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

Background: Poxvirus-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. Results: Five 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. Conclusion: Our 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

Genomic Sequencing and Analysis of a Novel Human Cowpox Virus With Mosaic Sequences From North America and Old World Orthopoxvirus

Orthopoxviruses (OPXVs) not only infect their natural hosts, but some OPXVs can also cause disease in humans. Previously, we partially characterized an OPXV isolated from an 18-year-old male living in Northern Norway. Restriction enzyme analysis and partial genome sequencing characterized this virus as an atypical cowpox virus (CPXV), which we named CPXV-No-H2. In this study, we determined the complete genome sequence of CPXV-No-H2 using Illumina and Nanopore sequencing. Our results showed that the whole CPXV-No-H2 genome is 220,276 base pairs (bp) in length, with inverted terminal repeat regions of approximately 7 kbp, containing 217 predicted genes. Seventeen predicted CPXV-No-H2 proteins were most similar to OPXV proteins from the Old World, including Ectromelia virus (ECTV) and Vaccinia virus, and North America, Alaskapox virus (AKPV). CPXV-No-H2 has a mosaic genome with genes most similar to other OPXV genes, and seven potential recombination events were identified. The phylogenetic analysis showed that CPXV-No-H2 formed a separate clade with the German CPXV isolates CPXV_GerMygEK938_17 and CPXV_Ger2010_MKY, sharing 96.4 and 96.3% nucleotide identity, respectively, and this clade clustered closely with the ECTV-OPXV Abatino clade. CPXV-No-H2 is a mosaic virus that may have arisen out of several recombination events between OPXVs, and its phylogenetic clustering suggests that ECTV-Abatino-like cowpox viruses form a distinct, new clade of cowpox viruses

The epidemiological trend of monkeypox and monkeypox-varicella zoster viruses co-infection in North-Eastern Nigeria

Background: Monkeypox (MPX) is endemic in Nigeria, but it was first reported in Adamawa state, North-Eastern Nigeria, in January 2022. There are currently 172 cases of MPX in Nigeria, with four reported deaths, and Adamawa has the second-highest case count. Therefore, this study was undertaken to evaluate the epidemiological profile of this viral disease. Methods: This is a cross-sectional study. The skin and blood samples were screened for the presence for Monkeypox virus (MPXV) and Varicella Zoster virus (VZV) DNA by real-time PCR; the clinical diagnosis was based on symptoms of visual signs of skin lesions and other clinical symptoms from January to July 2022. Results: A total of 33 suspected cases aged 1–57 years [26 (79%) males vs. 7 (21%) females] were screened for MPX and VZV. Twenty-four (72.7%) were positive (6.1% were MPX only, 39% were VZV only, and 27% were both MPX and VZV). Most cases of MPX (82%), VZV (69%) and MPX-VZV co-infection (78%) occurred in males. More than half (54%) of those infected were children and adolescents between 0 and 19 years. All patients experienced body rashes and itching, and other clinical symptoms included fever, headache, mouth sores, muscle aches and lymphadenopathy. Over 64 and 86% of patients had contact with livestock and rodents, respectively. Conclusion: MPXV, VZV and MPXV-VZV co-infections occurred predominantly among males and children in Adamawa state, Nigeria. Given the patient contact with rodents and livestock, further research on the animal reservoir is needed to highlight the transmission of MPXV in Adamawa

Genomic Sequencing and Phylogenomics of Cowpox Virus

Cowpox virus (CPXV; genus Orthopoxvirus; family Poxviridae) is the causative agent of cowpox, a self-limiting zoonotic infection. CPXV is endemic in Eurasia, and human CPXV infections are associated with exposure to infected animals. In the Fennoscandian region, five CPXVs isolated from cats and humans were collected and used in this study. We report the complete sequence of their genomes, which ranged in size from 220–222 kbp, containing between 215 and 219 open reading frames. The phylogenetic analysis of 87 orthopoxvirus strains, including the Fennoscandian CPXV isolates, confirmed the division of CPXV strains into at least five distinct major clusters (CPXV-like 1, CPXV-like 2, VACV-like, VARV-like and ECTV-Abatino-like) and can be further divided into eighteen sub-species based on the genetic and patristic distances. Bayesian time-scaled evolutionary history of CPXV was reconstructed employing concatenated 62 non-recombinant conserved genes of 55 CPXV. The CPXV evolution rate was calculated to be 1.65 × 10−5 substitution/site/year. Our findings confirmed that CPXV is not a single species but a polyphyletic assemblage of several species and thus, a reclassification is warranted

Monkeypox Virus in Nigeria: Infection Biology, Epidemiology, and Evolution

Monkeypox is a zoonotic disease caused by monkeypox virus (MPXV), which is a member of orthopoxvirus genus. The reemergence of MPXV in 2017 (at Bayelsa state) after 39 years of no reported case in Nigeria, and the export of travelers’ monkeypox (MPX) from Nigeria to other parts of the world, in 2018 and 2019, respectively, have raised concern that MPXV may have emerged to occupy the ecological and immunological niche vacated by smallpox virus. This review X-rays the current state of knowledge pertaining the infection biology, epidemiology, and evolution of MPXV in Nigeria and worldwide, especially with regard to the human, cellular, and viral factors that modulate the virus transmission dynamics, infection, and its maintenance in nature. This paper also elucidates the role of recombination, gene loss and gene gain in MPXV evolution, chronicles the role of signaling in MPXV infection, and reviews the current therapeutic options available for the treatment and prevention of MPX. Additionally, genome-wide phylogenetic analysis was undertaken, and we show that MPXV isolates from recent 2017 outbreak in Nigeria were monophyletic with the isolate exported to Israel from Nigeria but do not share the most recent common ancestor with isolates obtained from earlier outbreaks, in 1971 and 1978, respectively. Finally, the review highlighted gaps in knowledge particularly the non-identification of a definitive reservoir host animal for MPXV and proposed future research endeavors to address the unresolved questions

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

Abstract Background Poxvirus-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. Results Five 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. Conclusion Our 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.</p

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

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

Background: Poxvirus-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. Results: Five 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. Conclusion: Our 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