An insect cell line derived from the small brown planthopper supports replication of rice stripe virus, a tenuivirus

Abstract

A cell line from the small brown planthopper (SBPH; Laodelphax striatellus) was established to study replication of rice stripe virus (RSV), a tenuivirus. The SBPH cell line, which had been subcultured through 30 passages, formed monolayers of epithelial-like cells. Inoculation of cultured vector cells with RSV resulted in a persistent infection. During viral infection in the SBPH cell line, the viral non-structural protein NS3 co-localized with the filamentous ribonucleoprotein particles of RSV, as revealed by electron and confocal microscopy. The knockdown of NS3 expression due to RNA interference induced by synthesized double-stranded RNAs from the NS3 gene significantly inhibited viral infection in the SBPH cell line. These results demonstrated that NS3 of RSV might be involved in viral replication or assembly. The persistent infection of the SBPH cell line by RSV will enable a better understanding of the complex relationship between RSV and its insect vector.Plant viruses in the genera Tospovirus, Tenuivirus, Rhabdovirus, Phytoreovirus and Fijivirus are transmitted by their respective insect vectors in a persistent propagative manner (Ammar et al., 2009;Hogenhout et al., 2008). Insights into replication of these viruses in their vectors will help in development of new strategies to control the transmission of the viruses by the vectors. Continuous cultures of cells derived from insect vectors are powerful tools for studying the replication cycle of persistent propagative plant viruses because they are more uniform than primary cultures and thus provide more consistent data (Creamer, 1993). Continuous cultures of leafhopper cells provide a good system to study the replication cycle of phytoreoviruses in their insect vectors (Kimura & Omura, 1988;Omura & Kimura, 1994;Wei et al., 2006Wei et al., , 2007.Rice stripe virus (RSV), the type species of the genus Tenuivirus, has filamentous ribonucleoprotein particles (RNPs) that contain nucleoprotein, RNA-dependent RNA polymerase and four negative-sense single-stranded RNA segments that encode seven proteins (Toriyama, 1986;Falk & Tsai, 1998;Wei et al., 2009;Xiao et al., 2010). RSV is transmitted by the small brown planthopper (SBPH; Laodelphax striatellus Fallén) in a persistent propagative manner (Toriyama, 1986;Wei et al., 2009). Currently, the mechanism that enables efficient multiplication of RSV in its vector is unknown due to the lack of SBPH cell lines. In previous attempts to establish primary cell cultures, only a small number of cells migrated occasionally from the embryonic explants dissected from SBPH eggs, and eventually the subculture died off (Mitsuhashi, 1969(Mitsuhashi, , 2001Yamada et al., 1970). The present report describes the first successful establishment of continuous cultures of SBPHs for the study of RSV replication.The SBPH cell line was established by adapting the protocol described by Kimura & Omura (1988). Nonviruliferous SBPHs eggs that had been oviposited 8 days earlier in leaf sheaths of rice plants were surface-sterilized with 70 % ...

Rice Reoviruses in Insect Vectors

“…During the past 30 years, since insect cell lines were available from rice leafhoppers (N. cincticeps and R. dorsalis) and planthoppers (L. striatellus, S. furcifera, and N. lugens) (18,46,51,52,67,68,84,119), we have begun to understand the viral replication cycle, including entry, multiplication, and spread in insect vector cells (Figure 2). Primary cell cultures of leafhopper or planthopper, originally established from the embryonic fragments dissected from insect eggs, are subcultured to form continuous cultures of cells growing in a monolayer (Figure 2; 18,67,68,84). Such continuous insect cell lines support a uniform and synchronous viral infection, enabling the early replication process of viruses to be traced (18,25,67,68,84).…”

“…Primary cell cultures of leafhopper or planthopper, originally established from the embryonic fragments dissected from insect eggs, are subcultured to form continuous cultures of cells growing in a monolayer (Figure 2; 18,67,68,84). Such continuous insect cell lines support a uniform and synchronous viral infection, enabling the early replication process of viruses to be traced (18,25,67,68,84). We next describe cellular details about the various stages of the replication cycles of rice reoviruses in leafhopper or planthopper cell lines.…”

Small Interfering RNA Pathway Modulates Initial Viral Infection in Midgut Epithelium of Insect after Ingestion of Virus

“…Continuous cultures of vector cells grown in monolayers (VCMs) were developed from SBPH or WBPH and maintained in growth medium as described previously (30,32). SRBSDV was purified from infected cultured WBPH cells as described previously (29,30).…”

Proteomic Analysis of Interaction between a Plant Virus and Its Vector Insect Reveals New Functions of Hemipteran Cuticular Protein

“…Culture of L. striatellus Cell Line-The L. striatellus cell line was established by adapting the protocol already detailed (32). Nonviruliferous eggs of L. striatellus that had been oviposited 8 days earlier in leaf sheaths of rice plants were surface-sterilized with 70% ethanol, and embryonic fragments were dissected from the eggs in Tyrode's solution, treated with 0.25% trypsin in Tyrode's solution for 15 min, and then incubated with Kimura's insect medium at 25°C.…”