Evolutionary Analysis of Dengue Serotype 2 Viruses Using Phylogenetic and Bayesian Methods from New Delhi,India

Dengue fever is the most important arboviral disease in the tropical and sub-tropical countries of the world. Delhi, the metropolitan capital state of India, has reported many dengue outbreaks, with the last outbreak occurring in 2013. We have recently reported predominance of dengue virus serotype 2 during 2011–2014 in Delhi. In the present study, we report molecular characterization and evolutionary analysis of dengue serotype 2 viruses which were detected in 2011–2014 in Delhi. Envelope genes of 42 DENV-2 strains were sequenced in the study. All DENV-2 strains grouped within the Cosmopolitan genotype and further clustered into three lineages; Lineage I, II and III. Lineage III replaced lineage I during dengue fever outbreak of 2013. Further, a novel mutation Thr404Ile was detected in the stem region of the envelope protein of a single DENV-2 strain in 2014. Nucleotide substitution rate and time to the most recent common ancestor were determined by molecular clock analysis using Bayesian methods. A change in effective population size of Indian DENV-2 viruses was investigated through Bayesian skyline plot. The study will be a vital road map for investigation of epidemiology and evolutionary pattern of dengue viruses in India.     

Molecular characterization of dengue and chikungunya virus strains circulating in New Delhi,India

Dengue and chikungunya are acute viral infections with overlapping clinical symptoms. Both diseases are transmitted by common mosquito vectors resulting in their co‐circulation in a region. Molecular and serological tests specific for both dengue and chikungunya infections were performed on 87 acute phase blood samples collected from patients with suspected dengue/chikungunya infections in Delhi from September to December, 2011. RT‐PCR and IgM ELISA were performed to detect dengue virus (DENV) and chikungunya virus (CHIKV). NS1 and IgG ELISA were also performed to detect DENV specific antigen and secondary DENV infection. DENV infection was detected in 49%, CHIKV infection in 29% and co‐infection with DENV and CHIKV in 10% of the samples by RT‐PCR. DENV serotypes 1, 2 and 3 were detected in this study. Nine DENV‐1 strains, six DENV‐2 strains and 20 CHIKV strains were characterized by DNA sequencing and phylogenetic analysis of their respective envelope protein genes. DENV‐1 strains grouped in the American African genotype, DENV‐2 strains in the Cosmopolitan genotype and CHIKV strains in the East Central South African genotype by phylogenetic analysis. This is one of the few studies reporting the phylogeny of two dengue virus serotypes (DENV‐1 and DENV‐2) and CHIKV. Surveillance and monitoring of DENV and CHIKV strains are important for design of strategies to control impending epidemics.     

Natural Strain Variation and Antibody Neutralization of Dengue Serotype 3 Viruses

Dengue viruses (DENVs) are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs) have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII). It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge “type specific” epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII.     

Characterization, Genetic Diversity, and Evolutionary Link of Cucumber mosaic virus Strain New Delhi from India

The genome of Cucumber mosaic virus New Delhi strain (CMV-ND) from India, obtained from tomato, was completely sequenced and compared with full genome sequences of 14 known CMV strains from subgroups I and II, for their genetic diversity. Sequence analysis suggests CMV-ND shares maximum sequence identity at the nucleotide level with a CMV strain from Taiwan. Among all 15 strains of CMV, the encoded protein 2b is least conserved, whereas the coat protein (CP) is most conserved. Sequence identity values and phylogram results indicate that CMV-ND belongs to subgroup I. Based on the recombination detection program result, it appears that CMV is prone to recombination, and different RNA components of CMV-ND have evolved differently. Recombinational analysis of all 15 CMV strains detected maximum recombination breakpoints in RNA2; CP showed the least recombination sites.     

从贵州白纹伊蚊体内分离到一株2型登革病毒

从贵州省独山县麻尾镇白纹伊蚊标本中分离到一株病毒,用抗DEN 14型单克隆抗体经间接免疫荧光法和RTPCR产物酶切分型鉴定为DEN2,并对其 NS1和E基因RTPCR产物进行部分基因序列测定,与DEN2 NGC株比较,麻尾株的NS1基因区有7个碱基发生点突变,E基因区有1个碱基的插入,两个序列被GenBank收录,编号分别为AY277402、AY278226;麻尾分离株与其他9株DEN2型病毒进行系统发育关系的分析,结果显示与D243株系统进化关系最近;证明贵州省存在DEN感染的自然循环。     

Phylogenetic Analysis of the Dengue Virus Strains Causing the 2019 Dengue Fever Outbreak in Hainan,China

Dengue virus is an arthropod-borne pathogen that is transmitted to humans primarily by Aedes spp.mosquitos,causing the acute infectious disease,dengue fever (DF).Until 2019,no dengue outbreak had been reported in Hainan Province for over20 years.However,in early September of 2019,an increasing number of infected cases appeared and the DF outbreak lasted for over one month in Haikou City,Hainan Province.In our study,we collected 97 plasma samples from DF patients at three hospitals,as well as 1585 mosquito larvae samples from puddles in different areas of Haikou.There were 49(50.5%) plasma samples found to be strongly positive and 9 (9.3%) plasma samples were weakly positive against the NS1 antigen.We discovered DENV both in the patient's plasma samples and mosquito larvae samples,and isolated the virus from C6/36 cells inoculated with the acute phase serum of patients.Phylogenetic analysis revealed that the new strains were the most closely related to the epidemic strain in the southern regions of China,belonging to lineage IV,genotype I,DENV-1.Compared to the seven closest strains from neighboring countries and provinces,a total of 18 amino acid mutations occurred in the coding sequences (CDS) of the new isolated strain,DENV1 HMU-HKU-2.Our data shows that dengue virus is re-emerged in Hainan,and pose new threats for public health.Thus regular molecular epidemiological surveillance is necessary for control and prevention of DENV transmission.     

Molecular Cloning,Sequence, and Phylogenetic Analysis of T Helper 1 Cytokines of Pashmina Goats

Cytokines play an important role in regulation of immune responses either in health or disease. In the present study, the cDNAs encoding mature Interleukin (IL)-2, interferon gamma (IFN-γ), and IL-12 p35 and p40 of Pashmina goat were cloned and sequenced. The amino acid sequence was deduced from nucleotide sequence and compared with those available in GeneBank. Mature forms of goat IL-2, IFN-γ, IL-12 p35, and IL-12 p40 composed of 135, 143, 196, and 305 amino acid residues, respectively. Comparison of amino acid sequence of goat IL-2 with sheep, buffalo, cattle, pig, camel, cat, and human sequences showed homology percentages of 100, 97.8, 96.3, 72.4, 72.4, 67.2, and 64.7, respectively. Amino acid sequence of goat IFN-γ showed 98.6, 95.8, 81.1, 81.8, 80.4, and 62.9 percent homology with sheep, bovine, pig, horse, dog, and humans, respectively. Homology ranging from 81.6 to 99% for IL-12 p35 sequences and 85.6 to 100% for IL-12 p40 sequences at amino acid level were observed across these species. Multiple sequence alignment and phylogenetic analysis of goat cytokines revealed close relationship with sheep sequence.     

Molecular evidence of occurrence of Tomato Leaf Curl New Delhi Virus infecting cucurbits in several states in India

Abstract

Tomato Leaf Curl New Delhi Virus (ToLCNDV) is an important begomovirus constraint to the production of cucurbitaceous and other crops in India and its subcontinent. In this study first time, symptomatic samples of different cucurbits were collected from Telangana, Uttar Pradesh (Varanasi) and Madhya Pradesh location in India. The symptomatic samples were associated with begomovirus infection was confirmed by PCR amplification using specific universal primers Deng 540/541. Thirteen out of fifteen samples were amplified with newly designed bipartite specific primers JKR 58?F and JKR 59?R. In eleven out of thirteen bipartite samples contain betasatellite, confirmed by PCR amplification using specific primer CLB36F and CLB37R. The amplified PCR product with JKR58F and 59R of bitter gourd sample collected from Telangana was sequenced. The sequence was share 97.53% similarity with ToLCNDV infecting bottle gourd in Haryana in India (FN645905). Phylogenic analysis revealed that ToLCNDV infecting cucurbits are different, from ToLCNDV infecting tomato.     

Immature and Mature Dengue Serotype 1 Virus Structures Provide Insight into the Maturation Process

Dengue virus is a major human pathogen that has four serotypes (DENV1 to -4). Here we report the cryoelectron microscopy (cryo-EM) structures of immature and mature DENV1 at 6- and 4.5-Å resolution, respectively. The subnanometer-resolution maps allow accurate placement of all of the surface proteins. Although the immature and mature viruses showed vastly different surface protein organizations, the envelope protein transmembrane (E-TM) regions remain in similar positions. The pivotal role of the E-TM regions leads to the identification of the start and end positions of all surface proteins during maturation.     

Urban health and pharmaceutical consumption in Delhi, India

This paper interrogates the routine and unproblematic use of terms such as 'self-medication' in biomedical and anthropological discourse. A typical depiction of the social factors that explain the practice of 'self-medication' in India is to put together the supply side factors (such as protection offered by the government for the production of generic drugs, especially in the small scale sector, and expansion of the number of drug store outlets), with the increasing demand for allopathic drugs. The paper provides an ethnographic account of the intricate connections between households and biomedical practitioners in urban neighbourhoods in Delhi. It breaks away from the conventional opposition drawn between the practices of physicians and the beliefs of their patients, and suggests that what constitutes the medical environments of these neighbourhoods is the product of medical practices, household economies and concepts of disease. Thus pharmaceutical use is determined as much by practices of dispensation and by how practitioners understand what constitutes therapy as by household understanding of the normal and the pathological. This paper uses both quantitative data and narrative interviews to provide an in-depth understanding of the circulation of pharmaceuticals within the life worlds of the urban poor.     

Identification of Dengue Virus Serotype 3 Specific Antigenic Sites Targeted by Neutralizing Human Antibodies

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Molecular Phylogenetic Analysis of Candida krusei

Revealing the phylogenetic relationships of Candida krusei strains (sexual form Pichia kudriavzevii) is a prerequisite for understanding the evolution of its virulence-associated mechanisms and ecological lifestyles. Molecular phylogenetic analyses based on entire internal transcribed spacer region (ITS) and multilocus sequence typing (MLST) data were carried out with sequences available in public databases and Hungarian isolates from animals obtained for the study. The ITS haplotype network yielded a high frequency haplotype at the centre of the network (H1; n?=?204) indicating that various selective pressure might resulted in population expansion from H1. MLST analysis identified three new genotypes among animal-derived isolates, therefore overall 203 sequence types were investigated to determine the population structure of C. krusei. The most commonly encountered sequence types were ST 17 and ST 67. Phylogenetic analyses showed diverse genetic construction of C. krusei population. Evidence of potential recombination events were also observed that might play some role in high intraspecies genetic variability among strains, however, the limited data of C. krusei genotypes from different countries prevented us to identify accurate evolutionary routes of commensal and pathogenic strains or species-specific lineages. Further expansion of C. krusei MLST database may promote the better understanding of the mixed evolutionary history of this species.

     

Emission Estimates of Particulate PAHs from Biomass Fuels Used in Delhi,India

The emission factors for particulate-phase polycyclic aromatic hydrocarbons (PAHs) were evaluated for various biomass fuels (fuelwood, dung cakes, and agricultural residue) that are being commonly used in Delhi as a source of energy. Emission factors of total particulate PAHs varied from 35.9 ± 1.9 to 59.7 ± 4.4 mg/kg. Higher levels of total PAHs and particulate matter (PM) were found from dung cakes as compared to fuelwood and agricultural residue. The emission factors for PM from dung cake, fuelwood, and agricultural residue are 25 ± 8, 15 ± 3.2, and 12.1 ± 9.4 g/kg, respectively. The total PAH emissions showed an increase with high particulate matter emission rates and lower combustion efficiency. Fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, and benzo[a]pyrene constituted the major fraction of PAH emissions from all biomass fuels. The annual budget estimates (total emissions per year) for PAHs and PM from biomass fuels used in Delhi are 30.5 ± 2.3 Mg (Megagrams) and 11.6 ± 4.4 Gg (Gigagrams), respectively. An attempt has also been made to evaluate the preliminary budget estimates of PAHs and PM emitted from the use of biomass fuels as a source of energy in India by using the emission factors obtained in the present study.     

Molecular Phylogenetic Analysis Reveals the New Genus Hemisphaericaspora of the Family Debaryomycetaceae

Four strains of a novel ascomycetous yeast species were recovered from the frass of wood-boring beetles collected from the Baotianman Nature Reserve and the Laojieling Nature Reserve in Henan Province, China. This species produced unconjugated and deliquescent asci with hemispheroid or helmet-shaped ascospores. Analysis of gene sequences for the D1/D2 domain of the large subunit (LSU) rRNA, as well as analysis of concatenated gene sequences for the nearly complete small subunit (SSU) rRNA and D1/D2 domain of the large subunit (LSU) rRNA placed the novel species in a small clade including only one recognised species, Candida insectamans, in the family Debaryomycetaceae (Saccharomycotina, Ascomycota). DNA sequence analyses demonstrated that the novel species was distinct from all currently recognised teleomorphic yeast genus. The name Hemisphaericaspora nanyangensis gen nov., sp. nov. is proposed to accommodate the novel genus and species. The new genus can be distinguished from closely related teleomorphic genera Lodderomyces and Spathaspora through sequence comparison and ascospore morphology. The ex-type strain of H. nanyangensis is CBS 13020^T ( = CICC 33021 = NYNU 13717). Furthermore, based on phenotypic and genotypic characteristics, C. insectamans is transferred to the newly described genus as Hemisphaericaspora insectamans comb. nov., in accordance with the changes in the International Code of Nomenclature for algae, fungi and plants.     

铲形杰氏涡虫(单肠目,达氏科)新纪录的形态学描述及其分子系统学分析

本文对分布于广东省深圳市侨城湿地(113°58'E,22°31'N)咸淡水的一种单肠目Rhabdocoel涡虫进行了形态、发育及18S r DNA与28S r DNA分子系统学的分析与比较。结果表明,此涡虫为达氏科Dalyelliidae铲形杰氏涡虫Gieysztoria knipovici Beklemischev,1953,为中国新纪录种。该物种模式标本分布于欧洲黑海近岸。本研究详细描述了其外部形态与内部结构特征,首次发现该涡虫精巢超长,前端达咽部,后端至腹侧,呈长条形(该属涡虫2个精巢呈球形或椭圆形,位于肠后腹侧);除了骨质阴茎之外还具有可再生的骨质阴茎鞘。阴茎鞘位于骨质阴茎外侧,半包裹骨质阴茎,呈长条形骨质结构。交配后的个体常在交配囊内发现阴茎鞘1~5个。鉴于该物种特殊的形态学特征,其分类地位还有待进一步研究。     

The changing epidemiology of dengue in Delhi, India

The distribution of deformed wing virus infection within the honey bee reproductive castes (queens, drones) was investigated by in situ hybridization and immunohistology from paraffin embedded sections. Digoxygenin or CY5.5 fluorochrome end-labelled nucleotide probes hybridizing to the 3' portion of the DWV genome were used to identify DWV RNA, while a monospecific antibody to the DWV-VP1 structural protein was used to identify viral proteins and particles. The histological data were confirmed by quantitative RT-PCR of dissected organs. Results showed that DWV infection is not restricted to the digestive tract of the bee but spread in the whole body, including queen ovaries, queen fat body and drone seminal vesicles.