Molecular characterization of Indian Sugarcane streak mosaic virus isolates reveals recombination and negative selection in the P1 gene

Sugarcane streak mosaic virus (SCSMV), a member of the genus Poacevirus is an important viral pathogen affecting sugarcane production in India. The P1 gene of ten Indian isolates was sequenced and compared with previously reported SCSMV isolates. Comparative sequence analysis revealed a high level of diversity in the P1 gene (83–98% nucleotide sequence identity; 87–100% amino acid sequence identity), and the Indian SCSMV isolates were found to be the most variable (up to 9% diversity at the amino acid level). Phylogenetic tree analysis showed clustering of 17 SCSMV isolates into two groups: group I included isolates from India (except SCSMV-TPT) and Pakistan, and group II consisted of isolates from Japan, Indonesia, Thailand and SCSMV-TPT. The results obtained from phylogenetic study were further supported by the different in silico analysis viz. SNPs (single nucleotide polymorphism), INDELs (insertion and deletion) and evolutionary distance analysis. A significant proportion of recombination sites were observed at the N terminal region of P1 gene. Analysis of selection pressure indicated that the P1 gene of the Indian SCSMV isolates is under strong negative or purifying selection. It is likely that recombination identified in Indian SCSMV isolates, along with strong purifying selection, enhances the speed of elimination of deleterious mutations in the P1 gene. The evolutionary processes (recombination and selection pressure) together contributed to the observed genetic diversity and population structure of Indian SCSMV isolates.     

Epstein-Barr virus isolates retain their capacity to evade T cell immunity through BNLF2a despite extensive sequence variation

The Epstein-Barr virus (EBV)-encoded immune evasion protein BNLF2a inhibits the transporter associated with antigen processing (TAP), thereby downregulating HLA class I expression at the cell surface. As a consequence, recognition of EBV-infected cells by cytotoxic T cells is impaired. Here, we show that sequence polymorphism of the BNLF2a protein is observed with natural EBV isolates, with evidence for positive selection. Despite these mutations, the BNLF2a variants efficiently reduce cell surface HLA class I levels. This conservation of BNLF2a function during evolution of EBV implies an important role for the viral TAP inhibitor in preventing T cell recognition during viral infection.     

Shift of phylogenic position in megalocytiviruses based on three different genes

Major capsid protein (MCP), the adenosine triphosphatase (ATPase), and the PstI fragment genes from five Japanese and three Korean megalocytivirus isolates were sequenced and phylogenetically analyzed with known megalocytiviruses. Phylogenetic trees formed three major clusters (M1, M2, and M3 or P1, P2, and P3), and genogroup I was divided into two minor clusters (M1a/M1b and P1a/P1b) using three target genes. Sequence identity was >97% within each cluster, except cluster II of the PstI fragment (>94% of sequence identity). Interestingly, different genotyping patterns were observed for the same isolates depending on the gene analyzed. The JPN-YelTail and JPN-BfTuna isolates located in the minor M1a cluster, based on MCP and ATPase nucleotide sequences, appeared in the minor P1b cluster based on the PstI fragment, suggesting a shift of phylogenic position in megalocytiviruses. Further study will be conducted to compare the viral antigenicity and pathogenicity between the two isolates showing the shift of phylogenic position and the other isolates clustered within genogroup I.     

Intraspecies polymorphism of Cryptosporidium parvum revealed by PCR-restriction fragment length polymorphism (RFLP) and RFLP-single-strand conformational polymorphism analyses

A glycoprotein (Cpgp40/15)-encoding gene of Cryptosporidium parvum was analyzed to reveal intraspecies polymorphism within C. parvum isolates. Forty-one isolates were collected from different geographical origins (Japan, Italy, and Nepal) and hosts (humans, calves, and a goat). These isolates were characterized by means of DNA sequencing, PCR-restriction fragment length polymorphism (PCR-RFLP), and RFLP-single-strand conformational polymorphism (RFLP-SSCP) analyses of the gene for Cpgp40/15. The sequence analysis indicated that there was DNA polymorphism between genotype I and II, as well as within genotype I, isolates. The DNA and amino acid sequence identities between genotypes I and II differed, depending on the isolates, ranging from 73.3 to 82.9% and 62.4 to 80.1%, respectively. Those among genotype I isolates differed, depending on the isolates, ranging from 69.0 to 85.4% and 54.8 to 79.2%, respectively. Because of the high resolution generated by PCR-RFLP and RFLP-SSCP, the isolates of genotype I could be subtyped as genotypes Ia1, Ia2, Ib, and Ie. The isolates of genotype II could be subtyped as genotypes IIa, IIb, and IIc. The isolates from calves, a goat, and one Japanese human were identified as genotype II. Within genotype II, the isolates from Japan were identified as genotype IIa, those from calves in Italy were identified as genotype IIb, and the goat isolate was identified as genotype IIc. All of the genotype I isolates were from humans. The Japanese isolate (code no. HJ3) and all of the Nepalese isolates were identified as genotypes Ia1 and Ia2, respectively. The Italian isolates were identified as genotype Ib, and the Japanese isolate (code no. HJ2) was identified as genotype Ie. Thus, the PCR-RFLP-SSCP analysis of this glycoprotein Cpgp40/15 gene generated a high resolution that has not been achieved by previous methods of genotypic differentiation of C. parvum.     

Intraspecies Polymorphism of Cryptosporidium parvum Revealed by PCR-Restriction Fragment Length Polymorphism (RFLP) and RFLP-Single-Strand Conformational Polymorphism Analyses

A glycoprotein (Cpgp40/15)-encoding gene of Cryptosporidium parvum was analyzed to reveal intraspecies polymorphism within C. parvum isolates. Forty-one isolates were collected from different geographical origins (Japan, Italy, and Nepal) and hosts (humans, calves, and a goat). These isolates were characterized by means of DNA sequencing, PCR-restriction fragment length polymorphism (PCR-RFLP), and RFLP-single-strand conformational polymorphism (RFLP-SSCP) analyses of the gene for Cpgp40/15. The sequence analysis indicated that there was DNA polymorphism between genotype I and II, as well as within genotype I, isolates. The DNA and amino acid sequence identities between genotypes I and II differed, depending on the isolates, ranging from 73.3 to 82.9% and 62.4 to 80.1%, respectively. Those among genotype I isolates differed, depending on the isolates, ranging from 69.0 to 85.4% and 54.8 to 79.2%, respectively. Because of the high resolution generated by PCR-RFLP and RFLP-SSCP, the isolates of genotype I could be subtyped as genotypes Ia1, Ia2, Ib, and Ie. The isolates of genotype II could be subtyped as genotypes IIa, IIb, and IIc. The isolates from calves, a goat, and one Japanese human were identified as genotype II. Within genotype II, the isolates from Japan were identified as genotype IIa, those from calves in Italy were identified as genotype IIb, and the goat isolate was identified as genotype IIc. All of the genotype I isolates were from humans. The Japanese isolate (code no. HJ3) and all of the Nepalese isolates were identified as genotypes Ia1 and Ia2, respectively. The Italian isolates were identified as genotype Ib, and the Japanese isolate (code no. HJ2) was identified as genotype Ie. Thus, the PCR-RFLP-SSCP analysis of this glycoprotein Cpgp40/15 gene generated a high resolution that has not been achieved by previous methods of genotypic differentiation of C. parvum.     

Shared usage of the chemokine receptor CXCR4 by primary and laboratory-adapted strains of feline immunodeficiency virus

Strains of the feline immunodeficiency virus (FIV) presently under investigation exhibit distinct patterns of in vitro tropism. In particular, the adaptation of FIV for propagation in Crandell feline kidney (CrFK) cells results in the selection of strains capable of forming syncytia with cell lines of diverse species origin. The infection of CrFK cells by CrFK-adapted strains appears to require the chemokine receptor CXCR4 and is inhibited by its natural ligand, stromal cell-derived factor 1alpha (SDF-1alpha). Here we found that inhibitors of CXCR4-mediated infection by human immunodeficiency virus type I (HIV-1), such as the bicyclam AMD3100 and short peptides derived from the amino-terminal region of SDF-1alpha, also blocked infection of CrFK by FIV. Nevertheless, we observed differences in the ranking order of the peptides as inhibitors of FIV and HIV-1 and showed that such differences are related to the species origin of CXCR4 and not that of the viral envelope. These results suggest that, although the envelope glycoproteins of FIV and HIV-1 are substantially divergent, FIV and HIV-1 interact with CXCR4 in a highly similar manner. We have also addressed the role of CXCR4 in the life cycle of primary isolates of FIV. Various CXCR4 ligands inhibited infection of feline peripheral blood mononuclear cells (PBMC) by primary FIV isolates in a concentration-dependent manner. These ligands also blocked the viral transduction of feline PBMC by pseudotyped viral particles when infection was mediated by the envelope glycoprotein of a primary FIV isolate but not by the G protein of vesicular stomatitis virus, indicating that they act at an envelope-mediated step and presumably at viral entry. These findings strongly suggest that primary and CrFK-adapted strains of FIV, despite disparate in vitro tropisms, share usage of CXCR4.     

Analysis of polymorphic region of GAM-1 gene in Plasmodium vivax Korean isolates.

The identification, characterization and quantification of Plasmodium sp. genetic polymorphism are becoming increasingly important in the vaccine development. We investigated polymorphism of Plasmodium vivax GAM-1 (PvGAM-1) gene in 30 Korean isolates. The polymorphic region of the PvGAM-1 gene, corresponding to nt 3792-4029, was amplified using polymerase chain reaction (PCR) followed by sequencing. All of the P. vivax Korean isolates were one type of GAM-1 gene, which were identical to that of the Belem strain. It is suggested that PvGAM-1 could not be used as a genetic marker for identifying or classifying P. vivax Korean isolates. It revealed that the polymorphic pattern was acquired basically by duplication and modification or deletion event of a 33 bp-motif fragment ended by poly guanine (G) and that there were at least three complete and one partial 33 bp-motif sequences within the polymorphic region in the longest cases such as those of South Korean and Belem isolates. In addition, we clustered P. vivax isolates with parsimonious criteria on the basis of PvGAM-1 polymorphic patterns (insertion/deletion patterns).     

Enterovirus capsid interactions with decay-accelerating factor mediate lytic cell infection

The cellular receptor usage of numerous human enteroviruses can differ significantly between low-cell-culture-passaged clinical isolates and highly laboratory-passaged prototype strains. The prototype strain of coxsackievirus A21 (CVA21) displays a dual-receptor specificity as determined with a receptor complex consisting of decay-accelerating factor (DAF) and intercellular adhesion molecule 1 (ICAM-1). In this study, the cellular receptor interactions of low-cell-passage CVA21 clinical isolates with respect to their interactions with cell surface-expressed DAF and ICAM-1 were compared to those of the CVA21 prototype (Kuykendall) strain. Dual-receptor usage of DAF and ICAM-1 by CVA21 clinical isolates was confirmed by cell transfection and radiolabeled binding assays. The cellular attachment of clinical and prototype CVA21 strains to cells that coexpressed DAF and ICAM-1 was not additive compared to the viral binding to cells expressing one or other receptor. In fact, the binding data suggest there is an inhibition of CVA21 cellular attachment in environments where high-level coexpression of both DAF and ICAM-1 occurs. Antibody cross-linking of DAF rendered cells susceptible to lytic infection by the CVA21 clinical isolates. In a novel finding, three clinical isolates could, to various degrees, infect and lyse DAF-expressing cells in the absence of DAF-antibody cross-linking and ICAM-1 expression. Sequence analysis of the P1 region of clinical and prototype virus genomes identified a number of coding changes that may contribute to the observed enhanced DAF usage phenotype of the clinical CVA21 isolates. None of the amino acid changes was located in the previously postulated ICAM-1 footprint, a receptor-binding environment that was conserved on the capsid surface of all CVA21 clinical isolates. Taken together, the data suggest that community-circulating strains of CVA21 can infect target cells expressing either ICAM-1 or DAF alone and that such interactions extend tissue tropism and impact directly on viral pathogenesis.     

Genetic diversity and natural selection at the domain I of apical membrane antigen-1 (AMA-1) of Plasmodium falciparum in isolates from Iran

The apical membrane antigen-1 (AMA-1) of Plasmodium falciparum is a prime malaria asexual blood-stage vaccine candidate. Antigenic variation is one of the main obstacles in the development of a universal effective malaria vaccine. The extracellular region of P. falciparum AMA-1 (PfAMA-1) consists of three domains (I-III), of which the domain I is the most diverse region of this antigen. The objective of our study was to investigate and analyze the extent of genetic diversity and the effectiveness of natural selection at the AMA-1 domain I of P. falciparum in isolates from Iran. A fragment of ama-1 gene spanning domain I was amplified by nested PCR from 48 P. falciparum isolates collected from two major malaria endemic areas of Iran during 2009 to August 2010 and sequenced. Genetic polymorphism and statistical analyses were performed using DnaSP and MEGA software packages. Analysis of intrapopulation diversity revealed relatively high nucleotide and haplotype diversity at the PfAMA-1 domain I of Iranian isolates. Neutrality tests provided strong evidence of positive natural selection acting on the sequenced gene region. The findings also demonstrated that, in addition to natural selection, intragenic recombination may contribute to the diversity observed at the domain I. The results obtained will have significant implications in the design and the development of an AMA-1-based vaccine against falciparum malaria.     

Type 1, P and S fimbriae, and afimbrial adhesin I are not essential for uropathogenic Escherichia coli to adhere to and invade bladder epithelial cells

Fimbrial (type 1, P, and S) and afimbrial adhesins, the unique virulence traits of uropathogenic Escherichia coli (UPEC), are well recognized for their role in the initial step of uropathogenesis. In this study, we investigated whether these adhesins are dispensable for UPEC in adherence and invasion of uroepithelial cells by using E. coli isolates (n=40) from cystitis patients and T-24 cells, the bladder carcinoma cell line. We found all isolates adherent to T-24 cells within 15 min of infection. In invasion assay, all isolates could invade T-24 cells to a variable degree; 22.5% of them were found highly invasive. About 33% of isolates that do not have any recognized adhesins were as invasive as other isolates. The amplitude of invasiveness was also independent of the adhesins. In conclusion, this study demonstrates that type 1 fimbriae, P fimbriae, S fimbriae, and afimbrial adhesin I are not required for UPEC to adhere to and invade uroepithelial cells.     

Highly reliable heterologous system for evaluating resistance of clinical herpes simplex virus isolates to nucleoside analogues

Clinical resistance of herpes simplex virus (HSV) types 1 and 2 to acyclovir (ACV) is usually caused by the presence of point mutations within the coding region of the viral thymidine kinase (TK) gene. The distinction between viral TK mutations involved in ACV resistance or part of viral polymorphism can be difficult to evaluate with current methodologies based on transfection and homologous recombination. We have developed and validated a new heterologous system based on the expression of the viral TK gene by the protozoan parasite Leishmania, normally devoid of TK activity. The viral TK genes from 5 ACV-susceptible and 13 ACV-resistant clinical HSV isolates and from the reference strains MS2 (type 2) and KOS (type 1) were transfected as part of an episomal expression vector in Leishmania. The susceptibility of TK-recombinant parasites to ganciclovir (GCV), a closely related nucleoside analogue, was evaluated by a simple measurement of the absorbance of Leishmania cultures grown in the presence of the drug. Expression of the TK gene from ACV-susceptible clinical isolates resulted in Leishmania susceptibility to GCV, whereas expression of a TK gene with frameshift mutations or nucleotide substitutions from ACV-resistant isolates gave rise to parasites with high levels of GCV resistance. The expression of the HSV TK gene in Leishmania provides an easy, reliable, and sensitive assay for evaluating HSV susceptibility to nucleoside analogues and for assessing the role of specific viral TK mutations.     

Parallel phylogenetic analyses using the N, G or Nv gene from a fixed group of VHSV isolates reveal the same overall genetic typing

Different genetic regions representing the viral phospho- (P), nucleocapsid- (N) or glyco-protein (G) gene have been used for phylogenetic studies of viral haemorrhagic septicaemia virus (VHSV). Since these analyses were performed on different virus isolates using various genomic regions, it has been difficult to evaluate how the choice of target region affects the output of the analyses. To address this, we sequenced and performed parallel phylogenetic analysis of an N gene fragment, the entire Nv (non-structural protein) and G genes, and 4 different fragments of the G gene from a fixed virus panel. The overall genotyping of the selected isolates was identical for the 7 target regions, but separation of Genotype I sub-lineages was best when the analysis was performed on the full length G gene (1524 nucleotides, nt). Good resolution was furthermore obtained using smaller sequencing windows represented by a G gene fragment (nt 360 to 720) or the Nv gene (366 nt), although these regions had different characteristics with respect to resolution of Genotype I sublineages and resolution within Sub-lineage Ia. Phylogenetic analysis based on the deduced amino acid sequences was also performed. The phylogenetic relationship between the nucleotide and amino acid sequences of the isolates corresponded best in the case of the N gene/protein. For the 6 other genomic regions, genetically distant isolates occasionally grouped together when compared at protein levels. No clear relationship between the G gene genotyping and serotyping with neutralising (G protein specific) antibodies was observed, stressing that epidemiological analysis based on phenotypic characteristics such as serotype could be misleading.     

Replicative fitness of protease inhibitor-resistant mutants of human immunodeficiency virus type 1

The relative replicative fitness of human immunodeficiency virus type 1 (HIV-1) mutants selected by different protease inhibitors (PIs) in vivo was determined. Each mutant was compared to wild type (WT), NL4-3, in the absence of drugs by several methods, including clonal genotyping of cultures infected with two competing viral variants, kinetics of viral antigen production, and viral infectivity/virion particle ratios. A nelfinavir-selected protease D30N substitution substantially decreased replicative capacity relative to WT, while a saquinavir-selected L90M substitution moderately decreased fitness. The D30N mutant virus was also outcompeted by the L90M mutant in the absence of drugs. A major natural polymorphism of the HIV-1 protease, L63P, compensated well for the impairment of fitness caused by L90M but only slightly improved the fitness of D30N. Multiply substituted indinavir-selected mutants M46I/L63P/V82T/I84V and L10R/M46I/L63P/V82T/I84V were just as fit as WT. These results indicate that the mutations which are usually initially selected by nelfinavir and saquinavir, D30N and L90M, respectively, impair fitness. However, additional mutations may improve the replicative capacity of these and other drug-resistant mutants. Hypotheses based on the greater fitness impairment of the nelfinavir-selected D30N mutant are suggested to explain observations that prolonged responses to delayed salvage regimens, including alternate PIs, may be relatively common after nelfinavir failure.