Phylogenetic Analysis And Searching Bovine Papillomaviruses In Teat Papillomatosis Cases In Cattle By Histopathological, Immunohistochemical And Transmission Electron Microscopy Methods

Papillomaviruses are epitheliotropic viruses causing proliferations in skin, mucosa and various internal organs in different animal species. Especially due to lesions it causes in teats of cattle, it leads to important economical losses in milk sector. In this study, the aim was to diagnose bovine papillomaviruses (BPVs) causing teat papillomas in cattle by immunohistochemical, transmission electron microscopy (TEM) and molecular methods and to detect the defect on tissues by the virus using histopathological method. In addition to this, sequence analysis of the isolated field strains were to be carried out and their genetic and phylogenetic closeness with the strains within the literature were to be detected. After confirming teat papillomatosis in the collected samples using histopathological and immunohistochemical methods, other diagnosis methods were then used. During the TEM examination of teat lesions, intranuclear virus particles were seen in epithelium cells. After carrying out PCR using degenerate primers and type specific primers, 7 samples were detected as positive for BPV and these samples were used for typing using sequence analysis/PCR with type-specific primers. Within these analysis, three out of seven BPV isolates we collected from infected teat tissues of different cattle were detected as BPV-6, two as BPV-10, one as BPV-2 and one as BPV-8. Five isolates we isolated during sequence analysis of positive samples were found in Xipapillomavirus 1 genus, one in Epsilonpapillomavirus 1 genus and another in Deltapapillomavirus genus. As a result, in molecular diagnosis of BPV that takes place in etiology of teat papillomas, using type specific primers proved to be useful following the usage of genotyping in molecular diagnosis of BPV and generate primers in characterization. Detecting BPV types and their prevalence, taking biosafety measures in animal breeding and giving importance to vaccine studies was considered essential.

BBAP amplification profiles of apple varieties

Several types of allergies are currently known and are characterized by an exaggerated response of the immune system to substances from various sources called allergens. One of them is a food allergy, which is becoming more common in the population. For this reason, it is necessary to describe the issue from several aspects including genomic variability of plant allergens. The objective of this study was to analyse intraspecific variability of Bet v 1 of 10 different varieties of apple species (Malus domestica Borkh.). BBAP technique for genomic determination of the presence of Bet v 1 homologs at the DNA level was performed. Degenerate primers that anneal a variable and conserved part of PR-10 protein homologues genes were used in the analyse. Amplicons were generated and formed relatively monomorphic profiles, indicating the stability of the given isoforms of Bet v 1 within the selected apple varieties. To evaluate the potential allergenicity of selected varieties further studies on another molecular level such as a comparison of gene expression of the PR-10 family members and their protein expression levels are needed.

Red Clover Hydroxycinnamoyl-CoA:L-DOPA Hydroxycinnamoyl Transferase, a BAHD Hydroxycinnamoyl-Coenzyme A:L-3,4-Dihydroxyphenylalanine Hydroxycinnamoyl Transferase That Synthesizes Clovamide and Other N-Hydroxycinnamoyl-Aromatic Amino Acid Amides

Red clover leaves accumulate high levels (up to 1 to 2% of dry matter) of two caffeic acid derivatives: phaselic acid (2-O-caffeoyl-L-malate) and clovamide [N-caffeoyl-L-3,4-dihydroxyphenylalanine (L-DOPA)]. These likely play roles in protecting the plant from biotic and abiotic stresses but can also help preserve protein during harvest and storage of the forage via oxidation by an endogenous polyphenol oxidase. We previously identified and characterized, a hydroxycinnamoyl-coenzyme A (CoA):malate hydroxycinnamoyl transferase (HMT) from red clover. Here, we identified a hydroxycinnamoyl-CoA:L-DOPA hydroxycinnamoyl transferase (HDT) activity in unexpanded red clover leaves. Silencing of the previously cloned HMT gene reduced both HMT and HDT activities in red clover, even though the HMT enzyme lacks HDT activity. A combination of PCR with degenerate primers based on BAHD hydroxycinnamoyl-CoA transferase sequences and 5′ and 3′ rapid amplification of cDNA ends was used to clone two nearly identical cDNAs from red clover. When expressed in Escherichia coli, the encoded proteins were capable of transferring hydroxycinnamic acids (p-coumaric, caffeic, or ferulic) from the corresponding CoA thioesters to the aromatic amino acids L-Phe, L-Tyr, L-DOPA, or L-Trp. Kinetic parameters for these substrates were determined. Stable expression of HDT in transgenic alfalfa resulted in foliar accumulation of p-coumaroyl- and feruloyl-L-Tyr that are not normally present in alfalfa, but not derivatives containing caffeoyl or L-DOPA moieties. Transient expression of HDT in Nicotiana benthamiana resulted in the production of caffeoyl-L-Tyr, but not clovamide. Coexpression of HDT with a tyrosine hydroxylase resulted in clovamide accumulation, indicating the host species’ pool of available amino acid (and hydroxycinnamoyl-CoA) substrates likely plays a major role in determining HDT product accumulation in planta. Finally, that HDT and HMT proteins share a high degree of identity (72%), but differ substantially in substrate specificity, is promising for further investigation of structure-function relationships of this class of enzymes, which could allow the rational design of BAHD enzymes with specific and desirable activities.

First Report of Natural Infection of Zucchini by Tomato Chlorosis Virus and Cucurbit Chlorotic Yellows Virus in China

Zucchini (Cucurbita pepo) is an extensively cultivated and important economic cucurbit crop in China. In September 2018 and 2019, interveinal chlorosis and yellowing symptoms, suspected to be caused by either tomato chlorosis virus (ToCV; genus Crinivirus) or cucurbit chlorotic yellows virus (CCYV; genus Crinivirus) or by their co-infection, were observed on zucchini plants in a greenhouse in Shandong Province, China. The incidence of the disease in the greenhouse was 20–30%. To identify the causal agent(s) of the disease, leaf samples from 66 zucchini plants were collected in 14 greenhouses in the cities of Shouguang (n = 12), Dezhou (n = 36), Qingzhou (n = 12), and Zibo (n = 6) in Shandong. Four whitefly (Bemisia tabaci) samples and four symptomatic tomato samples were also collected from these sampling sites (one each for each site) because numerous whiteflies were observed in the sampling greenhouses and ToCV was previously reported in greenhouse tomato plants from these regions (Zhao et al. 2014). To determine whether the symptoms were associated with Crinivirus infection, reverse transcription polymerase chain reaction (RT-PCR) using Crinivirus-specific degenerate primers (CriniRdRp251F/CriniRdRp995R) (Wintermantel and Hladky 2010) was performed first on total RNA extracted using the TRIzol protocol (Jordon-Thaden et al. 2015). Thereafter, the RNA samples were subjected to RT-PCR with ToCV- or CCYV-specific primers (Sun et al. 2016; Gan et al. 2019). Of the 66 zucchini samples, 54 tested positive by the degenerate crinivirus primer pair; and among them, 10 tested positive for ToCV only, 40 positive for CCYV only, and 4 positive for both viruses. Interestingly, while both viruses were detected in all B. tabaci samples, only ToCV was detected in the tomato samples (n = 4). To confirm the identity of the viruses, the amplicons of ToCV (four samples each of tomato, B. tabaci and zucchini) and CCYV (four samples each of B. tabaci and zucchini) were Sanger sequenced (Tsingke Biotechnology Co., Ltd., Beijing, China) after cloning into pMD18-T vectors (Takara, Shiga, Japan). BLASTn analysis demonstrated that all sequences were identical to their respective amplicons. The ToCV sequences (GenBank accession numbers: tomato, MN944406; B. tabaci, MN944404; zucchini, MN944405) shared 100% sequence identity with isolates from Beijing (KT751008, KC887999, KR184675, and KP335046), Hebei (KP217196), and Shandong (KX900412). The CCYV sequence (GenBank accession number MT396249) shared 99.9% sequence identity with isolates China (JN126046, JQ904629, KP896506, KX118632, KY400633, and MK568545), Greece (LT716000, LT716001, LT716002, LT716005, and LT716006), and Cyprus (LT992909, LT992910, and LT992911). To assess the transmissibility of ToCV and CCYV, virus-free B. tabaci (n = 30) were placed in ToCV or CCYV-infected zucchini plants for one day for virus acquisition. Thereafter, the whiteflies were transferred into virus-free zucchini seedlings (cv. ‘Zaoqingyidai’, 4-leaf-stage, n = 6 for each of the control, ToCV and CCYV treatment) for one day. Three weeks after inoculation, all plants that were inoculated with either ToCV or CCYV displayed same symptoms as those observed in the greenhouses, whereas plants in the control group remained symptom free. RT-PCR analysis using ToCV- and CCYV-specific primers confirmed the infection of the plants with the respective virus, whereas control plants were free from the viruses. CCYV has been previously reported on zucchini in Algeria (Kheireddine et al. 2020), Iran (LR585225), and Cyprus (LT992910). To our knowledge, this is the first report of CCYV infection in zucchini in China, and moreover the first report of ToCV infection in zucchini in the world. Clearly, stringent management is needed to minimize the losses caused by these viruses in greenhouse operations in the region.

Phylogenetic Analysis and Searching Bovine Papillomaviruses in Teat Papillomatosis Cases in Cattle by Histopathological, Immunohistochemical and Transmission Electron Microscopy Methods

Papillomaviruses are epitheliotropic viruses causing proliferations in skin, mucosa and various internal organs in different animal species. Especially due to lesions it causes in teats of cattle, it leads to important economical losses in milk sector. In this study, the aim was to diagnose bovine papillomaviruses (BPVs) causing teat papillomas in cattle by immunohistochemical, transmission electron microscopy (TEM) and molecular methods and to detect the defect on tissues by the virus using histopathological method. In addition to this, sequence analysis of the isolated field strains were to be carried out and their genetic and phylogenetic closeness with the strains within the literature were to be detected. After confirming teat papillomatosis in the collected samples using histopathological and immunohistochemical methods, other diagnosis methods were then used. During the TEM examination of teat lesions, intranuclear virus particles were seen in epithelium cells. After carrying out PCR using degenerate primers and type specific primers, 7 samples were detected as positive for BPV and these samples were used for typing using sequence analysis/PCR with type-specific primers. Within these analysis, three out of seven BPV isolates we collected from infected teat tissues of different cattle were detected as BPV-6, two as BPV-10, one as BPV-2 and one as BPV-8. Five isolates we isolated during sequence analysis of positive samples were found in Xipapillomavirus 1 genus, one in Epsilonpapillomavirus 1 genus and another in Deltapapillomavirus genus. As a result, in molecular diagnosis of BPV that takes place in etiology of teat papillomas, using type specific primers proved to be useful following the usage of genotyping in molecular diagnosis of BPV and generate primers in characterization. Detecting BPV types and their prevalence, taking biosafety measures in animal breeding and giving importance to vaccine studies was considered essential.

Determination of Butyrate Synthesis Capacity in Gut Microbiota: Quantification of but Gene Abundance by qPCR in Fecal Samples

Butyrate is formed in the gut during bacterial fermentation of dietary fiber and is attributed numerous beneficial effects on the host metabolism. We aimed to develop a method for the assessment of functional capacity of gut microbiota butyrate synthesis based on the qPCR quantification of bacterial gene coding butyryl-CoA:acetate CoA-transferase, the key enzyme of butyrate synthesis. In silico, we identified bacteria possessing but gene among human gut microbiota by searching but coding sequences in available databases. We designed and validated six sets of degenerate primers covering all selected bacteria, based on their phylogenetic nearness and sequence similarity, and developed a method for gene abundance normalization in human fecal DNA. We determined but gene abundance in fecal DNA of subjects with opposing dietary patterns and metabolic phenotypes—lean vegans (VG) and healthy obese omnivores (OB) with known fecal microbiota and metabolome composition. We found higher but gene copy number in VG compared with OB, in line with higher fecal butyrate content in VG group. We further found a positive correlation between the relative abundance of target bacterial genera identified by next-generation sequencing and groups of but genecontaining bacteria determined by specific primers. In conclusion, this approach represents a simple and feasible tool for estimation of microbial functional capacity.

In Silico Analysis of Actin Gene as a Candidate for DNA Non-Halal Detection Base on Real-Time PCR

Actin genes are genes that are common in organisms, and their expression is constitutive. These genes are used for gene normalization and internal control of DNA extraction, but the actin gene is not widely used for halal certification tests. Bioinformatic studies help to analyze the experiment through in silico more deeply before the experiment is carried out in laboratory, making it more efficient and time effective. uMelt is an analysis to predict the melting curve of target amplification in real-time PCR. Real-time PCR has been widely used for screening and detection of pork content in a product. This research aimed to explore actin gene as a candidate for testing pork using qPCR. The study was carried out in two main stages, namely alignment of the DNA sequence and analysis of the melting curve using the uMelt approach. The results showed a set of actin genes containing conserved regions that can be used as degenerate primers with different family-type coverages. Melting curve prediction with uMelt shows differences in tm peaks so as the types of samples can be easily identified. The use of bioinformatic applications such as uMelt helps in the simulation of predicting the melting curve to increase the precision of the analysis.

First Report of Little cherry virus 1 Infecting Sweet Cherry in Ontario, Canada

The Niagara fruit belt is one of the richest fruit-producing areas in Canada, contributing to 90% of Ontario's tender fruits such as peach, plum and sweet cherry. Little cherry virus 1 (LCV1) of the genus Velarivirus is a causal agent of little cherry disease which has devastated cherry crops in many regions (Eastwell and Bernardy 1998, Jelkmann and Eastwell, 2011). From 2013 to 2018, foliar symptoms indicative of viral infection such as leaf deformation, ringspot, mottling, vein clearing, and reddening were found on sweet cherry trees grown in the Niagara region. To determine if these trees were infected by a virus, small RNAs (sRNAs) were isolated from separately pooled asymptomatic and symptomatic leaves using the mirPremier microRNA isolation kit (Sigma Aldrich Canada, Oakville, ON). The sRNAs were used to create two libraries (four leaves per library) with the TruSeq Small RNA Sample Prep Kit (Illumina, San Diego, CA). The sRNA libraries were separately sequenced with the MiSeq Desktop Sequencer (Illumina, San Diego, CA). In total, 5,380,196 reads were obtained and Trimmomatic (Bolger et al. 2014) was used to remove adaptors. The remaining 4,733,804 clean reads were assembled into contigs using Velvet 0.7.31 (Zerbino and Birney, 2008) and Oases 0.2.09 (Schulz et al. 2012) with minimum length of 75 nt (Supplementary Table 1). A BLASTn search (Altschul et al. 1997) of the contigs identified the presence of Cherry virus A (genus: Capillovirus), two members of the Ilarvirus genus (Prunus necrotic ringspot virus and Prune dwarf virus) in both libraries. LCV1 was only found in contigs derived from the symptomatic library. Of the clean reads, 22,016 were assembled into six contigs (with lengths ranging from 86 to 116 nt, Supplementary Table 1) mapping to LCV1, covering 7.07% of the viral genome. To confirm LCV1 infection, primers were designed from the assembled contigs and used for reverse transcription polymerase chain reaction (RT-PCR). Amplicons were sequenced and the terminal sequences were determined using 5’ and 3’ RACE Systems (Invitrogen, Burlington, ON). Degenerate primers were designed from multiple sequence alignments of published LCV1 genomes for amplification and primer walking to obtain the sequence of LCV1 (Table S2). The complete genome sequence of LCV1 has a length of 16,934 nt and was deposited in GenBank (accession no. MN508820). A BLASTn search showed that this isolate is nearly identical (99.6% sequence identity) to an isolate from California (accession no. MN131067). To determine the incidence of infection, a field survey was performed at the same location during spring months of 2014 to 2018 using RT-PCR with primers specific to the viral coat protein gene (Supplementary Tale 2). Among 46 cherry trees sampled, two (4.3%) trees were infected with LCV1 and showed negative results with CVA, PNRSV and PDV. Both trees displayed mild suturing of primary and secondary veins (Supplementary Figure 1). LCV1 has been identified in Western stone fruit producing regions (British Columbia in Canada, and Washington, California, and Oregon in the United States of America). To the best of our knowledge, this is the first report of LCV1 in any eastern region of Canada. The low incidence of LCV1 suggests that this virus is not widespread in this region. Routine monitoring and detection of LCV1 is required to prevent this devastating cherry disease from spreading in this region.

Evolution and diversity of bat and rodent Paramyxoviruses from North America

Paramyxoviruses are a diverse group of negative-sense, single-stranded RNA viruses of which several species cause significant mortality and morbidity. In recent years the collection of paramyxoviruses sequences detected in wild mammals has substantially grown, however little is known about paramyxovirus diversity in North American mammals. To better understand natural paramyxovirus diversity, host range, and host specificity, we sought to comprehensively characterize paramyxoviruses across a range of diverse co-occurring wild small mammals in Southern Arizona. We used highly degenerate primers to screen fecal and urine samples and obtained a total of 55 paramyxovirus sequences from 12 rodent species and 6 bat species. We also performed illumina RNA-seq and de novo assembly on 14 of the positive samples to recover a total of 5 near full-length viral genomes. We show there are at least 2 well-supported clades of rodent-borne paramyxoviruses, while bat-associated paramyxoviruses formed a putative single well-supported clade. Using structural homology modeling of the viral attachment protein, we infer that three of the five novel viruses likely bind sialic acid in a manner similar to other Respiroviruses, while the other two viruses from Heteromyid rodents likely bind a novel host receptor. We find no evidence for cross-species transmission, even among closely related sympatric host species. Taken together, these data suggest paramyxoviruses are a common viral infection in some bat and rodent species present in North America, and illuminate the evolution of these viruses.