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

Okeke, Malachy Ifeanyi; Okoli, Arinze S.; Nilssen, Øivind; Moens, Ugo; Tryland, Morten; Bøhn, Thomas; Traavik, Terje

doi:10.1186/1743-422x-11-119

Cited by 12 publications

(19 citation statements)

References 51 publications

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“…1 ) showed the best amplification of fish DNA in reactions using this amount (see below). This is also consistent with other studies using 2 ng of DNA per 20–25 µl PCR reaction, although amplification can be observed at much lower concentrations ( Andrade et al, 2012 ; Jin et al, 2012 ; Bernal-Martínez et al, 2013 ; Batmalle et al, 2014 ; Okeke et al, 2014 ).…”

Section: Methodssupporting

confidence: 92%

A localized PCR inhibitor in a porcelain crab suggests a protective role

Maklizi

Ouf

Ferreira

et al. 2014

PeerJ

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A number of polymerase chain reaction (PCR) inhibitors have been identified from biological and environmental samples. By and large, such substances are treated as random nuisances and contaminants with alternate functions; their inhibitory effects on DNA replication being a coincidental property of their molecular structure. Here, we demonstrate the presence of a localized PCR inhibitor in the foregut of the porcelain crab Petrolisthes rufescens (Anomura: Porcellanidae) from the Red Sea. The inhibitor precluded amplification of 28s, 16s and 18s gene sequences effectively but lost activity at 10−2 dilutions from initial concentration. Heat treatment was ineffective in arresting inhibition and spectrophotometric techniques suggested that the inhibitor was not a melanin-type compound. The compound was not detected from midgut, hindgut, or gills of the crab. Activity of the inhibitor was precluded when samples were treated with suspensions from the midgut, suggesting that enzymatic degradation of the inhibitor likely happens at that part of the gut. As many microbial pathogens invade their hosts via ingestion, we suggest the presence of the localized inhibitor could carry a defensive or immunological role for P. rufescens. The identity of the inhibitory molecule remains unknown.

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Section: Methodssupporting

confidence: 92%

A localized PCR inhibitor in a porcelain crab suggests a protective role

Maklizi

Ouf

Ferreira

et al. 2014

PeerJ

View full text Add to dashboard Cite

show abstract

“…1) showed the best amplification of fish DNA in reactions using this amount (see below). This is also consistent with other studies using 2 ng of DNA per 20-25 µl PCR reaction, although amplification can be observed at much lower concentrations (Andrade et al 2012;Jin et al, 2012;Bernal-Martínez et al, 2013;Batmalle et al, 2014;Okeke et al, 2014) To assess the presence and distribution of the inhibitor various PCR-based experiments were designed. All PCR reactions were developed on 1.0-1.7% agarose gels stained with ethidium bromide and the intensity of the bands obtained was observed under UV light in an ImageQuant 300 (General Electric) gel imaging system.…”

Section: Reviewing Manuscriptsupporting

confidence: 86%

Peer Review #2 of "A localized PCR inhibitor in a porcelain crab suggests a protective role (v0.1)"

Eichstaedt¹

2014

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A number of polymerase chain reaction (PCR) inhibitors have been identified from biological and environmental samples. By and large, such substances are treated as random nuisances and contaminants with alternate functions; their inhibitory effects on DNA replication being a coincidental property of their molecular structure. Here, we demonstrate the presence of a localized PCR inhibitor in the foregut of the porcelain crab Petrolisthes rufescens (Anomura: Porcellanidae) from the Red Sea. The inhibitor precluded amplification of 28s, 16s and 18s gene sequences effectively but lost activity at 10-2 dilutions from initial concentration. Heat treatment was ineffective in arresting inhibition and spectrophotometric techniques suggested that the inhibitor was not a melanin-type compound. The compound was not detected from midgut, hindgut, or gills of the crab. Activity of the inhibitor was precluded when samples were treated with suspensions from the midgut, suggesting that enzymatic degradation of the inhibitor likely happens at that part of the gut. As many microbial pathogens invade their hosts via ingestion, we suggest the presence of the localized inhibitor could carry a defensive or immunological role for P. rufescens. The identity of the inhibitory molecule remains unknown.

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“…Recently, the first poxvirus isolate, CPXV-No-H2, was described that produced V ϩ/ ATI bodies as a stable phenotype (48). Nevertheless, all other CPXVs isolated from Fennoscandia represent the V ϩ ATI phenotype and, hence, encode full-length ati and p4c genes (49). Since RatPox09, unlike FM2292, was isolated from rats and not a reservoir host, it is tempting to speculate that it may have lost the ability to form V ϩ ATIs.…”

Section: Discussionmentioning

confidence: 99%

Out of the Reservoir: Phenotypic and Genotypic Characterization of a Novel Cowpox Virus Isolated from a Common Vole

Hoffmann

Franke

Jenckel

et al. 2015

J Virol

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The incidence of human cowpox virus (CPXV) infections has increased significantly in recent years. Serological surveys have suggested wild rodents as the main CPXV reservoir. We characterized a CPXV isolated during a large-scale screening from a feral common vole. A comparison of the full-length DNA sequence of this CPXV strain with a highly virulent pet rat CPXV isolate showed a sequence identity of 96%, including a large additional open reading frame (ORF) of about 6,000 nucleotides which is absent in the reference CPXV strain Brighton Red. Electron microscopy analysis demonstrated that the vole isolate, in contrast to the rat strain, forms A-type inclusion (ATI) bodies with incorporated virions, consistent with the presence of complete ati and p4c genes. Experimental infections showed that the vole CPXV strain caused only mild clinical symptoms in its natural host, while all rats developed severe respiratory symptoms followed by a systemic rash. In contrast, common voles infected with a high dose of the rat CPXV showed severe signs of respiratory disease but no skin lesions, whereas infection with a low dose led to virus excretion with only mild clinical signs. We concluded that the common vole is susceptible to infection with different CPXV strains. The spectrum ranges from well-adapted viruses causing limited clinical symptoms to highly virulent strains causing severe respiratory symptoms. In addition, the low pathogenicity of the vole isolate in its eponymous host suggests a role of common voles as a major CPXV reservoir, and future research will focus on the correlation between viral genotype and phenotype/ pathotype in accidental and reservoir species. IMPORTANCEWe report on the first detection and isolation of CPXV from a putative reservoir host, which enables comparative analyses to understand the infection cycle of these zoonotic orthopox viruses and the relevant genes involved. In vitro studies, including whole-genome sequencing as well as in vivo experiments using the Wistar rat model and the vole reservoir host allowed us to establish links between genomic sequences and the in vivo properties (virulence) of the novel vole isolate in comparison to those of a recent zoonotic CPXV isolated from pet rats in 2009. Furthermore, the role of genes present only in a reservoir isolate can now be further analyzed. These studies therefore allow unique insights and conclusions about the role of the rodent reservoir in CPXV epidemiology and transmission and about the zoonotic threat that these viruses represent. C owpox virus (CPXV), a member of the genus Orthopoxvirus (OPV) in the Poxviridae family, is suspected to be widespread in Western Eurasian rodents, particularly vole species (1, 2). From the presumed reservoir hosts, spill-over infections to accidental hosts are regularly observed (3). The accidental hosts include domestic cats and also exotic animals in zoos, such as large felids and elephants, which regularly develop severe disease (3). As CPXV is a zoonotic virus, humans in direct contact wit...

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

Cited by 12 publications

References 51 publications

A localized PCR inhibitor in a porcelain crab suggests a protective role

A localized PCR inhibitor in a porcelain crab suggests a protective role

Peer Review #2 of "A localized PCR inhibitor in a porcelain crab suggests a protective role (v0.1)"

Out of the Reservoir: Phenotypic and Genotypic Characterization of a Novel Cowpox Virus Isolated from a Common Vole

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