Exploration of a new hepatitis a surveillance system in Beijing, China: based on molecular epidemiology

Background The incidence of hepatitis A virus (HAV) infection is low in Beijing, China, but the risk of outbreaks still exists. It is difficult to identify possible sources of infection among sporadic cases based on a routine surveillance system. Therefore, a more effective surveillance system needs to be established. Methods The epidemiological data of hepatitis A were obtained from a routine surveillance system. Patients with HAV confirmed at the local hospitals were asked to complete a questionnaire that included additional case information and possible sources of infection. Serum and fecal specimens were also collected for testing HAV RNA by polymerase chain reaction. In addition, the 321-nucleotide segment of the VP1/2A junction region was sequenced to determine the HAV genotype. Results In 2019, 110 HAV cases were reported in Beijing, with an incidence rate of 0.51/100,000. 61(55.5%) of these patients were male. The greatest proportion of these patients were aged from 30 to 60 years. The rate was lower in suburban and rural areas compared to urban areas. Contaminated food consumption, particularly seafood consumption, was the primary potential source of infection. Among the 16 specimens of confirmed HAV cases that could be sequenced, 93.8% were HAV IA, and 6.3% were HAV IB. In addition, the samples collected from all HAV sequences in this investigation showed 89.4–100% nucleotide homology. Two groups (each with three sporadic cases) showed 100% nucleotide homology. The three sporadic cases in one group had the same possible source of infection: contaminated salad with raw vegetables and seafood. In the other group, the three sporadic cases did not have an epidemiological connection. Conclusions In a low HAV prevalent area, such as in Beijing, incorporating molecular epidemiology into the routine surveillance system could help inform possible clusters of outbreaks and provide support for earlier control of HAV transmission. Nevertheless, increased sampling from detected cases and improved specimen quality are needed to implement such a system.

Molecular Characterization of Hemagglutinin-Neuraminidase Protein Virus Newcastle disease (ND) in Surabaya during 2003 and 2016

This study aimed to determine the mutation of amino acid, nucleotide homology, phylogenetic tree, epitope prediction of Hemagglutinin-Neuraminidase protein Newcastle Disease (ND) Virus isolated from traditional market around Surabaya. Samples were from 37 chicken with cloacal swab and one positive samples for control (LaSota). Samples were inoculated on embyonate chicken eggs and identified with HA test confirmed with HI test. Positive samples processed by PCR using forward and reverse primer with 503 bp RNA target. The PCR result then analyzed with sequencing. Result of sequencing analysis showed that theres similiarity between samples amino acid and vaccine isolate its effect the percentage of nucleotide homology and phylogenetic relation between isolate. Epitope NGAANNSGWGAPIHDPDYIGG have high immunogenic value at all of isolate which good as vaccine candidate.

Sequence phylogenetic analysis and associative genetic diversity of Sarcocystis hirsuta based on 18S rRNA gene

Background Sarcocystis hirsuta, a tissue cyst-forming coccidian parasite of cattle, is worldwide in distribution. In spite of its global presence, limited literature is available on its characterization studies. No literature is available from India on molecular aspects of S. hirsuta. The present study was designed to characterize the isolates of S. hirsuta on the 18S gene locus. A total of five isolates of S. hirsuta were characterized. PCR products were cloned, sequenced, and compared with other sequences across the world. A phylogenetic tree was constructed based on the maximum parsimony (MP) method with the tree–bisection–regrafting (TBR) algorithm. Results An appreciable genetic variability was noticed between various S. hirsuta isolates at the 18S gene locus. Sequences generated from the present study (MN121567–MN121571) represented two haplotypes with 99.74–100.00% nucleotide homology within themselves. Alongside, a nucleotide homology of 97.82–99.92% was observed between Indian isolates and isolates across the globe. The two haplotypes were markedly distinct from each other with 3 nucleotide substitutions within themselves. Overall, Indian isolates of S. hirsuta were close to those from China and Vietnam than to those from New Zealand, Brazil, and Germany. Conclusion The present communication describes the first report of phylogenetic characterization of S. hirsuta from India. The findings are very much important in delineating the evolutionary phylogenetics of S. hirsuta.

Distribution of CRISPR Types in Fluoroquinolone-Resistant Campylobacter jejuni Isolates

To aid development of phage therapy against Campylobacter, we investigated the distribution of the clustered regularly interspaced short palindromic repeats (CRISPR) systems in fluoroquinolone (FQ)-resistant Campylobacter jejuni. A total of 100 FQ-resistant C. jejuni strains from different sources were analyzed by PCR and DNA sequencing to determine resistance-conferring mutation in the gyrA gene and the presence of various CRISPR systems. All but one isolate harbored 1–5 point mutations in gyrA, and the most common mutation was the Thr86Ile change. Ninety-five isolates were positive with the CRISPR PCR, and spacer sequences were found in 86 of them. Among the 292 spacer sequences identified in this study, 204 shared 93–100% nucleotide homology to Campylobacter phage D10, 44 showed 100% homology to Campylobacter phage CP39, and 3 had 100% homology with Campylobacter phage CJIE4-5. The remaining 41 spacer sequences did not match with any phages in the database. Based on the results, it was inferred that the FQ-resistant C. jejuni isolates analyzed in this study were potentially resistant to Campylobacter phages D10, CP39, and CJIE4-5 as well as some unidentified phages. These phages should be excluded from cocktails of phages that may be utilized to treat FQ-resistant Campylobacter.

Detection of live attenuated influenza vaccine virus and evidence of reassortment in the U.S. swine population

Influenza vaccines historically have been multivalent, whole virus inactivated products. The first bivalent, intranasal, live attenuated influenza vaccine (LAIV; Ingelvac Provenza), with H1N1 and H3N2 subtypes, has been approved for use in swine. We investigated the LAIV hemagglutinin ( HA) sequences in diagnostic cases submitted to the Iowa State University Veterinary Diagnostic Laboratory and potential vaccine virus reassortment with endemic influenza A virus (IAV) in swine. From January 3 to October 11, 2018, IAV HA sequences demonstrating 99.5–99.9% nucleotide homology to the H1 HA or 99.4–100% nucleotide homology to the H3 HA parental strains in the LAIV were detected in 58 of 1,116 (5.2%) porcine respiratory cases (H1 HA A/swine/Minnesota/37866/1999[H1N1; MN99]; H3 HA A/swine/Texas/4199-2/1998[H3N2; TX98]). Nine cases had co-detection of HA genes from LAIV and wild-type IAV in the same specimen. Thirty-five cases had associated epidemiologic information that indicated they were submitted from 11 states representing 31 individual sites and 17 production systems in the United States. Whole genome sequences from 11 cases and another subset of 2 plaque-purified IAV were included in our study. Ten whole genome sequences, including 1 plaque-purified IAV, contained at least one internal gene from endemic IAV detected within the past 3 y. Phylogenetic analysis of whole genome sequences indicated that reassortment occurred between vaccine virus and endemic field strains circulating in U.S. swine. Our data highlight the need and importance of continued IAV surveillance to detect emerging IAV with LAIV genes in the swine population.

Molecular homology and multiple-sequence alignment: an analysis of concepts and practice

Sequence alignment is just as much a part of phylogenetics as is tree building, although it is often viewed solely as a necessary tool to construct trees. However, alignment for the purpose of phylogenetic inference is primarily about homology, as it is the procedure that expresses homology relationships among the characters, rather than the historical relationships of the taxa. Molecular homology is rather vaguely defined and understood, despite its importance in the molecular age. Indeed, homology has rarely been evaluated with respect to nucleotide sequence alignments, in spite of the fact that nucleotides are the only data that directly represent genotype. All other molecular data represent phenotype, just as do morphology and anatomy. Thus, efforts to improve sequence alignment for phylogenetic purposes should involve a more refined use of the homology concept at a molecular level. To this end, we present examples of molecular-data levels at which homology might be considered, and arrange them in a hierarchy. The concept that we propose has many levels, which link directly to the developmental and morphological components of homology. Of note, there is no simple relationship between gene homology and nucleotide homology. We also propose terminology with which to better describe and discuss molecular homology at these levels. Our over-arching conceptual framework is then used to shed light on the multitude of automated procedures that have been created for multiple-sequence alignment. Sequence alignment needs to be based on aligning homologous nucleotides, without necessary reference to homology at any other level of the hierarchy. In particular, inference of nucleotide homology involves deriving a plausible scenario for molecular change among the set of sequences. Our clarifications should allow the development of a procedure that specifically addresses homology, which is required when performing alignment for phylogenetic purposes, but which does not yet exist.

First Report of Stigmina palmivora Causing Leaf Spots on Phoenix roebelenii in Brazil

Phoenix roebelenii (Arecaceae), known as dwarf date (tamareira-anã in Brazil), is a palm native to Southeast Asia and widely cultivated worldwide because of its ornamental value and ease of adaptation to a broad range of climates and soil types (4). In June 2012, some individuals were observed in a private garden in the municipality of Viçosa (state of Minas Gerais, Brazil) bearing numerous necrotic lesions on its leaves. Representative samples were taken, dried in a plant press, and brought to the laboratory for examination. A fungus was regularly associated with the leaf spots. Fungal structures were mounted in lactophenol and slides were examined under a microscope (Olympus BX 51). Spores were taken from sporulating colonies with a sterile fine needle and plated on PDA for isolation. A pure culture was deposited in the culture collection of the Universidade Federal de Viçosa (accession COAD1338). A dried herbarium sample was deposited in the local herbarium (VIC39741). The fungus had the following morphology: conidiophores grouped on sporodochia, cylindrical, 12 to 29 × 5 to 6 μm, dark brown; conidiogenous cells, terminal, proliferating percurrently (annellidic), 8 to 20 × 5 to 6 μm, pale to dark brown; conidia obclavate to subcylindrical, straight, 58 to 147 × 5 to 6 μm, 6 to 16 septate, hila thickened and darkened with a thin-walled projecting papilla, dark brown, and verrucose. The morphology of the Brazilian collections agrees well with the description of Stigmina palmivora (2), a species known to cause leaf spots on P. roebelenii in the United States (Florida) and Japan (3). Pathogenicity was demonstrated through inoculation of leaves of healthy plants by placing 6 mm diameter cuture disks of COAD1338 on the leaf surface followed by incubation in a moist chamber for 48 h and then transferred to a greenhouse bench at 21 ± 3°C. Typical leaf spots were observed 15 days after inoculation. DNA was extracted from the isolate growing in pure culture and ITS and LSU sequences were generated and deposited in GenBank under the accession numbers KF656785 and KF656786, respectively. These were compared by BLASTn with other entries in GenBank, and the closest match for each region were Mycosphaerella colombiensis strain X215 and M. irregulariamosa strain CPC 1362 (EU514231, GU2114441) with 93% of nucleotide homology (over 100% query coverage) for ITS and 98% of nucleotide homology (over 100% query coverage) for LSU. There are no sequences for S. palmivora deposited in public databases for comparison, but for Stigmina platani, the type species in this genus, 86% and 96% nucleotide homology for ITS and LSU with S. palmivora were found. The genus Stigmina is regarded as being polyphyletic (1) and this is probably reflected by these low homology levels found in the BLASTn search. To our knowledge, this is the first report of Stigmina palmivora in Brazil. References: (1) P. W. Crous et al. Stud. Mycol. 75:37, 2012. (2) M. B. Ellis. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, UK, 1971. (3) D. F. Farr and A. Y. Rossman. Fungal Databases. Syst. Mycol. Microbiol. Lab. ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , 2013. (4) H. Lorenzi et al. Palmeira no Brasil: Exóticas e Nativas, 2nd ed. Editora Plantarum, Nova Odessa, Brazil, 2005.

First Report of Gray Mold (Amphobotrys ricini) on Copperleaf (Acalypha wilkesiana) in Brazil

Acalypha wilkesiana (Euphorbiaceae), common names copperleaf or Jacob's coat (in Brazil, crista-de-peru), is a popular ornamental native from the Pacific islands. It is widely used in gardens in Brazil (4). In January 2012, a group of diseased A. wilkesiana was found in a nursery at the municipality of Itaboraí (state of Rio de Janeiro, Brazil). Later, another group of individuals of the same plant species bearing identical disease symptoms were found in a botanic garden in the city of Rio de Janeiro (Jardim Botânico do Rio de Janeiro). Diseased plants had intense leaf blight. Such leaves dropped over healthy leaves of the same or other plants and necrosis was hence initiated on such leaves. Inflorescences were also affected by blight and after becoming necrotic a dieback of supporting stems also resulted. Abundant grayish sporulation was easily observed over necrotic tissues. Samples were collected, dried in a plant press, and representative specimens were deposited in the herbarium at the Universidade Federal de Viçosa. These were from Itaboraí (VIC 31822) and from Rio de Janeiro (VIC 31931). Structures were mounted in lactophenol for observation under a microscope and isolated in pure culture on PCA plates. Isolates were deposited in the culture collection of the Universidade Federal de Viçosa with accession numbers of COAD 1112 and COAD 1108, respectively. The fungus had the following morphology: conidiophores cylindrical, up to 1,200 μm branching dicotomously at mid-length in broad angles and then branching secondarily, light brown; conidiogenous cells ampulliform, terminal, denticulate; conidia globose, 6 to 11 μm diam, subhyaline to pale brown, smooth. This combination of features is typical of Amphobotrys ricini (2), a common pathogen of castor bean (1) and several other members of the Euphorbiaceae. DNA was extracted from each isolate growing in pure culture and ITS sequences were generated and deposited in GenBank under the accession numbers JX961613 (COAD 1108) and JX961614 (COAD 1112). These were compared by BLASTn with other entries in GenBank, and the closest match for both isolates was A. ricini (JF433374) with 97% nucleotide homology (over 97% query coverage) for COAD 1112 and 98% nucleotide homology (over 98% query coverage) for COAD 1112. Pathogenicity of the isolate from A. wilkesiana was demonstrated through brush inoculation of a conidial suspension (3 × 106 conidia. mL–1) onto healthy leaves of a A. wilkesiana individual followed by its transfer to a humid chamber for 48 h. Symptoms appeared after 3 days of inoculation and sporulation appeared over necrotic tissues after 10 days. Despite the importance of A. ricini as a plant pathogen, little has been investigated on its taxonomy with molecular tools. Although morphology and host-association are the basis for the delimitation of A. ricini, our preliminary results for ITS sequences suggest that this species may include cryptic taxa that are not properly discriminated on a morphological and pathological basis. This report follows other novel reports of A. ricini on ornamental Euphorbiaceae in Brazil (3) and, to our knowledge, represents the first report of A. ricini on A. wilkesiana worldwide. References: (1) G. H. Godfrey. J. Agric. Res. 23:679, 1923. (2) G. L. Hennebert. Persoonia 7:183, 1973. (3) B. V. Lima et al. Australas. Plant Dis. Notes 3:5, 2008. (4) H. Lorenzi and H. M. Souza. Plantas Ornamentais no Brasil - Arbustivas, Herbáceas e Trepadeiras. Nova Odessa: Instituto Plantarum, 1999.

First Report of Stagonosporiopsis cucurbitacearum Causing Fruit Rot of Luffa cylindrica in Brazil

Luffa cylindrica (Cucurbitaceae) is an Asian vine widely known as the source of loofah (4). In Brazil (local name bucha), it is cultivated by small scale producers as a cash crop. In January 2012, samples of fruits were collected in three areas in the municipality of Cipotânea, state of Minas Gerais (Brazil) bearing rot symptoms. These had large necrotic areas with a grayish epidermis and slightly sunken tissue. Internally, the fibrous parts were necrosed, darkened, and unmarketable. Isolations by surface sterilization of necrotic tissue with 10% bleach and plating onto potato dextrose agar yielded colonies with consistent morphology. A representative culture was deposited in the culture collection of the Universidade Federal de Viçosa (UFV) as COAD1119. Inoculations of seven healthy-appearing L. cylindrica fruits were performed with culture disks obtained from 4-day-old cultures grown on PDA, which were placed onto two points on the epidermis of each of seven fruits. Each point was either intact or previously injured with a sterile needle. Controls consisted of two fruits treated equally but with tap water agar disks in place of fungal inoculum. Fruits were then placed on trays with water-soaked cotton and the trays were wrapped in plastic bags and left over a bench at room temperature for 2 days. The plastic bags were then removed. After 5 days, necrosis was evident and fungal fruit bodies appeared at points with injury. No symptoms appeared on controls. Isolation from diseased tissue yielded colonies identical to those of the inoculated fungus. A dried sample was deposited in the local herbarium at UFV (VIC 32053). Slides were mounted in lactophenol and observed. The fungus had subepidermal perithecia, globose to subglobose, from 75.5 to 134 μm diam.; asci bitunicate, cylindrical, 8-spored; pseudoparaphyses filiform; ascospores fusoid to ellipsoidal, from 26 to 45 μm long and 8 to 11.5 μm wide, one septate, and hyaline. This morphology is consistent with Stagonosporopsis cucurbitacearum (syn. Didymella bryoniae) (3), a broad spectrum pathogen of cucurbits. Genomic DNA was extracted from the isolate growing in pure culture and ITS and LSU sequences were generated and deposited in GenBank under the accession numbers KC582022 and KC582021, respectively. Sequences were compared in BLASTn with other entries in GenBank, and the closest match for each region were S. cucurbitacearum strain CAP14C and D. bryoniae strain CBS 133.96 (JQ936326 and GU456335) with 100% of nucleotide homology for ITS and 100% of nucleotide homology for LSU. Cercospora citrullina and C. cucurbitae have been reported in Brazil on L. cylindrica and mistakenly indicated as synonyms of D. bryoniae (2). To our knowledge, this is the first valid report of S. cucurbitacearum causing fruit rot of loofah in Brazil and the first time pathogenicity to this host has been demonstrated. Losses due to the disease on the crop were reported to be high by growers and management to be difficult since there are no fungicides registered for this crop in Brazil. References: (1) M. M. Aveskamp et al. Stud. Mycol 65:1, 2010. (2) M. A. S. Mendes and A. F. Urben. Fungos em Plantas no Brasil. Brasília, Brazil: EMBRAPA-SPI. Retrieved from http://pragawall.cenargen.embrapa.br/aiqweb/michtml/micbanco01a.asp , 2012. (3) E. Puithalingam and P. Holliday. CMI Descriptions of Pathogenic Fungi and Bacteria 332:1, 1972. (4) J. W. Purseglove. Tropical Crops – Dicotyledons. Longman Group, London, 1968.

Genomic sequence of infectious bursal disease virus from Zambia suggests evidence for genome re-assortment in nature

Infectious bursal disease virus (IBDV) is a bi-segmented RNA virus, which belongs to the genus Avibirnavirus of the family Birnaviridae. Two serotypes, 1 and 2, exist in IBDV. The serotype 1 IBDVs are the causative agents of infectious bursal disease (IBD) in chickens worldwide and lead to immunosuppression in young birds. Genome re-assortment has been speculated to occur and contribute to the emergence of new IBDV strains. However, evidence was lacking until recently when two re-assortant viruses were detected in China. In this study, we determined the complete nucleotide sequence of an IBDV, designated KZC-104, from a confirmed natural IBD outbreak in Lusaka, Zambia in 2004. The genome consisted of 3074 and 2651 nucleotides in the coding regions of segments A and B, respectively. Alignment of both nucleotide and deduced amino acid sequences, and phylogenetic analysis revealed that the genome segment A of KZC-104 was derived from a very virulent strain, whereas its segment B was derived from a classical attenuated strain. On BLAST search, the full-length segments A and B sequences showed 98% closest nucleotide homology to the very virulent strain D6948 and 99.8% closest nucleotide homology to the classical attenuated strain D78, respectively. This is a unique IBDV reassortant strain, which has emerged in nature involving segment B of a live attenuated vaccine. This observation provides direct evidence for the involvement of vaccine strains in the emergence of reassortant IBDV in the field. Taken together, these findings suggest an additional risk of using live IBDV vaccines, which may act as genetic donors for genome re-assortment. Further studies are required to investigate the epidemiology and biological characteristics of reassortant strains so that the appropriate and safe IBDV vaccines can be recommended.