Nucleotide Sequence Analysis Research Articles

Genotyping, antifungal susceptibility testing, and biofilm formation of Trichosporon spp. isolated from urine samples in a University Hospital in Bangkok, Thailand.

The basidiomycetes yeast Trichosporon is widespread in the natural environment, but can cause disease, mainly in immunocompromised patients. However, there have been only few studies about this infection in Thailand. In this study, we characterized 53 Trichosporon spp. isolated from urine samples from patients admitted to a single hospital in Bangkok, Thailand over a one-year period from 2019 to 2020. The strains were identified using colony morphology, microscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and nucleotide sequence analysis of intergenic spacer 1 (IGS1). Fifty-one isolates were Trichosporon asahii, and the remaining isolates were Trichosporon inkin and other Trichosporon species. Three genotypes of IGS1-1, 3, and 7 were observed among T.asahii. The sensitivity of the yeasts to the antifungal drugs amphotericin B, fluconazole, and voriconazole ranged from 0.25 to >16 μg ml-1, 0.5-8 μg ml-1, and 0.01-0.25 μg ml-1, respectively. We investigated biofilm formation by the isolates, and no biofilm production was found in one isolate, low biofilm production in forty-four isolates, and medium biofilm production in six isolates. T. inkin produced biofilms at low levels, and Trichosporon spp. produced biofilms at medium levels. This research increases our understanding of the molecular epidemiology of Trichosporon spp. isolated from one university hospital in Bangkok, Thailand, and reveals their genetic diversity, antifungal susceptibility profiles, and capacity for in vitro biofilm production.

Abnormal overexpression of SoxD enhances melanin synthesis in the Ursa mutant of Bombyx mori

The pigment and structural color of insects play crucial roles in body protection, ecological adaptation, and signal communication. Epidermal melanization is a common and main coloring pattern, which results in broad phenotypic diversity. Melanin is one of the compounds contributing to dark brown-black pigmentation, which is synthesized from dopamine and tyrosine by the melanin metabolism pathway. The Ursa mutant of the silkworm Bombyx mori is a body-color mutant characterized by excessive melanin pigmentation in the larval epidermis. However, the exact gene responsible for this phenotype remains unclear. Here, we performed positional cloning of the gene responsible for Ursa, which was mapped to an 83-kb region on chromosome 14. The genomic region contains a protein-coding gene encoding a transcription factor, which was designated BmSoxD. The mutation site was determined by analysis of nucleotide sequences of the genomic region corresponding to BmSoxD, which identified a 449-bp transposable sequence similar to that of the B. mori transposon Helitron inserted into the sixth intron. BmSoxD was dramatically overexpressed in the epidermis of Ursa at the end of the molting stage compared with that of wild-type B. mori. Overexpression of BmSoxD led to upregulation of genes involved in the melanin metabolism pathway, whereas knocking down BmSoxD via small interfering RNAs blocked melanin pigment production in the larval epidermis. These data indicate that the mutation in BmSoxD is responsible for the Ursa mutant phenotype. We propose that the transposable sequence insertion causes abnormal overexpression of BmSoxD at the molting stage in the Ursa mutant, resulting in excessive melanin synthesis and its accumulation in epidermal cells.

Identification of four distinct ‘Candidatus Phytoplasma’ species in pomegranate trees showing witches' broom, little leaf and yellowing in Jordan, and preliminary insights on their putative insect vectors and reservoir plants

AbstractDuring field surveys conducted in northern Jordan from June to November 2020, phytoplasma‐like symptoms, including leaf yellowing/reddening and rolling, little leaf and witches' broom were observed in pomegranate. Disease incidence in 22 surveyed orchards ranged from 30% to 65%. Nested PCR‐based amplification of 16S rRNA gene detected phytoplasmas in 17% of collected symptomatic pomegranate trees. Amplicon nucleotide sequence analyses allowed attributing the detected phytoplasmas to ‘Candidatus Phytoplasma solani’, ‘Ca. P. aurantifolia’, ‘Ca. P. asteris’ and ‘Ca. P. ulmi’. These phytoplasmas were found in plants showing specific symptoms and differentially distributed in the considered locations. Additionally, three cicadellids (Macrosteles sexnotatus, Cicadulina bipunctata and Psammotettix striatus) and two non‐crop plants (Plantago major and Capsicum annuum) resulted hosting ‘Ca. P. asteris’ strains, and one cicadellid (Balclutha incisa) was carrying a ‘Ca. P. solani’ strain. A new pomegranate disease complex associated with multiple phytoplasmas, including ‘Ca. P. aurantifolia’ and ‘Ca. P. ulmi’, never reported before in this host plant, is described here. Moreover, preliminary indications are provided on its possible epidemiology in Jordan, involving two putative insect vectors (M. sexnotatus, B. incisa) first reported in the Country.

Infection of young foals with Equine Parvovirus-Hepatitis following a fatal non-biologic case of Theiler’s disease

Theiler’s disease (TD) is a (sub-)acute hepatitis in adult horses and one of the most common causes of acute hepatic failure. Recent findings indicate that equine parvovirus hepatitis (EqPV-H) likely causes TD and that its transmission occurs via iatrogenic and/or natural routes. Following the death of an EqPV-H positive mare with TD, close-contact mares and foals in the same paddock were monitored to evaluate if there was any evidence of EqPV-H. For this purpose, the serum of close contact horses was examined 6 and 42 days after the mare’s death for the presence of EqPV-H DNA and changes in liver-associated serum biochemical parameters. The foals had higher EqPV-H viral loads than the mares. Apart from the mare that was euthanized, none of the horses included in this study showed signs of severe disease and nor did they have particularly elevated liver enzymes. Nucleotide sequence analysis revealed no major differences between the viral DNA detected in the serum of the dead mare and any of the in-contact horses. In conclusion, our data confirmed previous findings that horizontal transmission of EqPV-H may occur through close contact between horses.

Genetic variability of the helix 54 of the 23S rDNA and its use as a molecular target for identification of species within the viridans group streptococci (VGS)

The aim of the present study is to establish a method, based on sequence analysis of the helix 54 of 23S rRNA gene, to identify clinical relevant strains belonging to viridans group streptococci (VGS). A set of 25 randomly selected clinical isolates of alpha-hemolytic streptococci from upper respiratory tract were characterized by the routine phenotypic methods (API 20 Strep test). Molecular characterization was assessed by genotypic analysis of the nucleotide sequence of the helix 54 of 23S rRNA and Intergenic spacer region 16S23S. Partial sequencing of the gdh gene was used on 10 strains of mitis group. Sequence variations of the helix 54 allowed the identification of strains to group level and even to species level for certain strains within sanguinis and anginosus groups. Infact, species identification was ambiguous for some strains belonged to the salivarius group (of VGS16 to VGS20) and the mitis group (of VGS1 to VGS14). These results are almost similar to those obtained by sequencing the 16S23S intergenic region. Thus, we use the gdh gene sequencing for the identification of strains, not recognized, within the mitis group. The results generated herein indicate that no single methodology can be used to provide an accurate identification to the species level of all VGS, although nucleotide sequence analysis of the helix 54 of 23S rRNA gene proved to be a reliable method for the identification of VGS to the group level and even to the species level within sanguinis and anginosus groups.

Construction and characterization of the full-length cDNA of an infectious clone of emerging porcine teschovirus-2.

Porcine teschovirus (PTV) is a causative agent of polioencephalomyelitis, encephalomyelitis, reproductive disorders and gastrointestinal and respiratory diseases in swine. In the present study, the PTV2 GX/2020 strain was isolated from pig intestinal tissue through the use of ST cells. Phylogenetic analysis of VP1 nucleotide sequences indicated that the GX/2020 isolate is closely related to PTV2. Furthermore, the full-length cDNA of an infectious GX/2020 clone was constructed using seamless ligation technology. The genome sequence of the rescued virus is largely consistent with the sequence of the parental virus, and it exhibits viral growth properties. The PTV2 virus was successfully isolated in the present study, and the reverse-genetic platform provides a foundation for studies of the pathogenic mechanisms of porcine teschovirus.

Detection of Hepatitis B Virus (HBV) Genotypes by Sequence in Diyala Province

Background: To put it simply, hepatitis is liver inflammation. Hepatitis B virus (HBV) infection is a public health problem because it can lead to serious liver conditions like hepatocellular carcinoma and cirrhosis. Ten HBV genotypes (A-J) were distinguished based on viral sequence similarity. Objective: detection of HBV utilizing polymerase chain reaction (PCR) technique, followed by viral nucleotide sequence and phylogenetic analysis. Methods: A cross-sectional study was conducted in170 reviewers (103 males and 67 females) who attended the Baquba Teaching Hospital, Teaching Laboratories, the Main Blood Bank, and Baladrooz General Hospital. Age ranging 13-75 years. The samples were collected from Diyala Province of Iraq; person data was collected by using questioner, including their name, age, gender, address, academic achievement, source of infection, take vaccine of HBV, and corona infection. Enzyme-linked immune sorbent assay test (ELISA) was used to detect hepatitis B surface antigen (HBsAg) in serum. Amplification Polymerase Chain Reaction (PCR) of HBV p gene of serum samples was performed to detect HBV from serum samples. The sequence of the PCR products of the polymerase gene was sent for Sanger sequencing using ABI3730XL, an automated DNA sequencer, by Macrogen Corporation – Korea for the study of mutations and genotype identification. The results were received by email and then analyzed using geneious software. Results: The positive samples of HBV by ELISA were 70 (41.20%), and 100 (58.80%) samples were negative. While positive samples for HBV-DNA PCR were 14 (8.20%). When compared the sequence of local isolates with NCBI-Blast Hepatitis B virus showed that all isolates were D genotype. Variation result showed that the highest type of mutation was Transition when compared with the NCBI referring sequences. Statistically: The present study revealed there was a significantly different (p<0.05) in the positivity of HBV detected by ELISA and PCR between study groups. Additionally, there was a significantly different (p<0.05) between the positivity of HBV detected by ELISA and PCR and age groups, gender, Social status, address, education , jaundice infection of patients, the HB vaccination, and infection source of patients. Keywords: HBV, ELISA, PCR, Sequence, Variation, Phylogenetic Tree, Diyala, Iraq.

Genetic diversity of porcine reproductive and respiratory syndrome virus and evaluation of three one-step real-time RT-PCR assays in Korea

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses in the global swine industry. Frequent genetic variations in this virus cause difficulties in controlling and accurately diagnosing PRRSV.MethodsIn this study, we investigated the genetic characteristics of PRRSV-1 and PRRSV-2 circulating in Korea from January 2018 to September 2021 and evaluated three one-step real-time reverse transcription polymerase chain reaction (RT-PCR) assays.ResultsA total of 129 lung samples were collected, consisting of 47 samples for PRRSV-1, 62 samples for PRRSV-2, and 20 PRRSV-negative samples. Nucleotide sequence analysis of open reading frames (ORFs) 5, ORF6, and ORF7 genes from PRRSV samples showed that PRRSV-1 belonged to subgroup A (43/47, 91.49%) and subgroup C (4/47, 8.51%), whereas PRRSV-2 was classified as lineage 1 (25/62, 40.32%), Korean lineage (Kor) C (13/62, 20.97%), Kor B (10/62, 16.13%), lineage 5 (9/62, 14.52%), and Kor A (5/62, 8.06%). Amino acid sequence analysis showed that the neutralizing epitope and T cell epitope of PRRSV-1, and the decoy epitope region and hypervariable regions of PRRSV-2 had evolved under positive selection pressure. In particular, the key amino acid substitutions were found at positions 102 and 104 of glycoprotein 5 (GP5) in some PRRSV-2, and at positions 10 and 70 of membrane protein (M) in most PRRSV-2. In addition, one-step real-time RT-PCR assays, comprising two commercial tests and one test recommended by the World Organization for Animal Health (OIE), were evaluated.ConclusionThe results revealed that two of the real-time RT-PCR assays had high sensitivities and specificities, whereas the real-time RT-PCR assay of the OIE had low sensitivity due to mismatches between nucleotides of Korean PRRSVs and forward primers. In this study, we genetically characterized recent PRRSV occurrences and evaluated three one-step real-time RT-PCR assays used in Korea.

Fungal endophytes affecting the health and recovery of Long’s Braya (Braya longii) and Fernald’s Braya (Braya fernaldii), endangered endemic species of Newfoundland, Canada

Long’s ( Braya longii Fernald) and Fernald’s ( B. fernaldii Abbe) Braya are endemic to the Great Northern Peninsula of Newfoundland, Canada. These herbaceous perennials are endangered due to habitat loss and degradation, as well as ongoing threats from fungal infections and insect herbivory. While recovery plans are being implemented, the identity of pathogenic fungi and their origins remain largely unknown. This study was initiated to describe the associated fungal community and infection symptoms. Plant tissues from three populations were sampled over 5 years yielding 326 fungal isolates identified primarily by nucleotide sequence analysis of the internal transcribed spacer rDNA region. Isolates included 36 taxa, 16 of which were detected once and 5 detected three times or less. In contrast, 12 taxa were detected across multiple sites, sampling intervals and years. These included two species of Alternaria, Boeremia exigua, closely related species of the Didymellaceae, Botrytis cinerea, Fusarium sp., Colletotrichum sp., Stagonosporopsis sp., Pleospora sp., and Truncatella angustata. Some common taxa showed seasonal trends. The isolated fungi included known pathogens of Brassicaceae. The plants in this study frequently showed symptoms indicative of fungal infection and insect herbivory. These findings improve our knowledge of Braya endophyte communities and will inform recovery efforts for these endangered species.

NtHash2: recursive spaced seed hashing for nucleotide sequences.

MotivationSpaced seeds are robust alternatives to k-mers in analyzing nucleotide sequences with high base mismatch rates. Hashing is also crucial for efficiently storing abundant sequence data. Here, we introduce ntHash2, a fast algorithm for spaced seed hashing that can be integrated into various bioinformatics tools for efficient sequence analysis with applications in genome research.ResultsntHash2 is up to 2.1× faster at hashing various spaced seeds than the previous version and 3.8× faster than conventional hashing algorithms with naïve adaptation. Additionally, we reduced the collision rate of ntHash for longer k-mer lengths and improved the uniformity of the hash distribution by modifying the canonical hashing mechanism.Availability and implementationntHash2 is freely available online at github.com/bcgsc/ntHash under an MIT license.Supplementary information Supplementary data are available at Bioinformatics online.

On the new classification of dermatophytes

According to the recent changes in the classification of the family Arthrodermataceae, it may already include 9 genera and 79 species. Dermatophytes associated with human and animal infections belong to the genera Trichophyton, Microsporum and Epidermophyton, whereas Arthroderma, Ctenomyces, Guarromyces, Lophophyton, Nannizzia and Paraphyton are mainly represented by geophilic species. Since morphological and physiological traits in fungi cannot provide reliable basis for classification, the modern system of dermatophytes was built on the basis of phylogenetic analysis of nucleotide sequences. The new dermatophyte classification employs the principle of one fungus, one name, according to which genetically similar perfect and imperfect forms belong to the same species.
 At the current state of genosystematics, the taxonomic identification of dermatophytes in most cases requires the use of molecular methods, in particular, DNA sequencing of the ITS region.
 In this review, we briefly outline the history of dermatophyte classification, summarize generic and species composition of the family and provide data on their ecology, accession numbers for reference sequences of ITS region, and references to major taxonomic publications.

Identification of mealybugs on Piper nigrum as vector of Piper yellow mottle virus (Badnavirus: Caulimoviridae)

Piper yellow mottle virus (PYMoV) is the dominant virus that causes mottle disease in black pepper (Piper nigrum). Two species of mealybugs, Ferrisia virgata and Planococcus minor have been reported as vectors of PYMoV. A different species of mealybug that has never been reported before was found in black pepper. Molecular approaches including total DNA isolation of mealybug collected from the field, mealybugs identification by DNA barcode, detection of PYMoV in single mealybugs, were conducted as an approach to identify the potential of mealybugs as PYMoV vector in the field. Mealybugs were collected from black pepper plants in Cimanggu (Bogor, West Java) and Sukamulya (Sukabumi, West Java). Characters of adult females were observed for morphological identification. Molecular-based identification of the mealybugs and PYMoV involved the following procedures: total DNA isolation, DNA amplification, nucleotide sequencing and sequence analysis. Three species of mealybugs, P. minor, F. virgata and Paracoccus marginatus were confirmed by morphological and molecular identification. This is the first report for the occurrence of P. marginatus in black pepper plants. PYMoV was successfully detected from field samples of F. virgata, P. minor and P. marginatus. This finding indicates the potential of insect vectors for disease spread and distribution.

New disease and first report of marasmioid fungus, Marasmius palmivorus (Sharples), causing white root rot in Arachis hypogaea L.

Abstract Groundnut (Arachis hypogaea L.) plants showing distinct symptoms of necrosis of leaves and severe rotting of roots were observed in an agricultural field at Arakkonam, Tamil Nadu, India. The rhizomorphs of the phytopathogenic fungus were obtained from the rotted roots of the diseased plants and were cultured in the laboratory, and based on the morphological characteristics and nucleotide sequencing analysis of ITS and nLSU region, the phytopathogen was identified as Marasmius palmivorus. The isolated fungus produced distinct fruiting bodies (basidiocarps) when grown under the laboratory conditions. The fungus grew as cottony white colonies on the potato dextrose agar medium and were found to contain septate and clamp connections when examined under light microscope. The pathogenicity of the isolated fungus was assessed by inoculating it on healthy groundnut plant under glasshouse conditions; this resulted in the establishment of typical disease symptoms, which confirmed the pathogenicity of the fungus. The fungal pathogen re-isolated from the artificially inoculated plants was used for molecular characterization and confirmed that the organism was M. palmivorus. To the best of our knowledge, this is the first report on the occurrence of M. palmivorus, causing white root rot disease in A. hypogaea L.

Genetic Markers for Detecting the DNA of Pathogenic Borrelia

The aim of the study was to analyze the genetic markers of Lyme disease pathogens, which can be used to specifically indicate maximum number of their strains and isolates. Materials and methods. The nucleotide sequences of various genes of Borrelia garinii, B. afzelii, B. burgdorferi were downloaded from the NCBI database (National Center for Biological Informatization). The occurrence of the analyzed nucleotide sequences in the genetic code of various organisms was determined in the nBLAST software utility. For the design of primers and probes, the Vector NTI 9.1.0 program (“Invitrogen Corporation”, Carlsbad, USA) was used. DNA was isolated using the MAGNO-sorb kit, version 100-200 (“AmpliSens”, Moscow, Russia), according to the manufacturer’s instructions. Primers and probes were synthesized at “Evrogen” company (Moscow, Russia). For PCR, reagents manufactured by “Synthol” company (Moscow, Russia) were applied.Results and discussion. In order to perform the reliable indication of pathogenic Borrelia, specific loci (genes) of B. garinii, B. afzelii, B. burgdorferi, which were significantly different from the genetic code of other representatives of the genus Borrelia and from the DNA of other organisms, have been determined by molecular-genetic methods. As a result of a preliminary determination of the analytical significance of the studied loci, the following genes and loci were selected for further work: pepX, clpA, ospA, p83/100, ospC and flaB, of which the flaB and ospA genes were selected for practical indication of pathogenic Borrelia DNA. The genetic markers of B. burgdorferi and B. afzelii are displayed during amplification of the flaB gene, while B. garinii and B. afzelii occur when the ospA gene is used as a genetic marker.

Squash Leaf Curl Virus: Species of Begomovirus as the Cause of Butternut Squash Yield Losses in Indonesia

Curling symptom was found in pumpkin plants in Bali, and the PCR detection result using Begomovirus universal primers indicated Begomovirus infection. Further research was conducted to determine the distribution, molecular character of SLCV, and yield loss of the pumpkins. Sampling was carried out at pumpkin plantations in 9 districts in Bali. Detection and identification were carried out using PCR method, followed by cloning and DNA sequencing. DNA band with the measurement of 900 bp was successfully amplified from several pumpkin samples from Denpasar, Gianyar, and Buleleng. Homology analysis of nucleotide sequences using the database in GenBank of SLCV of Balinese isolates showed the highest homology and kinship of 97.3-98.4% and 98.4-99.3% respectively with East Timor isolates from pumpkin plants. The phylogeny analysis showed that SLCV Indonesian isolates were in the same group as Asian isolates. The result of this study is the first report on infection and molecular characterization of SLCV in pumpkin plants in Indonesia. The yield loss caused by curling disease on individual pumpkin plants was 56.3%, and the disease caused a decrease in the quality of harvest fruits. Yield loss estimation caused by the disease in fields with different levels of disease intensity ranged from 10.02 to 25.83%. It was equal to yield loss ranging from IDR 878,400.00 to IDR 10,826,400.00 depending on the disease intensity. It is found that the correlation between curling disease severity and yield loss was high.

A novel nepovirus causing a chlorotic fleck disease on common bean (Phaseolus vulgaris L.).

Two common bean leaf samples from Ethiopia that had shown chlorotic fleck and veinal mosaic symptoms but tested ELISA-negative for known viruses were mechanically transmitted to herbaceous hosts to obtain virus isolates ET-773/4 and ET-779. Virus purification from Chenopodium quinoa systemically infected with ET-773/4 yielded icosahedral particles measuring ~ 30 nm in diameter and containing a single capsid protein of ~ 58 kDa, suggesting a nepovirus infection. Analysis of nucleotide sequences generated from RNA1 and RNA2 of the isolates indicated that they represent a distinct virus species in the genus Nepovirus. Surprisingly, the most closely related sequence in the GenBank database was that of Hobart nepovirus 3, an incompletely described metagenomic sequence obtained from honey bees in Tasmania. This new nepovirus from Ethiopia is provisionally named "bean chlorotic fleck virus".

Rickettsia felis and Other Rickettsia Species in Chigger Mites Collected from Wild Rodents in North Carolina, USA.

Chiggers are vectors of rickettsial pathogenic bacteria, Orientia spp., that cause the human disease, scrub typhus, in the Asian–Pacific area and northern Australia (known as the Tsutsugamushi Triangle). More recently, reports of scrub typhus in Africa, southern Chile, and the Middle East have reshaped our understanding of the epidemiology of this disease, indicating it has a broad geographical distribution. Despite the growing number of studies and discoveries of chigger-borne human disease outside of the Tsutsugamushi Triangle, rickettsial pathogens in chigger mites in the US are still undetermined. The aim of our study was to investigate possible Rickettsia DNA in chiggers collected from rodents in North Carolina, USA. Of 46 chiggers tested, 47.8% tested positive for amplicons of the 23S-5S gene, 36.9% tested positive for 17 kDa, and 15.2% tested positive for gltA. Nucleotide sequence analyses of the Rickettsia-specific 23S-5S intergenic spacer (IGS), 17 kDa, and gltA gene fragments indicated that the amplicons from these chiggers were closely related to those in R. felis, R. conorii, R. typhi, and unidentified Rickettsia species. In this study, we provide the first evidence of Rickettsia infection in chiggers collected from rodents within the continental USA. In North Carolina, a US state with the highest annual cases of spotted fever rickettsioses, these results suggest chigger bites could pose a risk to public health, warranting further study.

Isolation, Phenotypic and Molecular Identification of Actinomycetes From Soil and Evaluation of Their Efficiency in Control of the Pathogen Botrytis cinerea Caused Gray Rot Disease on Eggplant

The current study included the isolation of 29 isolates of Actinomycetes from soil samples that collected from different regions of Salah Al-Din Governorate, including districts (Samarra, Tikrit, Balad, Sharqat, Tuz and Ishaqi). The results of the antagonism of these isolates against the pathogenic fungus Botrytis cinerea showed the superiority of isolates S2, S5, Sh3, Sh1 and T2 on other isolates, the inhibition zone of the pathogenic fungus was 2.4, 2.2, 2.2, 2.1 and 1.9 cm, respectively. These isolates were identified using microscopic and biochemical tests, as well as their diagnosis molecularly to the species level using the nucleotide sequence analysis technique of 16S rRNA gene. All isolates belonged to the genus Streptomyces sp., the similarity percentage of the isolates ranged from (99.47-99.62%) with the Streptomyces spp. recorded in the database of the World GenBank. The above isolates were identified as Streptomyces kanamyceticus, S.hygroscopicus, S.achromogenes, S.atratus and S.griseus, respectively, and registered in the database of the World GenBank in NCBI. The filtrates of Streptomyces sp. grown in various media included; Glycerol Tyrosine Medium, Glycerol Yeast Extract Medium, Glycerol Aspargine Medium, Starch-Minerals Medium and Starch Peptone Yeast Extract Medium against pathogenic fungus B. cinerea, the results showed that the S. kanamyceticus filtrate recorded the highest inhibition rate in all media except Starch Mineral medium, which reached 8.13 and 8.66, 4.73, 4.46 and 5.46 mm, respectively. The effectiveness of some active substances with antifungal activity produced from the Streptomyces spp. was estimated, which included the antibiotic Fungichromin, chitinase and beta-glucanase, the results showed the production of fangichromin from S. kanamyceticus, S. hygroscopicus, and S.achromogenes only, which reched to 6.76, 6.66 and 5.4 μg/ml, respectively, on the othe hand, all isolates recorded the activity of chitinase and beta-glucanase, the highest activities were 0.895 and 1.254 units/ml in S. kanamyceticus, respectively.

A new strain of Papaya ring spot virus infecting ivy gourd (Coccinia grandis L.) in India

Ivy gourd (Coccinia grandis L.) plants showing virus likesymptoms such as mosaic mottling and thickening of leaflamina were collected from Tindivanam, Tamil Nadu. ThroughReverse transcription polymerase chain reaction, infectionof potyvirus was confirmed with the yield of desiredamplicon (~1.0 kb) using universal primer pair. Sequenceanalysis of amplified product showed the infection of Papayaring spot virus (PRSV). Further association of PRSV wasconfirmed with PRSV specific primer pair (GK PRSV F/R).The nucleotide sequence analysis showed identity of coatprotein region shared only 84% with PRSV isolate fromThailand in BLAST analysis. In phylogenetic analysis, PRSVinfecting ivy gourd formed a separate cluster from otherPRSV isolates. This confirms the association of distinct strainof PRSV on Ivy gourd in India.

Genetic Characterization of Co-circulated Classic and Very Virulent Infectious Bursal Disease Viruses in Commercial Broiler Flocks of Egypt

In recent years, the reintroduction of the infectious bursal disease virus (IBDV), particularly its severe strains, has imposed considerable cost on the Egyptian poultry industry. The goal of the current study was to investigate the molecular features of IBDV in Egypt from June 2019 to April 2021. A total of 30 field samples (bursa of Fabricius) were collected from broiler farms in which the chickens were vaccinated (Transmune 2512 s/c) at hatching. A highly variable region encompassing VP2 gene was targeted for IBDV screening utilizing reverse transcription-polymerase chain reaction (RT-PCR). Of 30 tested samples, 16 were positive by PCR. To isolate the virus, the bursal suspension was injected into 10-11 day embryonated chicken eggs via the chorioallantoic membrane. Five current positive isolates from 2021 were chosen for nucleotide and amino acid (aa) sequence analysis. Phylogenetically, three of the strains under study belonged to the very virulent (vvIBDV) strains, with 97-98% resemblance to Giza 2008 belonging to the (Genogroup 3) IBDV strain. The remaining two strains were identified as a vaccination strain (genotype 1) that matched the winter field 2512 vaccine strain by a similarity percentage of 98. Mutations in the antigenic locations of (P) domain loops were discovered when the sequencing samples were compared to the existing IBD vaccines. The circulating strains were found to be very similar to vvIBDV serotype 1 genotype 3 strains with mutations in the P domain loop providing a potential reason for the circulation of vvIBDV viruses in Egyptian broiler farms despite the vaccination program.