Amplified PCR Products Research Articles

Genotype of Varicella-zoster virus isolated in Jiangsu, China

ObjectiveTo analyze the genotypes of VZV in Jiangsu province to identify vaccine strains and wild strains, providing a molecular biological background for the effective prevention and control of varicella.MethodStratified sampling was used to collect herpes fluid or throat swab from patients diagnosed with varicella. ORF22 was carried out, and the restriction enzyme site of ORF38, ORF54 and ORF62 were detected.ResultsAll 207 virus strains were Clade 2 type by sequencing the PCR products of ORF22. The sequencing results showed that five SNP sites changed compared to the Dumas reference strain(Clade 1). From A to G at 37,902, from T to c at 38,055, from A to C at 38,081, and from G to A at 38,177, from G to A at 39,394. The prevalent VZV genotypes in Jiangsu is consistent with the P-Oka. The restriction enzyme site analysis of PCR amplification products from ORF38 (PstI), ORF54 (BglI), ORF62 (SmaI) showed that all 207 virus strains were wild-type. There were two different types of the wild strains, and 183 strains (88.4%) were PstI (+), BglI (+), SmaI (-). The wild strains between different regions showed no significant differences (χ2 = 0.05, P = 0.982).ConclusionsThe prevalent VZV genotypes are Clade 2 and the prevalent virus strains are wild strains in Jiangsu Province, the primary wild strain observed is mainly PstI (+), BglI (+), SmaI (-).

GWAS for root yield and its contributing traits in Withania somnifera (L.) dunal: First report

Ashwagandha [Withania somnifera (L.) Dunal] is a significant medicinal plant, with limited information on its genetic variability. This study evaluateed genetic diversity among 96 ashwagandha genotypes with the aim of developing strategies to improve root yield and secondary metabolites. A joint ANOVA indicated significant variability within the evaluated panel. Key traits, such as root girth, fresh root yield and dry root yield showed positive and significant correlations at both tested years. Molecular markers (SRAP, ISSR, and SSR) were used to assess genetic diversity. Amplified PCR products ranged from 210 bp (SRAP-22) to 1350 bp (SRAP-19), with PIC value ranged from 0.332 (SSR 20) to 0.494 (ISD 11), indicated moderate informativeness of markers. Genetic diversity analysis using Dice's coefficient formed six clusters, identifying two genotypes (86 and 87) as highly diverse. Population structure analysis revealed five clusters with an Fst range of 0.21–0.36, indicating allele flow among genotypes. The highest genetic differentiation was observed between clusters G2 and G4 (Fst = 0.1253). On the basis of structure analysis, genotypes in cluster G5 exhibited similar genetic make-up. A genome-wide association study (GWAS) identified a 490 bp locus of the SRAP 49 marker as highly effective and informatice. This locus was broadly associated with fresh root yield (FW) and dry root yield (DW) across individual and pooled environments. The positive correlation between these traits suggests simultaneous improvement is feasible. This study highlights the importance of genetic diversity for enhancing root yield and provides valuable insights for the genetic improvement of ashwagandha.

7030 Molecular Analysis Of Congenital Adrenal Hyperplasia-X Syndrome And Development Of A Pilot Panel For Analyzing Structural Variations With Long-Read Sequencing

Abstract Disclosure: J. Kim: None. S. Park: None. J. Yoon: None. D. Kim: None. S. Hwang: None. G. Kim: None. J. Kim: None. J. Choi: None. H. Yoo: None. Purpose: Genomic recombination events between the CYP21A2 and CYP21A1P genes are frequent due to their high sequence homology. Deletions in both the CYP21A2 and TNXB genes result in a chimeric TNXA/TNXB gene, leading to congenital adrenal hyperplasia (CAH)-X syndrome, characterized by a hypermobile form of Ehlers-Danlos syndrome. The aim of study was to determine the frequency of CAH-X syndrome and to develop a pilot panel for structural variation analysis using long-read sequencing technology. Methods: Among the 165 unrelated families with confirmed genetic mutations in the CYP21A2 gene, 44 probands had large deletions in one or both alleles. Long-range PCR was performed in 12 patients out of 44 probands using a forward primer for the 5’-UTR of the CYP21A2 and CYP21A1P and a reverse primer specific for exon 32 of TNXB. The amplified PCR products were then subjected to Sanger sequencing. We constructed a custom panel of 140 kb containing the RCCX modules arranged as STK19-C4A-CYP21A1P-TNXA-STK19B-C4B-CYP21A2-TNXB. In 4 patients with deletions in both the CYP21A2 and TNXB genes, long-read sequencing was performed using the custom panel on the PacBio Sequel II platform. Results: Out of 12 probands, eight harbored TNXA/TNXB chimeras. One patient was homozygous for CAH-X CH1 with a 120 bp deletion in exon 35 and had a Beighton score of 6 for joint hypermobility and multiple diverticula in the ascending colon. Two patients were heterozygous for CAH-X CH1, while three were heterozygous for the CAH-X CH2 with an intact exon 35 and the c.P4058W in exon 40. Two patients carried heterozygous mutations p.S4175N in exon 43, which is an unclassified type. Using long-read sequencing with a custom panel, missense mutations and TNXA/TNXB chimeras were detected in four patients previously identified by conventional direct sequencing and MLPA. Conclusions: This study identified 8 patients with CAH-X syndrome carrying four different TNXA/TNXB chimeras by long-range PCR. Long-read sequencing emerged as a comprehensive and efficient method for the molecular analysis of CAH, especially for the identification of structural variations. Biallelic deletion of TNXB was associated with severe clinical manifestations. Therefore, screening for CAH-X is essential for clinical management and prevention of the long-term consequences of CAH-X syndrome. Presentation: 6/1/2024

9266 Molecular Analysis Of Congenital Adrenal Hyperplasia-X Syndrome And Development Of A Pilot Panel For Analyzing Structural Variations With Long-Read Sequencing

Abstract Disclosure: J. Kim: None. S. Park: None. J. Yoon: None. D. Kim: None. S. Hwang: None. G. Kim: None. J. Kim: None. J. Choi: None. H. Yoo: None. Purpose: Genomic recombination events between the CYP21A2 and CYP21A1P genes are frequent due to their high sequence homology. Deletions in both the CYP21A2 and TNXB genes result in a chimeric TNXA/TNXB gene, leading to congenital adrenal hyperplasia (CAH)-X syndrome, characterized by a hypermobile form of Ehlers-Danlos syndrome. The aim of study was to determine the frequency of CAH-X syndrome and to develop a pilot panel for structural variation analysis using long-read sequencing technology. Methods: Among the 165 unrelated families with confirmed genetic mutations in the CYP21A2 gene, 44 probands had large deletions in one or both alleles. Long-range PCR was performed in 12 patients out of 44 probands using a forward primer for the 5’-UTR of the CYP21A2 and CYP21A1P and a reverse primer specific for exon 32 of TNXB. The amplified PCR products were then subjected to Sanger sequencing. We constructed a custom panel of 140 kb containing the RCCX modules arranged as STK19-C4A-CYP21A1P-TNXA-STK19B-C4B-CYP21A2-TNXB. In 4 patients with deletions in both the CYP21A2 and TNXB genes, long-read sequencing was performed using the custom panel on the PacBio Sequel II platform. Results: Out of 12 probands, eight harbored TNXA/TNXB chimeras. One patient was homozygous for CAH-X CH1 with a 120 bp deletion in exon 35 and had a Beighton score of 6 for joint hypermobility and multiple diverticula in the ascending colon. Two patients were heterozygous for CAH-X CH1, while three were heterozygous for the CAH-X CH2 with an intact exon 35 and the c.P4058W in exon 40. Two patients carried heterozygous mutations p.S4175N in exon 43, which is an unclassified type. Using long-read sequencing with a custom panel, missense mutations and TNXA/TNXB chimeras were detected in four patients previously identified by conventional direct sequencing and MLPA. Conclusions: This study identified 8 patients with CAH-X syndrome carrying four different TNXA/TNXB chimeras by long-range PCR. Long-read sequencing emerged as a comprehensive and efficient method for the molecular analysis of CAH, especially for the identification of structural variations. Biallelic deletion of TNXB was associated with severe clinical manifestations. Therefore, screening for CAH-X is essential for clinical management and prevention of the long-term consequences of CAH-X syndrome. Presentation: 6/1/2024

Reverse Transcription-polymerase Chain Reaction (RT-PCR) Based Detection and Identification of Monopartite Bean Common Mosaic Virus in Mungbean

Background: Bean common mosaic virus (BCMV) which belongs to the genus Potyvirus and the family Potyviridae, causes significant yield losses in legumes across the globe. Traditional methods for BCMV detection and diagnosis, such as visual inspection and serological assays, are often time-consuming, labour-intensive and may lack the sensitivity required for early detection. Advances in molecular techniques have made RT-PCR a powerful tool for the accurate detection of BCMV-infected plants. Methods: Three pairs of specific primers that flank the BCMV coat protein and polyprotein gene were designed using the Primer 3web tool to amplify fragments approximately ranging from 191 to 205 bp. The RT-PCR protocol was then standardized, including the optimization of annealing temperatures for all three primers, followed by the sequencing of the amplified PCR products. The, specificity and sensitivity of primer pair BCMV2 were then established. Result: Three pairs of specific primers were designed and RT-PCR protocol was standardized. The sequenced PCR products showed 98.03% to 99.50 % nucleotide similarity with the corresponding sequences of BCMV isolates used in primer designing. Further, the BCMV2 primer pair specifically amplified the target amplicon (200 bp) from the suspected leaf sample and no amplicon was observed when tested against other pathotypes of potyvirus such as BCMNV, BYMV, CABMV, SMV and PeMoV. The designed primer was sensitive enough to detect 0.05% of target cDNA. Additionally, the RT-PCR protocol was validated using field samples collected from Hyderabad, India. ELISA in combination with RT-PCR using the developed specific BCMV primer pair will ensure a foolproof, sensitive and rapid, procedure for diagnosis of BCMV in the quarantine processing of imported germplasm.

Clinical Characteristics and Prognostic Relevance of Co-Mutated Genes in Acute Myeloid Leukemia Patients with FLT3 Mutations

To investigate the clinical characteristics and influence of co-mutated gene on acute myeloid leukemia patients (AML) with FMS-like tyrosine kinase-3 (FLT3) mutations. A total of 273 FLT3+ AML patients were enrolled, and the co-mutation gene data of the patients were collected to further analyze the prognosis of the patients. FLT3 and other common mutations were quantified by PCR amplification products direct sequencing and second-generation sequencing (NGS). When patients were divided into FLT3- ITD +, FLT3- TKD +, FLT3- ITD ++TKD + and FLT3- ITD -+TKD - group according to the type of FLT3 mutations, it was found that the frequencies of TET2, GATA2, NRAS and ASXL1 mutation were significantly different among the 4 groups (all P < 0.05). When patients were divided into allelic ratio (AR) ≥0.5 and <0.5 group, it was found that the frequencies of FLT3- ITD +, FLT3 -ITD - +TKD -, NPM1, NRAS and C-kit were significantly different between the two groups (all P < 0.05). When patients were divided into normal and abnormal karyotype group, it was found that the frequencies of FLT3- ITD +, FLT3- TKD +, NPM1, GATA2 and C-kit were significantly different between the two groups (all P < 0.05). The median overall survival (OS) of AML patients with FLT3 -TKD + (including FLT3- ITD ++TKD +) was longer than that of patients with FLT3- ITD + alone (P < 0.05). The OS and relapse-free survival (RFS) of AML patients with FLT3++TET2+ were both shorter than those of patients with FLT3++TET2- (both P < 0.05). The mutation frequencies of co-mutated genes are correlated with subtypes of FLT3, karyotype and AR. AML patients with FLT3 -TKD + have longer OS than patients with FLT3- ITD + alone, and patients with co-mutation of TET2 have shorter median OS and RFS.

First report of two Pantoea species causing bacterial leaf blight on wheat in the United States.

Wheat (Triticum aestivum) is an important crop worldwide, contributing to about one third of the global caloric intake. In June 2021, leaves with bacterial blight symptoms, including yellow and necrotic lesions running parallel to veins, were found in several fields across five counties in eastern Colorado (Weld, Morgan, Sedgwick, Baca, and Kit Carson). Plants exhibiting these symptoms were scattered throughout fields, but symptoms appeared consistent across counties. To determine the causal agent and complete Koch's postulates, a 1 cm2 symptomatic leaf area was excised and macerated in 0.5 mL of sterilized water from four field samples. The lysate was spread on yeast extract dextrose calcium carbonate medium (YDC agar, 1% yeast extract, 2% dextrose, 2% calcium carbonate, 1.5% agar) to isolate bacteria. Single colonies of yellow, mucoid morphology were selected and streaked on new YDC plates. Isolate genomic DNA was extracted (Zymo Research Quick-DNA Fungal/Bacterial Miniprep Kit, #D6005), and ~30 ng of gDNA was used to amplify the 16S rRNA, gyrB, and rpoB genes of all four isolates (Barret et al., 2015; Delétoile et al., 2009; Krawczyk et al., 2020; Ogier et al., 2019). Amplified PCR products were cleaned (Zymo DNA Clean & Concentrator kit, #D4033) and Sanger sequenced, and all sequences have been deposited in NCBI (16S rRNA: OR707336, OR707337, OR707338, OR707339), (gyrB: PP407951, PP407952, PP407953, PP407954), (rpoB: PP407955, PP407956, PP407957, PP407958). A BLAST search against whole genomes identified one isolate from Kit Carson county (CO314) and two isolates from Baca county (CO316 and CO317) as Pantoea agglomerans with 100% identity for the 16S rRNA, gyrB, and rpoB genes, and one isolate from Weld county (CO315) was 100% identical to Pantoea allii for all three genes. To complete Koch's postulates and confirm Pantoea sp. as the causal disease agents, isolates were grown as lawns on DifcoTM Nutrient Agar (NA) medium (48h, 28℃), suspended in 10 mM MgCl2 using a final optical density of 0.1 (~109 colony forming units per milliliter (CFU/mL)), and syringe-infiltrated into the entire leaf area of 10-day-old wheat seedling leaves (var. Hatcher). Treatments of 10mM MgCl2 and a field isolate that does not cause symptoms, identified as Pseudomonas synxantha by 16S rRNA and gyrB sequencing, were negative controls. Inoculated wheat plants were transferred to a growth chamber (22℃, 90% relative humidity). Symptoms developed 14 days post inoculation (dpi), with the most severe appearing 21 dpi. Each of the four Pantoea isolates were re-isolated from symptomatic leaves by grinding them in a Tissue Lyser II (Qiagen) with two metal beads and diluting with 0.4 mL of sterile water. A 20 µL sample of each isolate was plated on NA (24h, 28℃). The colonies appeared phenotypically identical to the original isolates, and Sanger sequencing confirmed the identities of the isolates. To our knowledge, this is the first report of P. agglomerans causing disease in wheat in the United States, and the first report of P. allii as a wheat disease-causing agent. This report is consistent with previous communications showing P. agglomerans causing wheat disease in China (Gao et al., 2023), and P. ananatis in Poland (Krawczyk et al., 2020). The growing numbers of reports of Pantoea spp. as pathogens in recent years suggests increasing novel disease emergence on cereals worldwide.

Peptidoglycan recognition protein-1 gene polymorphism and its association with mastitis in Murrah buffalo

A wide variety of environmental or contagious microorganisms implicated in mastitis, impede the economic growth of dairy sector. Identification of polymorphism in candidate gene of host’s immune system and to rule out mastitis resistant allelic form of candidate gene usually remains prime focal point of research. Bovine peptidoglycan recognition protein-1 (PGLYRP-1), exclusively present in the granules of polymorphonuclear leukocytes has direct microbicidal properties. The present study was carried out to find the association between PGLYRP-1 polymorphic alleles with mastitis. Milk samples for somatic cell count and blood samples for PCR-RFLP analysis of PGLYRP-1 gene were collected from 20 mastitis negative and 20 mastitis positive Murrah buffaloes. There was significant difference in somatic cell count of mastitis and mastitis free animals. All amplified PCR products of ~862 bp size of partial region of PGLYRP-1 gene were subjected to each restriction enzyme (HincII or TaqαΙ or ApaI). Polymorphism in the partial region of PGLYRP-1 gene had not been established using PCR-RFLP as uniformity in pattern of digested fragments was seen. Target sequence PGLYRP-1 gene of Murrah buffalo was cloned and sequenced. BLAST analysis revealed sequence identity of PGLYRP-1 of Murrah buffalo with Bos taurus (JN085441.1) sequence at NCBI was 96%, 96% with Bos indicus (JN085440.1) and 96% with Bos indicus X Bos taurus (EU746454.1). In phylogenetic tree, the target sequence of PGLYRP-1 gene of Bubalus bubalis are found more closely related to Bos taurus than to Bos indicus.

Rapid detection of pathogenic E. coli based on CRISPR Cas system.

Access to safe and nutritious food is critical for maintaining life and supporting good health. Eating food that is contaminated with pathogens leads to serious diseases ranging from diarrhea to cancer. Many foodborne infections can cause long-term impairment or even death. Hence, early detection of foodborne pathogens such as pathogenic Escherichia coli strains is essential for public safety. Conventional methods for detecting these bacteria are based on culturing on selective media and following standard biochemical identification. Despite their accuracy, these methods are time-consuming. PCR-based detection of pathogens relies on sophisticated equipment and specialized technicians which are difficult to find in areas with limited resources. Whereas CRISPR technology is more specific and sensitive for identifying pathogenic bacteria because it employs programmable CRISPR-Cas systems that target particular DNA sequences, minimizing non-specific binding and cross-reactivity. In this project, a robust detection method based on CRISPR-Cas12a sensing was developed, which is rapid, sensitive and specific for detection of pathogenic E. coli isolates that were collected from the fecal samples from adult goats from 17 farms in Tennessee. Detection reaction contained amplified PCR products for the pathogenic regions, reporter probe, Cas12a enzyme, and crRNA specific to three pathogenic genes-stx1, stx2, and hlyA. The CRISPR reaction with the pathogenic bacteria emitted fluorescence when excited under UV light. To evaluate the detection sensitivity and specificity of this assay, its results were compared with PCR based detection assay. Both methods resulted in similar results for the same samples. This technique is very precise, highly sensitive, quick, cost effective, and easy to use, and can easily overcome the limitations of the present detection methods. This project can result in a versatile detection method that is easily adaptable for rapid response in the detection and surveillance of diseases that pose large-scale biosecurity threats to human health, and plant and animal production.

Viperidae snakes infected by mammalian-associated trypanosomatids and a free-living kinetoplastid

Trypanosomatids have achieved significant evolutionary success in parasitizing various groups, yet reptiles remain relatively unexplored. The utilization of advanced molecular tools has revealed an increased richness of trypanosomatids in vertebrate hosts. The aim of this study was to identify the trypanosomatid species infecting Bothrops moojeni and Crotalus durissus kept in captivity from 2000 to 2022. Blood samples were obtained from 106 snakes: 73C. durissus and 33 B. moojeni. Whole blood was collected for hemoculture, blood smears and centrifugated to obtain the blood clot that had its DNA extracted and submitted to Nested PCR (18S rDNA gene) to detect Trypanosomatidae. Positive samples were quantified and submitted to both conventional (Sanger) and next generation sequencing (NGS). Cloning of the amplified PCR product was performed for only one individual of C. durissus. To exclude the possibility of local vector transmission, attempts to capture sandflies were conducted using six CDC-LT type light traps. Molecular diagnosis revealed that 34% of the snakes presented trypanosomatid DNA, 47.94% in C. durissus and 3.9% in B. moojeni. The cloning process generated four colonies identified as a new MOTU named Trypanosomatidae sp. CROT. The presence of DNA of five trypanosomatids (Trypanosoma cruzi TcII/VI, Trypanosoma sp. DID, Trypanosoma cascavelli, Trypanosomatidae sp. CROT, Leishmania infantum and Leishmania sp.) and one free-living kinetoplastid (Neobodo sp.) was revealed through NGS and confirmed by phylogenetic analysis. The haplotypic network divided the T. cascavelli sequences into two groups, 1) marsupials and snakes and 2) exclusive to marsupials. Therefore, the diversity of Kinetoplastea is still underestimated. Snakes have the ability to maintain infection with T. cruzi and L. infantum for up to 20 years and the DNA finding of Neobodo sp. in the blood of a C. durissus suggests that this genus can infect vertebrates.

Interpretation and reporting considerations of low-level DNA profiles following MinElute® purification of PowerPlex® 21 amplified products

The generation of high-quality DNA profiles from trace amounts of DNA continues to be an issue in forensic casework. Several methods have been proposed over the years to increase recovery rates for low input DNA, including purification of PCR products, an increase in PCR cycle numbers and increasing injection time or voltage during electrophoresis. In this study, the characteristics of DNA profiles generated using QIAGEN MinElute® purification of Promega PowerPlex® 21 amplified products for low DNA input samples, ranging from 80 pg down to 4 pg, were evaluated. MinElute® purification was found to be a simple, effective and time efficient method, which can greatly improve the resolution of amplified PCR products, recovering 100% of donor concordant alleles from as little 16 pg of input template DNA and generating sufficient allelic information for interpretation from as low as 4 pg inputs. However, as is commonly observed with low template DNA samples, the results exhibited extensive disparity in the effects of stochastic variation in amplification, including increased heterozygote peak height imbalance, stutter ratios and instances of allelic drop-in and drop-out, both within and between replicates. As such, it is important that the extent and variability of these stochastic effects are appropriately incorporated in the development of robust profile interpretation guidelines for DNA profiles generated from purified PCR products.

Frequency of HBsAg variants in occult hepatitis B virus infected patients and detection by ARCHITECT HBsAg quantitative.

To analyze the amino acid substitution caused by mutations in the major hydrophilic region (MHR) of the S-region genes in the serum samples of occult hepatitis B virus infection (OBI), and to explore the reasons for the missed detection of HBsAg. The full-length gene of the S-region in hepatitis B virus(HBV) in the chronic hepatitis B virus(CHB)(10 samples) and OBI groups(42 samples) was amplified using a lab-developed, two-round PCR amplification technology. The PCR amplification products were sequenced/clone sequenced, and the nucleotide sequences of the S-region gene in HBV were compared to the respective genotype consensus sequence. Only 20 of the 42 samples in the OBI group had the S-region genes successfully amplified, with the lowest HBV DNA load of 20.1IU/ml. As S-region genes in HBV, 68 cloned strains were sequenced. In the OBI and CHB groups MHR region, with a mutation rate of 3.21% (155/4828) and 0.70% (5/710). The genetic mutation rate was significantly higher in the OBI group than in the CHB group (P<0.05). The common mutation types in the MHR region were: I126T, L162R, K122E, C124R, and C147Y.Mutations at s122, s126, and s162 were associated with subgenotypes, most of which being C genotypes. The high-frequency mutation sites L162R and K122E found in this study have not been reported in previous literature. The results of this study confirmed that MHR mutations can cause the missed detection of HBsAg, giving rise to OBI.

Evaluation of post-PCR purification methods and subsequent optimisation of the MinElute® PCR Purification Kit for low input DNA samples amplified with PowerPlex® 21

Weak and partial DNA profiles are commonly encountered within forensic casework due to amplification of low DNA input samples. One option for increasing allelic detection in such samples is the purification of amplified PCR product using commercially available column-based methods. In this study, four commercially available post-PCR purification methods, QIAGEN MinElute®, Independent Forensics Amplicon™ Rx, Millipore Microcon® and Thermo Fisher Scientific ExoSAP-IT™ were evaluated, comparing the quality of PowerPlex® 21 DNA profiles produced to the standard DNA profile generated prior to purification. An increased detection of alleles above the analytical threshold was observed following purification with the MinElute®, Amplicon™ Rx and Microcon® methods, allowing informative DNA profiles to be recovered using as little as 8 pg DNA. However, post-PCR purification using the ExoSAP-IT™ kit was unsuccessful, with no alleles detected above analytical threshold in samples with ≤16 pg DNA. The MinElute® kit was selected for optimisation on the basis of DNA profile quality, including increased detection of alleles and minimal artefacts. The MinElute® method was optimised by evaluating the number of washes and final elution buffer volume, resulting in a further increase in detection of alleles by reducing the elution buffer volume. Overall, this study showed that PowerPlex® 21 DNA profiles from low input DNA can be successfully enhanced by employing the MinElute® post-PCR purification method.

Detection and Phylogenetic Analysis of Orf Virus and Muskox Rhadinovirus 1 from Muskoxen (Ovibos moschatus) in the Canadian Arctic.

Orf virus (genus Parapoxvirus) has been associated with gross skin lesions on muskoxen (Ovibos moschatus) from Victoria Island, Nunavut, Canada, where muskox populations are experiencing population declines. Orf virus causes painful proliferative and necrotizing dermatitis upon viral replication and shedding, which may lead to animal morbidity or mortality through secondary infections and starvation. Herpesvirus, known to cause gross lesions on skin and mucosa during active viral replication, has also been documented in muskoxen but to date has not been associated with clinical disease. Our objective was to characterize the variation of orf virus and herpesvirus in wild muskoxen of the Canadian Arctic. Tissue samples including gross skin lesions from the nose, lips, and/or legs were opportunistically collected from muskoxen on Victoria Island, Nunavut and Northwest Territories, and mainland Nunavut, Canada, from 2015 to 2017. Sampled muskoxen varied in age, sex, location, hunt type, and body condition. Tissues from 60 muskoxen were tested for genetic evidence of orf virus and herpesvirus infection using PCR targeting key viral genes. Tissues from 38 muskoxen, including 15 with gross lesions, were also examined for histological evidence of orf virus and herpesvirus infection. Eleven muskoxen (10 from Victoria Island and one from mainland Nunavut) with gross lesions had microscopic lesions consistent with orf virus infection. Muskox rhadinovirus 1, a gammaherpesvirus endemic to muskoxen, was detected in 33 (55%) muskoxen including 17 with gross lesions. In all tissues examined, there was no histological evidence of herpesvirus-specific disease. Sequencing and characterization of amplified PCR products using phylogenetic analysis indicated that a strain of orf virus, which appears to be unique, is likely to be endemic in muskoxen from Victoria Island and mainland Nunavut. Many of the muskoxen are also subclinically infected with a known muskox-endemic strain of herpesvirus.

Behavioral and neurocognitive phenotypes in Crigler-Najjar syndrome in Tunisia

IntroductionCrigler-Najjar 1 (CN1) due to exon 3 mutations of the UGT1A1 gene is a not rare genetic disease in Tunisia with a founder effect. CN1 syndrome is very severe, and most of CN1 Tunisian patients die soon after birth, within a maximum of one year, due to kernicterus. Liver transplantation, which is the only available therapeutic method for CN1, remains unreachable.ObjectivesThe aim of this study was to report behavioral and neurocognitive phenotypes in CN1 patients who survived to school enrollment.Methods We have selected all patients evaluated from 2004 to 2010, both clinically and molecularly, for a deficiency of bilirubin-UGT enzyme activity leading to a pathological elevation of unconjugated bilirubin with a suspicion of CN1 syndrome. Direct sequencing of targeted PCR amplification products was performed for molecular analysis of UGT1A1. Behavioral and mental features of patients were studied through our genetic counselling.Results We identified 15 patients with the homozygous c.1070 A&gt;G Tunisian mutation. Their age at diagnosis ranged from one week to 9 months for 13 patients. Six of them died within a month of molecular investigation. Only two boys were of school age, i.e. 6 and 9 years. The first had been hospitalized at 3 months year-old for a prolonged jaundice treated with phenobarbital and phototherapy. His psychomotor and neurological development was normal, with school attendance at the age of six. The second patient presented with an unexplored jaundice at the age of 3 days, which was later complicated by seizures and treated with phenobarbital. Despite neurological and motor sequelae associated to language impairments with slurred speech, he attended school at the age of six.ConclusionsThe neurological and behavioral profile of CN1 patients depends on familial and medical management. Quick diagnosis, close follow up and early liver transplantation can improve prognosis.Disclosure of InterestNone Declared

Detection and Molecular Characterization of Canine Coronavirus Based on Partial Membrane Gene Sequences

Canine coronavirus (CCoV) infection in dogs is common all over the world and progresses with gastroenteritis findings. Infection as a result of complications with secondary factors may result in death, especially in puppies. The virus, which is excreted in the feces, spreads indirectly through the contamination of food, water, and the environment. This study, it was aimed at revealing the CCoV infection and obtaining current molecular information about the infection. In addition, molecular characterization of CCoV strains circulating in the region was made based on the M (membrane protein) gene. The study material consisted of stool samples from 12 dogs with gastroenteritis findings. The amplified PCR products were subjected to sequence analysis and a phylogenetic tree was constructed by comparing them with different reference CCoV isolates from GenBank. In the phylogenetic tree, 1 of the 5 positive samples was determined to be CCoV-I, and 4 samples were determined to be CCoV-IIa. It was determined that the strains obtained were 85.4 - 97.7% similar among themselves and 82.7-98% similar to other strains obtained from GenBank. As a result of study, current molecular information about CCoV circulating in the Balıkesir region was obtained. With this study, it is thought that new research on the existence and molecular epidemiology of CCoV infection in Türkiye will make important contributions to vaccine studies and the control of infection.

Molecular Characterization and Seasonal Variation in Expression of HSP70.1 Gene in Gangatiri Cattle and Its Comparison with Buffalo.

Under tropical climate heat stress is a major challenge for livestock production. HSP70.1 is a ubiquitously expressed protein maintaining cellular machinery through proper folding of denatured proteins and prevents cellular apoptosis and protect cell from heat stress. Therefore, present investigation was undertaken to explore genetic variability in HSP70.1 gene in Gangatiri cattle, its comparison with buffalo sequences and differential expression in different season. The allelic variant was identified by sequencing amplified PCR product of HSP70.1 gene by primer walking. Season-wise total RNA samples was prepared for differential expression study. Brilliant SYBR Green QPCR technique was used to study the expression kinetics of this gene. DNA sequencing by primer walking identified four allelic variants in Gangatiri cattle. Sequence alignment study revealed four, six and one substitutions in the 5' untranslated region (5'UTR), coding and 3' untranslated region ((3'UTR) of HSP70.1 gene, respectively. Comparative analysis of HSP70.1 gene revealed that Cattle has shorter 5'UTR and 3' UTR than the buffalo. In Gangatiri cattle, summer season has significantly higher (P ≤ 0.05) expression of HSP70.1 than the spring and winter. The relative expression of HSP70.1 was increased by more than six folds in summer and nearly 1.5 folds higher in winter in comparison to the spring season. Therefore, HSP70.1 may be considered to have a critical role in the development of thermal tolerance in Gangatiri cattle.

First Report of Fusarium flocciferum Causing Wilt Disease of Chili (Capsicum annuum) in Northern Himalayas

In August 2020 chili (Capsicum annuum L.) showing wilt symptoms were collected from different districts of the Kashmir: Pulwama, Srinagar, Baramulla, and Anantnag. From each district one location was selected for sample collection and a total of 23 chili isolates were isolated. The tissue bit technique was used to isolate fungus from the infected samples on potato dextrose agar (PDA) medium, purified using the single spore technique, maintained at 25⁰±1℃ and then stored at 4° C (Ferniah et al. 2014) . Initially cultural characteristics appeared as white colonies which gradually turned to pale white colored and attained a growth of 90 mm in 18 days of incubation at 25 ± 1°C. Microscopic observations revealed that mycelium was branched and cylindrical, 3.53–4.98 µm in width. Microconidia were ellipsoidal, hyaline, 0-1 septa werepresent, and 6-7 x 3-4 µm in size. Macroconidia were cylindrical, hyaline, 2-6 septa, measuring 20-60 x 40-45 µm in size. Molecular identification of the pathogens with ITS, TEF, and RPB2 was successfully carried out and the fungi was confirmed as Fusarium flocciferum infecting chili. Amplified PCR products were sequenced and were successfully submitted and accessioned in GenBank with accession number OM189458, OM441199, OR484037 for ITS, TEF, and RPB2 gene. To confirm Koch’s postulates pathogenicity test was carried out using rhizosphere inoculation technique (Najar et al. 2011, Parihar et al. 2022). In total 7 replications for sand maize meal medium (potting mixture) was prepared by autoclaving 90 g of sand and 10 g of maize meal in 250 ml of erlenmeyer flask comprising 40 ml of distilled water. The spore suspension at 100 µl per pot was inoculated and was mixed with the sterilized potting mixture in a ratio of (2:1) and up to seven days pathogen was allowed to infect the soil (Davey and Papavizas 1962; Hami et al. 2021). Then chili seeds (cv. Kashmir long-1) were sown in infected potting mixture and grown for three weeks to allow the pathogen to infect the host plants. F. flocciferum took six weeks for appearance of symptoms in the infected potted plants. Control mock inoculation of the potting mixture was carried out using water droplets instead of spore suspension at 100 µl per pot. Seven replications were kept for both inoculated and un-inoculated / control mock pots. The plants developed initial symptoms from light green to yellowish discoloration of leaves followed by the drooping, shriveling, and ultimately leading to death. The collar region of the plant was cut vertically and observed that vascular bundles showed brownish spots and discoloration, indicating wilt as the cause of death. The pathogens were re-isolated and inoculated from all infected plants, then compared with their original pure culture inoculated first, which completely resembled based on morphological, cultural, and pathogenic characteristics. No symptoms were observed on control plants. A phylogenetic analysis was also carried out using ClustalW software that grouped the species identified by different genes into different clades. F. flocciferum has been reported earlier in pea, faba bean and bamboo (Kainthola et al. 2022; Šišić et al. 2020) . In solanaceous crops, this species have been explored as wilt pathogens for the first time from India, indicating diversifying nature of Fusarium flocciferum across various hosts including solanaceous crops.

First report of grapevine asteroid mosaic-associated virus in grapevines in China.

Grapevine asteroid mosaic-associated virus (GAMaV), a member of the genus Marafivirus of the family Tymoviridae, was first described to infect grapevines in California (Abou Ghanem-Sabanadzovic et al. 2003). Since then, GAMaV has been reported from Greece, Japan, Canada, Uruguay, France, Hungary, Italy, Spain, Switzerland and Russia, and also in some free-living grapevines in North America (Kyriakopoulou, 1991; Morán et al., 2021; Reynard et al., 2022; Shvets et al., 2022; Thompson et al., 2021). GAMaV may be associated with grapevine asteroid mosaic disease (Martelli 2014). In August 2022, a grapevine cv. Cabernet Sauvignon exhibiting chlorotic mottling was collected in Ningxia, China. Total RNAs were extracted using RNAprep Pure Plant Plus Kit (DP441, TIANGEN BIOTECH, Beijing), and the ribosomal RNA were removed by the Epicentre Ribo-Zero rRNA Removal Kit (Epicentre, Madison, WI, USA). The ribosomal RNA-depleted RNAs were then used to construct a cDNA library using a TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, USA), which was sequenced on an Illumina NovaSeq 6000 platform (Biomarker Biology Technology), resulting in 39,297,567 paired-end clean reads (150 nt × 2). Reads mapping to the grapevine genome (GenBank accession no PN40024) were removed using hisat2 2.1.0 software. The 15,003,158 unmapped reads were de novo assembled into 70,512 contigs using the rnaviralSPAdes method in the SPAdes v3.15.3 software with default parameters and analyzed through BLASTn and BLASTx analysis. Five viruses and two viroids were identified: GAMaV (5 contigs), grapevine Pinot gris virus (3 contigs), grapevine berry inner necrosis virus (3 contigs) , grapevine rupestris stem pitting-associated virus (4 contigs), grapevine red globe virus (2 contigs), grapevine yellow speckle 1 viroid (4 contigs) and hop stunt viroid (3 contigs). The five contigs of GAMaV were 352 nt~2, 224 nt in length, which were assembled from 3, 308 reads and shared 85.56%~91.81% nt identity with the genome of the GAMaV isolate GV30 (KX354202) with 93.3% coverage. To further confirm the infection of GAMaV, we designed two pairs of primers, GAMaV-mel1a/1b (5'-CACCTCGCCCCCTACCTTGAC-3'/5'-AAGAGGACGCCTTTGCGGGAG-3') and GAMaV-cp1a/1b (5'-CTAGCGACGACCGCACTGATC-3'/5'-GTCGGTGTACGAGATTTGGTC-3'), which were used to amplify the 329-bp and 440-bp fragments in the helicase (Hel) domain and coat protein (CP) gene of GAMaV genome in RT-PCR, respectively. The amplified PCR products were cloned and sequenced and the two sequences (OQ676951 and OQ676958) showed 91.2% and 93.4% nt identity with the isolate GV30, respectively. Furthermore, 429 grapevine samples of 71 cultivars were collected from 21 provinces and tested by RT-PCR using the above primer pairs. The results showed that 1.4% (6/429) of the samples tested positive, including one 'Autumn seedless' grapevine (Liaoning province), two 'Dawuhezi' (Liaoning), one 'Cabernet Gernischt' (Liaoning) and two 'Cabernet Sauvignon' (Tianjing and Shandong respectively). The partial sequences of the Hel domain (OQ676952-57) and CP gene (OQ676959-61) obtained from the positive samples by sequencing showed 89.1% to 84.5% and 93.6% to 93.9% nt identity with the isolate GV30, respectively. Because these GAMaV-positive grapevines did not show distinct symptoms, GAMaV pathogenicity remains challenging to confirm. This is the first report of GAMaV in grapevines in China, extending the information on its geographical distribution.

End-Point RT-PCR Assays for Detection and Typing of Epizootic Hemorrhagic Disease Virus.

The emergence of EHDV in Europe during the autumn of 2022 reinforces the need for molecular tools (RT-PCR) for rapid detection of animals infected with this virus. Viral genome testing can be performed on whole blood under anticoagulant, spleen, and bloody organ homogenates from ruminants. It can also be performed on cell culture following viral isolation tests. Various so-called classical or end-point RT-PCRs will be described, which permit the amplification of a part of the viral genome (targeting segment 7) allowing the detection of EHDV whatever the serotype (pan-RT-PCR) and also to amplify a portion of the gene coding the viral protein (VP) 2 enabling serotyping. The PCR amplification products are visualized by agarose gel electrophoresis. Sequencing of the type-specific RT-PCR amplification products allows for the serotype of the virus to be determined.