Malachy Ifeanyi Okeke

Comparative sequence analysis of A-type inclusion (ATI) and P4c proteins of orthopoxviruses that produce typical and atypical ATI phenotypes.

Some orthopoxviruses produce large proteinaceous intracellular bodies, known as A-type inclusions (ATIs) during infection of host cells. Virions associate with ATIs resulting in distinct phenotypes referred to as V+, V+/ and V−. The phenotype V+ has the virions embedded in the ATI matrix; V− has no virions embedded within or on the surface of the ATI matrix, whereas an aberrant phenotype, the V+/ has virions only on the surface of ATIs. Viruses that do not produce ATI are designated as V0. Recombinant viruses generated from a V+ cowpox virus (CPXV) and a V0 transgenic vaccinia virus (VACV) produced aberrant V+/ ATIs. ATI phenotype is dependent on the A-type inclusion protein (Atip) and the P4c protein. We sequenced the atip and p4c genes of parental and progeny recombinant viruses as well as their flanking sequences. The atip and p4c open reading frames were identical in parental V+ CPXV and hybrid V+/ progenies. Our results suggest that additional viral gene(s) are required for the formation of wild type V+ ATI.

In vitro host range, multiplication and virion forms of recombinant viruses obtained from co-infection in vitro with a vaccinia-vectored influenza vaccine and a naturally occurring cowpox virus isolate

BackgroundPoxvirus-vectored vaccines against infectious diseases and cancer are currently under development. We hypothesized that the extensive use of poxvirus-vectored vaccine in future might result in co-infection and recombination between the vaccine virus and naturally occurring poxviruses, resulting in hybrid viruses with unpredictable characteristics. Previously, we confirmed that co-infecting in vitro a Modified vaccinia virus Ankara (MVA) strain engineered to express influenza virus haemagglutinin (HA) and nucleoprotein (NP) genes with a naturally occurring cowpox virus (CPXV-NOH1) resulted in recombinant progeny viruses (H Hansen, MI Okeke, Ø Nilssen, T Traavik, Vaccine 23: 499–506, 2004). In this study we analyzed the biological properties of parental and progeny hybrid viruses.ResultsFive CPXV/MVA progeny viruses were isolated based on plaque phenotype and the expression of influenza virus HA protein. Progeny hybrid viruses displayed in vitro cell line tropism of CPXV-NOH1, but not that of MVA. The HA transgene or its expression was lost on serial passage of transgenic viruses and the speed at which HA expression was lost varied with cell lines. The HA transgene in the progeny viruses or its expression was stable in African Green Monkey derived Vero cells but became unstable in rat derived IEC-6 cells. Hybrid viruses lacking the HA transgene have higher levels of virus multiplication in mammalian cell lines and produced more enveloped virions than the transgene positive progenitor virus strain. Analysis of the subcellular localization of the transgenic HA protein showed that neither virus strain nor cell line have effect on the subcellular targets of the HA protein. The influenza virus HA protein was targeted to enveloped virions, plasma membrane, Golgi apparatus and cytoplasmic vesicles.ConclusionOur results suggest that homologous recombination between poxvirus-vectored vaccine and naturally circulating poxviruses, genetic instability of the transgene, accumulation of non-transgene expressing vectors or hybrid virus progenies, as well as cell line/type specific selection against the transgene are potential complications that may result if poxvirus vectored vaccines are extensively used in animals and man.

A naturally occurring cowpox virus with an ectromelia virus A-type inclusion protein gene displays atypical A-type inclusions

Human orthopoxvirus (OPV) infections in Europe are usually caused by cowpox virus (CPXV). The genetic heterogeneity of CPXVs may in part be due to recombination with other OPV species. We describe the characterization of an atypical CPXV (CPXV-No-H2) isolated from a human patient in Norway. CPXV-No-H2 was characterized on the basis of A-type inclusion (ATI) phenotype as well as the DNA region containing the p4c and atip open reading frames. CPXV-No-H2 produced atypical V(+/) ATI, in which virions are on the surface of ATI but not within the ATI matrix. Phylogenetic analysis showed that the atip gene of CPXV-No-H2 clustered closely with that of ectromelia virus (ECTV) with a bootstrap support of 100% whereas its p4c gene is diverged compared to homologues in other OPV species. By recombination analysis we identified a putative crossover event at nucleotide 147, downstream the start of the atip gene. Our results suggest that CPXV-No-H2 originated from a recombination between CPXV and ECTV. Our findings are relevant to the evolution of OPVs.

Comparison and phylogenetic analysis of cowpox viruses isolated from cats and humans in Fennoscandia

Cowpox virus (CPXV), a member of the genus Orthopoxvirus (OPV), has reservoirs in small mammals and may cause disease in humans, felidae and other animals. In this study we compared CPXVs isolated from humans and cats in Fennoscandia by restriction enzyme and DNA sequence analysis. The HindIII restriction profiles clearly distinguished geographically distinct CPXV isolates, whereas only minor differences were found between the profiles of geographically linked isolates. The complete gene sequences encoding the cytokine response modifier B, the hemagglutinin and the Chinese hamster ovary host range protein were determined for the same isolates and included in phylogenetic analysis. By including representative OPV sequences from GenBank, detailed comparative analyses were performed showing pronounced heterogeneity among CPXVs compared to members of other OPV species. However, a close relationship between the Norwegian (3 of 4 isolates) and Swedish isolates was detected, whereas the isolate from Finland was more closely related to a Russian isolate for all three genes compared. We infer that the investigated CPXVs have distinct evolutionary histories in different rodent lineages.

Molecular characterization and phylogenetics of Fennoscandian cowpox virus isolates based on the p4c and atip genes.

BackgroundCowpox virus (CPXV), a rodent-borne Orthopoxvirus (OPV) that is indigenous to Eurasia can infect humans, cattle, felidae and other animals. Molecular characterization of CPXVs isolated from different geographic locations is important for the understanding of their biology, geographic distribution, classification and evolution. Our aim was to characterize CPXVs isolated from Fennoscandia on the basis of A-type inclusion (ATI) phenotype, restriction fragment length polymorphism (RFLP) profiles of atip gene fragment amplicon, and phylogenetic tree topology in conjunction with the patristic and genetic distances based on full length DNA sequence of the atip and p4c genes.MethodsATI phenotypes were determined by transmission electron microcopy and RFLP profiles were obtained by restriction enzyme digestion of the atip gene fragment PCR product. A 6.2 kbp region spanning the entire atip and p4c genes of Fennoscandian CPXV isolates was amplified and sequenced. The phylogenetic affinity of Fennoscandian CPXV isolates to OPVs isolated from other geographic regions was determined on the basis of the atip and p4c genes.ResultsFennoscandian CPXV isolates encoded full length atip and p4c genes. They produce wild type V+ ATI except for CPXV-No-H2. CPXVs were resolved into six and seven species clusters based on the phylogeny of the atip and p4c genes respectively. The CPXVs isolated from Fennoscandia were grouped into three distinct clusters that corresponded to isolates from Norway, Sweden and Finland.ConclusionCPXV is a polyphyletic assemblage of six or seven distinct clusters and the current classification in which CPXVs are united as one single species should be re-considered. Our results are of significance to the classification and evolution of OPVs.

Orthopoxvirus DNA in Eurasian Lynx, Sweden

Cowpox virus, which has been used to protect humans against smallpox but may cause severe disease in immunocompromised persons, has reemerged in humans, domestic cats, and other animal species in Europe. Orthopoxvirus (OPV) DNA was detected in tissues (lung, kidney, spleen) in 24 (9%) of 263 free-ranging Eurasian lynx (Lynx lynx) from Sweden. Thymidine kinase gene amplicon sequences (339 bp) from 21 lynx were all identical to those from cowpox virus isolated from a person in Norway and phylogenetically closer to monkeypox virus than to vaccinia virus and isolates from 2 persons with cowpox virus in Sweden. Prevalence was higher among animals from regions with dense, rather than rural, human populations. Lynx are probably exposed to OPV through predation on small mammal reservoir species. We conclude that OPV is widely distributed in Sweden and may represent a threat to humans. Further studies are needed to verify whether this lynx OPV is cowpox virus.

CRISPR/Cas9—Advancing Orthopoxvirus Genome Editing for Vaccine and Vector Development

The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (Cas9) technology is revolutionizing genome editing approaches. Its high efficiency, specificity, versatility, flexibility, simplicity and low cost have made the CRISPR/Cas9 system preferable to other guided site-specific nuclease-based systems such as TALENs (Transcription Activator-like Effector Nucleases) and ZFNs (Zinc Finger Nucleases) in genome editing of viruses. CRISPR/Cas9 is presently being applied in constructing viral mutants, preventing virus infections, eradicating proviral DNA, and inhibiting viral replication in infected cells. The successful adaptation of CRISPR/Cas9 to editing the genome of Vaccinia virus paves the way for its application in editing other vaccine/vector-relevant orthopoxvirus (OPXV) strains. Thus, CRISPR/Cas9 can be used to resolve some of the major hindrances to the development of OPXV-based recombinant vaccines and vectors, including sub-optimal immunogenicity; transgene and genome instability; reversion of attenuation; potential of spread of transgenes to wildtype strains and close contacts, which are important biosafety and risk assessment considerations. In this article, we review the published literature on the application of CRISPR/Cas9 in virus genome editing and discuss the potentials of CRISPR/Cas9 in advancing OPXV-based recombinant vaccines and vectors. We also discuss the application of CRISPR/Cas9 in combating viruses of clinical relevance, the limitations of CRISPR/Cas9 and the current strategies to overcome them.

Hazard Characterization of Modified Vaccinia Virus Ankara Vector: What Are the Knowledge Gaps?

Modified vaccinia virus Ankara (MVA) is the vector of choice for human and veterinary applications due to its strong safety profile and immunogenicity in vivo. The use of MVA and MVA-vectored vaccines against human and animal diseases must comply with regulatory requirements as they pertain to environmental risk assessment, particularly the characterization of potential adverse effects to humans, animals and the environment. MVA and recombinant MVA are widely believed to pose low or negligible risk to ecosystem health. However, key aspects of MVA biology require further research in order to provide data needed to evaluate the potential risks that may occur due to the use of MVA and MVA-vectored vaccines. The purpose of this paper is to identify knowledge gaps in the biology of MVA and recombinant MVA that are of relevance to its hazard characterization and discuss ongoing and future experiments aimed at providing data necessary to fill in the knowledge gaps. In addition, we presented arguments for the inclusion of uncertainty analysis and experimental investigation of verifiable worst-case scenarios in the environmental risk assessment of MVA and recombinant MVA. These will contribute to improved risk assessment of MVA and recombinant MVA vaccines.

Surface Engineering of the RNA Coliphage Q? to Display Plasmodium Falciparum Derived Asexual Blood Stage Antigens UB05 and Merozoite Surface Protein 3

Background: Naturally acquired immune responses to Plasmodium falciparum merozoite surface protein 3 (MSP3) and UB05 are implicated in semi immunity in populations living in malaria endemic areas. Thus designing chimeric malaria vaccine candidates involving MSP-3 and UB05 displayed upon the surface of a phage in its native form could potentiate their immunogenicity and antigenicity. In this study, we have engineered both MSP3 and UB05 upon the Qβ and assessed their antigenicity with plasma from children living in a high malaria transmission region of Cameroon. Methods: The surface of the RNA coliphage Qβ was genetically modified to display three Plasmodium falciparum derived immunogens including MSP3, UB05 and a chimera of the two UB05-MSP3. The resultant recombinant phages including QβMSP3, QβUB05 and QβUB05-MSP3 with surface displayed malaria immunogens were produced after transformation of the E. coli strain HB101. Plasma levels of antigen specific IgG antibody were then determined in samples from malaria positive and negative children living in a high malaria transmission region of Cameroon. Results: To improve yield each recombinant phage was scaled up to 1014 pfu/ml using production strategies previously optimized in our group. This was significantly higher (P<0.001) relative to the 108 pfu/ml of the wild type C l i n i c al M icr obio logy: pen cc e s s ISSN: 2327-5073 Clinical Microbiology: Open Access Waffo et al., Clin Microbiol 2018, 7:4 DOI: 10.4172/2327-5073.1000314 Research Article Open Access Clin Microbiol, an open access journal ISSN:2327-5073 Volume 7 • Issue 4 • 1000314 phage when produced routinely. Conformational integrity of the surface displayed antigens was confirmed in ELISA assays by testing for the specific recognition of the N and C-terminal parts of both UB05 and MSP3 using the recombinant QβMSP3, QβUB05 and chimeric QβUB05-MSP3 phages as antigens and the monoclonal antibodies XQ38G73-N, X-Q0KGH2-N, X-Q38G73-C, X-Q0KGH2-C targeting the Nand C-terminals of both UB05 and MSP3 respectively. Antigen specific naturally acquired IgG antibodies in plasma from both malaria negative and positive children living in a high transmission area of Cameroon recognized all three recombinant phages. However, plasma from children less than five years old contained significantly less plasma levels of antigen specific IgG antibodies. Conclusion: Thus, the novel recombinant phages QβMSP3, QβUB05 and QβUB05-MSP3 can be used as a tool for assessing natural or vaccine-induced antibody responses against malaria. The recombinant chimeric QβUB05MSP3 phage is validated as a multivalent antigen for tracking semi immunity to malaria.