Rinderpest virus expressing enhanced green fluorescent protein as a separate transcription unit retains pathogenicity for cattle

Abstract

A full-length DNA clone of a virulent strain of rinderpest virus was constructed with the gene for the enhanced green fluorescent protein (eGFP) inserted as a separate transcription unit between the P and M genes. Rescue of the virus from the modified clone using reverse genetics generated a virus that grew to the same levels as the virus rescued from the unmodified DNA clone in cell culture. The recombinant virus expressed eGFP to a high level and was used to follow virus replication in real-time using live-cell imaging. Cattle infected with both the recombinant wild-type virus and the recombinant eGFP expressing virus developed clinical disease similar to that of the wild-type natural virus isolate. Detection of virus in circulating peripheral blood leukocytes was equivalent to that of the animals infected with the wild-type virus. The high level of expression of soluble eGFP by this virus allowed us to detect viral replication in infected animals by confocal microscopy. Imaging vibrating microtome sections by confocal microscopy provided good preservation of tissue and cellular architecture as well as revealing the sites of replication of the virus in different tissues of infected animals.

Pathogenesis of wild-type- and vaccine-based recombinant peste des petits ruminants virus (PPRV) expressing EGFP in experimentally infected domestic goats

“…To our knowledge, no recombinant PPRV harbouring an additional transcription unit (ATU) has been used in pathogenesis studies. Although it is known that adding an ATU (especially before the nucleoprotein, position 1) may alter the morbillivirus-typical transcriptional gradient and therefore potentially attenuate the recombinant virus [32], an in vivo study in cattle using recombinant RPV expressing EGFP on position 3 of the genome did not show any differences in pathogenesis compared to the virus without EGFP expression [47]. Moreover, in vitro characterization and comparison performed in this study did not hint towards any difference based on the insertion of EGFP in rPPRV/Tbilisi-EGFP, suggesting that the used virus strain and/or age of the animal were responsible for the moderate clinical symptoms observed in our study.…”

Growth kinetics of a Vero cells adapted Bangladeshi strain of peste des petits ruminants (PPR) virus in cell culture

“…Growth curve analysis of recombinant rinderpest virus (RPVs) containing chimeric H proteins was conducted by Parida et al ( 2006 ) where Vero cells were infected with viruses such as RPV2C, PPRV, or RPV2C-PPRTm (at MOI 0.1) or virus RPV2C-PPRExt (at MOI 0.01), and the same titre was found on 72 hpi which was 3.5 log10 TCID 50 /ml. Marmoset B95a and Vero-SLAM cells were infected simultaneously with two rinderpest virus and titres were found between 5.0 log10 TCID 50 /ml and 6.0 log10 TCID 50 /ml by 96 hpi (Banyard et al 2010 ). In another study of rinderpest virus growth profile conducted by Nores and McCullough ( 1997 ) observed, the highest virus titres were obtained with the RPV-Saudi isolate at 10 dpi.…”

Development and application of reporter-expressing mononegaviruses: Current challenges and perspectives

“…Multiple paramyxoviruses expressing various fluorescent reporters have been used to gain insight into viral pathogenesis (Banyard et al, 2010; de Swart et al, 2007; de Vries et al, 2010; Ludlow et al, 2012; Rudd et al, 2006; von Messling et al, 2004), and a human metapneumovirus expressing green fluorescent protein (GFP) was used to evaluate the attenuating effect of altering N-glycosylation sites on various virus proteins both in vitro and in vivo for the development of live attenuated vaccines (Zhang et al, 2011). For Bornaviruses, dissemination throughout the nervous system was studied using a Borna disease virus expressing GFP (Ackermann et al, 2010).…”