Whole genome sequencing of recombinant viruses obtained from co-infection and superinfection of Vero cells with modified vaccinia virus ankara vectored influenza vaccine and a naturally occurring cowpox virus.

Modified vaccinia virus Ankara (MVA) has been widely tested in clinical trials as recombinant vector vaccine against infectious diseases and cancers in humans and animals. However, one biosafety concern about the use of MVA vectored vaccine is the potential for MVA to recombine with naturally occurring orthopoxviruses in cells and hosts in which it multiplies poorly and, therefore, producing viruses with mosaic genomes with altered genetic and phenotypic properties. We previously conducted co-infection and superinfection experiments with MVA vectored influenza vaccine (MVA-HANP) and a feline Cowpox virus (CPXV-No-F1) in Vero cells (that were semi-permissive to MVA infection) and showed that recombination occurred in both co-infected and superinfected cells. In this study, we selected the putative recombinant viruses and performed genomic characterization of these viruses. Some putative recombinant viruses displayed plaque morphology distinct of that of the parental viruses. Our analysis demonstrated that they had mosaic genomes of different lengths. The recombinant viruses, with a genome more similar to MVA-HANP (>50%), rescued deleted and/or fragmented genes in MVA and gained new host ranges genes. Our analysis also revealed that some MVA-HANP contained a partially deleted transgene expression cassette and one recombinant virus contained part of the transgene expression cassette similar to that incomplete MVA-HANP. The recombination in co-infected and superinfected Vero cells resulted in recombinant viruses with unpredictable biological and genetic properties as well as recovery of delete/fragmented genes in MVA and transfer of the transgene into replication competent CPXV. These results are relevant to hazard characterization and risk assessment of MVA vectored biologicals.

A comprehensive review of monkeypox virus and mpox characteristics.

Monkeypox virus (MPXV) is the etiological agent of monkeypox (mpox), a zoonotic disease. MPXV is endemic in the forested regions of West and Central Africa, but the virus has recently spread globally, causing outbreaks in multiple non-endemic countries. In this paper, we review the characteristics of the virus, including its ecology, genomics, infection biology, and evolution. We estimate by phylogenomic molecular clock that the B.1 lineage responsible for the 2022 mpox outbreaks has been in circulation since 2016. We interrogate the host-virus interactions that modulate the virus infection biology, signal transduction, pathogenesis, and host immune responses. We highlight the changing pathophysiology and epidemiology of MPXV and summarize recent advances in the prevention and treatment of mpox. In addition, this review identifies knowledge gaps with respect to the virus and the disease, suggests future research directions to address the knowledge gaps, and proposes a One Health approach as an effective strategy to prevent current and future epidemics of mpox.

Survey of attitude to human genome modification in Nigeria.

Gene editing and mitochondrial replacement therapy (MRT) are biotechnologies used to modify the host nuclear and mitochondrial DNA, respectively. Gene editing is the modification of a region of the host genome using site-specific nucleases, in particular the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. Heritable and somatic genome editing (HGE and SGE) are used in gene therapy. MRT is a technique used to substitute the defective mitochondria in the recipient embryo with a female donor healthy mitochondrion in order to prevent the inheritance of mothers' defective mitochondria resulting in the change of mitochondria of the entire generation to come. To evaluate the perception of the Nigerian citizens on human genome modification, two survey forms were created and distributed in-person and majorly online. There was a total of 268 responses, 188 from the public and 80 from health workers and bio-scientists. The results showed poor knowledge about gene editing and MRT by the Nigerian public, but its use to prevent and cure inherited diseases was supported. Morality and religion have great influence on the attitude of Nigerians towards genome modification, but the influence of religion and morality is not unequivocal. Multiple regression analysis of Nigerian public responses shows that gender (females), age (19-30 years), monthly income (NGN 0 to 30,000), and level of education (tertiary) are significantly associated with approval of human genome editing, but the survey of health workers and bio-scientists shows no significant association except for females who approve and Muslims who disapprove of human genome editing.

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

[This corrects the article DOI: 10.3389/fpubh.2022.1066589.].

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.

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.

A critical evaluation of Nigeria's response to the first wave of COVID-19.

BACKGROUND: The first wave of the Coronavirus Disease 2019 (COVID-19) pandemic began when the first index case was reported in Nigeria on the 27th of February 2020, and since then, more than 68,000 cases of the disease were confirmed, with 1173 deaths as of November 30, 2020. MAIN BODY: Daily situation reports from the Nigeria Centre for Disease Control spanning February 27-November 30, 2020, were fully considered in this review. Further literature search was performed using PubMed and Google Scholar databases for articles related to response measures adopted by Nigeria. The instantaneous reproduction number (R) was then estimated as a metric to investigate the non-pharmaceutical intervention measures. Nigeria responded to COVID-19 pandemic by implementing anti-COVID-19 mitigation strategies in travel restrictions, social distancing, source control, contact tracing, self-isolation, and quarantine, as well as in clinical interventions. Our epidemiological model estimated the R-value of more than 1.0 in Nigeria and in each of all the 36 states and the Federal Capital Territory. CONCLUSION: Nigeria implemented containment and mitigation measures in response to the first wave of COVID-19 and these measures may have contributed to the mild COVID-19 outcome in Nigeria compared to the global trend. However, inadequate PCR testing capacity, lack or suboptimal utilization of epidemic metrics like the virus reproduction number (R) to inform decision making, and premature easing of lockdown measures among others were major challenges to the effective implementation of the COVID-19 response measures.

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.

Phylogenetic and Mutational Analysis of Lassa Virus Strains Isolated in Nigeria: Proposal for an In Silico Study.

BACKGROUND: In 2018, the total number of Lassa fever cases in Nigeria was significantly higher than that observed in previous years. Hence, studies had attempted to determine the underlying cause. However, reports using phylogenetic methods to analyze this finding ruled out the emergence of potentially more transmissible Lassa virus strains or an increase in human-to-human viral transmission as the cause underlying the increase in cases. Two years later, the situation seems even worse as the number of confirmed cases has reached an all-time high according to situational reports released by the Nigerian Center for Disease Control. OBJECTIVE: Considering the increasing trend of Lassa fever cases and related mortality, the major objective of this study is to map mutations within the genomes of Lassa virus isolates from 2018 and 2019 using the reference sequence available at the National Center for Biotechnology Information as a benchmark and compare them to the genomes of viruses isolated during 1969-2017. This study would also attempt to identify a viral marker gene for easier identification and grouping. Finally, the time-scaled evolution of Lassa virus in Nigeria will be reconstructed. METHODS: After collecting the sequence data of Lassa virus isolates, Bayesian phylogenetic trees, a sequence identity matrix, and a single nucleotide polymorphism matrix will be generated using BEAST (version 2.6.2), Base-By-Base, and DIVEIN (a web-based tool for variant calling), respectively. RESULTS: Mining and alignment of Lassa virus genome sequences have been completed, while mutational analysis and the reconstruction of time-scaled maximum clade credibility trees, congruence tests for inferred segments, and gene phylogeny analysis are ongoing. CONCLUSIONS: The findings of this study would further the current knowledge of the evolutionary history of the Lassa virus in Nigeria and would document the mutations in Nigerian isolates from 1969 to 2019. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/23015.

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.

Antibacterial activity of Citrus limonum fruit juice extract.

The fruit juice extract of Citrus limonum was investigated for antibacterial activity. The antibacterial activity of the extract on ten strains of bacteria was determined by both agar well diffusion and macro-broth dilution methods. The extract was variously bacteriostatic and bactericidal against Bacillussubtilis ATCC 6051, Staphylococcus aureus ATCC 12600, Escherichia coli ATCC 11775, Pseudomonas aeruginosa ATCC 10145 as well as locally isolated clinical strains of the above bacteria and Salmonella kintambo (Human: 13, 23: mt:-), Salmonella typhi and Proteus sp. The MICs ranged from 0.78 mg/ml to 50mg/ml; MBCs, 25.0mg/ml to >100mg/ml and MBC/MIC ratios 2.0 to >16.0. These results provide scientific justification for the medicinal use of Citrus limonum fruit juice by Nigerian herbalists in the treatment of diseases in which strains of the test organisms have been implicated as etiologic agents.

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

BACKGROUND: Cowpox 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. METHODS: ATI 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. RESULTS: Fennoscandian 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. CONCLUSION: CPXV 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.

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.

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.

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.

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 V°. Recombinant viruses generated from a V+ cowpox virus (CPXV) and a V° 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.

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.

Modified vaccinia virus Ankara multiplies in rat IEC-6 cells and limited production of mature virions occurs in other mammalian cell lines.

Recombinant viruses based on modified vaccinia virus Ankara (MVA) are vaccine candidates against infectious diseases and cancers. Presently, multiplication of MVA has been demonstrated in chicken embryo fibroblast and baby hamster kidney (BHK-21) cells only. The multiplication and morphogenesis of a recombinant (MVA-HANP) and non-recombinant MVA strain in BHK-21 and 12 other mammalian cell lines have now been compared. Rat IEC-6 cells were fully permissive to MVA infection. The virus yield in IEC-6 cells was similar to that obtained in BHK-21 cells at low as well as high multiplicities of infection. Vero cells were semi-permissive to MVA infection. Mature virions were produced in supposedly non-permissive cell lines. The multiplication and morphogenesis of non-recombinant MVA and MVA-HANP were similar. These results are relevant to the production and biosafety of MVA-vectored vaccines.

Recombinant viruses obtained from co-infection in vitro with a live vaccinia-vectored influenza vaccine and a naturally occurring cowpox virus display different plaque phenotypes and loss of the transgene.

Some poxviruses are very attractive as transgenic vaccine vectors for humans, domestic animals and wildlife. Poxviridae family members circulate in different ecosystems and parts of the world, providing a pool of possible recombination partners for released or escaped genetically modified poxviruses. We performed in vitro double infections with a vaccinia virus strain Ankara (MVA) vectored influenza vaccine and a cowpox virus isolate from Norway, isolated hybrids, and further analyzed three hybrid viruses with different plaque phenotypes. One of the hybrids was genetically unstable, and during adaptation to new host cells its MVA derived influenza gene was deleted at a high frequency. This is significant in a risk assessment context, since the transgene would be the only logical tag for monitoring unwanted spread and non-target effects of a vaccine virus. Putative recombination events involving genetically modified and naturally occurring viruses should be included in health and environmental risk assessments.