Partial Sequence Analysis Research Articles

A global phylogeny of Elysia Risso, 1818 (Gastropoda: Heterobranchia): molecular systematic insights focusing on European taxa and description of a new species

Abstract The genus Elysia comprises about one-third of the species richness in Sacoglossa. However, the species diversity in the genus remains poorly characterized in some areas like the north-eastern Atlantic and Mediterranean waters. To clarify the systematics of this genus and to characterize the species diversity in undersampled regions, we performed an integrative study based on a thorough literature review, molecular and morphological analyses, and species delimitation approaches. We conducted phylogenetic analyses of partial sequences of two mitochondrial genes (COI and 16S) and two nuclear genes (H3, 28S) using Bayesian inference and maximum likelihood methods, which confirmed the presence of five of the recognized European Elysia species: Elysia viridis, E. timida, E. flava, E. margaritae, and E. rubeni. Moreover, a new species (Elysia azorica sp. nov.) was identified in the Azores, and E. gordanae, currently considered a junior synonym of E. margaritae, was recovered as a distinct species. In addition, we consider E. hetta as a junior synonym of E. gordanae, and E. translucens as a taxon inquirendum. Finally, the tropical E. evelinae is recorded along European coasts for the first time. Our results demonstrate the value of integrative approaches in resolving taxonomic uncertainty surrounding polymorphism and unravelling potential cases of cryptic or pseudocryptic species complexes.

Molecular Characterization and Antimicrobial Susceptibilities of Corynebacterium pseudotuberculosis Isolated from Caseous Lymphadenitis of Smallholder Sheep and Goats.

Caseous lymphadenitis (CLA) is a bacterial infection caused by Corynebacterium pseudotuberculosis (C. pseudotuberculosis) that affects sheep and goats, leading to abscess formation in their lymph nodes. The present study aimed to isolate and identify C. pseudotuberculosis from CLA in smallholder sheep and goats, and determine the resistance patterns, virulence, and resistance genes of the isolates. Additionally, genotypic and phylogenetic analysis of the isolates was conducted using ERIC-PCR and DNA sequencing techniques. A cross-sectional study examined 220 animals (130 sheep and 90 goats) from 39 smallholder flocks for clinical signs of CLA. Fifty-four (24.54%) animals showed CLA-compatible lesions, confirmed by C. pseudotuberculosis isolation and PCR identification. Sheep had a lower infection rate of CLA (18.46%) compared with goats (33.3%). Antimicrobial susceptibility testing of 54 C. pseudotuberculosis isolates to 24 antimicrobial drugs revealed that they were 100% resistant to bacitracin and florfenicol, while none of the isolates were resistant to norfloxacin. A high resistance rate was observed for penicillin and erythromycin (92.6% each). Interestingly, 16.7% of C. pseudotuberculosis isolates recovered from sheep showed vancomycin resistance. Molecular characterization of C. pseudotuberculosis isolates revealed that PLD, PIP, and FagA virulence genes were present in all examined isolates. However, the FagB, FagC, and FagD genes were detected in 24 (100%), 20 (83%), and 18 (75%) of the sheep isolates, and 26 (87%), 26 (87%), and 18 (60%) of the goat isolates, respectively. The β-lactam resistance gene was present in all isolates. Furthermore, 83% of the sheep isolates carried the aminoglycoside (aph(3″)-lb), chloramphenicol (cat1), and bacitracin (bcrA) resistance genes. Among the isolates recovered from goats, 73% were found to contain macrolides (ermX), sulfonamide (sul1), and bacitracin (bcrA) resistance genes. It is worrisome that the glycopeptide (vanA) resistance gene was detected in 8% of the sheep isolates as a first report. ERIC-PCR genotyping of 10 multi-drug-resistant C. pseudotuberculosis isolates showed a high similarity index of 83.6% between isolates from sheep and goats. Nucleotide sequence analysis of partial 16S rRNA sequences of C. pseudotuberculosis revealed 98.83% similarity with biovar Ovis of globally available reference sequences on the Genbank database. Overall, our findings might indicate that C. pseudotuberculosis infection in smallholders in Egypt might be underestimated despite the significant financial impact on animal husbandry and potential health hazards it poses. Moreover, this study highlights the importance of implementing a sustainable control strategy and increasing knowledge and awareness among smallholder breeders to mitigate the economic impact of CLA.

Three new species of Gromia (Protista, Rhizaria) identified from the Romanian Black Sea shelf

The protist genus Gromia was first described in 1835 by Dujardin and while gromiids are prominent in the marine environment, Gromia oviformis was, for a long time, the only valid species regularly recorded. To date, 16 species that are morphologically and/or genetically distinct have been described. While recent studies are documenting their diversity and their ecological importance, G. oviformis has been the sole gromiid species identified in the Black Sea, although unnamed Gromia species have also been recorded. We collected sediment samples from the Romanian continental shelf at varying depths (48 – 58 m) to study the morphological and genetic diversity of gromiids in this part of the Black Sea. Three new species, Gromia bugnae sp. nov., Gromia dianae sp. nov. and Gromia fabi sp. nov., were identified based on an integrative taxonomic approach, thus bringing the total described gromiid species to 19. Analysis of partial SSU rRNA gene sequences confirms that these are distinct species. Additionally, an undescribed species is represented by a sequence from the northern part of the Black Sea (Sevastopol, Kazachya Bay). The study provides further evidence of the diversity of gromiids in the Black Sea and underlines the importance of this little-known group in marginal seas.

16SrRNA sequencing analysis for identification of Klebsiella pneumoniae isolated from the extreme kitchen environment

Swabs from dishwasher samples were collected and cultured on different media, and then a gram stain was conducted on pure colonies to find whether they were Gram-positive or negative. 32-gram negative Isolates were obtained from the dishwasher; then, we chose isolates under study depending on morphological features on previous media for further investigation. 32 Gram-negative isolates were obtained from a dishwasher, and three isolates of Klebsiella pneumoniae were diagnosed by some phenotypic characteristics and approved by using 16 SrRNA partial sequencing analyses. The 3 isolates deposited in the NCBI database under accession number OK 254156.1 for K.pneumoniae strain NPK 323, OK 247423.1 for K. pneumonia as strain CUMB SAM-61, and OK245427.1 for K. pneumonia strain PD17. The phylogenetic tree for 3 isolates was done by using MEGA II software. Many experiments have been conducted on two isolates (OK 247423.1 and OK245427.1 ) because the OK 254156.1 strain was lost during laboratory work and repeated cultures. A hemolysis test on blood agar and a lipase test on egg–yolk agar were done; both isolates showed negative results for hemolysis blood and producing lipase enzyme, while both isolates showed their ability to produce lecithinase enzyme. The two isolates gave an excellent result in the tube method for the biofilm formation test. Also, a good candidate production test was obtained for these two isolates using L.B. acetate agar medium. Conclusion: Bacterial species differ according to the environments in which they live, as the species that are isolated from clinical sources and possess many virulence factors that make them more dangerous and pathogenic to humans differ about the same species if isolated from a variety of external environments, which makes them virulent or have new characteristics that make them adapted to live in the environments from which they are isolated. Keywords: Klebsiella pneumonia, virulence factor, extreme conditions, phylogenetic tree.

Mammalian orthoreoviruses exhibit rare genotype variability in genome constellations.

Mammalian orthoreoviruses (reoviruses) are currently classified based on properties of the attachment protein, σ1. Four reovirus serotypes have been identified, three of which are represented by well-studied prototype human reovirus strains. Reoviruses contain ten segments of double-stranded RNA that encode 12 proteins and can reassort during coinfection. To understand the breadth of reovirus genetic diversity and its potential influence on reassortment, the sequence of the entire genome should be considered. While much is known about the prototype strains, a thorough analysis of all ten reovirus genome segment sequences has not previously been conducted. We analyzed phylogenetic relationships and nucleotide sequence conservation for each of the ten segments of more than 60 complete or nearly complete reovirus genome sequences, including those of the prototype strains. Using these relationships, we defined genotypes for each segment, with minimum nucleotide identities of 77-88% for most genotypes that contain several representative sequences. We applied segment genotypes to determine reovirus genome constellations, and we propose implementation of an updated reovirus genome classification system that incorporates genotype information for each segment. For most sequenced reoviruses, segments other than S1, which encodes σ1, cluster into a small number of genotypes and a limited array of genome constellations that do not differ greatly over time or based on animal host. However, a small number of reoviruses, including prototype strain Jones, have constellations in which segment genotypes differ from those of most other sequenced reoviruses. For these reoviruses, there is little evidence of reassortment with the major genotype. Future basic research studies that focus on the most genetically divergent reoviruses may provide new insights into reovirus biology. Analysis of available partial sequences and additional complete reovirus genome sequencing may also reveal reassortment biases, host preferences, or infection outcomes that are based on reovirus genotype.

Molecular Epidemiology of Dengue Virus Circulating During 2021 Outbreak in Pakistan

Aim: The current study aimed to identify prevalent dengue serotypes during a major outbreak in 2021 in Pakistan. Materials & methods: From September-November 2021, 373 serum samples from different provinces were tested by RT-PCR for serotype identification, followed by partial sequence analysis of selected dengue-positive samples. Results: Out of 373 samples, 273 (73%) were positive for dengue virus infection with male predominance (61.9%). Complete predominance of DENV-2 serotype (99%) and a single case of DENV-1 was observed. Sequencing data revealed the presence of a cosmopolitan genotype within the DENV-2 Pakistani isolates. Conclusion: This study highlights the persistent predominance of DENV-2 during the 2021 outbreak, suggesting a need to improve surveillance strategies and vector breeding control programs in Pakistan.

Genetic characterization of dengue virus serotype 1 circulating in Reunion Island, 2019–2021, and the Seychelles, 2015–2016

BackgroundAn unprecedent increase in the number of cases and deaths reported from dengue virus (DENV) infection has occurred in the southwestern Indian ocean in recent years. From 2017 to mid-2021 more than 70,000 confirmed dengue cases were reported in Reunion Island, and 1967 cases were recorded in the Seychelles from 2015 to 2016. Both these outbreaks displayed similar trends, with the initial circulation of DENV-2 which was replaced by DENV-1. Here, we aim to determine the origin of the DENV-1 epidemic strains and to explore their genetic characteristics along the uninterrupted circulation, particularly in Reunion.MethodsNucleic acids were extracted from blood samples collected from dengue positive patients; DENV-1 was identified by RT-qPCR. Positive samples were used to infect VERO cells. Genome sequences were obtained from either blood samples or infected-cell supernatants through a combination of both Illumina or MinION technologies.ResultsPhylogenetic analyses of partial or whole genome sequences revealed that all DENV-1 sequences from Reunion formed a monophyletic cluster that belonged to genotype I and were closely related to one isolate from Sri Lanka (OL752439.1, 2020). Sequences from the Seychelles belonged to the same major phylogenetic branch of genotype V, but fell into two paraphyletic clusters, with greatest similarity for one cluster to 2016–2017 isolate from Bangladesh, Singapore and China, and for the other cluster to ancestral isolates from Singapore, dating back to 2012. Compared to publicly available DENV-1 genotype I sequences, fifteen non-synonymous mutations were identified in the Reunion strains, including one in the capsid and the others in nonstructural proteins (NS) (three in NS1, two in NS2B, one in NS3, one in NS4B, and seven in NS5).ConclusionIn contrast to what was seen in previous outbreaks, recent DENV-1 outbreaks in Reunion and the Seychelles were caused by distinct genotypes, all likely originating from Asia where dengue is (hyper)endemic in many countries. Epidemic DENV-1 strains from Reunion harbored specific non-synonymous mutations whose biological significance needs to be further investigated.

Diversity and phylogeography of begomoviruses and DNA satellites associated with the leaf curl and mosaic disease complex of eggplant

Eggplant is one of the important vegetable crops grown across the world, and its production is threatened by both biotic and abiotic stresses. Diseases caused by viruses are becoming major limiting factors for its successful cultivation. A survey for begomovirus-like symptoms in 72 eggplant fields located in six different Indian states revealed a prevalence of disease ranging from 5.2 to 40.2%, and the symptoms recorded were mosaic, mottling, petiole bending, yellowing, and upward curling, vein thickening, and enation of the leaves, and stunting of plants. The causal agent associated with these plants was transmitted from infected leaf samples to healthy eggplant seedlings via grafting and whiteflies (Bemisia tabaci). The presence of begomovirus was confirmed in 72 infected eggplant samples collected from the surveyed fields exhibiting leaf curl and mosaic disease by PCR using begomovirus specifc primers (DNA-A componet), which resulted in an expected amplicon of 1.2 kb. The partial genome sequence obtained from amplified 1.2 kb from all samples indicated that they are closely related begomovirus species, tomato leaf Karnataka virus (ToLCKV, two samples), tomato leaf curl Palampur virus (ToLCPalV, fifty eggplant samples), and chilli leaf curl virus (ChLCuV, twenty samples). Based on the partial genome sequence analysis, fourteen representative samples were selected for full viral genome amplification by the rolling circle DNA amplification (RCA) technique. Analyses of fourteen eggplant isolates genome sequences using the Sequence Demarcation Tool (SDT) indicated that one isolate had the maximum nucleotide (nt) identity with ToLCKV and eight isolates with ToLCPalV. Whereas, four isolates four isolates (BLC1-CH, BLC2-CH, BLC3-CH, BLC4-CH) are showing nucleotide identity of less than 91% with chilli infecting viruses begomoviruses with chilli infecting begomoviruses and as per the guidelines given by the ICTV study group for the classification of begomoviruses these isolates are considered as one novel begomovirus species, for which name, Eggplant leaf curl Chhattisgarh virus (EgLCuChV) is proposed. For DNA-B component, seven eggplant isolates had the highest nt identity with ToLCPalV infecting other crops. Further, DNA satellites sequence analysis indicated that four betasatellites identified shared maximum nucleotide identity with the tomato leaf curl betasatellite and five alphasatellites shared maximum nucleotide identity with the ageratum enation alphasatellite. Recombination and GC plot analyses indicated that the bulk of begomovirus genome and associated satellites presumably originated from of previously known mono and bipartite begomoviruses and DNA satellites. To the best of our knowledge, this is India's first report of ToLCKV and a noval virus, eggplant leaf curl Chhattisgarh virus associated with eggplant leaf curl disease.

Occurrence of canine parvovirus type 2c in diarrhoeic faeces of dogs in Kolkata, India.

Canine parvovirus-2(CPV-2) causes a highly contagious disease of dogs characterised by acute hemorrhagic gastroenteritis, lethargy, vomiting, fever and usually bloody or mucoid diarrhoea. In the present study, 41 faecal samples collected from dogs exhibiting the signs of fever, vomition, bloody or mucoid diarrhoea in Kolkata, India were screened by haemagglutination test and PCR for detection of capsid protein coding VP2 gene. The viral genotype was detected by multiplex PCR and analysis of partial VP2 gene nucleotide sequences of selected PCR products with bioinformatics tool. Thirteen (31.71%) samples were found positive with HA titre ≥ 32 whereas 28 (68.29%) samples were positive by PCR of VP2 gene indicating higher sensitivity of PCR. Highest occurrence of CPV-2 was observed in the age group of 1-6 months (80.65%) and non-descript breeds with no history of vaccination (85%). Three samples were antigenic type CPV-2a, rest were CPV-2b/CPV 2c. Six CPV sequences were found to be highly similar to published CPV 2c sequences in BLAST analysis revealing a maximum identity of 99-100% with other CPV-2c strains and clustered together with CPV-2c strains of India and other countries in phylogenetic analysis. The present study highlights the need for continuous monitoring of samples to detect gradual changes in circulating CPV-2 genotypes in India.

Purification and characterization of a serine protease from the fruit of Ficus carica cultivar Masui Dauphine.

Ficus carica produces, in addition to the cysteine protease ficin, a serine protease (FSP). Here, we purified FSP to homogeneity from the fruit of F. carica cultivar Masui Dauphine. An 81-fold enrichment in specific activity of FSP with 2.1% recovery was attained. Three protein bands (70, 62, and 60kDa) were identified on SDS-PAGE. Each band was identified as a subtilisin-like protease (661 amino acids) by trypsin digestion and LC-MS/MS analysis, and the partial N-terminal amino-acid sequence analysis. Gelatin zymography revealed that the active FSP exists as a dimer. The optimum hydrolysis pH of FSP was 7.5, and the pHs at which the enzyme retained its initial activity by 70% in 24h were 8.0-11.0. The optimum hydrolysis temperature of FSP was 50-60ºC, and the temperature required to reduce the initial activity by 50% in 15min was 70ºC. These results will inform the industrial use of FSP.

Molecular detection and characterization of Anaplasma spp. in cattle and sable antelope from Lusaka and North-Western provinces of Zambia

Rickettsiales of the genus Anaplasma are globally distributed tick-borne pathogens of animals and humans with complex epidemiological cycles. Anaplasmosis is an important livestock disease in Zambia but its epidemiological information is inadequate. This study aimed to detect and characterize the species of Anaplasma present in domestic and wild ruminants in Zambia with a focus on the infection risk posed by the translocation of sable antelope (Hippotragus niger) from North-Western Province to Lusaka Province. Archived DNA samples (n=100) extracted from whole blood (sable n=47, cattle n=53) were screened for Anaplasmataceae using 16S rRNA partial gene amplification followed by species confirmation using phylogenetic analysis. Out of the 100 samples, Anaplasma species were detected in 7% (4/57) of the cattle and 24% (10/43) of the sable antelope samples. Of the 14 positive samples, five were determined to be A. marginale (four from cattle and one from sable), seven were A. ovis (sable) and two were A. platys (sable). Phylogenetic analysis of the 16S rRNA partial gene sequences revealed genetic proximity between A. ovis and A. marginale, regardless of host. The detection of Anaplasma in wildlife in Zambia shows the risk of transmission of Anaplasma species associated with wildlife translocation.

Morphological characterization, pathogenicity screening, and molecular identification of Fusarium spp. isolates causing post-flowering stalk rot in maize.

Post flowering stalk rot (PFSR) of maize caused by the Fusarium species complex is a serious threat to maize production worldwide. The identification of Fusarium species causing PFSR based on morphology traditionally relies on a small set of phenomic characteristics with only minor morphological variations among distinct Fusarium species. Seventy-one isolates were collected from 40 sites in five agro-climatic zones of India to assess the diversity of Fusarium spp. associated with maize crops showing symptoms of PFSR in the field. To investigate the pathogenicity of Fusarium spp. causing PFSR sixty isolates were toothpick inoculated between the first and second node at 55 days after sowing during the tassel formation stage of the crop in Kharif (Rainy season), and Rabi (Winter season) season field trials. Ten most virulent Fusarium isolates, based on the highest observed disease index, were identified by homology and phylogenetic analyses of partial sequences of the translation elongation factor 1 α (Tef-1α). Based on morphological traits such as mycelial growth patterns and mycelial pigmentation, Fusarium isolates were divided into nine clusters. The isolates were judged to be virulent based on their ability to decrease seedling vigour in in-vivo situations and high disease severity in field experiments. Pathogenicity test during the Kharif season showed 12 isolates with virulent disease symptoms with a mean severity ranging between 50 to 67 percent disease index (PDI) whereas in Rabi season, only five isolates were considered virulent, and the mean severity ranged between 52 to 67 PDI. Based on pathological characterization and molecular identification, 10 strains of Fusarium species namely, Fusarium acutatum (2/10), Fusarium verticillioides (Syn. Gibberella fujikuroi var. moniliformis) (7/10), Fusarium andiyazi (2/10) recorded the highest diseases index. All these species are part of the Fusarium fujikuroi species complex (FFSC). The distribution of virulent isolates is specific to a geographical location with a hot humid climate. Increased knowledge regarding the variability of Fusarium spp. responsible for PFSR of maize occurring across wide geographical locations of India will enable more informed decisions to be made to support the management of the disease, including screening for resistance in maize-inbred lines.

Isolation and characterization of Priestia megaterium KD7 for the biological control of pear fire blight.

Erwinia amylovora is a plant pathogen that causes fire blight disease in Rosaceous plants, such as pear and apple. To develop an effective biocontrol method to suppress E. amylovora, a total of 16 bacteria were isolated from pear orchard soil in China and screened for antagonistic activity in vitro. Among them, 9 isolates that exhibited antagonistic activity against E. amylovora were identified, including Bacillus atrophaeus, Priestia megaterium (previously known as Bacillus megaterium) and Serratia marcescens based on the partial 16S rDNA sequence analysis and similarity search. The plate confrontation experiments showed that strain 8 (P. megaterium strain KD7) had strong antagonistic activity against E. amylovora. The methanolic extract from cell-free supernatant of strain KD7 displayed high antibacterial activities against E. amylovora. Furthermore, the active compounds of strain KD7 were separated by thin layer chromatography (TLC) and the amino acids were detected by the presence of a spot with retention factor (Rf) of 0.71. Next, three lipopeptides were identified with high-resolution mass spectrometry (HRMS), including C13-surfactin [M+H]+ at m/z 1008.14, C15-surfactin [M+H]+ at m/z 1036.50, and C14-iturin A [M+H]+ at m/z 1043.17. Strain KD7 showed multiple antibiotic resistance, such as ampicillin, erythromycin, penicillin and tetracycline. The detached pear leaves, twigs and fruits assay showed that both protective and curative action with strain KD7 had the ability to decrease the development of fire blight. Taken together, P. megaterium strain KD7 is a potential effective biocontrol agent against fire blight.

Gathered from the vine: a survey of seven grapevine viruses within New England vineyards.

Vineyards in the Southeastern New England American Viticultural Area were surveyed for the incidence of seven major viruses: grapevine leafroll-associated viruses (GLRaV-1, GLRaV-2, GLRaV-3, and GLRaV-4), grapevine fanleaf virus (GFLV), tomato ringspot virus (ToRSV), and tobacco ringspot virus (TRSV). Viruses were detected by ELISA and confirmed by RT-PCR and Sanger sequencing. Multiple viruses were present in 19 out of the 25 vineyards surveyed between 2018 to 2020. GLRaV-3 (27.59%) was the most prevalent virus followed by GLRaV-4 (14.90%), GLRaV-1 (13.52%), GLRaV-2 (11.03%), ToRSV (6.34%), GFLV (5.24%), and TRSV (2.62%). Furthermore, phylogenetic analyses of the viral partial genome sequences acquired in this study revealed that the grapevine viruses present in this area are diverse, indicating that they may have been introduced from different sources. Our findings stress the need for improving the sanitary status of planting materials to avoid the introduction and dissemination of viruses to vineyards in this important wine-producing region of New England.

Genetic Diversity of Norovirus in Children with Acute Gastroenteritis in Southwest Nigeria, 2015–2017

Norovirus (NoV) is a leading cause of viral gastroenteritis globally, especially in children below five years. Epidemiological studies on the diversity of NoV in middle- and low-income countries, including Nigeria, are limited. This study aimed to determine the genetic diversity of NoV in children below five years with acute gastroenteritis at three hospitals in Ogun State, Nigeria. A total of 331 fecal samples were collected from February 2015 to April 2017, while 175 were randomly selected and analyzed using RT-PCR, partial sequencing and phylogenetic analyses of both the polymerase (RdRp) and capsid (VP1) genes. NoV was detected in 5.1% (9/175; RdRp) and 2.3% (4/175; VP1) of samples, with 55.6% (5/9) co-infection with other enteric viruses. A diverse genotype distribution was identified, and GII.P4 was the dominant RdRp genotype detected (66.7%), with two genetic clusters, followed by GII.P31 (22.2%). The rare GII.P30 genotype (11.1%) was detected at a low rate for the first time in Nigeria. Based on the VP1 gene, GII.4 was the dominant genotype (75%), with two variants, Sydney 2012 and possibly New Orleans 2009, co-circulating during the study. Interestingly, both intergenotypic, GII.12(P4) and GII.4 New Orleans(P31), and intra-genotypic, GII.4 Sydney(P4) and GII.4 New Orleans(P4), putative recombinant strains were observed. This finding suggests the first likely report of GII.4 New Orleans(P31) in Nigeria. In addition, GII.12(P4) was first described in Africa and globally in this study, to the best of our knowledge. This study provided insights into the genetic diversity of NoV circulating in Nigeria, which would be useful for ongoing and future vaccine design and monitoring of emerging genotypes and recombinant strains.

Is Xenopus laevis introduction linked with Ranavirus incursion, persistence and spread in Chile?

Ranaviruses have been associated with amphibian, fish and reptile mortality events worldwide and with amphibian population declines in parts of Europe. Xenopus laevis is a widespread invasive amphibian species in Chile. Recently, Frog virus 3 (FV3), the type species of the Ranavirus genus, was detected in two wild populations of this frog near Santiago in Chile, however, the extent of ranavirus infection in this country remains unknown. To obtain more information about the origin of ranavirus in Chile, its distribution, species affected, and the role of invasive amphibians and freshwater fish in the epidemiology of ranavirus, a surveillance study comprising wild and farmed amphibians and wild fish over a large latitudinal gradient (2,500 km) was carried out in 2015-2017. In total, 1,752 amphibians and 496 fish were tested using a ranavirus-specific qPCR assay, and positive samples were analyzed for virus characterization through whole genome sequencing of viral DNA obtained from infected tissue. Ranavirus was detected at low viral loads in nine of 1,011 X. laevis from four populations in central Chile. No other amphibian or fish species tested were positive for ranavirus, suggesting ranavirus is not threatening native Chilean species yet. Phylogenetic analysis of partial ranavirus sequences showed 100% similarity with FV3. Our results show a restricted range of ranavirus infection in central Chile, coinciding with X. laevis presence, and suggest that FV3 may have entered the country through infected X. laevis, which appears to act as a competent reservoir host, and may contribute to the spread the virus locally as it invades new areas, and globally through the pet trade.

Marine flatworm Acanthozoon sp.-associated bacteria with antibiotic property from the Java Sea

Neglected invertebrates, marine flatworms, have attracted global research interest due to their biological and chemical potential properties. The marine flatworms (Turbellaria), Phylum Platyhelminthes, belong to the Polycladida group. There are about 3000 species of free-living flatworms that make a living by hunting and eating other animals. A marine flatworm Acanthozoon sp. was used in this study due to its abundant presence in the site location. Staphylococcus epidermidis is an opportunistic pathogenic bacterium that was previously considered a harmless skin disease bacterium. This species is now considered to be in the first rank among the causative agents of nosocomial infection, specifically in the form of infections of the urinary tract, respiratory tract, and surgical site wounds. The aims of this study were to explore the biological diversity of marine flatworm-associated bacteria with antipathogenic properties and to detect the presence of polyketide synthase (PKS) and nonribosomal peptide synthetase NRPS gene clusters through a molecular approach. Recent studies have shown that S. epidermidis undergoes functional changes in the pro-inflammatory peptide family so that it has functions in immune evasion and biofilm development. Therefore, the search for new antimicrobial compounds is urgently needed due to the limited choice of antibiotic use. In the preliminary screening by overlay test, 7 out of 17 (41.2%) isolates showed antibacterial activities. These isolates were reselected and their activity confirmed by using plug agar and disk-diffusion methods. The FA02, FA03, FA05, FA07, FA13, FA16, and FA17 isolates demonstrated their inhibitory activities consistently against the causative agent of nosocomial infection S. epidermidis. Based on the morphological and 16S rRNA partial sequencing analysis, these isolates were closely related to the genus Virgibacillus, Brevibacterium, Alcanivorax, and Vibrio. None of these seven antibacterial strains possesses PKS-I and PKS-II, except NRPS genes for Virgibacillus salarius strain FA02, V. salarius strain FA16, and V. salarius strain FA17. The results of this study showed that bacteria associated with marine flatworms have future potential as a source of promising natural products for the development of antibiotics.

Incidence and detection of cucurbit mosaic disease in Assam

The cucurbits belong to the family Cucurbitaceous (gourd family) under the order Cucurbit ales which include many economically important vegetable and fruit species. Among various factors, viruses are one of the important factors reducing in quantity as well as quality of cucurbits. These crops are severely affected by the cucurbit mosaic disease worldwide, which is the major viral disease of the cucurbits. Considering the importance of the disease, the present investigation was taken up for incidence, detection, host range study and characterization of viruses associated with cucurbit mosaic disease in Assam. Survey was conducted in 5 districts of Assam (Sonitpur, Biswanath, Jorhat, Sivasagar, Dibrugarh) during 2021-22. Symptomatology revealed presence of various mosaic symptoms in the surveyed areas. Among the five surveyed locations, the symptoms observed in Sonitpur district were severe, whereas Biswanath district were moderate and mild symptoms were observed in Jorhat, Sivasagar, and Dibrugarh districts. Vector incidence study showed that aphid is the predominant vector. Molecular assay confirmed the presence of Cucumber Mosaic Virus in the collected cucurbit samples of surveyed areas; with severe incidence of mosaic disease in Sonitpur (90.91%), moderate in Biswanath (66.67%), mild in Jorhat (14.29%), Sivasagar (11%) and Dibrugarh (25%) districts. Molecular characterization of Cucumber mosaic virus cucurbit isolate from Jorhat district of Assam, was done through partial sequencing and similarity analysis showed 90.94 to 94.74 per cent sequence homology with different worldwide isolates of Cucumber mosaic virus.

Phosphate Solubilizing and Phytate Degrading Streptomyces Isolates Stimulate the Growth and P Accumulation of Maize (Zea mays) Fertilized with Different Phosphorus Sources

Actinobacteria are involved in many soil processes related to plant growth, including the secretion of various bioactive products and nutrient cycling. The aim of this study was to characterize the plant growth promoting traits (phosphate solubilizing activity, phytate degradation ability and indolic compounds production, and organic acids secretion) of seven actinobacteria isolates and evaluate the effect of two selected rhizospheric Streptomyces on growth and phosphorus content of maize in presence of different phosphorus (P) sources (tri-calcium phosphate, triple super phosphate and calcium phytate). Phylogenetic analysis of partial 16S rRNA sequences showed that the Streptomyces isolates were clustered in two distinct separated groups. All Streptomyces isolates produced 2-ketogluconic acid and oxalic acid in fermentation medium supplemented with inorganic P sources, while in the presence of phytate, all isolates secreted 2-ketogluconic acid and malic acid. All the isolates were able to release inorganic P from insoluble phosphate sources and this behavior was negatively correlated with pH reduction. Phytate degradation raged from 1.65% to 95.2%, and total indole compound production ranged from 6.3 to 27.8 µg ml−1. The shoot dry weight, root dry weight, plant height, chlorophyll content and tissue P concentration were significantly (p < 0.05) increased by both inoculations with Streptomyces isolates and by the addition of different phosphorus sources, compared to their appropriate controls. The inoculation of Streptomyces isolates resulted in a significant increase in plant phosphorus content from different phosphate sources. Streptomyces sp. UTMC 1478 (Streptomyces sp. 63) and Streptomyces sp. 47 were suitable inoculants for increasing maize growth and P from soil amended with different phosphorus sources, indicating their potential use as plant growth-promoting phospho-bacteria.

Detection of West Nile Virus Lineage 2 in Eastern Romania and First Identification of Sindbis Virus RNA in Mosquitoes Analyzed using High-Throughput Microfluidic Real-Time PCR.

The impact of mosquito-borne diseases on human and veterinary health is being exacerbated by rapid environmental changes caused mainly by changing climatic patterns and globalization. To gain insight into mosquito-borne virus circulation from two counties in eastern and southeastern Romania, we have used a combination of sampling methods in natural, urban and peri-urban sites. The presence of 37 mosquito-borne viruses in 16,827 pooled mosquitoes was analyzed using a high-throughput microfluidic real-time PCR assay. West Nile virus (WNV) was detected in 10/365 pools of Culex pipiens (n = 8), Culex modestus (n = 1) and Aedes vexans (n = 1) from both studied counties. We also report the first molecular detection of Sindbis virus (SINV) RNA in the country in one pool of Culex modestus. WNV infection was confirmed by real-time RT-PCR (10/10) and virus isolation on Vero or C6/36 cells (four samples). For the SINV-positive pool, no cytopathic effectwas observed after infection of Vero or C6/36 cells, but no amplification was obtained in conventional SINV RT-PCR. Phylogenetic analysis of WNV partial NS5 sequences revealed that WNV lineage 2 of theCentral-Southeast European clade, has a wider circulation in Romania than previously known.