Partial Nucleotide Sequences Research Articles

Distribution and phylogenetics of hepatitis E virus genotype 4 in humans and animals.

Worldwide, hepatitis E virus (HEV) infection is considered a significant public health concern. In particular, HEV genotype 4 (HEV-4) has spread to more areas and host species. In this study, we describe the global distribution of HEV-4 and characterize HEV-4 subtypes by host, country and year of isolation. We retrospectively collected HEV-4 sequences available before December 31, 2019, in GenBank. HEV-4 and its subtypes were determined using phylogenetic comparison with HEV reference sequences. Information on the isolation of the sequences was extracted from the GenBank or original publications. Temporal, spatial and host characteristics of the sequences were summarized and nucleotide similarity was calculated based on five amplified fragments within HEV genome, stratified by host, country and year. A total of 2295 HEV-4 complete and partial nucleotide sequences were studied. The majority (92.7%) was isolated in China's mainland, Japan, Hong Kong and France. A total of 20 animal hosts were documented, though swine remained predominant (71.7%). Globally, prevalent HEV-4 subtypes changed remarkably over the last 18years. Subtypes 4a, 4b, 4d and 4h were most commonly isolated (80.3%). Subtypes 4c, 4e, 4f, 4g and 4i remained limited in temporal distribution. High nucleotide similarities were observed between the sequences amplified in HEV ORF2, in the same and neighbouring countries, and in similar animal hosts. China and Japan are endemic for HEV-4, and have all the subtypes. In Europe, France has a high prevalence of HEV-4. Increases in affected areas and animal hosts imply consistent cross-border and cross-species transmission.

Determination of virulence and genetic variability of three Spodoptera littoralis nucleopolyhedrovirus isolates from Egypt

BackgroundThe genetic diversity is naturally occurring in baculovirus population, which may affect the potency of the bio-agent. The Spodoptera littoralis nucleopolyhedrovirus is a promising bio-control agent that can be used to control the cotton leafworm, S. littoralis in Egypt. This study described the virulence and genetic variability of 3 S. littoralis nucleopolyhedrovirus isolates named Cab2, P2 and P3 previously recovered from diseased S. littoralis larvae of cabbage, pepper and eggplant cultivation fields.ResultsThe viral genomic DNA restriction analysis using PstI and ScaI endonucleases showed identical Pst1 restriction pattern of 10 fragments in both Cab2 and P3 isolates, while there are 7 extra fragments present in P2 isolate of about 25, 19.8, 15, 14, 11, 9 and 6.7 (kbp). On the other hand, the restriction pattern using ScaI revealed 10 common fragments in all three isolates, in addition to an extra marker fragment of about 25 kb in P3 isolate absent in both P2 and Cab3 isolates. The phylogenetic analysis of the partial nucleotide sequence of the polyhedrin gene confirmed an extensive degree of co-linearity among the genomes of the 3 tested isolates, with pairwise sequence identities of 97.04% with S. littoralis nucleopolyhedrovirus isolate A26-5 (Acc. No. AY706717). The median lethal concentration (LC50) value revealed that Cab2 had the best virulence toward the first instar of S. littoralis larvae with LC50 value of 1.59 × 104 Obs/ml compared to the virulence recorded by P3 and P2 which were 1.23 × 106 and 2.7 × 104 OBs/ml, respectively. The median survival time (LT50) value of Cab2 isolate was the highest speed of treated larval mortality with 120 hpi compared to P2 and P3, which recorded 132 hpi for both isolates.ConclusionsAlthough all three S. littoralis nucleopolyhedrovirus isolates showed considered genotypic variation and insecticidal activity against S. littoralis larvae, the variant Cab2 showed the highest virulence and speed mortality against the first instar larvae of S. littoralis. The new variant proved to possess promising insecticidal properties for the development of virus-based biopesticide for the control of S. littoralis population in Egypt.

Experimental Cross-Species Transmission of Rat Hepatitis E Virus to Rhesus and Cynomolgus Monkeys.

Rat hepatitis E virus (rat HEV) was first identified in wild rats and was classified as the species Orthohepevirus C in the genera Orthohepevirus, which is genetically different from the genotypes HEV-1 to HEV-8, which are classified as the species Orthohepevirus A. Although recent reports suggest that rat HEV transmits to humans and causes hepatitis, the infectivity of rat HEV to non-human primates such as cynomolgus and rhesus monkeys remains controversial. To investigate whether rat HEV infects non-human primates, we inoculated one cynomolgus monkey and five rhesus monkeys with a V-105 strain of rat HEV via an intravenous injection. Although no significant elevation of alanine aminotransferase (ALT) was observed, rat HEV RNA was detected in fecal specimens, and seroconversion was observed in all six monkeys. The partial nucleotide sequences of the rat HEV recovered from the rat HEV-infected monkeys were identical to those of the V-105 strain, indicating that the infection was caused by the rat HEV. The rat HEV recovered from the cynomolgus and rhesus monkeys successfully infected both nude and Sprague-Dawley rats. The entire rat HEV genome recovered from nude rats was identical to that of the V-105 strain, suggesting that the rat HEV replicates in monkeys and infectious viruses were released into the fecal specimens. These results demonstrated that cynomolgus and rhesus monkeys are susceptible to rat HEV, and they indicate the possibility of a zoonotic infection of rat HEV. Cynomolgus and rhesus monkeys might be useful as animal models for vaccine development.

Paraphoma garibaldii sp. nov. causing leaf spot disease of Campanula rapunculoides in Italy.

Leaf and stem spots are among the most important diseases compromising ornamental plants worldwide. In this study, Paraphoma garibaldii sp. nov. is described from leaf lesions on Campanula rapunculoides in Piedmont, Northern Italy. The new species was characterised using a polyphasic approach including morphological characterisation and a multilocus molecular phylogenetic analysis based on partial nucleotide sequences of the translation elongation factor 1-α (tef1), the internal transcribed spacers (ITS) region and the β-tubulin (tub2) markers. Pathogenicity tests and the fulfilment of Koch's postulates confirm P. garibaldii as a novel foliar pathogen of Campanula rapunculoides. Presently, the fungal infection due to Paraphoma garibaldii is known from a single location in Italy, and further surveys are required to determine its distribution and relative importance. Citation: Guarnaccia V, Martino I, Tabone G, Crous PW, Gullino ML (2022). Paraphoma garibaldii sp. nov. causing leaf spot disease of Campanula rapunculoides in Italy. Fungal Systematics and Evolution 9: 19-26. doi: 10.3114/fuse.2022.09.03.

Detection of a New Variant in Outbreak of Bovine Ephemeral Fever in Turkey, 2020

Background: In this study, partial nucleotide sequence analysis of the G gene was performed for the molecular characterization of the virus that caused the bovine ephemeral fever virus (BEFV) epidemic in Turkey in 2020. Phylogenetic analysis of these nucleotide sequences was performed with the virus nucleotide sequences of the epidemics seen in 2008 and 2012. These sequences were announced in GenBank. Phylogenetic analysis of these nucleotide sequences was performed with the virus nucleotide sequences of the epidemics seen in 2008 and 2012. Methods: The study was conducted in dairy cattle holdings located in Diyarbakır Sur, Çınar and Dicle regions in South-eastern Turkey in August-November 2020. The number of animals in the holdings consisted of 750 (n=750), 150 (n=150) and 200 (n=200) cattle, respectively. Result: Severe respiratory symptoms and high mortality in the affected animals were notable symptoms. As a result of the phylogenetic analysis, it was determined that the virus that caused the epidemic in Turkey in 2020 was formed by a new variant in the Turkey-2 group, which was similar to the Indian isolates, unlike the Turkey-1 group, which was close to the Middle East variants in 2008 and 2012 isolates.

Genetic Variation Analysis between Wild and Cultured Pangasianodon hypopthalmus Using COI and Cytochrome b Among Asian Countries

One of the top species in the aquaculture sector, known as striped catfish or Pangasianodon hypophthalmus, is an important and valuable freshwater fish in many countries. Due to the high demand for this species, their number has declined to "threatened" levels. Hence, the purpose of this study is to analyse the genetic variation of wild and cultured striped catfish collected from five producers in Asian countries; Thailand, Vietnam, Indonesia, India, and Philippines, by using mitochondrial DNA partial region data sequence; CO1 and cytochrome b gene. Population analyses using 395 base pairs length for CO1 and 275 base pairs length of cytochrome b partial region nucleotide sequence have shown no significance difference between wild and cultured striped catfish. Vietnam species had shown a wide range of genetic distance of the intrapopulation compared with other countries in the range of 0.000-0.040 for CO1 gene and 0.003-0.008 for cytochrome b gene. The Neighbour-joining method has also been used to construct phylogenetic trees using CO1 gene; the tree formed few subclades with mixed populations, and the tree using cytochrome b showed only Vietnam species divided into a few sub-populations. For the other four countries, Thailand, Indonesia, India, and Philippines were in the same group. Hence, this study's findings may provide a reference for inter and intra-relationships of P. hypophthalmus that may help in the aquaculture activity of this striped catfish.

Root Nodule Microsymbionts of Native Coriaria myrtifolia in Algeria.

Coriaria myrtifolia occurs as natural flora of warm temperate climates of northern Algeria which commonly found in hedges, forest and ravine edges. This actinorhizal species was known to establish a mutualistic symbiosis with members of phylogenetic cluster 2 (including strains associated to Coriaria spp., Ceanothus, Datiscaceae, and Dryadoideae) within the genus Frankia. Attempts to isolate C. myrtifolia microsymbionts from native plants growing in 4 locations in Algeria permitted to only recover asymbiotic Frankia strains (unable to reestablish nodulation and to fix nitrogen) from phylogenetic cluster 4 and several non-Frankia actinobacteria including members of Micrococcus, Micromonospora, Nocardia, Plantactinospora, and Streptomyces genera. The biodiversity of Frankia microsymbionts of C. myrtifolia root nodules was assessed using PCR-amplification followed by partial nucleotide sequencing of glnA1 (glutamine synthetase type 1) gene. On the 12 different glnA1 gene sequences obtained in this study, 9 were detected for the first time, and were mainly closelyrelated to Mediterranean genotypes previously described in the Grand Maghreb countries (Morocco and Tunisia) and in Europe (France) but without clear separations from other cluster 2 genotypes.

Identification, serotyping and antimicrobial susceptibility of Riemerella anatipestifer isolated from ducks in Vietnam

Background:Septicemia caused by Riemerella anatipestifer (R. anatipestifer) is a serious problem in the duck industry worldwide, and it is currently one of the major concerns for duck farming in Vietnam..Aim:This study was conducted to identify the causative agent of septicemia in ducks in Vietnam. The antimicrobial susceptibility and serotypes of R. anatipestifer isolates were also determined to provide valuable information for disease treatment and vaccine development.Methods:Riemerella anatipestifer was isolated using blood agar and chocolate agar media. The commercial API 20NE microtest system and the partial nucleotide sequence analysis of the 16s rRNA were used to identify R. anatipestifer strains. Serotypes were determined by slide agglutination test using standard antisera against R. anatipestifer. The disk diffusion method was utilized to investigate the antimicrobial susceptibility of R. anatipestifer isolated strains.Results:A total of 408 samples were collected from ducks with typical symptoms of septicemia for R. anatipestifer isolation. Sixty-nine R. anatipestifer strains were identified. Serotyping results showed that 30 out of 69 bacterial strains were classified as serotypes 1, 6, 8, 10, and 20, with serotype 10 being the most prevalent. The antimicrobial susceptibility test revealed that 100% of the bacterial isolates were susceptible to Amoxicillin/clavulanic acid and Imipenem. On the contrary, the majority of R. anatipestifer strains were resistant to Nalidixic acid (89.9%), Streptomycin (75.4%), and Norfloxacin (72.5%).Conclusion:This is the first ever report in terms of identification, serotyping, and antimicrobial susceptibility tests of R. anatipestifer causing septicemia in ducks of Vietnam, providing useful scientific information for treatment as well as vaccine development to control the disease.

Molecular Phylodiagnosis of Taenia hydatigena from Stray Dogs in Erbil Province-Kurdistan Region/Iraq

Taenia hydatigena is one of the most prevalent taeniid species of livestock and dogs. A total of 52 fecal samples were collected from September 2019 to May 2020. The highest prevalence rate was observed in Rural area (21.15%, 11/52), as compared to Urban area (15.38%, 8/52). Nineteen (9 males and 10 females) out of 52 dogs were infected with T. hydatigena; there were no significant differences in prevalence based on molecular technique was observed between females (19.23%) and males (17.31%). The DNA’s of taeniid eggs were extracted, amplified, and sequenced by targeting of mitochondrial cytochrome c oxidase subunit 1 (cox1). The partial nucleotide sequence was submitted to the GenBank and recorded under accession number (MT298194-MT298197). Molecular analysis indicates that the specimens characterized in this study belong to the same species and shared greater 98.71–100% identity with cox1 sequences of T. hydatigena. This is the first molecular diagnosis of T. hydatigena in the stray dogs at Erbil Province. The data are a useful for health and educational authorities responsible for designing and implementing effective measures for disease control.

Genomic Epidemiology and Evolution of Scallion Mosaic Potyvirus From Asymptomatic Wild Japanese Garlic.

Scallion mosaic virus (ScaMV) belongs to the turnip mosaic virus phylogenetic group of potyvirus and is known to infect domestic scallion plants (Allium chinense) in China and wild Japanese garlic (Allium macrostemon Bunge) in Japan. Wild Japanese garlic plants showing asymptomatic leaves were collected from different sites in Japan during 2012–2015. We found that 73 wild Japanese garlic plants out of 277 collected plants were infected with ScaMV, identified by partial genomic nucleotide sequences of the amplified RT-PCR products using potyvirus-specific primer pairs. Sixty-three ScaMV isolates were then chosen, and those full genomic sequences were determined. We carried out evolutionary analyses of the complete polyprotein-coding sequences and four non-recombinogenic regions of partial genomic sequences. We found that 80% of ScaMV samples have recombination-like genome structure and identified 12 recombination-type patterns in the genomes of the Japanese ScaMV isolates. Furthermore, we found two non-recombinant-type patterns in the Japanese population. Because the wild plants and weeds may often serve as reservoirs of viruses, it is important to study providing the exploratory investigation before emergence in the domestic plants. This is possibly the first epidemiological and evolutionary study of a virus from asymptomatic wild plants.

Origin of Pathogens of Grapevine Crown Gall Disease in Hokkaido in Japan as Characterized by Molecular Epidemiology of Allorhizobium vitis Strains.

Crown gall is a globally distributed and economically important disease of grapevine and other important crop plants. The causal agent of grapevine crown gall is tumorigenic Allorhizobium vitis (Ti) strains that harbor a tumor-inducing plasmid (pTi). The epidemic of grapevine crown gall has not been widely elucidated. In this study, we investigated the genetic diversity of 89 strains of Ti and nonpathogenic A. vitis to clarify their molecular epidemiology. Multi-locus sequence analysis (MLSA) of the partial nucleotide sequences of pyrG, recA, and rpoD was performed for molecular typing of A. vitis strains isolated from grapevines with crown gall symptoms grown in 30 different vineyards, five different countries, mainly in Japan, and seven genomic groups A to F were obtained. The results of MLSA and logistic regression indicated that the population of genetic group A was significantly related to a range of prefectures and that the epidemic of group A strains originated mainly in Hokkaido in Japan through soil infection. Moreover, group E strains could have been transported by infected nursery stocks. In conclusion, this study indicates that both soil infection and transporting of infected nursery stocks are working as infection source in Hokkaido.

Phylogenetic analysis of West Nile virus: first report of lineage 1 in donkey in Turkey.

West Nile virus (WNV), a member of the Flaviviridae, is a major arbovirus that causes West Nile fever. Previous data showed the prevalence of the WNV serologically and molecular in Turkey, and the presence of lineage 1 in horses and humans has been reported. This is the first notification of partial phylogeny of WNV detected in donkeys in the northeast of Turkey (on the Iranian border). Blood serum samples collected from 25 donkeys without clinical symptoms were tested by RT-PCR. Sequence analysis of the sample detected as positive was performed. Multiple sequence alignments of reference sequences taken from GenBank were performed using the ClustalW method using the MEGA6 program. Partial nucleotide sequences of the capsid gene coding region revealed that the strains are closely related to viruses of lineage 1, clade 1a. According to the phylogenetic tree, the TUR/Igdir/donkey strain was included in the same cluster as the strain (KJ958922) previously obtained from horses in Turkey and the strain (GQ851658) from the Central African Republic. This study is the first report to show the circulation of WNV lineage 1 in donkeys in Turkey.

First genetically confirmed records of the little gulper shark Centrophorusuyato (Squaliformes: Centrophoridae) from Cypriot waters.

The taxonomy within the genus Centrophorus has been controversial almost since its origin, raising uncertainties about the identification, the phylogenetic placement and the geographical distribution of several species. The partial nucleotide sequences of two mitochondrial DNA gene regions, the cytochrome c oxidase subunit I and the 16S ribosomal RNA, genetically confirmed the presence of the little gulper shark in Cypriot waters. The species presence in the Mediterranean Sea is revised and discussed.

Molecular characterization and pathological identification of a novel strain of delta papillomavirus-4 (bovine papillomavirus-2) in Egypt.

Background and Aim:Bovine papillomaviruses (BPV) are a heterogeneous group of oncoviruses, distributed globally, which produce major economic losses. In the current study, we compared the results of different diagnostic approaches and compared the strains identified in this study with previously characterized strains at local and international levels.Materials and Methods:Samples of skin warts were collected from five bovines with generalized papillomatosis from two Egyptian provinces, Menya and Ismailia, in 2020. Electron microscopy, molecular characterization, histopathological, and immunohistochemical examination were performed.Results:BPV was detected using electron microscopy in the collected samples. Using molecular characterization, BPV-2 was successfully identified for 1st time in Egypt. The strain has 99.6% identity with the BPV-2 reference strains obtained from GenBank. These results were supported by histopathology and immunohistochemistry examination. Partial nucleotide sequences of the L1 gene were submitted to GenBank with accession numbers MW289843 and MW289844.Conclusion:BPV-2 was reported for 1st time in the current study. The strain was identified grossly, microscopically, and pathologically and confirmed using molecular approaches. All results were consistent. The sequence analysis revealed that this strain has high sequence similarity to the reference Deltapapillomavirus-4, BPV-2 strains from Brazil and China.

First report of Pythium aristosporum causing corn stalk rot in China.

Corn (Zea mays L.) stalk rot, caused by various pathogens, is one of the most prevalent corn diseases worldwide. In October 2019, a survey was carried out to determine pathogenic fungi causing corn stalk rot in 3 fields (~120 ha) in Harbin city (44.04°N 125.42°E), Heilongjiang Province, China. In each field, 100 plants at 5 sampling points were assessed at the milk stage (R3) of development. Disease incidence was 12%. Symptomatic plants showed rapid death of the upper leaves, drooping ears and stalks were soft, hollow, watersoaked with white hyphae present on teh outside of the stalk. Pieces of tissue (0.25 cm2) from 15 individual diseased stalks (5 plants/field) were surface disinfested in 0.5% NaOCl for 5 min, rinsed three times in sterile distilled water and cultured on potato dextrose agar (PDA) containing streptomycin (50 μg/mL). After three days of incubation, a total of twelve fungal cultures with uniform characteristics were isolated and subcultured by transferring hyphal tips onto V8. Colonies on V8 selective medium were creamy white and floccus, with a growth rate of 20 mm/day at 26°C in darkness. Oospores were mostly plerotic, and oogonia walls were 1.3 to 2.7 μm thick (n = 50); globose oogonia, 23.9 to 30.5 μm in diameter (n = 50), and had 1 to 8 antheridia. Based on these characteristics, the isolates were identified as Pythiumsp. (van der Plaats-Niterink 1981). Genomic DNA was extracted from single conidial cultures of representative isolates (MZYJF1, MZYJF3 and MZYJF7), and the internal transcribed spacer (ITS) region and cytochrome coxidase subunit II (CoxII) gene were amplified and sequenced using the primers ITS1/ITS4 (Yin et al. 2012) and COX2f/COX2r (Hudspeth et al. 2000), respectively. Partial nucleotide sequences of 796 bp and 573 bp for the ITS and COX11 amplicons, respectively, were obtained and deposited in GenBank (accession no. MW447501 for ITS, and MW471006 for COXII). MegaBLAST analysis of the ITS and CoxII sequences of MZYJF1 isolate showed 100% similarity with sequences from P. aristosporum strain ATCC 11101. The isolates were identified asP. aristosporumbased on the fact thatP. aristosporumhas aplerotic oospores and less antheridia per oogonium than P. arrhenomanes(van der Plaats-Niterink 1981).A pathogenicity test was performed on corn cv. Xianyu 335 at tasseling stage (VT) in the field. An oospore suspension, obtained from isolate MZYJF1 grown on V8 agar media for 4 weeks (Green and Jensen, 2000) and diluted to 1×104 oospores/mL using blood cell counting method, was injected into the base of the maize stems of 6 healthy plants (1.5 ml/plant ) using a syringe. Control plants were injected with distilled sterile water. All inoculated plants showed symptoms 25 days after inoculation that were similar to those observed in the field. The oomycete of P. aristosporum was reisolated from symptomatic plants on V8 agar media and identified according to morphological and molecular characteristics. No symptoms were observed on the control plants. P. aristosporum has previously been reported on causing damping-off of pea in the Columbia basin of Central Washington (Alcala et al. 2016) and on soybean in North Dakota (Zitnick-Anderson and Nelson 2015). To our knowledge, this is the first report of P. aristosporum causing corn stalk rot in China. Corn stalk rot caused by P. aristosporum poses a threat to significantly reduce the quality of corn. Thus, its distribution needs to be investigated and effective disease management strategies developed.

Antagonistic Potential of Soil Streptomyces Isolates from Southern Thailand to Inhibit Foodborne Bacterial Pathogens.

Streptomyces are well known for their competence to produce thousands of bioactive secondary metabolites and enzymes. This study aimed to assess the inhibitory activities of crude extracts from diverse Streptomyces collected from rice soils in Narathiwat, Thailand, against foodborne bacterial pathogens. In total, 136 Actinomycete isolates were screened using a cross-streak method for the ability to produce effective metabolites against 5 pathogenic bacteria. Out of these, 19 (13.97%) isolates had antibacterial activity against at least one tested bacterium. Most of the isolates could strongly suppress the growth of S. aureus ATCC25923 and B. cereus MTCC430 except P. aeruginosa ATCC27853. On the basis of morphological, cultural, and biochemical characteristics, all potent isolates exhibited typical features that fitted the genus Streptomyces. Two of the 7 selected ethyl acetate crude extracts had good antagonistic activity against S. aureus ATCC25923 and B. cereus MTCC430 when tested using the agar well diffusion assay. Furthermore, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of the 2 extracts evaluated using the colorimetric broth microdilution method ranged from 256 to >1,024 μg/ml against the tested bacteria. The partial nucleotide sequences of the 16S rRNA gene led to identifying both active isolates as Streptomyces species. These active Streptomyces isolates could provide an interesting source for generating innumerable natural compounds with antibacterial activity that can presumably be developed to fight bacterial pathogens in the near future.

An emerging orthopneumovirus detected from dogs with canine infectious respiratory disease in China.

Canine infectious respiratory disease (CIRD) is a major cause of morbidity in dogs and is associated with several viral pathogens. The viral diversity associated with CIRD was investigated by analyzing the viral communities from nine CIRD-affected dogs using metagenomics. The results identified 10 mammalian viruses, including canine parvovirus, canid alphaherpesvirus 1, canine kobuvirus, Felis catus papillomavirus 3, canine respiratory coronavirus, canine adenovirus 2, Canis familiaris polyomavirus (DogPyV), canine coronavirus, human papillomavirus and canine pneumovirus (CPnV). Interestingly, CPnV and DogPyV were first discovered in China. Further investigation in 107 samples in China using specific PCR found only two CPnV positive strains in 51 CIRD samples and none in 56 healthy samples. Furthermore, a complete nucleotide sequence of CPnV strain SMU-2020-CB19 and a partial nucleotide sequence of strain SMU-2020-CB14 were obtained. Sequence comparisons and phylogenetic analysis showed that both novel CPnV strains were a close match to the detected swine orthopneumovirus strain in USA, but distantly related to other CPnV strains. Here, the first discovery and characterization of orthopneumovirus in dogs with CIRD in China were reported, highlighting the need for further research on pathogenicity and transmission in China.

Molecular characterization of Deformed wing viruses identified in honeybee (Apis mellifera L.) colonies in Erzincan province of Turkey

Deformed wing virus (DWV), which belongs to the Iflaviridae family, is a devastating virus causing severe financial losses in honey bee colonies all over the world. In 2020 (November), virus-suspected honey bee specimens collected from Erzincan province were screened by molecular assays against DWV infection. In 6 of 12 samples, an expected approximately 700 bp DNA bands were achieved, indicating probably virus-positive. Two randomly selected amplified DNA band suggesting the presence of the virus was further cloned into a pGEM-T Easy vector and electrically transformed into E. coli competent bacteria. Recombinant plasmids obtained from cloning were purified from E. coli and sequenced by next-generation sequencing (NGS). The partial nucleotide sequences of DWV were ascertained to contain 711 nucleotides and these sequences were recorded in the NCBI GenBank with MW962981 and MW962982 accession number. The highest sequence similarity of both isolates ranged from 98.03% to 98.17%, at the nucleotide level. Furthermore, phylogenetic relationships of Erzincan DWV sequences were revealed using 15 unique sequences from different ecological regions registered in the GenBank. Phylogenetic tree displayed that DWV Erzincan isolates are clustered with UK (HM067438), Spain (MK262743), and Sweden (MN746311) isolates. According to our best knowledge, DWV has been reported for the first time in the honey bees from Erzincan province of Turkey by this study.

Ubiquitous influenza A virus in Chilean swine before the H1N1pdm09 introduction.

Influenza A virus (IAV) was a neglected swine pathogen in South America before the 2009 H1N1 pandemic (A(H1N1)pdm2009). The A(H1N1)pdm2009 strain has widely spread among the Chilean swine population and co-circulates with endemic H1N2 and H3N2 viruses. The presence of IAV as a swine pathogen in Chilean swine before the 2009 pandemic is unknown. To understand the IAV in swine prior to 2009, aY retrospective study of samples from pigs affected with respiratory diseases was conducted. Ninety formalin-fixed and paraffin-embedded lung tissues belonging to 21 intensive pig production companies located in five different administrative regions of Chile, collected between 2005 and 2008, were evaluated. The tissues were tested by immunohistochemistry (IHC), identifying that 9 out of 21 farms (42.8%) and 31 out of 90 (34.4%) samples were IAV positive. Only three out of the 31 IHC-positive samples were positive upon RNA extraction and rtRT-PCR analysis. Partial nucleotide sequences were obtained from one sample and characterized as an H3N2 subtype closely related to a human seasonal H3N2 IAVs that circulated globally in the mid-90s. These results indicate that IAV was circulating in swine before 2009 and highlight the value of conducting retrospective studies through genomic strategies to analyse historical samples.

Virological Characterization of Roof-Harvested Rainwater of Densely Urbanized Low-Income Region.

Roof-harvested rainwater (RHRW) is considered relatively clean water, even though the possible presence of pathogens in the water may pose human health risks. In this study, we investigated the occurrence of enteric viruses in the first flush (10mm) of RHRW from a densely populated and low-income urbanized region of Rio de Janeiro. One hundred samples (5 L) were collected from 10 rainfall events between April 2015 and March 2017. RNA and DNA viruses were concentrated using the skimmed milk flocculation method and analyzed using the TaqMan® quantitative RT-qPCR and qPCR. Human adenoviruses, noroviruses, rotaviruses A, and avian parvoviruses were detected in 54%, 31%, 12%, and 12% of the positive samples. JC polyomavirus, also targeted, was not detected. Virus concentrations ranged from 1.09 × 101 to 2.58 × 103 genome copies/Liter (GC/L). Partial nucleotide sequence confirmed the presence of HAdV type 41, norovirus genotype GII.4, and avian parvovirus 1. The results suggest that the first flush diversion devices may not adequately remove enteric virus from the rainwater. Additional treatment of RHRW is required to mitigate potential health risks from potable use of captured water.