Pathotypic and Sequence Characterization of Newcastle Disease Viruses from Vaccinated Chickens Reveals Circulation of Genotype II, IV and XIII and in India.

Newcastle disease virus (NDV) causes a highly contagious disease which continuously haunts the global poultry industry. The nature and molecular epidemiology of NDVs prevalent in recent outbreaks in India is poorly understood. This study aimed to characterize NDVs prevalent in vaccinated flocks in India using whole-genome sequencing and biological pathotyping. Twelve field isolates were collected from outbreaks which occurred in different parts of India and characterized as velogenic based on their intracerebral pathogenicity index (ICPI) and amino acid sequence at the F protein cleavage site. All 12 of the field isolates and five commonly used vaccine strains were selected for whole-genome sequencing using Ion Torrent PGM technology, yielding complete genome sequences for ten field isolates and all vaccine strains. The genome of all isolates was found to be 15 192 nt long with a high level of conservation across multiple genomic features with APMV-I viruses. Phylogenetic analysis and evolutionary distance calculations placed the isolates in genotypes II, IV and XIII. Revisiting other recently reported strains provided preliminary evidence of genotypes VI, VII and XVIII circulating in India. Comparison between the field and vaccine virus sequences revealed unique genomic and amino acid differences in important antigenic regions of the F and hemagglutinin-neuraminidase (HN) genes which can be targeted for site directed mutagenesis to evaluate the impact of these substitutions on virus pathogenicity. This study highlights the requirement to evaluate current vaccines through systematic protection assays to determine protection efficacy against field isolates.

Mechanisms of human immunodeficiency virus type 2 RNA packaging: efficient trans packaging and selection of RNA copackaging partners.

Human immunodeficiency virus type 2 (HIV-2) has been reported to have a distinct RNA packaging mechanism, referred to as cis packaging, in which Gag proteins package the RNA from which they were translated. We examined the progeny generated from dually infected cell lines that contain two HIV-2 proviruses, one with a wild-type gag/gag-pol and the other with a mutant gag that cannot express functional Gag/Gag-Pol. Viral titers and RNA analyses revealed that mutant viral RNAs can be packaged at efficiencies comparable to that of viral RNA from which wild-type Gag/Gag-Pol is translated. These results do not support the cis-packaging hypothesis but instead indicate that trans packaging is the major mechanism of HIV-2 RNA packaging. To further characterize the mechanisms of HIV-2 RNA packaging, we visualized HIV-2 RNA in individual particles by using fluorescent protein-tagged RNA-binding proteins that specifically recognize stem-loop motifs in the viral genomes, an assay termed single virion analysis. These studies revealed that >90% of the HIV-2 particles contained viral RNAs and that RNAs derived from different viruses were copackaged frequently. Furthermore, the frequencies of heterozygous particles in the viral population could be altered by changing a 6-nucleotide palindromic sequence at the 5'-untranslated region of the HIV-2 genome. This finding indicates that selection of copackaging RNA partners occurs prior to encapsidation and that HIV-2 Gag proteins primarily package one dimeric RNA rather than two monomeric RNAs. Additionally, single virion analyses demonstrated a similar RNA distribution in viral particles regardless of whether both viruses had a functional gag or one of the viruses had a nonfunctional gag, providing further support for the trans-packaging hypothesis. Together, these results revealed mechanisms of HIV-2 RNA packaging that are, contrary to previous studies, in many respects surprisingly similar to those of HIV-1.

Genetic diversity of HIV type 1 subtype C env gene sequences from India.

Considering the severity of the HIV-1 subtype C epidemic, data on the epidemiology and distribution of HIV subtypes in India are relatively sparse. Keeping this in view, 28 env gene sequences from patients were sequenced and analyzed. The samples were collected over a period of 10 years from 1995 to 2004. Assessment of the interisolate genetic distances of the study isolates, which were all subtype C, showed interisolate distances varying from 2 to 19% (mean: 14%) with the maximum diversity observed in the samples collected in 2003-2004. Analysis of the phylogenetic relationships among subtype C env sequences from six different countries and our study isolates revealed an overall star-like phylogeny with almost all sequences from India forming a monophyletic lineage. A lower diversity within the immunodominant epitopes was found. The data generated from this study should prove valuable for the production of vaccine against subtype C.

Molecular mechanisms of simian immunodeficiency virus SIV(agm) RNA encapsidation.

Primate lentiviruses are composed of several distinct lineages, including human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus SIVagm. HIV-1 and HIV-2 have significant differences in the mechanisms of viral RNA encapsidation. Therefore, the RNA packaging mechanisms of SIVagm cannot be predicted from the studies of HIV-1 and HIV-2. We examined the roles of the nucleocapsid (NC) zinc finger motifs on RNA packaging by mutating the conserved zinc finger (CCHC) motifs, and whether SIVagm has a preference to package RNA in cis by comparing the RNA packaging efficiencies of gag mutants in the presence of a wild-type vector. Our results indicate that the SIVagm NC domain plays an important role in Gag-RNA recognition; furthermore SIVagm is distinct from the other currently known primate lentiviruses as destroying either zinc finger motif in the NC causes very drastic RNA packaging defects. Additionally, trans-packaging is a major mechanism for SIVagm RNA encapsidation.

Analysis of HIV type 1 subtype C full-length gag gene sequences from India: novel observations and plausible implications.

Twenty-four HIV-1 gag genes from patients in India were sequenced and analyzed. All measured 1476-1491 nucleotides with an average of 1483 nucleotides in length. Phylogenetic analysis revealed a homogeneous epidemic of HIV-1 subtype C. Intragenotypic divergence of up to 6.6% was present. Fourteen novel conserved signature pattern residues were delineated for HIV-1 subtype C strains. Each of the 15 nucleocapsid (NC) basic residues was highly conserved p6 gag LXSLFG and PT/SAPP motifs were highly conserved except for PTVPT in three and PTAPT in two strains. Zinc finger motifs were conserved in all. Documented HIV-1 subtype C gag immunodominant CTL epitopes were conserved. The evolving predominance and the change in nature of the epidemic from Thai B to that of subtype C in the Northeastern regions of India were observed. Tracking the evolution of the Indian epidemic has implications for developing a vaccine.

Reduced susceptibility to ciprofloxacin and gyra gene mutation in North Indian strains of Salmonella enterica serotype Typhi and serotype Paratyphi A.

The emergence of reduced susceptibility to ciprofloxacin among Salmonella enterica serotype Typhi and serotype Paratyphi A leading to clinical failure of treatment poses a great therapeutic challenge. The mechanism of fluoroquinolone resistance in clinical isolates of S. Typhi and S. Paratyphi A is not very well documented. The present study was carried out with the objective of molecular characterization of reduced quinolone susceptibility amongst the strains of S. Typhi and S. Paratyphi A isolated from the patients with enteric fever during January, 2000, to April, 2003, in a North Indian hospital. A total of 422 culture-positive cases of enteric fever were reported to the hospital during the period of study, of which S. Typhi was isolated from 350 cases and S. Paratyphi A from 72 cases. The antimicrobial susceptibility of these strains was determined by disk diffusion and agar dilution method according to NCCLS guidelines, and E-test method. A total of 140 randomly selected strains, isolated during the years 1993-1999, that were available from the laboratory stocks were also studied to compare with the present strains. To study the quinolone susceptibility, the strains were divided into nalidixic acid sensitive (NAS), nalidixic acid intermediate resistant, (NAI) and nalidixic acid resistant (NAR) on the basis of susceptibility to nalidixic acid. Clinical history was available from 174 patients, of which 93 needed hospitalization due to severe disease. Of these, 82 patients were infected with NAR strains and 22 patients had a documented evidence of clinical failure to ciprofloxacin therapy. The patients infected with NAR strains were younger and had a significantly longer duration of fever (p value < 0.05) than those infected with NAS strains. It was observed that the proportion of NAR strains increased gradually over the years. These strains had a significantly higher range of MIC of ciprofloxacin (0.023-1.0 microg/ml) as compared to the NAS strains (0.002-0.125 microg/ml) (p value < 0.05). The sequencing of quinolone resistance determining region (QRDR) of the gyrA gene showed the presence of mutation at either Ser 83 or at Asp 87 in all the NAR and NAI strains. None of the NAS strains had a mutation, suggesting that the gyrA gene mutation is sufficient to confer resistance to nalidixic acid and reduced susceptibility to ciprofloxacin. This mutation, although phenotypically expressed as decreased susceptibility to ciprofloxacin, goes undetected by the disk diffusion method using the present NCCLS guidelines. Hence, it can increase morbidity and mortality due to delay in appropriate antibiotic treatment.

Genomic diversity of human immunodeficiency virus type-1 in India.

Surveillance of HIV-1 subtypes has important implications for the development of candidate vaccine and understanding the possible differences in the transmission and natural history of different subtypes. In this study, HIV-1 subtypes were determined for homologies in the C2-V3-V5 region by heteroduplex mobility assay (HMA) in HIV-1 seropositive patients referred to the National HIV/AIDS Reference Centre, All India Institute of Medical Sciences in New Delhi, India. Of the 125 samples analysed, 98 (78.4%) were HIV-1 subtype C, 11 (8.8%) were subtype B', 3 (2.4%) were subtype A and 2 (1.6%) were subtype E. In 11 samples, subtype determination was not clear-cut. It is possible that these individuals may be infected with recombinant strains of HIV-1. These findings may have significant implications for the designing and testing of effective HIV-1 vaccine candidate in India.