Identification and characterization of phage display-selected peptides having affinity to Peste des petits ruminants virus.

Phage display is a well-established technique used for selecting novel ligands having affinity to a plethora of targets including proteins, viruses, whole bacterial and mammalian cells as well as lipid targets. In the present study, phage display technology was used to identify peptides having affinity to PPRV. The binding capacity of these peptides was characterized through various formats of ELISA using phage clones, linear and multiple antigenic peptides. The whole PPRV was used as an immobilized target in a surface biopanning process using a 12-mer phage display random peptide library. After five rounds of biopanning, forty colonies were picked and amplified followed by DNA isolation and amplification for sequencing. Sequencing suggested 12 different clones expressing different peptide sequence Phage-ELISA was performed using all 12 phage clones. Results indicated that four phage clones i.e., P4, P8, P9 and P12 had a specific binding activity to PPR virus. Linear peptides displayed by all 12 clones were synthesized using solid phase peptide synthesis and subjected to virus capture ELISA. No significant binding of the linear peptides with PPRV was evident which may be due to loss of conformation of linear peptide after coating. When the four selected phage clones displayed peptide sequences were synthesized in Multiple antigenic peptide (MAP) format and used in virus capture ELISA, the results indicated significant binding of PPRV to the MAPs. It may be due to increased avidity and/or better projection of binding residues in 4-armed MAPs as compared to linear peptides. MAP-peptides were also conjugated on gold nanoparticles (AuNPs). Visual colour change from wine red to purple was observed on addition of PPRV in MAP-conjugated AuNPs solution. This colour change may be attributable to the networking of PPRV with MAP -conjugated AuNPs resulting in aggregation of AuNPs. All these results supported the hypothesis that the phage display selected peptides were capable of binding to the PPRV. The potential of these peptides to develop novel diagnostic or therapeutic agents remains to be investigated.

Development of multiplex PCR assay for species-specific detection and identification of Saprolegnia parasitica.

Saprolegnia parasitica is the most important pathogen under the genus, Saprolegnia which causes devastating oomycete diseases in freshwater fish. At present, the most common molecular method for identification of Saprolegnia species is sequencing of ribosomal DNA internal transcribed spacer (rDNA-ITS) region. In this study, a highly sensitive multiplex PCR targeting rDNA-ITS region and a hypothetical protein gene was developed using two sets of primer pair. In this PCR, two amplicons of different size of 750 bp and 365 bp are produced only in case of S. parasitica while other Saprolegnia species had single amplicon. This protocol could also differentiate Saprolegnia species from other fungus based on the size of rDNA-ITS region. The protocol does not require sequencing and can identify S. parasitica in a single reaction. Therefore, the multiplex PCR developed in this study may prove to be an easier, faster and cheaper molecular method for identification of S. parasitica.

Neuraminidase inhibitors susceptibility profiles of highly pathogenic influenza A (H5N1) viruses isolated from avian species in India (2006-2015).

We tested 65 highly pathogenic avian influenza (HPAI) A(H5N1) viruses, isolated from avian species in India between 2006 and 2015, for susceptibility to the FDA approved neuraminidase (NA) inhibitors (NAIs), oseltamivir and zanamivir using a phenotypic fluorescence-based assay. The overall incidence of resistant variants among HPAI A(H5N1) viruses was 7.69% (5/65). The NA inhibition assay identified 3 viruses resistant to oseltamivir (N294S substitution, N2 numbering) and 2 cross-resistant to oseltamivir and zanamivir (E119A or I117V+E119A substitutions), all of which belonged to hemagglutinin (HA) clade 2.2 (5/17) and predominantly circulated in Indian poultry during 2006-2010. In comparison to E119A substitution alone, viruses with I117V+E119A double substitutions showed greater reduction in susceptibility to both oseltamivir and zanamivir. The NAI resistance-associated NA markers, identified in this study, were as a result of naturally occurring mutations. Of note, 48 viruses of HA clade 2.3.2.1 that circulated in Indian poultry during 2011-2015 were susceptible to both oseltamivir and zanamivir. It is essential to monitor NAI susceptibility among human and avian HPAI A(H5N1) viruses that would provide baseline data to develop strategies for pandemic preparedness and therapeutic interventions.

Antimicrobial peptides of buffalo and their role in host defenses.

Antimicrobial peptides (AMPs) are highly conserved components of the innate immune system found among all classes of life. Buffalo (Bubalus bubalis), an important livestock for milk and meat production, is known to have a better resistance to many diseases as compared to cattle. They are found to express many AMPs such as defensins, cathelicidins, and hepcidin which play an important role in neutralizing the invading pathogens. Buffalo AMPs exhibit broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. Similar to its natural form, synthetic analogs of buffalo AMPs are also antimicrobial against bacteria and even fungus making them a good target for the development of therapeutic antimicrobials. In addition to its antimicrobial effect, AMPs have been demonstrated to have a number of immunomodulatory functions, and their genes are responsive to infections. Further, induction of their gene expression by external factors may help in preventing infectious diseases. This review briefly discusses the AMPs of buffalo identified to date and their possible role in innate immunity.

Structure-activity relationship of buffalo antibacterial hepcidin analogs.

Hepcidin is an anti-microbial peptide expressed predominantly in the liver of many species. Based on the amino acid sequence deduced from buffalo (Bubalus bubalis) hepcidin cDNA (Accession no. EU399814), six peptides Hepc(1-25), Hepc(6-25), Hepc(7-25), Hepc(9-25), Hepc(11-25) and Hepc(15-25) were synthesized using solid-phase fluorenylmethoxycarbonyl (Fmoc) chemistry. CD spectroscopy revealed different spectra of the peptides in different solvents and in all the cases beta-structure was found to be dominant with less alpha-helix as predicted. Quantitation of secondary structure indicated the highest beta-structure for all the six peptides in SDS solution, when used as mimetic for membrane-like environment. The CD spectra of all the peptides taken in water showed that degree of randomness decreased with increase in chain length of the peptide. Out of the six peptides, only Hepc(1-25), Hepc(6-25) and Hepc(7-25) showed antibacterial activity against Staphylococcus aureus (Gram-positive bacteria). The peptides did not show any sensitivity toward E. coli (Gram-negative bacteria). Minimum inhibitory concentration (MIC) showed the lowest value for Hepc(7-25) as an antibacterial agent, followed by Hepc(6-25) and Hepc(1-25). The peptides Hepc(9-25), Hepc(11-25) and Hepc(15-25) with more random structure did not show any antimicrobial activity The study demonstrated that 5 amino acids at N-terminal in buffalo hepcidin can be truncated without loss of antimicrobial activity and further reduction of length of the analog from 20 to 19 amino acids resulted increase in the activity because of increase in beta-structure of the peptide shown by CD spectroscopy.