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September 14 2022
Electron microscopy and a sequence-independent, single-primer amplification (SISPA) viromics approach for monkeypox virus genome determination
Nicola Fletcher 1,2*,†, Gabriel Gonzalez 3,4†, Luke Meredith 5†, Tiina O’Neill 2, Kevin Purves 1,2, Daniel Hare 3, Brian Keogan 3, Charlene Bennett 3, Joanne Moran 3, Sophie O’Reilly 6, Lili Gu 6, Matthew Angeliadis 6, Marion Pouget 6, Dimitri Scholz 2, Virginie Gautier 6, Patrick Mallon 6,7, Stephen Gordon 1,2, Jonathan Dean 3, Jeff Connell 3, Michael Carr 3,4, Cillian De Gascun 3*.
Monkeypox is a viral zoonosis endemic in West and Central Africa and presents clinically in humans as a smallpox-like illness (Bunge et al., 2022). Monkeypox virus (MPXV) is an enveloped virus with a particle size of ca. 300×240 nm containing a double-stranded DNA genome of ~197 kb taxonomically classified in the family Poxviridae, genus Orthopoxvirus (Weaver and Isaacs, 2008).
There is increasing public health concern about the geographical spread and re-emergence of MPXV arising from the recent multi-country outbreaks, with more than 47 206 cases reported in 92 non-endemic countries since the first case was laboratory confirmed in the United Kingdom on the 7th of May 2022 (updated August 28th 2022 (CDC, 2022)). The MPXV genome reported (GenBank ON872184) here was from a laboratory-confirmed Irish case (Orthopox group RNA and MPXV-specific RNA detection by real-time PCR) identified in June 2022. Transmission electron microscopy (TEM) of a lesion swab sample collected visualised multiple characteristic brick-shaped poxvirus virions with lattice-like surface detail (Fig. 1). We performed a sequence-independent, single-primer amplification (SISPA) viromics approach to achieve a non-selective (unbiased) amplification of viral RNA transcripts and here report horizontal genome coverage over 90% with an average vertical coverage (depth) between 9x and 21x. Phylogenetic analysis with 35 publicly available international MPXV genome sequences from multiple countries (UK, Israel, Nigeria, USA, Singapore and Portugal) sampled between 2017-2019 and 2022 (Happi et al.), demonstrated high sequence similarity to other cases circulating in the 2022 outbreak in Europe and USA clustered together in clade 3 (Happi et al.); with the Irish MPXV genome further clustered in the B.1 lineage (Fig. 2). ...
https://virological.org/t/electron-m...ermination/899
Electron microscopy and a sequence-independent, single-primer amplification (SISPA) viromics approach for monkeypox virus genome determination
Nicola Fletcher 1,2*,†, Gabriel Gonzalez 3,4†, Luke Meredith 5†, Tiina O’Neill 2, Kevin Purves 1,2, Daniel Hare 3, Brian Keogan 3, Charlene Bennett 3, Joanne Moran 3, Sophie O’Reilly 6, Lili Gu 6, Matthew Angeliadis 6, Marion Pouget 6, Dimitri Scholz 2, Virginie Gautier 6, Patrick Mallon 6,7, Stephen Gordon 1,2, Jonathan Dean 3, Jeff Connell 3, Michael Carr 3,4, Cillian De Gascun 3*.
Monkeypox is a viral zoonosis endemic in West and Central Africa and presents clinically in humans as a smallpox-like illness (Bunge et al., 2022). Monkeypox virus (MPXV) is an enveloped virus with a particle size of ca. 300×240 nm containing a double-stranded DNA genome of ~197 kb taxonomically classified in the family Poxviridae, genus Orthopoxvirus (Weaver and Isaacs, 2008).
There is increasing public health concern about the geographical spread and re-emergence of MPXV arising from the recent multi-country outbreaks, with more than 47 206 cases reported in 92 non-endemic countries since the first case was laboratory confirmed in the United Kingdom on the 7th of May 2022 (updated August 28th 2022 (CDC, 2022)). The MPXV genome reported (GenBank ON872184) here was from a laboratory-confirmed Irish case (Orthopox group RNA and MPXV-specific RNA detection by real-time PCR) identified in June 2022. Transmission electron microscopy (TEM) of a lesion swab sample collected visualised multiple characteristic brick-shaped poxvirus virions with lattice-like surface detail (Fig. 1). We performed a sequence-independent, single-primer amplification (SISPA) viromics approach to achieve a non-selective (unbiased) amplification of viral RNA transcripts and here report horizontal genome coverage over 90% with an average vertical coverage (depth) between 9x and 21x. Phylogenetic analysis with 35 publicly available international MPXV genome sequences from multiple countries (UK, Israel, Nigeria, USA, Singapore and Portugal) sampled between 2017-2019 and 2022 (Happi et al.), demonstrated high sequence similarity to other cases circulating in the 2022 outbreak in Europe and USA clustered together in clade 3 (Happi et al.); with the Irish MPXV genome further clustered in the B.1 lineage (Fig. 2). ...
https://virological.org/t/electron-m...ermination/899