Indian Strains Research Articles

Molecular detection and characterization of Influenza ‘C’ viruses from western India

Since 2003, India has had a well-established influenza surveillance network, though Influenza C virus was not the focus of study. We therefore retrospectively analyzed clinical samples from Pune, western India collected during January 2009 to August 2015, by real-time RT-PCR. Three of 2530 samples of patients with influenza-like illness (ILI) or severe acute respiratory illness (SARI) showed positivity for Influenza C virus infection, while 105 and 31 samples were positive for Influenza A and B viruses respectively. Influenza C viruses were successfully isolated using the embryonated egg system and whole genomes were sequenced and analyzed phylogenetically. HE gene-based phylogeny showed that two viruses C/India/P119564/2011 and C/India P121719/2012 clustered with the C/Sao Paulo/378/82 (SP82) lineage, whereas C/India/P135047/2013 clustered with the C/Kanagawa/1/76 (KA76) lineage. The internal gene of these viruses grouped in two lineages. The PB1, PB2, M and NS genes of the study viruses grouped with C/Yamagata/26/81 (YA81), while the P3 (PA) and NP genes grouped with C/Mississippi/80 (MS80). Bayesian clock studies conclude that the Indian strains may have emerged through multiple reassortment events.

Molecular epidemiology of beta-lactamase producing nosocomial Gram-negative pathogens from North and South Indian hospitals.

Resistant Gram-negative bacterial (GNB) infections, apart from tremendously escalating the cost of treatment, are a cause for substantial morbidity and mortality among hospitalized patients. Such bacteria are rapidly acquiring resistance to many antimicrobial agents, especially the beta-lactams which are the most frequently prescribed antimicrobials in hospital and community patient care settings, and now also to colistin; a last-line drug to treat infections with such bacteria. The greatest threat to antimicrobial treatment is the production of metallo beta-lactamases, and plasmid-mediated serine carbapenemases. We conducted a two-year study to observe the pattern of beta-lactamase enzyme production (extended spectrum beta-lactamases (ESBLs), AmpC and carbapenemases) among the nosocomial GNB isolated from intensive care units (ICUs) of North and South Indian hospitals. A total of 761 non-duplicate GNB were included in the study from North (554; 73 %) and South India (207; 27 %). All strains were subjected to Clinical and Laboratory Standards Institute (CLSI) recommended screening tests for detection of beta-lactamase production, followed by polymerase chain reaction (PCR)-based detection of clinically important beta-lactamase genes mediating resistant phenotypes among these isolates. Out of the 761 GNB, Acinetobacter spp., Klebsiella spp., Pseudomonas spp., Enterobacter spp. and others were 27, 23 , 21 , 17 , 5 and 7 % respectively. A high prevalence of ESBL was found across all genera in these strains. The carbapenem resistance was higher in North than in South Indian GNB. The level of AmpC production was comparatively lower in both North and South Indian strains. Beta-lactamases showed tremendous variation in geographic distribution. Thus, their detection and characterization is important from a clinical-epidemiological, laboratory and infection control point of view. Knowledge of this epidemiology can predict the empiric antimicrobial treatment.

Comparison of different inactivation methods on the stability of Indian vaccine strains of foot and mouth disease virus

In this study, the efficiency of binary ethyleneimine (BEI) in combination with formaldehyde (FA) and glutaraldehyde (GTA) in inactivating the Indian FMDV vaccine strains is compared. The acceptable safety of virus inactivation was faster and the inactivation rates were increased many-folds with combination of inactivants than BEI alone. FMDV A was inactivated rapidly than the other two serotypes with BEI + FA combination. Inactivation plots were linear for all the serotypes irrespective of inactivation process. Further, the integrity studies on 146S using serotype specific ELISA indicated no significant change in the antigenic mass of all the serotypes throughout the inactivation process. However, the loss of 146S antigen occurred in the subsequent steps of downstream processing. Further, the studies on intactness of viral RNA using real time PCR indicated the amplification of 1D gene sequences in all the preparations of timed samples irrespective of serotypes/inactivation process. Further, inactivated virus preparation (146S) was more stable at lower temperatures for all the serotypes/inactivation process. Among the combinations of inactivants, BEI + FA out performed compared to BEI + GTA and BEI in terms of inactivation rates, 146S yield and its storage stability, irrespective of the serotypes.

Genetic signatures coupled with lineage shift characterise endemic evolution of Dengue virus serotype 2 during 2015 outbreak in Delhi, India.

In 2015, New Delhi witnessed a massive outbreak of Dengue virus (DENV) resulting in high morbidity and mortality. We report the molecular characterisation of the dominant circulating DENV strain to understand its evolution and dispersal. DENV infections were diagnosed by detection of IgM/NS1 antigen, and serotyping was performed by C-PrM PCR. Envelope gene was amplified, and variation(s) in envelope gene were analysed. Phylogenetic tree construction, time-based phylogeny and origin of DENV were analysed. Site-specific selection pressure of envelope gene variants was analysed. Confirmed DENV infection was observed in 11.34% (32 of 282) cases, while PCR positivity for C-PrM region was observed in 54.16% (13 of 24) of NS1 antigen-positive cases. All samples belonged to serotype 2 and cosmopolitan genotype. Phylogenetic analysis using envelope gene revealed segregation of cosmopolitan genotype strains into specific lineages. The Indian strains clustered separately forming a distinct monophyletic lineage (lineage III) with a signature amino acid substitution viz., I162V and R288K. Selection pressure analysis revealed that 215D, 288R and 304K were positively selected sites. The rate of nucleotide substitution was 6.93×10-4 substitutions site-1 year-1 with time to most common ancestor was around 10years with JX475906 (Hyderabad strain) and JN030345 (Singapore strain) as its most probable ancestor. We observed evolution of a distinct lineage of DENV-2 strains on the Indian subcontinent with possible changes in endemic circulating dengue strains that might give rise to more pathogenic strains.

Identification and physiological properties of a Nigerian strain of Volvariella sp. isolated from oil palm waste.

Over the years several species of edible mushrooms have been collected for consumption from different agro-wastes in Nigeria. Identification of most of these mushrooms was often by morphological descriptive methods. This study reports the morphological study, physiological study and identification of a Nigerian wild strain of Volvariella sp. (VNW) isolated from discarded oil palm waste and three Indian commercial strains V11, V245 and V247 of V. volvacea. Effect of incubation temperatures and medium pH was investigated. Molecular characterization of the strains was carried out using ITS-1 and ITS-4 primers. Results obtained showed close similarities of the Nigerian strain to the Indian strains with few morphological variations in colour, shape and appearance. Growth was observed at temperature range of 20-40 °C and pH range of 4.0-8.0 for all strains with optimum mycelia extension at 35 °C and pH 6.0. VNW recorded a significantly higher (p ≤ 0.05) mycelia extension rate at 35 °C (25.20 ± 1.80mm/day) and pH 6.0 (40.20 ± 0.34mm/day). Highest biomass yield was observed at pH 6.0 with V11 recording a significantly (p ≤ 0.05) higher yield (1.74 ± 0.07g/100mL). Increasing percentage (w/v) of CaSO4·H2O increased biomass yield of all the strains. NJ phylogenetic tree showed the Nigerian and Indian strains in the same cluster indicating evolutionary closeness than with other species of Volvariella from GenBank in a separate cluster even though they share a common ancestor. This successfully proves the identity of a Nigerian strain of Volvariella sp. VNW from oil palm waste as V. volvacea with GenBank accession number KC894923.

Genetic characterization of enterovirus strains identified in Hand, Foot and Mouth Disease (HFMD): Emergence of B1c, C1 subgenotypes, E2 sublineage of CVA16, EV71 and CVA6 strains in India

Hand, Foot and Mouth disease (HFMD) is a common childhood disease and caused due to Enterovirus-A (EV-A), EV-B and EV-C species worldwide. Cases of HFMD were reported from, Ahmedabad (Gujarat, 2012) and Pune (Maharashtra, 2013–2014) in India. The present study highlights the identification of EV strains (CVA16, CVA6, CVA4 and Echo12), characterization of subgenotypes of CVA16, CVA6 strains during 2012–14 and CVA16, CVA6, EV71 strains reported from the earlier study (2009–10) in HFMD cases from India. A total 158 clinical specimens collected from 64 HFMD cases (2012–2014) were included in the study. EV detection was carried out by 5′NCR based RT-PCR, molecular typing and subgenotyping was by VP1/2A junction or VP1, full VP1 gene amplification respectively followed by phylogenetic analysis. The present study reports 63.92% (101/158) EV positivity by RT-PCR. Ninety four of the 101 (93.06%) EV positive strains were amplified by VP1/2A junction or VP1 regions. Sequence analysis revealed the presence of CVA16 (61.7%), CVA6 (34.04%), CVA4 and Echo12 (4.3%). A total of 114 EV positive strains were genotyped using full and partial VP1 region. All CVA16 Indian strains (n=70) clustered with rarely reported B1c subgenotype, CVA6 (n=43) and EV71 (n=1) strains clustered with sub-lineage E2 and C1 subgenotypes respectively. In summary, the study reports genetic characterization of CVA16, CVA6, CVA4 and Echo12 strains in HFMD cases from India. Circulation of B1c subgenotype of CVA16, E2 sub-lineage of CVA6 and C1 subgenotype of EV 71 strains in HFMD cases were reported for the first time from India. This study helps to understand the genotype distribution, genetic diversity of EV strains associated with HFMD from Eastern, Western and Southern regions in India.

Molecular characterization and phylogenetic analyses of virulent infectious bronchitis viruses isolated from chickens in Eastern Saudi Arabia.

Infectious bronchitis virus (IBV) is one of the major respiratory viral threats for chickens. Despite the intensive application of IBV vaccines, several outbreaks have been reported worldwide. Here, we report several IBV outbreaks in thirteen poultry farms in Eastern Saudi Arabia (ESA) from 2013 to 2014. The main goals of the current study were as follows: (1) isolation and molecular characterization of the currently circulating strains in ESA (Al-Hasa, Dammam, and Buqayq) and (2) evaluation of the immune status of these birds to IBV. To achieve our goals, tissue specimens (trachea, lungs, liver, kidney and cecal tonsils) and sera were collected. High morbidity up to 100% and mortality ranging from 18 to 90% were reported. Severe infection was observed in the trachea, bronchi, and kidneys of the infected birds. IBV strains were isolated using embryonated chicken eggs. The isolated viruses induced hemorrhage, dwarfing and death of the inoculated embryos 3–5 days post-infection. The circulating IBV strains were identified by sequencing the partial IBV-N and IBV-S1 genes. These viruses showed 95% sequence identity to Indian, Italian, Egyptian and Chinese strains and were quite distinct from the locally used vaccines on the genomic level. Interestingly, high antibody titers against IBV were reported in some of these farms, suggesting the presence of new virulent strains in ESA. The seroconversion of infected birds was reported among the affected flocks. In conclusion, very virulent IBV strains are currently circulating in ESA. Further studies are currently in progress to molecularly characterize these IBV strains. Electronic supplementary materialThe online version of this article (doi:10.1007/s13337-017-0375-7) contains supplementary material, which is available to authorized users.

Voacamine alters Leishmania ultrastructure and kills parasite by poisoning unusual bi-subunit topoisomerase IB

Indole alkaloids possess a large spectrum of biological activities including anti-protozoal action. Here we report for the first time that voacamine, isolated from the plant Tabernaemontana coronaria, is an antiprotozoal agent effective against a large array of trypanosomatid parasites including Indian strain of Leishmania donovani and Brazilian strains of Leishmania amazonensis and Trypanosoma cruzi. It inhibits the relaxation activity of topoisomerase IB of L. donovani (LdTop1B) and stabilizes the cleavable complex. Voacamine is probably the first LdTop1B-specific poison to act uncompetitively. It has no impact on human topoisomerase I and II up to 200μM concentrations. The study also provides a thorough insight into ultrastructural alterations induced in three kinetoplastid parasites by a specific inhibitor of LdTop1B. Voacamine is also effective against intracellular amastigotes of different drug unresponsive field isolates of Leishmania donovani obtained from endemic zones of India severely affected with visceral leishmaniasis. Most importantly, this is the first report demonstrating the efficacy of a compound to reduce the burden of drug resistant parasites, unresponsive to SAG, amphotericin B and miltefosine, in experimental BALB/c mice model of visceral leishmaniasis. The findings cumulatively provide a strong evidence that voacamine can be a promising drug candidate against trypanosomatid infections.

Differential sensitivity to infections and antimicrobial peptide-mediated immune response in four silkworm strains with different geographical origin

The domesticated silkworm Bombyx mori has an innate immune system, whose main effectors are the antimicrobial peptides (AMPs). Silkworm strains are commonly grouped into four geographical types (Japanese, Chinese, European and Tropical) and are generally characterised by a variable susceptibility to infections. To clarify the genetic and molecular mechanisms on which the different responses to infections are based, we exposed one silkworm strain for each geographical area to oral infections with the silkworm pathogens Enterococcus mundtii or Serratia marcescens. We detected a differential susceptibility to both bacteria, with the European strain displaying the lowest sensitivity to E. mundtii and the Indian one to S. marcescens. We found that all the strains were able to activate the AMP response against E. mundtii. However, the highest tolerance of the European strain appeared to be related to the specific composition of its AMP cocktail, containing more effective variants such as a peculiar Cecropin B6 isoform. The resistance of the Indian strain to S. marcescens seemed to be associated with its prompt capability to activate the systemic transcription of AMPs. These data suggest that B. mori strains with distinct genetic backgrounds employ different strategies to counteract bacterial infections, whose efficacy appears to be pathogen-dependent.

Emergence of West Nile virus in West Bengal, India: a new report.

The ICMR virus unit in Kolkata functions as an Appex Referral Laboratory for the detection of dengue (DENV) and chikungunya (CHIKV) infections in the eastern part of India. In spite of efforts for confirmatory diagnosis, some samples remain undiagnosed every year. West Nile virus (WNV) infection may mimic either dengue (flavivirus) or chikungunya (alphavirus) like illness. WNV is endemic in the tropical region where its principal/potential vectors are Aedes and Culex. We explored the existence of WNV within undiagnosed samples to identify the emergence of a new public health problem. Of 1278 sera samples, 574 were negative for DENV and CHIKV either by ELISA or by reverse transcriptase (RT)-PCR. Of these 574 negative samples, 83 (14.5%) and 141 (24.56%) were positive for WNV by ELISA and RT-PCR, respectively; no samples were positive for WNV by both methods. After assembling raw sequencing data, partial envelope genome sequence of West Bengal isolates, WNV was compared through BLAST with other WNV Indian strains and 98% homology detected. Phylogenetic analysis of one West Bengal isolates (Accession No. KY421790) and 28 Indian isolates available in GenBank, indicated close clustering. The serological and molecular approaches have clearly established the emergence of WNV in West Bengal. Hence, for proper case management, detection of WNV in common febrile illness is strongly recommended.

Recombinant human adenovirus-5 expressing capsid proteins of Indian vaccine strains of foot-and-mouth disease virus elicits effective antibody response in cattle

Recombinant adenovirus-5 vectored foot-and-mouth disease constructs (Ad5- FMD) were made for three Indian vaccine virus serotypes O, A and Asia 1. Constructs co-expressing foot-and- mouth disease virus (FMDV) capsid and viral 3C protease sequences, were evaluated for their ability to induce a neutralizing antibody response in indigenous cattle (Bos indicus). Purified Ad5-FMD viruses were inoculated in cattle as monovalent (5×109 pfu/animal) or trivalent (5×109 pfu/animal per serotype) vaccines. Animals vaccinated with monovalent Ad5-FMD vaccines were boosted 63days later with the same dose. After primary immunization, virus neutralization tests (VNT) showed seroconversion in 83, 67 and 33% of animals vaccinated with Ad5-FMD O, A and Asia 1, respectively. Booster immunization elicited seroconversion in all of the animals (100%) in the monovalent groups. When used in a trivalent form, the Ad5-FMD vaccine induced neutralizing antibodies in only 33, 50 and 16% of animals against serotypes O, A and Asia 1, respectively on primo-vaccination, and titers were significantly lower than when the same vectors were used in monovalent form. Neutralizing antibody titers differed by serotype for both Ad5-FMD monovalent and trivalent vaccines, with Asia 1 serotype inducing the lowest titers. Antibody response to Ad5 vector in immunized cattle was also assessed by VNT. It appeared that the vector immunity did not impact the recall responses to expressed FMDV antigens on booster immunization. In summary, the study suggested that the recombinant Ad5-FMD vaccine has a potential use in monovalent form, while its application in multivalent form is not currently encouraging.

Impact of Ocimum Basilicum Leaf Essential Oil on The Survival and Behaviour of An Indian Strain of Dengue Vector, Aedes aegypti (L.)

Objective: Aedes aegypti (L.) is a well-known widely spread disease vector transmitting several diseases of concern. Current investigations were undertaken to discover the larvicidal and repellent potential of essential oil from the basil plant, Ocimum basilicum leaves against Ae. aegypti as a suitable and eco-safe alternate to chemical insecticides. Methods: The larvicidal potential of basil oil was evaluated against Ae. aegypti early fourth instars using WHO protocol and LC50 and LC90 values were calculated. The efficacy of basil oil as mosquito repellent was assessed using the human-bait technique for 3 hours and the percent protection was calculated. Results: Exposure of early fourth instars of Ae. aegypti with various concentrations of O. basilicum essential oil for 24 h revealed a moderate LC50 and LC90 value of 141.95 ppm and 100.82 ppm, respectively. The investigations also showed that oil did not have any hormono-mimetic effects as it did not result in the formation of any larval-pupal intermediates. However, the basil essential oil exhibited significant effective repellency against adult Ae. aegypti. Introduction of the arms of human volunteers applied with basil oil in the cages of dengue vector resulted in 100% protection till 120 min as compared to control arm which experienced 3 to 8 bites. Remarkably, the experimental arm was bitten just once in every 15 minutes in next 45 minutes after which only 2 bites were recorded until 180 min of exposure. Conclusion: Our results recommended the efficient use of basil leaf essential oil as an efficient repellent and as a moderate larvicide agent against Ae. aegypti. Identification of bioactive components in the oil, their individual mode of action and synergistic effects, along with investigations on their impact on the environment and nontarget organisms would help in formulating strategies for mosquito management.

Genetic Characterization of Circulating 2015 A(H1N1)pdm09 Influenza Viruses from Eastern India.

In 2015, the swine derived A(H1N1)pdm09 pandemic strain outbreak became widespread throughout the different states of India. The reported cases and deaths in 2015 surpassed the previous years with more than 39000 laboratory confirmed cases and a death toll of more than 2500 people. There are relatively limited complete genetic sequences available for this virus from Asian countries. In this study, we describe the full genome analysis of influenza 2015 A(H1N1)pdm09 viruses isolated from West Bengal between January through December 2015. The phylogenetic analysis of the haemagglutinin sequence revealed clustering with globally circulating strains of genogroup 6B. This was further confirmed by the constructed concatenated tree using all eight complete gene segments of Kolkata A(H1N1)pdm09 isolates with the other strains from different timeline and lineages. A study from Massachusetts Institute of Technology (MIT) in 2015 reported novel mutations T200A and D225N in haemagglutinin gene of a 2014 Indian strain (A/India/6427/2014). However, in all the pandemic strains of 2014–2015 reported from India, so far including A(H1N1)pdm09 strains from Kolkata, D225N mutation was not observed, though the T200A mutation was found to be conserved. Neuraminidase gene of the analyzed strains did not show any oseltamivir resistant mutation H275Y suggesting continuation of Tamiflu® as drug of choice. The amino acid sequences of the all gene segments from 2015 A(H1N1)pdm09 isolates identified several new mutations compared to the 2009 A(H1N1)pdm09 strains, which may have contributed towards enhanced virulence, compared to 2009 A(H1N1)pdm09 strains.

Phylogenetic relationship and genotypic variability in Anaplasma marginale strains causing anaplasmosis in India

Anaplasma marginale is a tick borne rickesttsial parasite known to cause bovine anaplasmosis. There are prevalence reports from different parts of India, however, information regarding genetic diversity and phylogenetic association of the Indian strains are unknown. In the current study, 965 cattle blood samples from two states of India, Seemandhra and Telangana, were investigated for the presence of A. marginale by PCR using major surface protein 4 gene (msp4). We found an overall infection of 16.4%, with 3.4% prevalence in Seemandhra and 22.2% in Telangana. Sequence analysis of the 24 cloned msp4 gene indicated genetic diversity among Indian clinical strains of A. marginale which may be due to evolutionary pressure or migration of strains. Phylogenetic association analysis revealed that most of the strains showed close proximity with strains from Mexico and other strains showed closeness to strains reported from countries like Brazil, Zimbabwe, Prico and Hungary. This is the first report from India, identifying heterogeneous population of A. marginale strains causing anaplasmosis, and such data can play an important role in designing new control policies.

Development of a novel rapid micro-neutralization ELISA for the detection of neutralizing antibodies against Chandipura virus

BackgroundChandipura virus (CHPV) is a leading cause of acute encephalitis with high mortality in paediatric population in India. A micro-neutralization ELISA (MN-ELISA) assay was developed for the detection of neutralizing antibodies (Nab) against CHPV. This novel method gives read-out in the form of ELISA optical density (OD) values and has a shorter turn-around time (TAT) as compared to the conventional cytopathic effect (CPE)-based neutralization assay (MN-CPE). The assay was developed using an Indian strain of CHPV. During the development of the assay different parameters such as cell count, dilution of primary and secondary antibodies and time point for the test termination were optimized. The new and conventional assays were run in parallel where known positive and negative human serum samples were used as test controls. The conventional MN-CPE was terminated at 48h post-infection (p.i.) and stained with Amido black, while in the new assay, MN-ELISA was terminated at pre-determined 18h p.i. and the infected cells were fixed with acetone, followed by in-situ ELISA. Results of both the assays were compared.The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the new test was 100% when compared with the conventional MN-CPE method as a ‘gold standard’. The MN-ELISA showed two-fold higher antibody titer in one sample and one sample was additionally positive than MN-CPE ELISA. ConclusionThe MN-ELISA is rapid, more sensitive and read-out of results is by measurement of OD, which could be more accurate than manual observation of reduction in CPE. This novel test could be used as an alternative to the conventional MN-CPE based assay in sero-surveillance and in future vaccine studies.

Measles virus genotypes circulating in India, 2011-2015.

The Government of India is accepted to participate in the measles elimination and rubella control goal 2020, hence genetic characterization of measles viruses (MeV) becomes essential. At National Reference Laboratory (National Institute of Virology, Pune), the throat swabs/urine specimens (n = 380) or PCR products (n = 219) obtained from the suspected measles cases were referred for the molecular testing and subsequently, MeV nucleoprotein (N) gene sequencing/genotyping. In addition, 2,449 suspected measles cases, mainly from the Maharashtra state were referred for the laboratory diagnosis. A detailed study was performed on N gene sequences obtained during last two decades. Indian MeV sequences obtained during 2011-2015 were compared with 1996-2010 sequences and genetic divergence was studied. Circulation of measles genotypes B3 (n = 3), D4 (n = 49), and D8 (n = 351) strains were observed in 19 States and three Union Territories of India. In addition, 64 measles viruses were isolated from 253 throat swab or urine specimens obtained from the suspected measles cases. During 2011-2015, 67.9% (1,663/2,449) suspected measles cases were laboratory confirmed. Molecular studies showed circulation of measles genotype B3 in India along with prominently circulating genotypes D4 and D8 except D7 strains. The genetic diversion within Indian B3, D4, and D8 genotypes was 0.3%, 1.1%, and 2.1%, respectively. The genetic divergence of Indian B3, D4, and D8 measles strains with the WHO reference sequences was 2.5%, 2.6%, and 1.8%, respectively. It is crucial data for national immunization program. More measles/rubella genotyping studies are necessary to track transmission and to support measles elimination and rubella control. J. Med. Virol. 89:753-758, 2017. © 2016 Wiley Periodicals, Inc.

Whole Genome Sequencing Demonstrates Limited Transmission within Identified Mycobacterium tuberculosis Clusters in New South Wales, Australia.

Australia has a low tuberculosis incidence rate with most cases occurring among recent immigrants. Given suboptimal cluster resolution achieved with 24-locus mycobacterium interspersed repetitive unit (MIRU-24) genotyping, the added value of whole genome sequencing was explored. MIRU-24 profiles of all Mycobacterium tuberculosis culture-confirmed tuberculosis cases diagnosed between 2009 and 2013 in New South Wales (NSW), Australia, were examined and clusters identified. The relatedness of cases within the largest MIRU-24 clusters was assessed using whole genome sequencing and phylogenetic analyses. Of 1841 culture-confirmed TB cases, 91.9% (1692/1841) had complete demographic and genotyping data. East-African Indian (474; 28.0%) and Beijing (470; 27.8%) lineage strains predominated. The overall rate of MIRU-24 clustering was 20.1% (340/1692) and was highest among Beijing lineage strains (35.7%; 168/470). One Beijing and three East-African Indian (EAI) clonal complexes were responsible for the majority of observed clusters. Whole genome sequencing of the 4 largest clusters (30 isolates) demonstrated diverse single nucleotide polymorphisms (SNPs) within identified clusters. All sequenced EAI strains and 70% of Beijing lineage strains clustered by MIRU-24 typing demonstrated distinct SNP profiles. The superior resolution provided by whole genome sequencing demonstrated limited M. tuberculosis transmission within NSW, even within identified MIRU-24 clusters. Routine whole genome sequencing could provide valuable public health guidance in low burden settings.

Molecular Confirmation of Conserved Nature of Genes Encoding MPB53 and MPB63 Immune Dominant Proteins in Indian Strain of Mycobacterium bovis

The genes encoding MPB53 and MPB63 immuno dominant proteins of Mycobacterium bovis field strain 3/86-RV were amplified by polymerase chain reaction (PCR). The recombinant plasmids were constructed via inserting the mpb53 and mpb63 genes into the pET vector (named pET28b-mpb53 and pET28b-mpb63). The nucleotide sequences of the insert PCR products were determined by vector specific primer. Both genes contained an open reading frame of 402 and 390 base pairs for mpb53 and mpb63, respectively. The sequenced genes and its deduced amino acid sequences were compared with the published sequences of reference M. tuberculosis complex strains. The sequences of the mpb53 and mpb63 genes share more than 99 % nucleotide homology and deduced protein sequence homology pointing towards conserved molecular nature of these immunodominant proteins among M. tuberculosis complex species.

Molecular Characterization and Viral Origin of the 2015 Dengue Outbreak in Xishuangbanna, Yunnan, China.

A total of 1067 serum samples were collected from febrile patients in Xishuangbanna, Yunnan, 2015. Of these, 852 cases were confirmed to be dengue NS1-positive. 76 structural protein genes were sequenced through RT-PCR based on the viral RNAs extracted from serum samples. Phylogenetic analysis revealed that all strains were classified as cosmopolitan genotype of DENV-2. After comparing with the DENV-2SS, 173 base substitutions were found in 76 sequences, resulting in 43 nonsynonymous mutations, of which 22 mutations existed among all samples. According to secondary structure prediction, 8 new possible nucelotide/protein binding sites were found and another 4 sites were lost among the 775 amino acids of DENV structural proteins as compared with DENV-2SS. Meanwhile, 6 distinct amino acid changes were found in the helix and strand regions, and the distribution of the exposed and buried regions was slightly altered. The results indicated that the epidemic dengue strains of Xishuangbanna in 2015 are most similar to the Indian strain in 2001 and the Sri Lankan strain in 2004. Moreover, it also show a very strong similarity to the epidemic strains of Fujian province in 1999 and 2010, which show that there is an internal recycling epidemic trend of DENV in China.

Mitochondrial DNA from the eradicated European Plasmodium vivax and P. falciparum from 70-year-old slides from the Ebro Delta in Spain

Phylogenetic analysis of Plasmodium parasites has indicated that their modern-day distribution is a result of a series of human-mediated dispersals involving transport between Africa, Europe, America, and Asia. A major outstanding question is the phylogenetic affinity of the malaria causing parasites Plasmodium vivax and falciparum in historic southern Europe-where it was endemic until the mid-20th century, after which it was eradicated across the region. Resolving the identity of these parasites will be critical for answering several hypotheses on the malaria dispersal. Recently, a set of slides with blood stains of malaria-affected people from the Ebro Delta (Spain), dated between 1942 and 1944, have been found in a local medical collection. We extracted DNA from three slides, two of them stained with Giemsa (on which Plasmodium parasites could still be seen under the microscope) and another one consisting of dried blood spots. We generated the data using Illumina sequencing after using several strategies aimed at increasing the Plasmodium DNA yield: depletion of the human genomic (g)DNA content through hybridization with human gDNA baits, and capture-enrichment using gDNA derived from P. falciparum Plasmodium mitochondrial genome sequences were subsequently reconstructed from the resulting data. Phylogenetic analysis of the eradicated European P. vivax mtDNA genome indicates that the European isolate is closely related to the most common present-day American haplotype and likely entered the American continent post-Columbian contact. Furthermore, the European P. falciparum mtDNA indicates a link with current Indian strains that is in agreement with historical accounts.