Continuous Cell Lines from the European Biting Midge Culicoides nubeculosus (Meigen, 1830).

Lesley Bell-Sakyi,Houssam Attoui,Lisa Luu,Fauziah Mohd Jaafar,Eric Denison,Baptiste Monsion,Peter P C Mertens,Simon Carpenter

doi:10.3390/microorganisms8060825

Abstract

Culicoides biting midges (Diptera: Ceratopogonidae) transmit arboviruses of veterinary or medical importance, including bluetongue virus (BTV) and Schmallenberg virus, as well as causing severe irritation to livestock and humans. Arthropod cell lines are essential laboratory research tools for the isolation and propagation of vector-borne pathogens and the investigation of host-vector-pathogen interactions. Here we report the establishment of two continuous cell lines, CNE/LULS44 and CNE/LULS47, from embryos of Culicoides nubeculosus, a midge distributed throughout the Western Palearctic region. Species origin of the cultured cells was confirmed by polymerase chain reaction (PCR) amplification and sequencing of a fragment of the cytochrome oxidase 1 gene, and the absence of bacterial contamination was confirmed by bacterial 16S rRNA PCR. Both lines have been successfully cryopreserved and resuscitated. The majority of cells examined in both lines had the expected diploid chromosome number of 2n = 6. Transmission electron microscopy of CNE/LULS44 cells revealed the presence of large mitochondria within cells of a diverse population, while arrays of virus-like particles were not seen. CNE/LULS44 cells supported replication of a strain of BTV serotype 1, but not of a strain of serotype 26 which is not known to be insect-transmitted. These new cell lines will expand the scope of research on Culicoides-borne pathogens.

Highlights

Biting midges of the genus Culicoides are vectors of a variety of pathogens of veterinary or medical importance, including viruses, protozoa and helminths [1,2,3,4,5]
The European midge species Culicoides nubeculosus (Meigen 1830), which is found across the Western Palearctic region from Spain and the United Kingdom in the west to Poland and Turkey in the east [15,16,17,18], is considered to have a low vectorial capacity for bluetongue virus (BTV) and Schmallenberg virus (SBV) [10,11]
Biting midge cell lines derived from the North American species C. sonorensis, previously known as Culicoides variipennis [22,23,24], have been used in studies on orbivirus replication and transmission [25,26,27,28], Culicoides antiviral immunity [29] and ability to support infection and growth of the bacterial symbiont Wolbachia [30]

Summary

Introduction

Biting midges of the genus Culicoides are vectors of a variety of pathogens of veterinary or medical importance, including viruses, protozoa and helminths [1,2,3,4,5]. Biting midge cell lines derived from the North American species C. sonorensis, previously known as Culicoides variipennis [22,23,24], have been used in studies on orbivirus replication and transmission [25,26,27,28], Culicoides antiviral immunity [29] and ability to support infection and growth of the bacterial symbiont Wolbachia [30]. One of these lines, KC, is used routinely in reference laboratories to isolate emerging arbovirus strains, in particular of BTV. The ability of one of the cell lines was tested to support the replication of strains of two serotypes of BTV, BTV-1 known to be transmitted by midges and BTV-26 with no known arthropod vector

Materials and Methods

Results

Discussion