Orthopoxvirus

Monkeypox virus is a zoonotic orthopoxvirus (OPX) of west and central sub-Saharan Africa. We conducted a cross-sectional serosurvey in Likouala region, Republic of Congo to assess exposure to OPX. Whole blood was collected using Nobuto blood filter strips (NBFS). Titers of IgM and IgG to OPX were assessed using an enzyme-linked immunosorbent assay. Demographic and clinical characteristics were compared with serostatus using the chi-square test or Fisher's exact test. Multivariate logistic regression was performed to evaluate factors for independent association with serostatus. A total of 994 specimens were analyzed; the overall seroprevalence for OPX IgM was 1.7%. Age < 25 years reduced the likelihood of OPX exposure, and persons living in Ngangania village had independently higher odds (odds ratio = 33.5, 95% confidence interval = 7.2-166). Blood collection for serosurveys using NBFS is feasible and practical. Adult activities such as hunting and carcass preparation may play...

An oligonucleotide microarray for detection and identification of orthopoxviruses was developed. Genus specific and orthopoxvirus species-specific regions of the genes encoding chemokine binding and alpha/beta-interferon binding proteins were used as a target. The developed microarray allows the variola, monkeypox, cowpox, vaccinia, camel-pox and ectromelia (mousepox) viruses to be distinguished with a high degree of reliability.

Human monkeypox has never been reported in Ghana, but rodents captured in forested areas of southern Ghana were the source of the monkeypox virus introduced into the United States in 2003. Subsequent to the outbreak in the United States, 204 animals were collected from two commercial trapping sites in Ghana. Animal tissues were examined for the presence of orthopoxvirus (OPXV) DNA using a real-time polymerase chain reaction, and sera were assayed for antibodies against OPXV. Animals from five genera (Cricetomys, Graphiurus, Funiscirus, and Heliosciurus) had antibodies against OPXV, and three genera (Cricetomys, Graphiurus, and Xerus) had evidence of OPXV DNA in tissues. Additionally, 172 persons living near the trapping sites were interviewed regarding risk factors for OPXV exposure, and their sera were analyzed. Fifty-three percent had IgG against OPXV; none had IgM. Our findings suggest that several species of forest-dwelling rodents from Ghana are susceptible to naturally occurri...

PCR following two methods of DNA extraction was used to confirm the growth of camel pox virus (CPV) and vaccinia virus in cell culture and chorioallantoic membrane. Results were compared with the commonly used neutralization test. The first method of DNA extraction was accomplished by using viral DNA in tissue culture supernatant and Chorioallantoic membrane, which was released by initial heating for 15 min at 99 degrees C followed by ordinary PCR. In the second method DNA was extracted by using DNA Isolation Kit from tissue culture supernatant and used as a template. Rapid identification and differentiation of CPV and Vaccinia virus were achieved by PCR and this assay proved to be fast and feasible, and can be an alternative to orthodox serological methods.

Monkeypox virus is a zoonotic orthopoxvirus (OPX) of west and central sub-Saharan Africa. We conducted a cross-sectional serosurvey in Likouala region, Republic of Congo to assess exposure to OPX. Whole blood was collected using Nobuto blood filter strips (NBFS). Titers of IgM and IgG to OPX were assessed using an enzyme-linked immunosorbent assay. Demographic and clinical characteristics were compared with serostatus using the chi-square test or Fisher's exact test. Multivariate logistic regression was performed to evaluate factors for independent association with serostatus. A total of 994 specimens were analyzed; the overall seroprevalence for OPX IgM was 1.7%. Age < 25 years reduced the likelihood of OPX exposure, and persons living in Ngangania village had independently higher odds (odds ratio = 33.5, 95% confidence interval = 7.2-166). Blood collection for serosurveys using NBFS is feasible and practical. Adult activities such as hunting and carcass preparation may play...

In January 2015, during a 3-week period, 12 captive Tonkean macacques at a sanctuary in Italy died. An orthopoxvirus infection was suspected because of negative-staining electron microscopy results. The diagnosis was confirmed by histology, virus isolation, and molecular analysis performed on different organs from all animals. An epidemiologic investigation was unable to define the infection source in the surrounding area. Trapped rodents were negative by virologic testing, but specific IgG was detected in 27.27% of small rodents and 14.28% of rats. An attenuated live vaccine was administered to the susceptible monkey population, and no adverse reactions were observed; a detectable humoral immune response was induced in most of the vaccinated animals. We performed molecular characterization of the orthopoxvirus isolate by next-generation sequencing. According to the phylogenetic analysis of the 9 conserved genes, the virus could be part of a novel clade, lying between cowpox and ect...

A strain of camelpox virus (CMLV) isolated in the Sudan was attenuated by serial passage in Vero cell monolayers for use as a future vaccine strain. The safety and potency of passage 115 virus (designated Sudan CMLV/115) was tested. Camels inoculated with CMLV/115 showed no clinical disease or skin lesions, developed low-level antibodies and cell-mediated immune response and resisted challenge with virulent wild-type CMLV. Field testing of the candidate vaccine showed that the developed vaccine induces immune response and is safe for young and pregnant camels.

Orthopoxvirus species like cowpox, vaccinia and monkeypox virus cause zoonotic infections in humans worldwide. Infections often occur in rural areas lacking proper diagnostic infrastructure as exemplified by monkeypox, which is endemic in Western and Central Africa. While PCR detection requires demanding equipment and is restricted to genome detection, the evidence of virus particles can complement or replace PCR. Therefore, an easily distributable and manageable antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection of orthopoxviruses was developed to facilitate particle detection. By comparing the virus particle binding properties of polyclonal antibodies developed against surface-exposed attachment or fusion proteins, the surface protein A27 was found to be a well-bound, highly immunogenic and exposed target for antibodies aiming at virus particle detection. Subsequently, eight monoclonal anti-A27 antibodies were generated and characterized by peptide epitope...

Alastrim variola minor virus, which causes mild smallpox, was first recognized in Florida and South America in the late 19th century. Genome linear double-stranded DNA sequences (186,986 bp) of the alastrim virus Garcia-1966, a laboratory reference strain from an outbreak associated with 0.8% case fatalities in Brazil in 1966, were determined except for a 530-bp fragment of hairpin-loop sequences at

A microarray method was developed for simultaneous detection and identification of six species of Orthopoxvirus (OPV) including Variola, Monkeypox, Cowpox, Camelpox, Vaccinia, and Ectromelia viruses. The method allowed us to discriminate OPV species from varicella-zoster virus (VZV), Herpes Simplex 1 virus (HSV-1), and Herpes Simplex 2 virus (HSV-2) that cause infections with clinical manifestations similar to OPV infections. The nucleotide sequences of the C23L/B29R and the B19R genes identified for 86 and 72 different OPV strains, respectively, were used to design species-specific microarray oligonucleotide probes (oligoprobes). The microarray also contained several oligoprobes selected from the ORF31, US4, and US5 genes of VZV, HSV-1, and HSV-2, respectively. The samples (from HSVs or OPVs) of ssDNAs for analyses were prepared by using asymmetric PCR followed by chemical labeling of ssDNA with Cy3 dye. DNA from 52 samples of various OPV species, two isolates of VZV, two of HSV-1, and three of HSV-2 were tested using the developed microarray assay; all tested viruses were accurately identified. To ensure the robustness of the microarray assay, three additional unrelated variola virus strains with unknown sequences of the C23L/B29R and the B19R genes were tested. In each instance the microarray unambiguously identified them as Variola virus species. The results obtained in this study demonstrated that this new microarray method is a valuable tool for the rapid and accurate detection and differentiation of these important viral pathogens.

A method for species-specific detection of orthopoxviruses pathogenic for humans and animals is described. The method is based on hybridization of a fluorescently labeled amplified DNA specimen with the oligonucleotide DNA probes immobilized on a microchip (MAGIChip). The probes identify species-specific sites within the crmB gene encoding the viral analogue of tumor necrosis factor receptor, one of the most important determinants of pathogenicity in this genus of viruses. The diagnostic procedure takes 6 h and does not require any sophisticated equipment (a portable fluorescence reader can be used).

Monkeypox virus is a zoonotic orthopoxvirus (OPX) of west and central sub-Saharan Africa. We conducted a cross-sectional serosurvey in Likouala region, Republic of Congo to assess exposure to OPX. Whole blood was collected using Nobuto blood filter strips (NBFS). Titers of IgM and IgG to OPX were assessed using an enzyme-linked immunosorbent assay. Demographic and clinical characteristics were compared with serostatus using the chi-square test or Fisher's exact test. Multivariate logistic regression was performed to evaluate factors for independent association with serostatus. A total of 994 specimens were analyzed; the overall seroprevalence for OPX IgM was 1.7%. Age < 25 years reduced the likelihood of OPX exposure, and persons living in Ngangania village had independently higher odds (odds ratio = 33.5, 95% confidence interval = 7.2-166). Blood collection for serosurveys using NBFS is feasible and practical. Adult activities such as hunting and carcass preparation may play...

Although smallpox was eradicated over 30 years ago, the disease remains a major threat. High mortality, high infectivity and low resistance of the contemporary population make the smallpox virus very attractive to terrorists. The possible presence of illegal stocks of the virus or risk of deliberate genetic modifications cause serious concerns among experts. Hence, it is reasonable to seek effective drugs that could be used in case of smallpox outbreak. This paper reviews studies on compounds with proven in vitro or in vivo antipoxviruses potential, which show various mechanisms of action. Nucleoside analogues, such as cidofovir, can inhibit virus replication. Cidofovir derivatives are developed to improve the bioavailability of the drug. Among the nucleoside analogues under current investigation are: ANO (adenozine N1-oxide) and its derivatives, N-methanocarbothymidine [(N)-MCT], or derivatitives of aciklovir, peninclovir and brivudin. Recently, ST-246 - which effectively inhibits ...

A screening assay for real-time LightCycler (Roche Applied Science, Mannheim, Germany) PCR identification of smallpox virus DNA was developed and compiled in a kit system under good manufacturing practice conditions with standardized reagents. In search of a sequence region unique to smallpox virus, the nucleotide sequence of the 14-kDa fusion protein gene of each of 14 variola virus isolates of the Russian World Health Organization smallpox virus repository was determined and compared to published sequences. PCR primers were designed to detect all Eurasian-African species of the genus Orthopoxvirus . A single nucleotide mismatch resulting in a unique amino acid substitution in smallpox virus was used to design a hybridization probe pair with a specific sensor probe that allows reliable differentiation of smallpox virus from other orthopoxviruses by melting-curve analysis. The applicability was demonstrated by successful amplification of 120 strains belonging to the orthopoxvirus sp...