Identification of Non-Haemagglutinating Influenza A/H3 Virus and Characterization of Haemagglutinin (HA) and Neuraminidase (NA) Strain Mutations in Influenza-Like-Illness Cases in Egypt on MDCK Cell Line.

Background: The present study was undertaken to diagnose and characterize canine distemper virus (CDV) isolated from dogs of southern Gujarat, India. CDV is lethal disease of canines and felines. Total of 40 different samples were collected from 18 suspected stray dogs having different clinical signs which were processed for diagnosis and characterization of CDV.Methods: All samples were processed by employing different methods like, Immunochromatography based lateral flow test (LFA), IgG based indirect enzyme linked immunosorbent assay (i-ELISA), one step RT-PCR, nested one step RT-PCR and virus isolation in MDCK cell line. Restriction endonuclease (RE) analysis was used to characterize CDV Nucleocapsid (N) gene. Conclusion: Only 04 samples (02 nasal and 02 ocular swabs) of 02 dogs found positive for LFA, while 14 serum samples out of 17 samples of 18 dogs found positive for IgG antibody. As all dogs were unvaccinated, serum samples found positive in IgG based ELISA considered for confirmative positive for CDV infection. Whereas 13 samples of 10 dogs found positive for one step RT-PCR and nested one step RT-PCR. In RE digestion, characteristic two bands were found. All representative CDV positive samples of 10 dogs showed characteristic cytopathic effect in MDCK cell line. On age group wise percent positivity was found 71.42 % (05/07) in 0 - ≤6 months, while 77.77% (07/09) in 6- ≤12 months of age group, whereas, both samples were found positive in 12 months and above group. Overall 77.77% (14/18) dogs found positive for CDV infection. To the best of our knowledge, this is the first report on study of CDV infection in dogs from Gujarat state, India.

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In-vitro validation of self designed siRNA targeting non-structural 1 gene of Influenza A virus

South Asian Journal of Experimental Biology ◽

10.38150/sajeb.4(6).p315-322 ◽

2015 ◽

Vol 4 (6) ◽

pp. 315-322

Author(s):

Bhawana Jain ◽

Amita Jain ◽

Om Prakash ◽

Ajay K. Singh ◽

Tanushree Dangi ◽

...

Keyword(s):

Cell Line ◽

Influenza A Virus ◽

Mdck Cell ◽

Influenza A ◽

Screening Method ◽

Viral Gene ◽

Mdck Cell Line ◽

Ns1 Gene ◽

Conserved Region

The genomic variability makes Influenza A virus (IAV) difficult to be con-trolled by existing vaccines or anti-influenza drugs. Viral gene targeting siRNA induces the RNAi mechanism in the host and silents the gene by cleaving mRNA. Objective was to develop siRNA targeting non-structural 1 gene and to validate its efficiency in vitro. siRNA was designed rationally, targeting the most conserved region (delineated with the help of multiple sequence align-ment) of NS1 gene of IAV strains. To choose the most efficient siRNA, three levels screening method was developed. Ultimately one siRNA duplex was selected on the basis of its unique position in conserved region. siRNA effica-cy was confirmed in vitro on commonly used Madin Darby Canine Kidney (MDCK) cell line for IAV propagation using two clinical isolates i.e. Influenza A/H3N2 [A/India/LKO864/2011(H3N2)] and Influenza A/pdmH1N1 [A/India/LKO2151/2012(H1N1)]. Of total 173 strains worldwide and 30 strains from India, 32 bp long (position 561 - 592) conserved region was identified. The longest ORF of NS1 gene was targeted by the selected siRNA, which showed 65.5% inhibition in replication of Influenza A/pdmH1N1 and 67.2% inhibition in replication of Influenza A/H3N2 at 48 hpi on MDCK cell line. This study shows that siRNA targeting NS1 may be quite effective in controlling IAV rep-lication so can be used as anti-IAV therapeutic agent.

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