Pathological Features and Molecular Phenotype of MMTV Like-Positive Feline Mammary Carcinomas

In the last few years MMTV-like nucleotide sequences were detected in some feline and canine mammary tumours. Due to the confirmed role of cats in the epidemiology of the MMTV-like virus, the aim of this study was to investigate the main pathological features of positive feline mammary carcinomas (FMCs). Twenty-four FMCs were collected at the University of Bologna, submitted to laser microdissection and analysed by nested fluorescence-PCR using primer sets specific for MMTV env sequence. For immunohistochemistry, an antibody against MMTV protein 14 (p14) was used. MMTV-like sequences were detected in three out of 24 FMCs (12.5%), one tubular carcinoma, one tubulopapillary carcinoma and one ductal carcinoma. All PCR-positive tumours were also positive for p14. Multiple nucleotide alignment has shown similarity to MMTV ranging from 98% to 100%. All the 102 examined FMCs were submitted to immunohistochemistry for molecular phenotyping. Of the nine MMTV-like positive FMCs, six were basal-like and three luminal-like. Our results demonstrate MMTV-like sequences and protein in FMCs of different geographic areas. Molecular phenotyping could contribute to understand the possible role of MMTV-like virus in FMC tumor biology.

VADR: validation and annotation of virus sequence submissions to GenBank

BackgroundGenBank contains over 3 million viral sequences. The National Center for Biotechnology Information (NCBI) previously made available a tool for validating and annotating influenza virus sequences that is used to check submissions to GenBank. Before this project, there was no analogous tool in use for non-influenza viral sequence submissions.ResultsWe developed a system called VADR (Viral Annotation DefineR) that validates and annotates viral sequences in GenBank submissions. The annotation system is based on the analysis of the input nucleotide sequence using models built from curated RefSeqs. Hidden Markov models are used to classify sequences by determining the RefSeq they are most similar to, and feature annotation from the RefSeq is mapped based on a nucleotide alignment of the full sequence to a covariance model. Predicted proteins encoded by the sequence are validated with nucleotide-to-protein alignments using BLAST. The system identifies 43 types of “alerts” that (unlike the previous BLAST-based system) provide deterministic and rigorous feedback to researchers who submit sequences with unexpected characteristics. VADR has been integrated into GenBank’s submission processing pipeline allowing for viral submissions passing all tests to be accepted and annotated automatically, without the need for any human (GenBank indexer) intervention. Unlike the previous submission-checking system, VADR is freely available (https://github.com/nawrockie/vadr) for local installation and use. VADR has been used for Norovirus submissions since May 2018 and for Dengue virus submissions since January 2019. Other viruses with high numbers of submissions will be added incrementally.ConclusionVADR improves the speed with which non-flu virus submissions to GenBank can be checked and improves the content and quality of the GenBank annotations. The availability and portability of the software allow researchers to run the GenBank checks prior to submitting their viral sequences, and thereby gain confidence that their submissions will be accepted immediately without the need to correspond with GenBank staff. Reciprocally, the adoption of VADR frees GenBank staff to spend more time on services other than checking routine viral sequence submissions.

Similarity and Phylogenetic Analysis of Herbicide-Resistant Goosegrass (Eleusine indica) Biotypes

Molecular approach as a herbicide-resistant agent detector is an initial solution before controlling of <em>Eleusine indica </em>weed. This review article is aimed to obtain the basic data of similarity and phylogenetic values among herbicide-resistant <em>E. indica</em><em> </em>biotypes. This research used a descriptive analytical method. The analysis involved 14 nucleotide sequences of herbicide-resistant <em>E.indica </em>biotypes obtained from the National Center for Biotechnology Information. The nucleotide alignment of herbicide-resistant <em>E. indica </em>biotypes was conducted with ClustaLW using the Molecular Evolutionary Genetics Analysis (MEGA) v. 5.05 software based on a method of neighbor-joining tree construct/test. The results showed that two nucleotides of 7,921 herbicide-resistant <em>E. indica</em> biotypes were homologous (sequence 1,231 and sequence 1,408). The similarity values among herbicide-resistant <em>E. indica</em> biotypes ranged from 0.00 to 1.19. The information of phylogenetic pattern is needed in the selection of the herbicides mode of action rotation in order to control herbicide-resistant <em>E. indica </em>biotypes.

First Report of Trichoderma oblongisporum Causing Green Mold Disease on Lentinula edodes (shiitake) in China

Lentinula edodes (shiitake) is well known for its delicious taste and valuable medical functions, and ranked as the second most important mushroom in terms of total world production. In March 2012, a serious green mold epidemic occurred on many mushroom farms in Suizhou County of China. The infected mycelia of L. edodes in cultivated bags became rotten, yellow, wilted, and finally died, with the surfaces of the cultivated bags covered with dark green fungal colonies. At a temperature above 20°C, disease incidence was nearly 100% on some mushroom farms. Three diseased cultivated bags were collected from three different mushroom farms, and two portions at the junction of the diseased and healthy portions of the bag were plated individually on potato dextrose agar (PDA) and incubated at 25°C for 4 days. Following incubation, agar discs cut from the growing front of colonies were inoculated onto fresh PDA and subcultured to obtain putative pathogens. Three purified isolates were all whitish initially, followed by the emergence of greenish conidial clusters at the outer margin of the colony. The underside of the colony appeared pale yellow. The growth rate of the isolates was about 0.95 to 1.02 cm/day in PDA at 25°C. Aerial mycelia were floccose, white, and septate. Chlamydospores were sub-globose to broadly ellipsoidal. Conidiophore branches arose at right angles, and primary branches arose singly or in pairs. Phialides were ampulliform, 3.1 to 6.7 × 2.7 to 4.0 μm, slightly constricted at the base, swollen in the middle, and narrowed abruptly at the apex. Conidia were produced on the top of the phialides with the shape varying from ellipsoidal to oblong, 3.3 to 4.7 × 2.4 to 3.2 μm. These observations were consistent with the description of Trichoderma oblongisporum by Bissett (1). The ITS and partial tef1 were amplified from the three isolates as previously reported (2) and sequenced (KM110064 to KM110069). Nucleotide alignment showed 99% sequence identity (ITS) with two T. oblongisporum isolates (FJ623268 and DQ083020), and 88% similarity (tef1) with T. oblongisporum (AY750884). Neighbor joining tree of ITS and tef1 nucleotide sequences also showed that our three isolates had the closest relationship with the aforementioned three T. oblongisporum strains. To determine pathogenicity, a sawdust substrate was sterilized for 2 h in polyethylene bags and subsequently inoculated separately with three isolates and L. edodes strain Qiu7, which was cultivated widely in Suizhou. When the mycelia of Qiu7 colonized the bags, 10 cm3 of substrate was withdrawn from each bag. The substrate was then exposed to 10 cm3 of mycelium from each pathogen in 10 bags. A parallel inoculation with 10 cm3 of sterilized sawdust substrate without pathogen mycelia was performed as a control. The inoculated cultivated bags were kept at 25°C. After 2 months, all of the mycelia in the bags became wilted and dead, and the cultivated bags became soft, rotten, and covered with dark green fungal colonies. The controls remained uninfected. The symptoms were similar to those observed on mushroom farms. Pathogens re-isolated from all the inoculated culture bags were confirmed to be T. oblongisporum through morphological characteristics, molecular identification, and phylogenetic analysis. To our knowledge, this is the first report of T. oblongisporum causing green mold disease on L. edodes in the world. References: (1) J. Bissett. Can. J. Bot. 69:2398, 1991. (2) N. Sadfi-Zouaoui et al. Can. J. Microbiol. 55:154, 2009.

The 3′ untranslated regions of Kamiti River virus and Cell fusing agent virus originated by self-duplication

Previously, it was shown that the 3′ untranslated region (3′UTR) of Kamiti River virus (KRV) is nearly twice as long as the 3′UTR of other flaviviruses (1208 nucleotides compared with 730 nucleotides for the longest 3′UTR of any virus in the Tick-borne encephalitis virus species). Additionally, KRV and the closely related Cell fusing agent virus (CFAV) were shown to contain two short, almost perfect repeat sequences of 67 nucleotides. However, the construction of a robust comparative nucleotide alignment has now revealed that the double-length 3′UTR and the direct repeats resulted from the virtually complete duplication of a primordial KRV 3′UTR. We also propose that the CFAV 3′UTR was derived from a KRV-like precursor sequence with a large deletion that nevertheless preserved the two direct repeat sequences. These data provide new insights into the evolution of the flavivirus 3′UTR.