The Studies of Perina nuda Picorna-like Virus, PnPV

• Main Authors: Chih-Yu Wu, 吳治宇
• Other Authors: Chung-Hsiung Wang
• Format: Others
• Language: en_US
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/88354229174729380693

博士 === 國立臺灣大學 === 昆蟲學研究所 === 91 === Acute disease outbreaks causing mass mortality of laboratory-reared larvae of ficus transparent wing moth, Perina nuda (Fabricius) had occurred. Moribund larvae exhibited a syndrome similar to the silkworm infected with infectious flacherie virus (IFV). Two viruses, P. nuda nucleopolyhedrovirus (PenuNPV) and a new picorna-like virus (named P. nuda picorna-like virus, PnPV), had been isolated from the infected larvae with flacherie disease. Negatively stained preparations showed that PnPV particles were icosahedral symmetry and 30 nm in diameter without envelope and distinct surface structure. PnPV had a buoyant density of 1.381 g/ml in caesium chloride. The viral genome is a single-stranded RNA molecule consisting of 9,476 nucleotides (nts) excluding the poly(A) tail, and contains an open reading frame (ORF) of 8,958 nts flanked by 473 and 45 nts non-coding regions on the 5’ and 3’ ends, respectively. The PnPV genome encodes four structural proteins (CP1-4). Their N-terminal sequences determined by Edman degradation demonstrate that they are located in the 5’ region of the genome. The 3’ part of the PnPV genome contains consensus sequence motifs for picornavirus RNA helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp) in an order from the 5’ to 3’ end. Take together these characters, the genome organization of PnPV resembles the mammalian picornaviruses and two insect picorna-like viruses, IFV of the silkworm and Sacbrood virus (SBV) of the honeybee. Based on the phylogenetic analysis of the eight conserved domains of the RdRp sequences, PnPV, IFV, and SBV form a separate cluster suggesting that these three insect picorna-like viruses might represent a novel group of the insect RNA viruses. A new cell line, designated NTU-Pn-HF, was established from the pupal ovary of P. nuda and characterized by morphological properties and molecular markers. Under electron microscopy, most of the Pn-HF cells were found to be infected by a picorna-like virus. A specific anti-PnPV antiserum and RT-PCR with a primer set (named as P51-F3 / P179-R4a) designed from the sequence of a putative PnPV’s helicase gene confirmed that the picorna-like virus in Pn-HF cells was PnPV. In addition, PnPV can persistently infect Pn-HF cells. This finding implies that Pn-HF cell line is not only a PnPV supporter for further work, but also a convenient tool for studying the pathogenesis of insect picornavirus and the mechanism of the picornavirus-persistent infection. In the study of tissue tropism, the existence of PnPV in the organs or tissues isolated from the 6th-instar larvae and newly emerged adult moths were orally infected with PnPV at their 2nd-instar stage were detected by RT-PCR. Results showed that all examined organs and tissues (including epidermis, muscle, nerve, fat body, tracheae, foregut, midgut, hindgut, gonad, and silk gland) were positive. This result implied that the PnPV’s infection is a systemic infection. Under electron microscopy, PnPV was found in cells of tracheae, midgut, and muscle, which confirmed again the results of RT-PCR detection. Furthermore, an in situ hybridization was used to examine the tissues of the 3th-end-instar larvae that were inoculated with PnPV previously at their 2nd-instar stage (about 3-5 days postinoculation), and the PnPV-positive signal was only shown in the epithelial cells of the midgut. Therefore, we suggest that the epithelial cells of the midgut are the first PnPV target site at the early infection.