HIV-1 is extensively studied; however scarce data exists on HIV-2 and HIV-D (HIV-1 and HIV-2 dual infections) from the endemic regions . In this context, while studying HIV-D we established an improved indirect mmunofluorescence Assay (IFA) for virus identification, that can be used both in experimental and diagnostic virology .
Indirect Immunofluorescence is tricky when simultaneous detection of two viruses is attempted . The challenge is to reduce non-specific interactions without impairing antibody-epitope binding with improved techniques to reduce background and other problems in visualizing the antigen of interest . We optimized an IFA for HIV-1 and HIV-2 detection in dual infection, where independent unlabeled primary antibodies for HIV-1 and HIV-2 were detected with a fluorophore-labelled secondary antibody to confirm dual infection; simultaneously yet independently for HIV-1 and HIV-2 on the dually infected PBMCs .
Previous studies have used IFA for studying HIV-1 and HIV-2 antibodies and their differences . To our knowledge this is the first report of an Indirect Immunofluorescence assay performed for assessing and confirming dual HIV-1 and HIV-2 infection during the virus isolation . However, simultaneous detection the two viruses in HIV-D, obviating independent processing for the two viruses remains a goal to achieve.
Infected PBMC’s Stained Using HIV-2 Positive Serum Followed by CY3 Conjugated to Anti-Human IgG (Red). The images were taken at 100 X using Olympus IX51 Microscope with pE excitation system and were merged using Cell F software.
Keywords: Antibody; HIV-1; HIV-2;HIV-1 and HIV-2 Dual Infections (HIV-D)Indirect Immunofluorescence Assay (IFA); Virus Isolation
This work was funded by National AIDS Research Institute-Indian Council of Medical Research Intramural funds. The authors would like to thank Director-in-charge, National AIDS Research Institute, Pune for support and encouragement; Indian Council of Medical Research, New Delhi for providing Travel grant support to PK to attend the Frontiers of Retro Virology Conference 2016 held at Erlangen Germany between 12th to 14th September 2016; National AIDS Control Organization for facilitating study enrolments. The authors are also grateful to the support of staff members from the NARI Clinics, Virology and Immunology laboratory. PK is supported by a Senior Research Fellowship from the Indian Council of Medical Research, Govt of India and is a Ph.D. Scholar with Symbiosis International University (SIU), Symbiosis School of Biomedical Sciences, Lavale, and Pune.
Figure 1: Indirect ImmunoFluroscence images of HIV-1 and HIV-2 infected patient’s PBMC’s. Nucleus of the PBMC’s is Stained with DAPI [2-(4-amidinophenyl)- 1H -indole-6-carboxamidine] (Blue); HIV-1 infected PBMC’s Stained with HIV-1 Positive Serum Followed by FITC Conjugated to Anti-Human IgG (Green); HIV-2 Infected PBMC’s Stained Using HIV-2 Positive Serum Followed by CY3 Conjugated to Anti-Human IgG (Red). The images were taken at 100 X using Olympus IX51 Microscope with pE excitation system and were merged using Cell F software.
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