Isolation and characterization of emerging Mpox virus from India.

Mpox (previously known as Monkeypox) has recently re-emerged, primarily through human-to-human transmission in non-endemic countries including India. Virus isolation is still considered as the gold standard for diagnosis of viral infections. Here, the qPCR positive skin lesion sample from a patient was inoculated in Vero E6 cell monolayer. Characteristic cytopathic effect exhibiting typical cell rounding and detachment was observed at passage-02. The virus isolation was confirmed by qPCR. The replication kinetics of the isolate was determined that revealed maximum viral titre of log 6.3 PFU/mL at 72 h postinfection. Further, whole genome analysis through next generation sequencing revealed that the Mpox virus (MPXV) isolate is characterized by several unique SNPs and INDELs. Phylogenetically, it belonged to A.2 lineage of clade IIb, forming a close group with all other Indian MPXV along with few from USA, UK, Portugal, Thailand and Nigeria. This study reports the first successful isolation and phenotypic and genotypic characterization of MPXV from India.

Clinical Manifestations of Monkeypox.

Monkeypox is a global health issue caused by the monkeypox virus. It can spread from person to person through respiratory secretions, direct exposure to dermatological lesions of infected patients, or exposure to contaminated objects. It is more common in homosexual men, and most patients are asymptomatic. The gold standard for diagnosis is a real-time polymerase chain reaction. In the absence of testing facilities, clinicians rely upon detailed history to exclude other causes of fever with rashes. Initially, there is a prodrome phase of a few days, which is followed by the appearance of rashes. The dermatological manifestations are in the form of an exanthematous rash, which transforms through a macular, papular, and vesicular phase and disappears after crusting in approximately 3 weeks. There can be associated lymphadenopathy in these patients. Respiratory manifestations include nasal congestion and shortness of breath that may result in secondary bacterial infections. Additionally, patients can have neurological involvement in the form of encephalitis. Furthermore, ocular involvement can occur in the form of conjunctivitis, keratitis, and corneal ulceration. Other symptoms can include diarrhea, vomiting, myalgia, and backache. Since most patients do not require hospitalization, the approach to treatment is mainly vigilant monitoring, antiviral therapy, and management of associated complications.

Susceptibility of Marmosets (Callithrix jacchus) to Monkeypox Virus: A Low Dose Prospective Model for Monkeypox and Smallpox Disease.

Although current nonhuman primate models of monkeypox and smallpox diseases provide some insight into disease pathogenesis, they require a high titer inoculum, use an unnatural route of infection, and/or do not accurately represent the entire disease course. This is a concern when developing smallpox and/or monkeypox countermeasures or trying to understand host pathogen relationships. In our studies, we altered half of the test system by using a New World nonhuman primate host, the common marmoset. Based on dose finding studies, we found that marmosets are susceptible to monkeypox virus infection, produce a high viremia, and have pathological features consistent with smallpox and monkeypox in humans. The low dose (48 plaque forming units) required to elicit a uniformly lethal disease and the extended incubation (preclinical signs) are unique features among nonhuman primate models utilizing monkeypox virus. The uniform lethality, hemorrhagic rash, high viremia, decrease in platelets, pathology, and abbreviated acute phase are reflective of early-type hemorrhagic smallpox.

Sequence of pathogenic events in cynomolgus macaques infected with aerosolized monkeypox virus.

UNLABELLED: To evaluate new vaccines when human efficacy studies are not possible, the FDA's "Animal Rule" requires well-characterized models of infection. Thus, in the present study, the early pathogenic events of monkeypox infection in nonhuman primates, a surrogate for variola virus infection, were characterized. Cynomolgus macaques were exposed to aerosolized monkeypox virus (10(5) PFU). Clinical observations, viral loads, immune responses, and pathological changes were examined on days 2, 4, 6, 8, 10, and 12 postchallenge. Viral DNA (vDNA) was detected in the lungs on day 2 postchallenge, and viral antigen was detected, by immunostaining, in the epithelium of bronchi, bronchioles, and alveolar walls. Lesions comprised rare foci of dysplastic and sloughed cells in respiratory bronchioles. By day 4, vDNA was detected in the throat, tonsil, and spleen, and monkeypox antigen was detected in the lung, hilar and submandibular lymph nodes, spleen, and colon. Lung lesions comprised focal epithelial necrosis and inflammation. Body temperature peaked on day 6, pox lesions appeared on the skin, and lesions, with positive immunostaining, were present in the lung, tonsil, spleen, lymph nodes, and colon. By day 8, vDNA was present in 9/13 tissues. Blood concentrations of interleukin 1ra (IL-1ra), IL-6, and gamma interferon (IFN-γ) increased markedly. By day 10, circulating IgG antibody concentrations increased, and on day 12, animals showed early signs of recovery. These results define early events occurring in an inhalational macaque monkeypox infection model, supporting its use as a surrogate model for human smallpox. IMPORTANCE: Bioterrorism poses a major threat to public health, as the deliberate release of infectious agents, such smallpox or a related virus, monkeypox, would have catastrophic consequences. The development and testing of new medical countermeasures, e.g., vaccines, are thus priorities; however, tests for efficacy in humans cannot be performed because it would be unethical and field trials are not feasible. To overcome this, the FDA may grant marketing approval of a new product based upon the "Animal Rule," in which interventions are tested for efficacy in well-characterized animal models. Monkeypox virus infection of nonhuman primates (NHPs) presents a potential surrogate disease model for smallpox. Previously, the later stages of monkeypox infection were defined, but the early course of infection remains unstudied. Here, the early pathogenic events of inhalational monkeypox infection in NHPs were characterized, and the results support the use of this surrogate model for testing human smallpox interventions.

Virulence and pathophysiology of the Congo Basin and West African strains of monkeypox virus in non-human primates.

Monkeypox virus is divided into Congo Basin and West African strains. The virulence and pathophysiology of two strains, Zr-599 (a Congo Basin monkeypox virus) and Liberia (a West African monkeypox virus), were evaluated in non-human primates. Four monkeys were infected by the subcutaneous (SC) and two by the intranasal (IN) inoculation routes for Zr-599 and Liberia at a dose of 10(6) p.f.u. One monkey in the Liberia/SC group was demonstrated to be co-infected with Gram-positive cocci and was excluded from analyses. Infections in three of the four Zr-599/SC monkeys and in one of the three Liberia/SC monkeys were fatal. Virus genome levels in blood in the Zr-599/SC monkeys were approximately 10 times higher than those in the Liberia/SC monkeys. Zr-599 affected respiratory, genito-urinary and gastrointestinal tract organs more severely than Liberia. Zr-599 was more virulent than Liberia and one of the factors might be the difference in organ tropism.

Clinical manifestations of human monkeypox influenced by route of infection.

BACKGROUND: In April 2003, an outbreak of monkeypox occurred in the United States following the importation of monkeypox virus (MPXV)-infected animals in a consignment of exotic pets from West Africa. Transmission of the virus to non-African captive species, including prairie dogs, preceded human disease. METHODS: We evaluated the influence of the route of infection on clinical illness for persons with confirmed and probable cases of human monkeypox. Exposures were categorized as being "noninvasive" (e.g., the person touched an infected animal, cleaned an infected animal's cage, and/or stood within 6 feet of an infected animal) or "complex" (e.g., invasive bite or scratch from an ill prairie dog plus potential noninvasive exposure), and associations between exposure, illness manifestation, and illness progression (i.e., elapsed time from first exposure to an ill prairie dog through various benchmarks of illness) were assessed. RESULTS: Patients with complex exposures were more likely than patients with noninvasive exposures to have experienced pronounced signs of systemic illness (49.1% vs. 16.7%; P=.041) and to have been hospitalized during illness (68.8% vs. 10.3%; P<.001). Complex exposures were also associated with shorter incubation periods (9 days for complex exposures vs. 13 days for noninvasive exposures) and the absence of a distinct febrile prodrome. CONCLUSIONS: The findings of this study indicate that route of infection can influence monkeypox illness manifestations.

Monkeypox transmission and pathogenesis in prairie dogs.

During May and June 2003, the first cluster of human monkeypox cases in the United States was reported. Most patients with this febrile vesicular rash illness presumably acquired the infection from prairie dogs. Monkeypox virus was demonstrated by using polymerase chain reaction in two prairie dogs in which pathologic studies showed necrotizing bronchopneumonia, conjunctivitis, and tongue ulceration. Immunohistochemical assays for orthopoxviruses demonstrated abundant viral antigens in surface epithelial cells of lesions in conjunctiva and tongue, with less amounts in adjacent macrophages, fibroblasts, and connective tissues. Viral antigens in the lung were abundant in bronchial epithelial cells, macrophages, and fibroblasts. Virus isolation and electron microscopy demonstrated active viral replication in lungs and tongue. These findings indicate that both respiratory and direct mucocutaneous exposures are potentially important routes of transmission of monkeypox virus between rodents and to humans. Prairie dogs offer insights into transmission, pathogenesis, and new vaccine and treatment trials because they are susceptible to severe monkeypox infection.

Immunogenicity of a highly attenuated MVA smallpox vaccine and protection against monkeypox.

The potential use of smallpox as a biological weapon has led to the production and stockpiling of smallpox vaccine and the immunization of some healthcare workers. Another public health goal is the licensing of a safer vaccine that could benefit the millions of people advised not to take the current one because they or their contacts have increased susceptibility to severe vaccine side effects. As vaccines can no longer be tested for their ability to prevent smallpox, licensing will necessarily include comparative immunogenicity and protection studies in non-human primates. Here we compare the highly attenuated modified vaccinia virus Ankara (MVA) with the licensed Dryvax vaccine in a monkey model. After two doses of MVA or one dose of MVA followed by Dryvax, antibody binding and neutralizing titres and T-cell responses were equivalent or higher than those induced by Dryvax alone. After challenge with monkeypox virus, unimmunized animals developed more than 500 pustular skin lesions and became gravely ill or died, whereas vaccinated animals were healthy and asymptomatic, except for a small number of transient skin lesions in animals immunized only with MVA.