Study on the Epidemiology and Clinical Picture of Human Parainfluenza Virus in Chennai. Standardization of Rapid Diagnostic Tool and to Study the Effect of Hemagglutinin Neuraminidase Inhibitors on the Isolates
Human parainfluenza viruses are a group of viruses that cause different types of respiratory infections and are most common in children and infants.
Throat and nasal swabs were collected from symptomatic patients in Chennai within three days onset of illness were determined the prevalence of HPIV by Multiplex reverse transcription PCR. Epidemiology of specific viral etiology in patients was observed throughout the years. The age wise distribution of HPIV cases were analyzed and divided into 0-10, 11-20, 21-30, 31-40, 41-50 and above 50. The prevalence in different age groups was statistically analyzed by standard error mean. Their positivity was observed in all the years during monsoon months of August to September and post monsoon months of November to February. Among the four serotypes HPIV type 3 is highly predominant in all the years (2011-2014). HPIV-2 positivity were occurred rarely in 2014. This study validates the prevalence of HPIV infection in Chennai and indicates the circulating serotypes and HPIV strains.
The PCR products were sequenced and submitted to genbank and assigned the accession number. Different sequences were retrieved from NCBI and aligned as FASTA format. Mutations were identified by multiple sequence alignment of HPIV by ClustalW tool. Amino acid alterations were identified in HPIV-3 (HN gene) at residue 295 which Histidine replaced by Tyrosine and at 297 which Serine replaced by Glycine. Another mutations were identified in HPIV-2 (N gene) at residue 138 which Histidine replaced by Tyrosine and at 140th residue identified amino acid alteration which Histidine replaced by Glutamine. The phylogenetic analysis were identified the homology of Chennai strains with other strains. HPIV type 3 (HN) strain was clustered with Fukuoka /2009, Nagasaki 2009 and Wash 64979. HPIV type 3 (NP) strain was grouped with Switzerland/2013, US/2000, and South Africa/2000. HPIV type 2 was compared with Greer strain and HPIV2/V94.
All clinical samples were cultured in the LLC-MK2, A549 and MDCK cell lines. Then specificity and sensitivity of the cell passages were characterized for clinical isolates. The number of positive cases were highly significant in the year 2011 followed by 2013. Out of 931 samples, 38 were isolated by LLC-MK2, 15 samples identified CPE in A549 and only 5 samples were grew in MDCK cells. Among the three cell lines LLC-MK2 was highly predominant for the isolation of
HPIV. Positive percentage remained very small in MDCK cell line thus for further confirmation were not studied, whereas other two cell lines LLC-MK2 and A549 performed further confirmation. The isolated Human parainfluenza virus type 2 were more sensitive in the early passages of 8 and 9 at day five for LLC-MK2, highly compatible in the 5th passage at day 7 for A549 and 8th passage at day 8 for MDCK passage at day 8 for rest of the passages were less sensitive and specificity. HPIV-3 was more accustomed in the 9th passage at day 5 for LLC-MK2.
The virus isolated samples were performed by hemadsorption assay (HAD) was aimed at confirmation of cytopathic effect were identified in LLC-MK2, A549 cell lines. Among the two cell lines LLC-MK2 was highly predominant to detect erythrocytes adhered on the monolayers and followed A549 cell lines. Cells infected with HPIV with C.Perfringens treatment by HAD assay to enhance erythrocyte binding for HPIV-2 (82%) and HPIV-3 (90%) in 24 well plate. Virus isolated samples were confirmed by plaque assay. Plaque was observed after 8-10 days of
incubation. Interactions of receptors between HPIV-2 and 3 with m.o.i. in LLC-MK2 appeared as fusion were not blocked in HPIV-2 whereas HPIV-3 achieved fusion were blocked syncytium was not formed. Similar outcomes remained in A549 cells. Further the study evaluated the neuraminidase enzyme activity of HPIV. 4-MU concentrations and used determined the percentage of substrate expended during the reaction. The signal to background ratio were determined as the fluorescence intensities restrained after 20 minutes. The substrate concentrations were used at 25 μM. Various concentration of HPIV- 2 and 3 (m.o.i.) with bacterial neuraminidase were performed by neuraminidase activity which are statistically significant.
Cytotoxicity of 4-GU-DANA at the concentrations less than 400 μM in A549 and greater than 641 μM in LLC-MK2 by MTT assay. Cytotoxicity of Ribavirin at the concentrations less than 405 μM in A549 and greater than 476 μM in LLC-MK2 by MTT assay. Cytotoxic percentage of Ribavirin were appears as high when compared with HN inhibitor. Cytotoxicity of glycyrrhizic acid form
Licorice at concentration 31 μM in LLC-MK2 and 45 μM in A549 were identified. Indicates that MTT obtained better results compared with other dyes.
Antiviral activity of neuraminidase inhibitor (4-GU-DANA) against HPIV by Hemadsorption inhibition assay was performed and ability to interfere with receptor interaction of HPIV-2 and 3 blocks hemadsorption activity at 600 μM seemed as 77% and 78% respectively. 4-GU-DANA inhibits receptor binding for HPIV-2 and 3 at 500 µM (60%) inhibit plaque formation in LL-CMK2 and (67%) and (79%) inhibit plaque reduction in A549 cells. In neuraminidase inhibition assay, less concentrations which inhibit the HPIV-2 and 3. The IC50 concentration for HPIV-2 at 2.5 μM and HPIV-3 at 1.6 μM. 4-GU-DANA concentrations were obtained less deliberation for HPIV-3 when compared to HPIV-2.
Antiviral activity of nucleoside inhibitor (Ribavirin) against HPIV type 2 and 3 by hemadsorption inhibition assay was performed to inhibit the adherence of erythrocytes to the monolayer of HPIV type 2 and 3 at 400 μM (77%) and (75%) individually. Ribavirin inhibits replication for HPIV-2 and 3 at 400 μM during pre and post adsorption period in LLC-MK2 and A549 cells. There was no significant reduction in plaque number due to existence of ribavirin during the adsorption period of 90 minutes. The plaque area was reduced by addition after the adsorption period. Further confirmed inhibition of HPIV-2 by molecular characterized, observed infected cells without drug showed band by molecular characteristics. RBV treated infected cells, band cannot be seen and indicating that RBV inhibited transcription of viral genome.
Cytotoxicity of Glycyrrihic acid from Licorice generated at 31 μM for LLC-MK2 and 45 μM for A549 by MTT assay. Antiviral activity of Glycyrrihic acid against HPIV-2 and 3 by hemadsorption inhibition assay was achieved to inhibit the erythrocyte adherence to the monolayer of HPIV-2 and 3 at 100 μM inhibition percentage was occurred 90% and 95% respectively. Glycyrrihic acid compound inhibits plaque formation at 70 μM replication of viral growth percentage was 86% for LLC-MK2 and 87% for A549 cells. Further confirmed by neuraminidase inhibition assay were performed at less concentration for HPIV-2 and 3 at 1.5 μM and 1.2 μM respectively.
Neuraminidase inhibitor (Zanamivir) and nucleoside inhibitor (Ribavirin) were performed for antiviral activity against HPIV-2 and 3. Among these inhibitors Ribavirin has highly preferable with less concentration when compared to HN inhibitor. Among these three compounds natural glycyrrhizic acid from Licorice root were performed and observed very minimum concentration to inhibit both serotype of Human parainfluenza virus-2 and 3. The following inhibitors namely
Neuraminidase inhibitor (Zanamivir) and nucleoside inhibitors (Ribavirin) maximum concentration was used to inhibit the HPIV-2 and 3. Finally, the phytal compound glycyrrhizic acid from licorice showed comparatively high inhibition on the viral growth in invitro screening. The Zanamivir nucleoside analog was elucidated for the mechanism of antiviral activity. The HN receptor of HPIV was docked with ligand Zanamivir using Autodock programme. All the HN receptor was significantly docked by Zanamivir. The 1V2i receptor was prominently docked with (95%) high frequency and good dock score
