Analysis of the structural proteins from the Musca domestica hytrosavirus with an emphasis on the major envelope protein.

The Musca domestica hytrosavirus (MdHV), a member of the family Hyrosaviridae, is a large, dsDNA, enveloped virus that infects adult house flies and causes a diagnostic hypertrophy of the salivary gland. Herein, studies were directed at identifying key structural components of the viral envelope and nucleocapsid. SDS-PAGE of detergent-treated virus fractions identified protein bands unique to the envelope and nucleocapsid components. Using prior LC-MSMS data we identified the viral ORF associated with the major envelope band, cloned and expressed recombinant viral antigens, and prepared a series of polyclonal sera. Western blots confirmed that antibodies recognized the target viral antigen and provided evidence that the viral protein MdHV96 underwent post-translational processing; antibodies bound to the target high molecular weight parent molecule as well as distinct sets of smaller bands. Immuno gold electron microscopy demonstrated that the anti-MdHV96 sera recognized target antigens associated with the envelope. The nucleocapsids migrated from the virogenic stroma in the nucleus through the nuclear membrane into the cytoplasm, where they acquired an initial envelope that contained MdHV96. This major envelope protein, appeared to incorporate into intracellular membranes of both the caniculi and rough endoplasmic reticulum membranes and mediate binding to the nucleocapsids. Oral infection bioassays demonstrated that the anti-HV96 polyclonal sera acted as neutralizing agents in suppressing the levels of orally acquired infections.

First Report of Pseudomonas cichorii Causing Leaf Spot of Stevia Detected in Florida.

During a routine nursery inspection in Hernando County, Florida in July 2011, leaf spot symptoms were observed on the popular sugar substitute stevia (Stevia rebaudiana). Spots were roughly circular to irregular in shape, variable in size, and dark brown with a yellow halo. White to cream-colored, circular, convex, gram-negative bacterial colonies were isolated on nutrient agar and identified as Pseudomonas cichorii based on the LOPAT scheme (2). DNA from four individual colonies inoculated in nutrient broth was extracted using DNeasy columns (Qiagen Inc., Valencia, CA). The PCR-amplified product from four different genes, 16S rRNA (1.5 Kbp), gyrB (0.9 Kbp), rpoB (1.2 Kbp), and rpoD (0.7 Kbp), was sequenced (GenBank Accession Nos. JQ994483, JQ994484, JQ994485, and JQ994486). Nucleotide and translated amino acid sequences for each gene were compared to the nucleotide and protein databases, respectively. The best matches were always with P. cichorii with nucleotide identities ranging from 98 to 99% and amino acid identities from 99 to 100%. Four healthy stevia plants were spray inoculated with 20 ml of a 108 CFU ml-1 suspension prepared from a 24-hour-old culture, of which two were sprayed with carborundum (silicon carbide) immediately prior to inoculation. Two additional plants were sprayed with carborundum only or sterile tap water only and served as healthy controls. Symptoms began to develop 4 days after inoculation. Spots originated at the tips or edges of the leaves and enlarged over time. Enlarging lesions progressed to encompass the entire leaf, accompanied by water soaking, curling, and necrosis. Blighting typically spread down to the stem and caused dieback. The pathogen was successfully reisolated from the lesions and produced identical LOPAT scheme results. Based on the information collected, it is believed that this is the first confirmed report of bacterial leaf spot caused P. cichorii on stevia worldwide. This find is significant due to the rising popularity of stevia cultivation for its sweetness and medicinal properties (1). To our knowledge, stevia is not currently being grown commercially in Florida; however, there is commercial acreage being developed elsewhere in the United States. References: (1) D. Patil et al. Asian J. Pharm. Clin. Res. 5:1, 2011. (2) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd Edition. The American Phytopathological Society. St. Paul, MN, 2001.

Impact of house fly salivary gland hypertrophy virus (MdSGHV) on a heterologous host, Stomoxys calcitrans.

The effect of Musca domestica salivary gland hypertrophy virus (MdSGHV) on selected fitness parameters of stable flies, Stomoxys calcitrans (L.), was examined in the laboratory. Virus-injected stable flies of both genders suffered substantially higher mortality than control flies. By day 9, female mortality was 59.3 +/- 10.1% in the virus group compared with 23.7 +/- 3.7% in the controls; mortality in virus-injected males was 78.1 +/- 3.1% compared with 33.3 +/- 9.3% for controls. Fecundity of control flies on days 6-9 was 49-54 eggs deposited per live female per day (total, 8,996 eggs deposited), whereas virus-injected flies produced four to five eggs per female on days 6-7 and less then one egg per female per day thereafter (total, 251 eggs). Fecal spot deposition by virus-injected flies was comparable to controls initially but decreased to approximately 50% of control levels by day 4 after injection; infected flies produced only 26% as many fecal spots as healthy flies on days 6 and 7. None of the virus-injected stable flies developed symptoms of salivary gland hypertrophy. Quantitative real-time polymerase chain reaction demonstrated virus replication in injected stable flies, with increasing titers of virus genome copies from one to four days after injection. MdSGHV in stable flies displayed tissue tropism similar to that observed in house fly hosts, with higher viral copy numbers in fat body and salivary glands compared with ovaries. Virus titers were approximately 2 orders of magnitude higher in house fly than in stable fly hosts, and this difference was probably due to the absence of salivary gland hypertrophy in the latter species.

Physical maps and virulence of Anticarsia gemmatalis nucleopolyhedrovirus genomic variants.

Seventeen plaque purified isolates of two viral preparations of Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), were analyzed in terms of the genomic changes after digestion of their DNAs with HindIII and PstI restriction enzymes. The 1979 AgMNPV wild type preparation (AgMNPV-'79) resulted in six different variants and the 1985 viral commercial preparation (AgMNPV-'85), in eleven. The genomic variation of all the isolates was mapped showing that those from 1985 presented more heterogeneity with changes mapped in additional sites in comparison to the AgMNPV-'79 variants. Their virulence was compared by infecting two Lepidopteran cell lines, Spodoptera frugiperda (IPLB-SF-21AE) and Anticarsia gemmatalis (UFL-AG-286). The results indicated that there was some difference in virulence within the AgMNPV-'85 variants. This commercial preparation had been applied in soybean fields in Brazil over several years to control the velvetbean caterpillar defoliation.

A variable region of Anticarsia gemmatalis nuclear polyhedrosis virus contains tandemly repeated DNA sequences.

A variable region (PstI-T fragment) of two genotypic variants of the Anticarsia gemmatalis multiple nucleocapsid nuclear polyhedrosis virus (AgMNPV) was sequenced and compared. This region, which is known to have deletions and duplications in AgMNPV variants, was shown to be formed of units of a 127 bp tandemly repeated sequence containing two 30 bp imperfect palindromes. Southern blot experiments showed that the PstI-T fragment contained one of the four homologous regions mapped interspersed in the AgMNPV genome. The comparison of the nucleotide sequences of the two variants, AgMNPV-2D and AgMNPV-D7, showed that the difference between these two variants in one of these regions (hr4) was caused by the addition or elimination of 381 bp corresponding exactly to three of the 127 bp repeated sequences. An analysis of the sequence showed homology to the homologous regions (hr) of other baculoviruses. The sequence upstream of the repetitive region contained a sequence homologous to the N-terminal portion of the Autographa californica MNPV and Choristoneura fumiferana MNPV p74 gene.