Production of Recombinant African Swine Fever Viruses: Speeding Up the Process.

Anusyah Rathakrishnan,Linda K Dixon,Ana Luisa Reis,Katy Moffat

doi:10.3390/v12060615

Abstract

African swine fever (ASF) is a devastating disease in pigs, with no vaccines for control. The genetic manipulation of African swine fever virus (ASFV) is often tedious and time consuming. Here, we describe a method to manipulate the virus genome to produce gene deletion viruses in a much-reduced time. This method combines the conventional homologous recombination with fluorescent-activated cells sorting (FACS), to isolate and purify viruses expressing fluorescent reporter genes. With three rounds of single cell isolation via FACS and two rounds of limiting dilution, we deleted two additional genes, EP153R and EP402R, from Benin 97/1 ASFV lacking the DP148R gene. By combining different fluorescent markers, this method has the potential to greatly facilitate studies on understanding ASFV gene functions and develop candidate live-attenuated vaccines.

Highlights

African swine fever virus (ASFV), the sole member of the family Asfaviridae, causes African swine fever (ASF) disease in European wild boar and domestic pigs
The buffy coat was collected, red blood cells lysed with 1X RBC Lysis Buffer (BioLegend, San Diego, CA, USA) and washed with PBS, to obtain purified porcine bone marrow cells (PBMs) that could be frozen down in freezing medium constituted of 90% FBS and 10% DMSO (Sigma-Aldrich, Dorset, United Kingdom (UK))
fluorescent-activated cells sorting (FACS) enables the sorting of a heterogeneous cell population into a relatively homogenous population, depending on their light scatters and fluorescent features

Summary

Introduction

African swine fever virus (ASFV), the sole member of the family Asfaviridae, causes African swine fever (ASF) disease in European wild boar and domestic pigs. After infection with virulent ASFV strains, the acute form of disease is characterized by fever, loss of appetite, lethargy and haemorrhage. The less virulent strains can lead to mild clinical signs, subacute forms of disease, or even chronic infections. Warthogs and bush pigs develop mild or no clinical signs, even when infected with highly virulent isolates and, they can act as reservoirs for infection [1]. ASF is endemic in many sub-Saharan African countries. ASF has a huge socio-economic impact, as pigs are important sources of protein. ASFV infection in these countries has led to destabilization of the pork industry throughout the world [2]

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