infectivity in whole blood and ≥ 3.47 +/-0.19 log10 pfu/mL of infectivity in plasma under standard operating conditions for those products. CONCLUSION: Based on this data and corresponding studies on infectivity in patients with monkeypox infections, use of Mirasol PRT would be expected to significantly reduce the risk of transfusion transmission of monkeypox.

Comparison of Monkeypox Virus Clade Kinetics and Pathology within the Prairie Dog Animal Model Using a Serial Sacrifice Study Design.

Monkeypox virus (MPXV) infection of the prairie dog is valuable to studying systemic orthopoxvirus disease. To further characterize differences in MPXV clade pathogenesis, groups of prairie dogs were intranasally infected (8 × 10(3) p.f.u.) with Congo Basin (CB) or West African (WA) MPXV, and 28 tissues were harvested on days 2, 4, 6, 9, 12, 17, and 24 postinfection. Samples were evaluated for the presence of virus and gross and microscopic lesions. Virus was recovered from nasal mucosa, oropharyngeal lymph nodes, and spleen earlier in CB challenged animals (day 4) than WA challenged animals (day 6). For both groups, primary viremia (indicated by viral DNA) was seen on days 6-9 through day 17. CB MPXV spread more rapidly, accumulated to greater levels, and caused greater morbidity in animals compared to WA MPXV. Histopathology and immunohistochemistry (IHC) findings, however, were similar. Two animals that succumbed to disease demonstrated abundant viral antigen in all organs tested, except for brain. Dual-IHC staining of select liver and spleen sections showed that apoptotic cells (identified by TUNEL) tended to colocalize with poxvirus antigen. Interestingly splenocytes were labelled positive for apoptosis more often than hepatocytes in both MPXV groups. These findings allow for further characterization of differences between MPXV clade pathogenesis, including identifying sites that are important during early viral replication and cellular response to viral infection.

Optimization of peptide-based ELISA for serological diagnostics: a retrospective study of human monkeypox infection.

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.

Application of the Ibis-T5000 pan-Orthopoxvirus assay to quantitatively detect monkeypox viral loads in clinical specimens from macaques experimentally infected with aerosolized monkeypox virus.

Monkeypox virus (MPXV), a member of the family Poxviridae and genus Orthopoxvirus, causes a smallpox-like disease in humans. A previously described pan-Orthopoxvirus assay, based on a broad-range polymerase chain reaction (PCR) coupled with electrospray ionization mass spectrometry (PCR/ESI-MS), was evaluated for its ability to detect MPXV from spiked human and aerosol-infected cynomolgous macaque (Macaca fascicularis) samples. Detection of MPXV DNA from macaque tissue, blood, and spiked human blood by the PCR/ESI-MS pan-Orthopoxvirus assay was comparable, albeit at slightly higher levels, to the current gold standard method of real-time PCR with the pan-Orthopoxvirus assay and had a limit of detection of 200 plaque-forming units. Furthermore, the platform was able to distinguish MPXV and vaccinia viruses that were spiked into macaque blood samples at various concentrations. This platform provides a new tool for the diagnosis and monitoring of orthopoxviral loads during vaccine or antiviral studies, but also could provide rapid identification during natural outbreaks or bioterrorism attacks.

Clonal vaccinia virus grown in cell culture fully protects monkeys from lethal monkeypox challenge.

The potential use of smallpox as an agent of bioterrorism has renewed interest in the development of a modern vaccine capable of replacing the standard Dryvax vaccine. Vaccinia virus (ACAM2000), clonally isolated from Dryvax and manufactured in cell culture, was tested for immunogenicity and protective activity in a non-human primate model. Cynomolgus monkeys vaccinated with ACAM2000, Dryvax, or ACAM2000 diluent (control) were challenged 2 months post-vaccination with a lethal, intravenous dose of monkeypox virus. ACAM2000 proved immunogenic and efficacious in protecting against lethal monkeypox challenge, as evident from a lack of post-challenge viral replication, and the absence of any significant clinical signs attributable to monkeypox infection. This protection correlated (with) neutralizing antibody titers equivalent to those generated in the Dryvax group post-vaccination, as well as a similar significant increase in the presence of neutralizing antibodies post-challenge. Control animals showed no signs of vaccine-induced seroconversion, displayed post-challenge tissue-associated viral replication and viremia, and developed severe monkeypox-specific clinical symptoms. The protective efficacy of ACAM2000 was found to be equivalent to the currently approved vaccine, Dryvax.

Antiviral treatment is more effective than smallpox vaccination upon lethal monkeypox virus infection.

There is concern that variola virus, the aetiological agent of smallpox, may be used as a biological weapon. For this reason several countries are now stockpiling (vaccinia virus-based) smallpox vaccine. Although the preventive use of smallpox vaccination has been well documented, little is known about its efficacy when used after exposure to the virus. Here we compare the effectiveness of (1) post-exposure smallpox vaccination and (2) antiviral treatment with either cidofovir (also called HPMPC or Vistide) or with a related acyclic nucleoside phosphonate analogue (HPMPO-DAPy) after lethal intratracheal infection of cynomolgus monkeys (Macaca fascicularis) with monkeypox virus (MPXV). MPXV causes a disease similar to human smallpox and this animal model can be used to measure differences in the protective efficacies of classical and new-generation candidate smallpox vaccines. We show that initiation of antiviral treatment 24 h after lethal intratracheal MPXV infection, using either of the antiviral agents and applying various systemic treatment regimens, resulted in significantly reduced mortality and reduced numbers of cutaneous monkeypox lesions. In contrast, when monkeys were vaccinated 24 h after MPXV infection, using a standard human dose of a currently recommended smallpox vaccine (Elstree-RIVM), no significant reduction in mortality was observed. When antiviral therapy was terminated 13 days after infection, all surviving animals had virus-specific serum antibodies and antiviral T lymphocytes. These data show that adequate preparedness for a biological threat involving smallpox should include the possibility of treating exposed individuals with antiviral compounds such as cidofovir or other selective anti-poxvirus drugs.

characterization of acute-phase humoral immunity to monkeypox: use of immunoglobulin M enzyme-linked immunosorbent assay for detection of monkeypox infection during the 2003 North American outbreak.

A monkeypox outbreak occurred in the United States in 2003. Patient's sera were sent to the Centers for Disease Control and Prevention as a part of outbreak response measures. Clinical and epidemiologic information was abstracted from the case investigation forms. Serum samples from patients were tested by using an immunoglobulin M (IgM)-capture and an IgG enzyme-linked immunosorbent assay ELISA against Orthopoxvirus antigen. The detection of antiviral IgG and IgM antibodies and the kinetics of the antiviral IgG and IgM antibody responses were evaluated. Patients were classified as confirmed, probable, or suspect cases or were excluded as cases based on laboratory test results and epidemiologic and clinical criteria. A total of 37 confirmed case patients with monkeypox were identified, and 116 patients were excluded as case patients based on molecular testing or insufficient epidemiology and clinical data to warrant classification as a suspect or probable case. Of 37 confirmed case patients, 36 had a known history (presence or absence) of smallpox vaccination. Of those, 29 of the 36 either had or developed an IgG response, while 34 of the 36 developed an IgM response, regardless of vaccination status. Serum collected > or =5 days for IgM detection or serum collected > or =8 days after rash onset for IgG detection was most efficient for the detection of monkeypox virus infection. IgM ELISA detects recent infection with orthopoxviruses and, in this case, recent infection with monkeypox virus. In addition, analysis of paired sera for IgG and IgM detected seroconversion, another indicator of recent infection. The ELISA results correlated with the virologic PCR and viral culture results, indicating its diagnostic capabilities for monkeypox and potentially other orthopoxvirus infections due to zoonotic transmission or bioterrorism events.