South Indian Isolates Research Articles

New record of mungbean yellow mosaic India virus in Desmodium laxiflorum and association of bhendi yellow vein mosaic betasatellite in Abelmoschus moschatus in Andhra Pradesh, India.

Mungbean yellow mosaic India virus (MYMIV) is one of the most serious commonly occurring yellow mosaic virus (YMV's) group in majority of the pulses especially black gram and green gram in southern India compared to previously reported mungbean yellow mosaic virus. In January 2020 Desmodium laxiflorum and Abelmoscus moschatus showing mosaic symptoms and vein yellowing were collected from Guntur and Prakasam districts respectively in Andhra Pradesh. PCR analysis using MYMIV and betasatellite specific primers gave desired expected amplification from the infected samples of A. moschatus (YMV-ABEL) whereas only MYMIV specific amplification was obtained in D. laxiflorum (YMV-DES). However, no PCR amplification was obtained in respective healthy leaf samples of both plants. Sequence analysis showed that the CP sequence of YMV-ABEL and YMV-DES showed a similarity of 99.19% with MYMIV (KP677496) and 99.75% with MYMIV (JN181003) respectively. The full-length betasatellite (1356bp) showed highest identity of 90% with bhendi yellow vein mosaic betasatellite (BYVMB) (GU111977). Phylogenetic analysis clustered the test isolates with south Indian isolates of MYMIV whereas the betasatellite sequence clustered with various isolates of BYVMB, tomato leaf curl New Delhi virus betasatellite and okra leaf curl betasatellite reported from India and Pakistan. To the best of our knowledge, this is the first report of a MYMIV in D. laxiflorum and A. moschatus and MYMIV betasatellite complex in A. moschatus.

Genetic variability in VP1 gene of infectious bursal disease virus from the field outbreaks of Kerala, India.

Infectious bursal disease (IBD) is considered as menace as it affects poultry industry globally causing immunosuppression, high mortality and heavy economic loss. Outbreaks of IBD were reported in many states of India including Kerala. VP1 gene acts as an important factor in the process of virus encapsidation and its involvement in viral virulence and viral replication indicates its importance in infectious bursal disease virus (IBDV). The present study was conducted to carry out the molecular characterization of VP1 gene of virulent IBDV in Kerala. A total of 42 samples were processed for the detection and analysis of VP1 gene of IBDV. Out of 42 samples, 21 samples were positive for VP1 gene of IBD. The phylogenetic analysis of the partial VP1 gene sequences reveals the clustering of IBDV isolates into very virulent IBDV (vvIBDV) and non-virulent IBDV (vIBDV). Eighteen isolates (11 isolates from vaccinated flock and 7 from non-vaccinated flocks) clustered with very virulent strains. Three isolates (2 isolates were from vaccinated flock and 1 from non-vaccinated flock) clustered with non-virulent IBDV strains, showing more evolutionarily similarity to south Indian strain VCN14/ABT/MVC/India. It is observed that vvIBDV isolates from this study have common ancestor with the south Indian strain PY12 but showed 9-10% divergence from this strains. The amino acid analysis of these 21 isolates revealed that 17 isolates possessed the characteristic vvIBDV TDN amino acid triplet, while the three isolates had non-vIBDV NEG amino acid triplet at 145/146/147 position. The remaining isolate 1/CVASP/IBDV/VP1 shows unique PDN triplet instead of TDN. Two vvIBDV isolates (15/CVASP/IBDV/VP1 and 18/CVASP/IBDV/VP1) showed 100% nucleotide and amino acid similarity with intermediate plus vaccine strain. Four vvIBDV isolates showed neutral amino acid substitution K251R which was earlier reported in Indian strains but first time in south Indian isolates. The most common unique amino acid substitution observed in our study was neutral E269D amino acid substitution in 12 isolates, neutral amino acid substitution T329S in five isolates, neutral T174N and non-polar to polar amino acid substitution A178T in isolate 10/CVASP/IBDV/VP1, non-polar to polar amino acid substitution P360R in isolate 17/CVASP/IBDV/VP1 and non-polar to polar amino acid substitution P188S in isolate 1/CVASP/IBDV/VP1. These novel mutations in our study reveal the role of genetic drift in the evolution of vvIBDV strains. The isolate 2/CVASP/IBDV/VP1 from non-vaccinated flock shows VP1 gene of non-vIBDV, but possessing VP2 of vvIBDV type indicates this is evolved by genetic shift of segments A and B. This is the first genetic characterization study of field VP1 gene of IBDV isolates in Kerala, India.

Identification and genetic analysis of infectious bursal disease viruses from field outbreaks in Kerala, India.

Recurrent infectious bursal disease (IBD) outbreaks were reported in different regions of Kerala, India. This paper reports the comparative genetic analysis of the hypervariable region of the VP2 gene of IBD virus isolates from the field outbreaks in Kerala. In phylogenetic analysis, the obtained field isolates fall into genogroup 1 and 3. In genogroup 3, all vvIBDV isolates shared a common ancestor with other south Indian isolates but isolates 9/CVASP/IBDV, 10/CVASP/IBDV, 12/CVASP/IBDV, 14/CVASP/IBDV and 17/CVASP/IBDV are most recently evolved and are diverged from the south Indian isolates. The amino acid sequence of 22 isolates was analysed, out of which 18 had conserved amino acids which were characteristic of vvIBDV. All the vvIBDV isolates obtained in the study had phenylalanine and valine at the position 240 and 294, respectively, similar to recently evolved Indian IBDV isolate (MDI14). But we observed T269A and S299N mutations in the isolate 6/CVASP/IBDV, and it is the first report of such mutations at these positions in India IBDV isolates. The isolate 11/CVASP/IBDV had a unique mutation of V225A which is not yet reported in IBDV isolates. Two isolates (15/CVASP/IBDV and 18/CVASP/IBDV) were 100% amino acid similar to intermediate plus vaccine strain. The isolates 8/CVASP/IBDV/VP2 and 19/CVASP/IBDV had amino acids unique for the intermediate vaccine with mutations observed at H253Q and V256I in 19/CVASP/IBDV, T270A and novel mutation N279Y in isolate 8/CVASP/IBDV. These two isolates had non-virulent classical heptapeptide sequence 'SWSARGS'; nevertheless, they produce field outbreaks of IBD. This is the first report of genetic characterisation of IBDV in Kerala, India.

South Indian Isolates of the Fusarium solani Species Complex From Clinical and Environmental Samples: Identification, Antifungal Susceptibilities, and Virulence.

Members of the Fusarium solani species complex (FSSC) are the most frequently isolated fusaria from soil. Moreover, this complex solely affects more than 100 plant genera, and is also one of the major opportunistic human pathogenic filamentous fungi, being responsible for approximately two-third of fusariosis cases. Mycotic keratitis due to Fusarium species is among the leading causes of visual impairment and blindness in South India, but its management is still challenging due to the poor susceptibility of the isolates to conventional antifungal drugs. Aims of the present study were to isolate South Indian clinical and environmental FSSC strains and identify them to species level, to determine the actual trends in their susceptibilities to antifungal therapeutic drugs and to compare the virulence of clinical and environmental FSSC members. Based on the partial sequences of the translation elongation factor 1α gene, the majority of the isolates—both from keratomycosis and environment—were confirmed as F. falciforme, followed by F. keratoplasticum and F. solani sensu stricto. In vitro antifungal susceptibilities to commonly used azole, allylamine and polyene antifungals were determined by the CLSI M38-A2 broth microdilution method. The first generation triazoles, fluconazole and itraconazole proved to be ineffective against all isolates tested. This phenomenon has already been described before, as fusaria are intrinsically resistant to them. However, our results indicated that despite the intensive agricultural use of azole compounds, fusaria have not developed resistance against the imidazole class of antifungals. In order to compare the virulence of different FSSC species from clinical and environmental sources, a Drosophila melanogaster model was used. MyD88 mutant flies having impaired immune responses were highly susceptible to all the examined fusaria. In wild-type flies, one F. falciforme and two F. keratoplasticum strains also reduced the survival significantly. Pathogenicity seemed to be independent from the origin of the isolates.

Genetic Diversity and Allelic Variation in South Indian Isolates of Group A Streptococci Causing Invasive Disease

Background: Reported literature on invasive group A streptococcal isolates in India is very scanty. This study was undertaken to determine the molecular heterogeneity of such isolates as seen in a tertiary care center. Materials and Methods: Thirty two blood culture isolates and 18 from other sterile body fluids were characterized by emm gene sequencing and multilocus sequence typing. Results: Forty two emm types were identified including 25 from 32 blood isolates and 17 from 18 other body fluid isolates. Types 110, 74, 63, 85, 102, 105, 124 and st854.1 were common to both groups and accounted for 40% of the isolates. Two types namely, stKNB6 and stKNB9 were newly identified types. MLST identified forty eight sequence types (MLST - ST) of which 31 were from 32 blood isolates and 17 from 18 body fluid isolates; thirty three of them were hitherto unrecognized at the time of identification. Two blood isolates of emm 85 had the same MLST - ST 484 while three blood isolates of emm 110 had three different STs namely, ST 493, 494 and 497. Two types, ST 493 and ST497 had single locus variation while ST 497 had a double locus variation. Conclusions: Our study shows that subtle allelic variations in the house keeping genes results in the development of new strains in a given emm type and contribute significantly to the existing high diversity of strains circulating in the community.

Coat protein sequence comparison of south Indian isolates of Papaya ringspot virus with other Indian subcontinent isolates.

Papaya ringspot virus (PRSV) is an economically important pathogen causing the most devastating disease in papaya worldwide. Isolates of PRSV originating from different geographical regions in south India were collected and maintained on natural host papaya. The 3’ terminal region of about 1.7 kb comprising a part of the nuclear inclusion b (NIb) gene, complete coat protein (CP) gene and the 3’ untranslated region (3’UTR) of seven Papaya ringspot virus (PRSV) isolates from south India were cloned and sequenced. The CP nucleotide and deduced amino acid sequences of seven isolates were compared to each other and to sequences of 22 other PRSV isolates from different geographical locations in the Indian subcontinent. The sequence comparisons revealed greater sequence divergence (up to 18.4% and 15% at nucleotide and amino acid levels, respectively) within Indian PRSV populations. All south Indian isolates were clearly separated from isolates of other geographical regions and formed a major group in phylogenetic trees, and the clustering pattern of isolates did not correlate well with their geographical origins.

Genetic diversity in strains of Helicobacter pylori from India and their relatedness to strains from other parts of the world

Helicobacter pylori is predominantly transmitted within families and infection occurs mostly in early childhood, frequently leading to persistent infection lifelong. In the present study, genetic diversity of Helicobacter pylori among North and South Indian isolates was evaluated. 16S rDNA, cagA, vacA and iceA genes were amplified followed by sequencing of respective amplicons for diversity analysis. Result of PCR assay showed that status of pathogenicity genes varied among strains from Varanasi and Hyderabad. Phylogenetic analysis based on 16S rDNA sequences showed clustering of Hyderabad and Varanasi strains in separate groups, pointing to significant diversity. Additionally, phylogenetic analysis revealed that most of the Varanasi strains shared homology with the strains from Taiwan except for two isolates which matched with an isolate from Brazil. On the other hand majority of the Hyderabad strains showed relatedness with strains from Brazil except one which showed homology with one strain from Taiwan. In conclusion our results show that genetic diversity among H. pylori isolates is widely prevalent regardless of the region from which they are isolated. More interestingly, phylogenetic analysis suggests that the Indian strains of H. pylori show close homology to those from Taiwan and/or Brazil.

Diversity in the cag pathogenicity island of Helicobacter pylori isolates in populations from North and South India

The cag pathogenicity island (cagPAI) has been reported to be the major virulence determinant in Helicobacter pylori-related diseases. In the present study, the diversity of the cagA gene and the integrity of the cagPAI in 158 H. pylori strains from Varanasi (North India) and Hyderabad (South India) were studied by amplifying the cagA gene (approximately 3.5 kb), followed by PCR-RFLP analysis. The results revealed significant differences in the cagA gene and the integrity of the cagPAI between North and South Indian isolates. Of 158 isolates, 40 (34.8 %) from Varanasi and 20 (46.5 %) from Hyderabad were found to carry an intact cagPAI. A partially deleted cagPAI was present in 75 (65.2 %) isolates from Varanasi and 23 (53.5 %) from Hyderabad. None of the isolates showed complete deletion of the cagPAI. Differences in the cagA 5' and 3' regions were also noted, and 11 isolates (8 from Varanasi and 3 from Hyderabad) that were cagA negative with primers for the 5' region turned out to be cagA positive with primers for the 3' variable region. It is tentatively concluded that the 3' variable region may be a better marker for cagA typing. The results also showed that the majority of the isolates harboured the Western-type EPIYA motif. PCR-RFLP analysis of the cagA gene showed 29 distinguishable digestion patterns, and cluster analysis of RFLP types from a random selection of 32 isolates placed all of the isolates into 5 groups. These results demonstrate that significant differences in the cagPAI occur among isolates from North and South India, and that RFLP of cagA could be employed for elucidating genetic variations among various isolates of H. pylori.

Phylogeny based on 16S rDNA and nifH sequences of Ralstonia taiwanensis strains isolated from nitrogen-fixing nodules of Mimosa pudica, in India.

Bacterial symbionts present in the indeterminate-type nitrogen (N)-fixing nodules of Mimosa pudica grown in North and South India showed maximum similarity to Ralstonia taiwanensis on the basis of carbon-source utilization patterns and 16S rDNA sequence. Isolates from the nodules of M. pudica from North India and South India showed identical ARDRA (Amplified Ribosomal DNA Restriction Analysis) patterns with Sau3AI and RsaI, but AluI revealed dimorphy between the North Indian and South Indian isolates. Alignment of 16S rDNA sequences revealed similarity of North Indian isolates with an R. taiwanensis strain isolated from M. pudica in Taiwan, whereas South Indian isolates showed closer relatedness with the isolates from Mimosa diplotricha. Alignment of nifH sequences from both North Indian and South Indian isolates with that of the related isolates revealed their closer affinity to alpha-rhizobia, suggesting that nif genes in the beta-rhizobia might have been acquired from alpha-rhizobia via lateral transfer during co-occupancy of nodules by alpha-rhizobia and progenitors of R. taiwanensis, members of the beta-subclass of Proteobacteria. Immunological cross-reaction of the bacteroid preparation of M. pudica nodules showed strong a positive signal with anti-dinitrogenase reductase antibody, whereas a weak positive cross-reaction was observed with free-living R. taiwanensis grown microaerobically in minimal medium with and without NH4Cl. In spite of the expression of dinitrogenase reductase under free-living conditions, acetylene reduction was not observed under N-free conditions even after prolonged incubation.