Standardization of an in-house multiplex real-time polymerase chain reaction for the simultaneous detection of Toxoplasma gondii, Rubella virus, cytomegalovirus, herpes simplex Virus 1 and 2, and Treponema pallidum infection among pregnant women.

BACKGROUND: An in-house multiplex real-time polymerase chain reaction (PCR) was developed in two cocktails for the identification of six Toxoplasma gondii, Rubella virus, cytomegalovirus, herpes simplex virus (1 and 2), and Treponema pallidum (syphilis) (TORCH-S) agents, which causes congenital infection among pregnant women. OBJECTIVE: Standardization and validation of an in-house multiplex real-time PCR assay for the detection of TORCH-S infection. METHODS: This study was conducted from February 2017 to February 2019. Primers specific for T. gondii, Rubella virus, cytomegalovirus, herpes simplex virus (1 and 2), and T. pallidum were designed using Primer3 software (https://bioinfo.ut.ee/primer3-0.4.0/). The primer sequences obtained were subjected to BLAST analysis using BLAST database. Synthetic DNA was obtained to use as positive control templates for all the six TORCH-S agents. The lower limit of the detection was performed using plasmid construct for each virus serially diluted from 10-1 to 10-9. RESULTS: An in-house multiplex real-time PCR was standardized and validated in two cocktails for TORCH-S agents, cocktail-1 (HSV1, rubella, and T. gondii), and cocktail-2 (HSV2, CMV, and T. pallidum). The lower limit of the detection for HSV1, rubella, and Toxoplasma were 60.7 copies/10 µl input, 76.4 copies/10 µl input, and 34.4 copies/10 µl input and for HSV2, CMV, and T. pallidum were 80.8 copies/10 µl input, 166 copies/10 µl input, and 43.7 copies/10 µl input, respectively. CONCLUSION: TORCH-S infection is one of the significant reasons for irregular pregnant outcomes. It is absolutely important to screen TORCH-S infection for women who had the histories of abnormal pregnancies to prevent birth defects and perinatal complications. This multiplex real-time PCR assay provides a rapid, sensitive, and specific technique to detect these six TORCH-S agents.

B cell epitopes in the intrinsically disordered regions of neuraminidase and hemagglutinin proteins of H5N1 and H9N2 avian influenza viruses for peptide-based vaccine development.

Avian influenza viruses (AIV) are very active in several parts of the globe and are the cause of huge economic loss for the poultry industry and also human fatalities. Three dimensional modeling was carried out for neuraminidase (NA) and hemagglutinin (HA) proteins of AIV. The C-score, estimated TM-Score, and estimated root-mean-square deviation (RMSD) score for NA of H5N1 were -1.18, 0.57 ± 0.15, and 9.8 ± 7.6, respectively. The C-score, estimated TM-Score, and estimated RMSD score for NA of H9N2 were -1.43, 0.54 ± 0.15, and 10.5 ± 4.6, respectively. The C-score, estimated TM-Score, and estimated RMSD score for HA of H5N1 were -0.03, 0.71 ± 0.12, and 7.7 ± 4.3, respectively. The C-score, estimated TM-Score, and estimated RMSD score for HA of H9N2 were -0.57, 0.64 ± 0.13, and 8.9 ± 4.6, respectively. Intrinsically disordered regions were identified for the NA and HA proteins of H5N1 and H9N2 with the use of PONDR program. Linear B cell epitope was predicted using BepiPred 2 program for NA and HA of H5N1 and H9N2 avian influenza strains. Discontinuous epitopes were predicted by Discotope 2 program. The linear epitopes that were considered likely to be immunogenic and within the intrinsically disordered region for the NA of H5N1 was TKSTNSRSGFEMIWDPNGWTGTDSSFSVK, and for H9N2 it was VGDTPRNDDSSSSSNCRDPNNERGAP. In the case of HA of H5N1, it was QRLVPKIATRSKVNGQSG and ATGLRNSPQRERRRKK; for H9N2 it was INRTFKPLIGPRPLVNGLQG and SLKLAVGLRNVPARSSR. The discontinuous epitopes of NA of H5N1 and H9N2 were identified at various regions of the protein structure spanning from amino acid residue positions 90 to 449 and 107 to 469, respectively. Similarly, the discontinuous epitopes of HA of H5N1 and H9N2 were identified in the amino acid residue positions 27 to 517 and 136 to 521, respectively. This study has identified potential and highly immunogenic linear and conformational B-cell epitopes towards developing a vaccine against AIV both for human and poultry use.

Interaction of human immunodeficiency virus-1 and human immunodeficiency virus-2 capsid amino acid variants with human tripartite motif 5α protein SPRY domain and its association with pathogenesis.

Purpose: The sequence variation of human immunodeficiency virus (HIV) capsid region may influence and alter the susceptibility to human tripartite motif 5α protein (huTRIM5α). Materials and Methods: Molecular docking was carried out with huTRIM5α SPRY domain by the use of ClusPro and Hex docking program for HIV-1 and HIV-2 capsid sequences. Results: The sequence analysis on HIV-1 and HIV-2 capsid gag gene identified 35 (19.7%) single-nucleotide polymorphisms (SNPs) in HIV-1 and 8 (4.5%) SNPs in HIV-2. The variations observed in the HIV-2 capsid region were significantly lower than HIV-1 (P < 0.001). The molecular docking analysis showed that HIV-1 wild type used V1 loop, while HIV-2 used V3 loop of huTRIM5α for interaction. HIV-1 with A116T SNP and HIV-2 with V81A SNP use V3 and V1 loop of huTRIM5α for interaction respectively. The reduced huTRIM5α inhibition may lead to a faster progression of disease among HIV-1-infected individuals. However, in case of HIV-2, increased inhibition by huTRIM5α slows down the disease progression. Conclusion: Polymorphisms in the capsid protein with both HIV-1- and HIV-2-monoinfected individuals showed the difference in the docking energy from the wild type. This is the first study which documents the difference in the usage of loop between the two HIV types for interaction with huTRIM5α. Variations in the capsid protein result in alteration in the binding to the restriction factor huTRIM5α.

Identification of B- and T-cell epitopes on HtrA protein of Orientia tsutsugamushi.

Orientia tsutsugamushi, a cause of scrub typhus is emerging as an important pathogen in several parts of the tropics. The control of this infection relies on rapid diagnosis, specific treatment, and prevention through vector control. Development of a vaccine for human use would be very important as a public health measure. Antibody and T-cell response have been found to be important in the protection against scrub typhus. This study was undertaken to predict the peptide vaccine that elicits both B- and T-cell immunity. The outer-membrane protein, 47-kDa high-temperature requirement A was used as the target protein for the identification of protective antigen(s). Using BepiPred2 program, the potential B-cell epitope PNSSWGRYGLKMGLR with high conservation among O. tsutsugamushi and the maximum surface exposed residues was identified. Using IEDB, NetMHCpan, and NetCTL programs, T-cell epitopes MLNELTPEL and VTNGIISSK were identified. These peptides were found to have promiscuous class-I major histocompatibility complex (MHC) binding affinity to MHC supertypes and high proteasomal cleavage, transporter associated with antigen processing prediction, and antigenicity scores. In the I-TASSER generated model, the C-score was -0.69 and the estimated TM-score was 0.63 ± 0.14. The location of the epitope in the 3D model was external. Therefore, an antibody to this outer-membrane protein epitope could opsonize the bacterium for clearance by the reticuloendothelial system. The T-cell epitopes would generate T-helper function. The B-cell epitope(s) identified could be evaluated as antigen(s) in immunodiagnostic assays. This cocktail of three peptides would elicit both B- and T-cell immune response with a suitable adjuvant and serve as a vaccine candidate.

Design of peptide epitope from the neuraminidase protein of influenza A and influenza B towards short peptide vaccine development.

Influenza viruses A and B are important human respiratory pathogens causing seasonal, endemic and pandemic infections in several parts of the globe with high morbidity and considerable mortality. The current inactivated and live attenuated vaccines are not effective. Therefore, it is of interest to design universal influenza virus vaccines with high efficacy. The peptide GQSVVSVKLAGNSSL of pandemic influenza, the peptide DKTSVTLAGNSSLCS of seasonal influenza and the peptide DILLKFSPTEITAPT of influenza B were identified as potential linear cell mediated epitopes. The epitopes predicted by BepiPred (B-cell epitope designer) program was subjected to docking experiment-using HexDock and CABS dock programs. The epitopes of pandemic H1N1 influenza A gave similar score of high affinity in docking. The epitope DKTSVTLAGNSSLCS of seasonal influenza A and epitope DILLKFSPTEITAPT of influenza B had high binding energy. It is further observed that the peptides GQSVVSVKLAGNSSL (pandemic influenza), DKTSVTLAGNSSLCS (seasonal influenza) DILLKFSPTEITAPT (influenza B) are found to interact with some known MHC class II alleles. These peptides have high-affinity binding with known MHC class II alleles. Thus, they have the potential to elicit cell immune response. These vaccines have to be further evaluated in animal models and human volunteers. These findings have application in the development of peptide B-cell epitope vaccines against influenza viruses.

Detection of parvovirus B19 in selected high-risk patient groups & their phylogenetic & selection analysis.

Background & objectives: Human parvovirus B19V (B19V) is known to be associated with erythema infectiosum commonly in children, aplastic crisis, especially in persons with underlying haemolytic disorders, hydrops fetalis in pregnancies and arthritis. This cross-sectional study was aimed to determine the presence of B19V infection in childhood febrile illnesses, association of B19V with arthropathies and in adult patients with end-stage renal disease (ESRD) on dialysis. The genetic diversity among the sequences was also analysed. Methods: A nested polymerase chain reaction (nPCR) assay was used for B19V DNA targeting VP1/VP2 region and used for testing 618 patients and 100 healthy controls. Phylogenetic analysis on nucleotide and amino acid sequences was carried out to compare our sequences with other Indian strains and global strains. Results: Among 618 samples tested, seven (1.13%) were found positive. The phylogenetic analysis revealed that all the seven sequences belonged to genotype 1 and showed low genetic diversity. The clustering pattern of seven sequences was similar both by nucleotide and by predicted amino acid sequences. The fixed effects likelihood analysis showed no positive or negatively selected sites. Interpretation & conclusions: Seven samples (4 from non-traumatic arthropathies, 2 from patients with ESRD and 1 from febrile illness patient) were found positive by nPCR. When our seven sequences were compared with global strains, the closest neighbour was other Indian strains followed by the Tunisian strains.

In Silico Validation of D7 Salivary Protein-derived B- and T-cell Epitopes of Aedes aegypti as Potential Vaccine to Prevent Transmission of Flaviviruses and Togaviruses to Humans.

Mosquito (Aedes aegyptii) salivary proteins play a crucial role in facilitating viral transmission from vector-to-host due to their role in facilitating the "blood meal" of the vector. Three main proteins, D7, aegyptin and Sialokinin play a role in this process. Using in-silico programs, we identified B- and T-cell epitopes in the mosquito salivary proteins D7 long and short form. T-cell epitopes with high affinity to the most prevalent HLA MHC class-I supertypes among different population groups was chosen. It is our postulate that these epitopes could be successful in eliciting B and T cell responses, which would decrease the vector blood meal efficiency and hence protect against host infection by certain viruses. These include causative agents like Dengue viruses, Chikungunya virus, Zika and Yellow fever viruses. These viruses are of major public health importance in several countries in the Americas, Asia and Africa. Experimental evidence exists in previously published literature showing the protective effect of antibodies to certain salivary proteins in susceptible hosts. A novel approach of immunizing humans against the vector proteins to reduce transmission of viruses is now under investigation in several laboratories. We have identified the following two B cell epitopes LAALHVTAAPLWDAKDPEQF one from D7L and the other TSEYPDRQNQIEELNKLCKN from D7S. Likewise, two T cell epitopes MTSKNELDV one from D7L and the other YILCKASAF from D7S with affinity to the predominant MHC class-I supertypes were identified towards evaluation as potential vaccine.

Short peptide epitope design from hantaviruses causing HFRS.

Several genotypes of the hantavirus cause hemorrhagic fever with renal syndrome (HFRS) and is an important public health problem worldwide. There is now growing interest to develop subunit vaccines especially focused to elicit cytotoxic T lymphocyte responses which are important against viral infection. We identified candidate T-cell epitopes that bind to Class I HLA supertypes towards identifying potential subunit vaccine entity. These epitopes are conserved in all 5 hantavirus genotypes of HFRS (Hantaan, Dobrava- Belgrade, Seoul, Gou virus and Amur). The epitopes identified from S and M segment genomes were analyzed for human proteasome cleavage, transporter associated antigen processing (TAP) efficiency and antigenicity using bioinformatic approaches. The epitope MRNTIMASK which had the two characteristics of high proteasomal cleavage score and TAP score, also had high antigenicity score. Our results indicate that this epitope from the nucleocapsid protein may be considered the most favorable moiety for the development of subunit peptide vaccine.

T-cell epitopes predicted from the Nucleocapsid protein of Sin Nombre virus restricted to 30 HLA alleles common to the North American population.

Hantavirus cardiopulmonary syndrome in North America is caused by Sin Nombre virus (SNV) and poses a public health problem. We identified T-cell epitopes restricted to HLA alleles commonly seen in the N. American population. Nucleocapsid (N) protein is 428 aminoacid in length and binds to RNA and functions also as a key molecule between virus and host cell processes. The predicted epitopes from N protein that bind to class I MHC were analyzed for human proteasomes cleavage, TAP efficiency, immunogenicity and antigenicity. We identified 8 epitopes through MHC binding prediction, proteasomal cleavage prediction and TAP efficiency. Epitope VMGVIGFSF had highest Vaxijen score and the epitope, TNRAYFITR had highest immunogenicity score. Epitope AAVSALETK and TIACGLFPA had 100% homology to many HCPS causing viruses. Our study focused on T-cell epitope prediction specific to restricted HLA haplotypes of racial groups in North America for the potential vaccine development. Among the candidate epitopes, FLAARCPFL was conserved in SNV, which is suitable for vaccine specific to the virus genotype. Peptide-based vaccines can be designed to include multiple determinants from several hantavirus genotypes, or multiple epitopes from the same genotype. Thereby, immune response will focus solely on relevant epitopes, avoiding non-protective responses or immune evasion. The other advantages include absence of infectious material unlike in live or attenuated vaccines. There is no risk of reversion or formation of adverse reassortants leading to virulence and no risk of genetic integration or recombination forming a rationale for vaccine design including for distinct geographical regions.

Prediction of Pan-Specific B-Cell Epitopes From Nucleocapsid Protein of Hantaviruses Causing Hantavirus Cardiopulmonary Syndrome.

Hantaviruses are emerging viral pathogens that causes hantavirus cardiopulmonary syndrome (HCPS) in the Americas, a severe, sometimes fatal, respiratory disease in humans with a case fatality rate of ≥50%. IgM and IgG-based serological detection methods are the most common approaches used for laboratory diagnosis of hantaviruses. Such emerging viral pathogens emphasizes the need for improved rapid diagnostic devices and vaccines incorporating pan-specific epitopes of genotypes. We predicted linear B-cell epitopes for hantaviruses that are specific to genotypes causing HCPS in humans using in silico prediction servers. We modeled the Andes and Sin Nombre hantavirus nucleocapsid protein to locate the identified epitopes. Based on the mean percent prediction probability score, epitope IMASKSVGS/TAEEKLKKKSAF was identified as the best candidate B-cell epitope specific for hantaviruses causing HCPS. Promiscuous epitopes were identified in the C-terminal of the protein. Our study for the first time has reported pan-specific B-cell epitopes for developing immunoassays in the detection of antibodies to hantaviruses causing HCPS. Identification of epitopes with pan-specific recognition of all genotypes causing HCPS could be valuable for the development of immunodiagnositic tools toward pan-detection of hantavirus antibodies in ELISA. J. Cell. Biochem. 118: 2320-2324, 2017. © 2017 Wiley Periodicals, Inc.

Application of viromics: a new approach to the understanding of viral infections in humans.

This review is focused at exploring the strengths of modern technology driven data compiled in the areas of virus gene sequencing, virus protein structures and their implication to viral diagnosis and therapy. The information for virome analysis (viromics) is generated by the study of viral genomes (entire nucleotide sequence) and viral genes (coding for protein). Presently, the study of viral infectious diseases in terms of etiopathogenesis and development of newer therapeutics is undergoing rapid changes. Currently, viromics relies on deep sequencing, next generation sequencing (NGS) data and public domain databases like GenBank and unique virus specific databases. Two commonly used NGS platforms: Illumina and Ion Torrent, recommend maximum fragment lengths of about 300 and 400 nucleotides for analysis respectively. Direct detection of viruses in clinical samples is now evolving using these methods. Presently, there are a considerable number of good treatment options for HBV/HIV/HCV. These viruses however show development of drug resistance. The drug susceptibility regions of the genomes are sequenced and the prediction of drug resistance is now possible from 3 public domains available on the web. This has been made possible through advances in the technology with the advent of high throughput sequencing and meta-analysis through sophisticated and easy to use software and the use of high speed computers for bioinformatics. More recently NGS technology has been improved with single-molecule real-time sequencing. Here complete long reads can be obtained with less error overcoming a limitation of the NGS which is inherently prone to software anomalies that arise in the hands of personnel without adequate training. The development in understanding the viruses in terms of their genome, pathobiology, transcriptomics and molecular epidemiology constitutes viromics. It could be stated that these developments will bring about radical changes and advancement especially in the field of antiviral therapy and diagnostic virology.

Development & standardization of an in-house IgM indirect ELISA for the detection of parvovirus B19 infections.

BACKGROUND & OBJECTIVES: Parvovirus B19 infections occur worldwide; the infection is acquired early in childhood but could occur later. B19 is reported to cause infection in childhood febrile illnesses, and arthropathies in adults and children and in end-stage renal disease (ESRD) seen in adults. This study was designed to develop an in-house IgM indirect ELISA for serological screening among patients and controls, and to compare ELISA results with those of nested polymerase chain reaction (nPCR) assay. METHODS: An in-house IgM indirect ELISA was standardized using peptide sequence of VP1/VP2 region of parvovirus B19. A total of 201 children and adult with febrile illnesses, 216 individuals with non-traumatic arthropathies, 201 cases of chronic anaemia associated with ESRD and 100 healthy controls were tested. Serum was separated from the blood and subsequently used for DNA extraction. The nested polymerase chain reaction (nPCR) for the detection of B19V DNA was performed using primers targeting the overlapping region of VP1/VP2 capsid protein genes. RESULTS: A total of 618 samples were tested for parvovirus B19 by an in-house IgM indirect ELISA. Among these samples, six were positive by in-house ELISA. The inter-rater agreement between ELISA and PCR assays was calculated using kappa coefficient analysis. The value of κ was 0.77 and the strength of agreement was 'good' (P<0.001). INTERPRETATION & CONCLUSIONS: The in-house IgM indirect ELISA was found to be simple with high sensitivity and specificity when compared with nPCR and could be used as an alternative to expensive commercial kits in resource-poor settings.

Prediction of B Cell Epitopes Among Hantavirus Strains Causing Hemorragic Fever With Renal Syndrome.

Hantavirus infections are now recognized to be a global problem. The hantaviruses include several genotypic variants of the virus with different distributions in varying geographical regions. The virus genotypes seem to segregate in association with certain manifestations specific for each syndrome. They primarily include HFRS, HCPS, febrile illness with or without mild involvement of renal diseases. In the course of our study on hantavirus etiology of febrile illnesses, we recovered a hantavirus strain identified by nPCR. This has been sequenced to be Hantaan-like virus (partial S segment). The current manuscript is focused on understanding the N protein coded by S segment in terms of variation of amino acid sequences of the virus genotypes associated with HFRS. The diagnosis of this infection is achieved by PCR testing of serum/plasma or demonstration of IgM/IgG in serum. The limitations of PCR are temporal often not positive after 7 days of onset of infection. IgM detection is possible around this period and up to 21 days. IgG detection is less definitive in acute infections. Here, we report characterization of the sequence diversity of HFRS strains, 3D structure of Hantaan N protein, and B-cell epitopes on this molecule. We predicted a 20 amino acid sequence length peptide by using BepiPred online server in IEDB analysis resource program. We suggest this peptide may be used for development of geographic region-specific immunoassays like EIAs for antibody detection, monoclonal antibody development, and immunoblots (line immunoassay). J. Cell. Biochem. 118: 1182-1188, 2017. © 2016 Wiley Periodicals, Inc.

Comparison of Three Different Hepatitis C Virus Genotyping Methods: 5'NCR PCR-RFLP, Core Type-Specific PCR, and NS5b Sequencing in a Tertiary Care Hospital in South India.

BACKGROUND: Based on genetic heterogeneity, hepatitis C virus (HCV) is classified into seven major genotypes and 64 subtypes. In spite of the sequence heterogeneity, all genotypes share an identical complement of colinear genes within the large open reading frame. The genetic interrelationships between these genes are consistent among genotypes. Due to this property, complete sequencing of the HCV genome is not required. HCV genotypes along with subtypes are critical for planning antiviral therapy. Certain genotypes are also associated with higher progression to liver cirrhosis. METHODS: In this study, 100 blood samples were collected from individuals who came for routine HCV genotype identification. These samples were used for the comparison of two different genotyping methods (5'NCR PCR-RFLP and HCV core type-specific PCR) with NS5b sequencing. RESULTS: Of the 100 samples genotyped using 5'NCR PCR-RFLP and HCV core type-specific PCR, 90% (κ = 0.913, P < 0.00) and 96% (κ = 0.794, P < 0.00) correlated with NS5b sequencing, respectively. Sixty percent and 75% of discordant samples by 5'NCR PCR-RFLP and HCV core type-specific PCR, respectively, belonged to genotype 6. All the HCV genotype 1 subtypes were classified accurately by both the methods. CONCLUSION: This study shows that the 5'NCR-based PCR-RFLP and the HCV core type-specific PCR-based assays correctly identified HCV genotypes except genotype 6 from this region. Direct sequencing of the HCV core region was able to identify all the genotype 6 from this region and serves as an alternative to NS5b sequencing.

Effect of HIV-1 Subtype C integrase mutations implied using molecular modeling and docking data.

The degree of sequence variation in HIV-1 integrase genes among infected patients and their impact on clinical response to Anti retroviral therapy (ART) is of interest. Therefore, we collected plasma samples from 161 HIV-1 infected individuals for subsequent integrase gene amplification (1087 bp). Thus, 102 complete integrase gene sequences identified as HIV-1 subtype-C was assembled. This sequence data was further used for sequence analysis and multiple sequence alignment (MSA) to assess position specific frequency of mutations within pol gene among infected individuals. We also used biophysical geometric optimization technique based molecular modeling and docking (Schrodinger suite) methods to infer differential function caused by position specific sequence mutations towards improved inhibitor selection. We thus identified accessory mutations (usually reduce susceptibility) leading to the resistance of some known integrase inhibitors in 14% of sequences in this data set. The Stanford HIV-1 drug resistance database provided complementary information on integrase resistance mutations to deduce molecular basis for such observation. Modeling and docking analysis show reduced binding by mutants for known compounds. The predicted binding values further reduced for models with combination of mutations among subtype C clinical strains. Thus, the molecular basis implied for the consequence of mutations in different variants of integrase genes of HIV-1 subtype C clinical strains from South India is reported. This data finds utility in the design, modification and development of a representative yet an improved inhibitor for HIV-1 integrase.

Frequency of transmitted drug resistance mutations among treatment-naïve HIV-1-infected individuals at a tertiary care centre in South India.

INTRODUCTION: Morbidity and mortality among HIV-1-infected individuals has been dramatically reduced by the implementation of combinational antiretroviral therapy (ART). However, the efficiency of these therapies is compromised due to HIV-1 transmitted drug resistance mutations (TDRMs). METHODS: We collected a total of 127 samples from ART-naïve HIV-infected individuals and sequenced the pol gene and analysed for drug resistance mutations using the Calibrated Population Resistance (CPR) tool in the Stanford database. RESULTS: All the 127 clinical samples (100 %) were identified as HIV-1 subtype C. Based on the CPR tool, three strains (2.4 %) had TDRMs, and these were K101E, Y181C and G190A. Our findings correlated well with the WHO surveys conducted in Asia, including India, which consistently reported <5 % TDRM among the specific populations assessed. CONCLUSION: In countries like India, regular monitoring of TDRMs will provide better information for clinical practice improvement and policy making.

Novel Insights on Hantavirus Evolution: The Dichotomy in Evolutionary Pressures Acting on Different Hantavirus Segments.

BACKGROUND: Hantaviruses are important emerging zoonotic pathogens. The current understanding of hantavirus evolution is complicated by the lack of consensus on co-divergence of hantaviruses with their animal hosts. In addition, hantaviruses have long-term associations with their reservoir hosts. Analyzing the relative abundance of dinucleotides may shed new light on hantavirus evolution. We studied the relative abundance of dinucleotides and the evolutionary pressures shaping different hantavirus segments. METHODS: A total of 118 sequences were analyzed; this includes 51 sequences of the S segment, 43 sequences of the M segment and 23 sequences of the L segment. The relative abundance of dinucleotides, effective codon number (ENC), codon usage biases were analyzed. Standard methods were used to investigate the relative roles of mutational pressure and translational selection on the three hantavirus segments. RESULTS: All three segments of hantaviruses are CpG depleted. Mutational pressure is the predominant evolutionary force leading to CpG depletion among hantaviruses. Interestingly, the S segment of hantaviruses is GpU depleted and in contrast to CpG depletion, the depletion of GpU dinucleotides from the S segment is driven by translational selection. Our findings also suggest that mutational pressure is the primary evolutionary pressure acting on the S and the M segments of hantaviruses. While translational selection plays a key role in shaping the evolution of the L segment. Our findings highlight how different evolutionary pressures may contribute disproportionally to the evolution of the three hantavirus segments. These findings provide new insights on the current understanding of hantavirus evolution. CONCLUSIONS: There is a dichotomy among evolutionary pressures shaping a) the relative abundance of different dinucleotides in hantavirus genomes b) the evolution of the three hantavirus segments.

A multiplex nested PCR for the simultaneous detection of Salmonella typhi, Mycobacterium tuberculosis, and Burkholderia pseudomallei in patients with pyrexia of unknown origin (PUO) in Vellore, South India.

BACKGROUND AND OBJECTIVES: Salmonella typhi, Mycobacterium tuberculosis, and Burkholderia pseudomallei are among the most important monocyte-tropic bacterial agents causing pyrexia of unknown origin (PUO), with a significant number of endemic infections in both South and Southeast Asian regions. These infections pose a major risk to travelers to these regions as well. METHODS: We developed and evaluated a multiplex nested polymerase chain reaction (PCR) for the simultaneous detection of the three pathogens in 305 patients' buffy coat samples. RESULTS: The assay for S. typhi and B. pseudomallei was able to detect down to 1 colony forming unit/5 µL PCR input and M. tuberculosis was detected down to 20 genome copies/5 µL PCR input. S. typhi was detected in 10 (3.3 %) individuals, B. pseudomallei in 10 individuals (3.3 %), and M. tuberculosis in 18 individuals (5.9 %). Co-infections of M. tuberculosis and B. pseudomallei were detected in three individuals and S. typhi and B. pseudomallei in two individuals. CONCLUSION: This protocol is efficient for PUO diagnosis especially in Asian countries.

Molecular detection of human metapneumovirus and human bocavirus on oropharyngeal swabs collected from young children with acute respiratory tract infections from rural and peri-urban communities in South India.

BACKGROUND: Acute respiratory tract infections (ARTIs) are one of the major causes of morbidity and mortality among young children in developing countries. Information on the incidence of human metapneumovirus (hMPV) and human bocavirus (HBoV) infections in developing countries, especially among rural children, is very limited. OBJECTIVES: This study was conducted to identify whether these viruses were associated with ARTI among children ≤5 years of age in rural and peri-urban populations in South India. METHODS: The study was cross-sectional with prospective sample collection. Oropharyngeal swabs were collected from children ≤5 years of age presenting with ARTI. None of the children in this study were known to have any immunosuppressive conditions. The two viruses, hMPV and HBoV, were identified using semi-nested polymerase chain reaction (PCR) assays and one-step PCR assays, respectively. The lower limits of detection of hMPV and HBoV were 6.69 × 10(5) plasmid copies and 5.77 × 10(3) plasmid copies, respectively, per 5 µL PCR reaction input. RESULTS: The frequency of hMPV infection in children was higher than that of HBoV infection. The different frequencies of hMPV in patients in various age groups with upper and lower respiratory tract infections were compared, and the variance was found to be insignificant. In the 38 children who were hMPV positive, the majority (73.7%) were from rural communities. The overall hMPV-positive rate was higher in the rural population than in the peri-urban population, but the difference was statistically insignificant. The youngest age at which hMPV-positive status was recorded was 5 months. CONCLUSION: This study demonstrated that hMPV was associated with a significant number (i.e. >10%) of ARTIs in children in South India, whereas a relatively smaller number of HBoV infections was observed.

Analysis of sequence diversity of human metapneumovirus collected from young children with acute respiratory tract infections in South India.

BACKGROUND: Human metapneumovirus (hMPV), which has a global distribution, is an important cause of acute respiratory tract infections, especially in children and immunocompromised patients. METHODS: We investigated the genetic variability of partial nucleoprotein (N) gene sequences of hMPV strains identified among young children in South India. The sequences of the N gene were compared with previously reported sequences available in the GenBank repository. RESULTS: The results showed that strains are localized in a geographically circumscribed area (topotype). The results also demonstrates that viruses from the same genetic lineage can circulate concurrently within a given location during a given season. The close clustering of the majority of our hMPV isolates indicates that the N gene sequences in the virus population are relatively homogeneous, and suggests temporal rather than geographic variations in the evolutionary pattern. In our study, the majority of the strains belonged to genetic lineage B2 (71.1 %), followed by A2b (18.4 %), A2a (7.9 %), and B1 (2.6 %), demonstrating the presence of 4 of the 5 known genotypes of hMPV. Global alignment of the nucleotide sequences showed that the strains are closely related to sequences from Canada, The Netherlands, and Australasia. Differences at the nucleotide level and the amino acid level were identified. The results provide evidence for the diversity of the N gene of hMPV in samples collected from South India compared with global strains. When investigated for selective pressure, the sequences showed 1 positively selected site and 19 negatively selected sites. CONCLUSION: These data should prove useful in further investigations of the evolutionary dynamics of hMPV infection.