Persistent Infection of a Nonvector Mosquito Cell Line (TRA-171) with Dengue Viruses

Abstract

Dengue viruses often caused an apparent, persistent infection in a cell line derived from a nonvector mosquito, Toxorhynchites amboinensis. The characteristics of the viruses were modified during the course of persistent infection, as demonstrated by the changes in neutralizing titers of hyperimmune ascitic fluids against dengue serotypes, increased ability to induce syncytia, and increased temperature sensitivity. Although neurovirulence in suckling mice was reduced for all serotypes, considerable levels of neurovirulence were detected for DEN-1, DEN-2, and DEN-4, after 1 year in culture. Replication of the original viruses was not significantly inhibited when the cell cultures were treated with virus-free supernatant fluids from persistently infected cultures.

Pathobiology of the Flaviviruses

“…Huang (1982) speculated that the main- tenance of neurovirulent strains in nature may require cycling through biological hosts, such as young birds (ducklings), which sustain brain infections, and that attenuated strains arise by persistent inherited (transovarial) infection of mosquitoes. The latter hypothesis is given weight by laboratory (Stollar and Shenk, 1973;Stollar, 1980;Kuno, 1982) and field studies (Reeves et al, 1958) with mosquito-borne togaviruses, which indicate that viruses from persistently infected arthropods or arthropod cells develop markers of attenuation (reduced neurovirulence, small plaque size, temperature lability or sensitivity). Oligonucleotide mapping of JE strains from Thailand (Burke et al, 1985a) indicated a high level of genotypic conservation among isolates from brains of fatal human cases compared to isolates from mosquito vectors.…”

Replication of Flaviviruses

“…Under these circumstances, it is possible to establish persistent infections simply by routine subculturing of surviving infected cells. This was the case in the establishment of persistent infections with dengue viruses in either HeLa cells (Maguire and Miles, 1965), rhesus monkey testis cells (Hotta and Evans, 1956), green monkey kidney cells (Shimazu et al, 1966), or an A. aibopictus cell line (Sinarachatanant and Olson, 1973), a line of Toxorhynchites amboinensis cells (Kuno, 1982) or human KB cells (Beasley et ai., 1960;Schulze and Schlesinger, 1963); WN virus in either murine L-929 cells (Jarman et al, 1968), lines of murine embryo fibroblasts (Brinton, 1982), or monkey LLC-MK2 cells (Katz and Goldblum, 1968); and SLE virus in a turtle heart (TH-l) cell line (Mathews and Vorndam, 1982).…”

Insect-transmitted vertebrate viruses: Flaviviridae

“…Such infections occur with or without the appearance of cytopathic effect (CPE). Kuno (64) showed that dengue virus infection of TRA-171 cells (from Toxorhynchites amboinensis) often resulted in an apparent, persistent infection. Titers of infectious extracellular virus varied extensively over 12 mo.…”

“…CPE was routinely visible in cells infected with both laboratory-adapted and -unadapted strains within the first 6 wk PI, but gradually diminished over time in the adapted strains, and was eliminated altogether in the unadapted strains. Some characteristics of viral replication in culture were altered during the course of persistent infection, as demonstrated by changes in neutralization characteristics, syncytia formation, and temperature sensitivity (64) (65) showed that of three mosquito cell lines tested, the TRA-284 line from T, amboinensis mosquitoes was the most sensitive to dengue virus and suggested that this line be used for routine isolation and identification procedures. In many cases, flavivirus isolation in insect cell culture is as, or more, sensitive than isolation techniques in animal hosts (15,61).…”

“…The observation that persistent dengue virus infections can be established in a non-vector mosquito cell line associated with changes in several of the important biological characteristics, such as temperature sensitivity and antigenic structure (64), presents the possibility that genetic selection of attenuated virus strains may be possible with insect cell lines. Pilot studies looking at the feasibility of producing flavivirus vaccines in insect cells should be initiated.…”