Morphological and molecular evidence that Culex nigripalpus baculovirus is an unusual member of the family Baculoviridae

Abstract

We present evidence that a newly discovered mosquito virus from Culex nigripalpus is an unusual member of the family Baculoviridae. Development of this virus was restricted to nuclei of midgut epithelial cells in the gastric caeca and posterior stomach. The globular occlusion bodies were not enveloped, measured around 400 nm in diameter, occurred exclusively in nuclei of infected cells and typically contained four, sometimes up to eight, virions. The developmental sequence involved two virion phenotypes : an occluded form (ODV) that initiated infection in the midgut epithelial cells, and a budded form that spread the infection in the midgut. Each ODV contained one rodshaped enveloped nucleocapsid (40i200 nm). The double-stranded DNA genome was approximately 105-110 kbp with an estimated GC content of 52 %. We have sequenced approximately one-third of the genome and detected 96 putative ORFs of 50 amino acids or more including several genes considered to be unique to baculoviruses. Phylogenetic analysis of the amino acid sequences of DNApol and p74 placed this virus in a separate clade from the genera Nucleopolyhedrovirus and Granulovirus. We provisionally assign this virus in the genus Nucleopolyhedrovirus, henceforth abbreviated as CuniNPV (for Culex nigripalpus nucleopolyhedrovirus), and suggest that, awaiting additional data to clarify its taxonomic status, it may be a member of a new genus within the family Baculoviridae.

Induction of reaper ortholog mx in mosquito midgut cells following baculovirus infection

“…However, by 48 h p.i., most cells at the posterior midgut and gastric caeca were positive for viral gene expression and had swollen nuclei. 23 This indicates that the virus initially infects only a small group of cells but later spreads horizontally to infect other cells, presumably via budded virus. Interestingly, while most of the cells in the posterior midgut were positive for viral gene expression, none of the cells at the anterior midgut was positive for viral gene expression.…”

“…Previous histological analysis has shown that many cells at the gastric caeca and posterior midgut of infected C. nigripalpus or C. quinquefasciatus larvae were heavily infected with CuniNPV occlusion bodies (OBs) and display signs of necrosis at the final stages of infection. 23,28 When TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) was applied to monitor possible apoptotic response to viral infection in C. quinquefasciatus larvae, there was no significant level of apoptosis at either early stage (2-8 h p.i.) or late stages (24 and 48 h p.i.)…”

“…CuniNPV viral OBs were purified from infected C. quinquefasciatus larvae as described previously. 23 The standard assay involves groups of 100 three-to four-day-old C. quinquefasciatus and A. aegypti larvae obtained from the laboratory colony. Larvae are exposed at a dose of 1.2 Â 10 9 OBs per ml in 100 ml of deionized water with 15 mM MgCl 2 plus 50 mg alfalfa and potbelly pig chow mixture (2 : 1).…”

“…Not all larval midgut cells are receptive to CuniNPV infection, which is limited to a particular group of resorbing/ secreting cells in the gastric caeca and the posterior midgut. 23 Once inside the midgut, the virus can spread from infected cells to neighboring cells via the budded form.…”

“…22 It is related to lepidopteran and hymenopteran baculoviruses, but genomic sequence comparison indicated that there is a large evolutionary distance between CuniNPV and lepidopteran baculoviruses. 23 CuniNPV infects only epithelial cells of the larval midgut, has a restricted host range, and mainly infects Culex spp. within the subgenus Culex.…”