Cell Culture for Production of Insecticidal Viruses

Abstract

While large-scale culture of insect cells will need to be conducted using bioreactors up to 10,000 l scale, many of the main challenges for cell culture-based production of insecticidal viruses can be studied using small-scale (20-500 ml) shaker/spinner flasks, either in free suspension or using microcarrier-based systems. These challenges still relate to the development of appropriate cell lines, stability of virus strains in culture, enhancing virus yields per cell, and the development of serum-free media and feeds for the desired production systems. Hence this chapter presents mainly the methods required to work with and analyze effectively insect cell systems using small-scale cultures. Outlined are procedures for quantifying cells and virus and for establishing frozen cells and virus stocks. The approach for maintaining cell cultures and the multiplicity of infection (MOI) and time of infection (TOI) parameters that should be considered for conducting infections are discussed.The methods described relate, in particular, to the suspension culture of Helicoverpa zea and Spodoptera frugiperda cell lines to produce the baculoviruses Helicoverpa armigera nucleopolyhedrovirus, HearNPV, and Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, AgMNPV, respectively, and the production of the nonoccluded Oryctes nudivirus, OrNV, using an adherent coleopteran cell line.

Cell lines from diamondback moth exhibiting differential susceptibility to baculovirus infection and expressing midgut genes

“…Among the P. xylostella cell lines, we selected both cell lines growing in attachment (FAFU&CU‐Px‐1, FAFU&CU‐Px‐2, FAFU&CU‐Px‐3, FAFU&CU‐Px‐4 and FAFU&CU‐Px‐5) and growing in suspension (FAFU&CU‐Px‐6), to provide attached cells and suspension cells for applications in research and biotechnology. The P. xylostella cell lines grew relatively fast in culture with the cell population doubling time from 18 to 23 h. Based on such a cell population growth rate, these P. xylostella cells can be appropriate choices of host cell lines for baculovirus and recombinant protein production (Reid et al ., ).…”

Transcriptomics Reveal Several Novel Viruses from Canegrubs (Coleoptera: Scarabaeidae) in Central Queensland, Australia

“…The effective control of the coconut rhinoceros beetle, Oryctes rhinoceros (Coleoptera: Scarabaeidae), in southeast Asia and the South Pacific islands by a double stranded DNA virus (OrNV: Oryctes rhinoceros nudivirus) [ 25 ] is widely considered to be a landmark example of classical biological control, and it represents one of the few examples of successful classical biological control involving an entomopathogen [ 33 ]. OrNV can infect and replicate in other coleopteran cell lines, including DSIR-HA-1179 (an embryonic cell line originally derived from the black African beetle, Heteronychus arator [ 34 ], which is a pest of sugarcane and other crops) and FRI-AnCu-35 (an embryonic cell line of the cupreous chafer, Anomala cuprea (Coleoptera: Scarabaeidae)) [ 35 ]. Currently, very few insect nudiviruses have been described [ 36 ], but members of the family Nudiviridae have great potential for use as biocontrol agents of insect pests, including canegrubs.…”