April 11, 2017 kribs

Research- Department of Infection and Immunology

 

Current Research at the Department of Infection and Immunology

A) Dengue virus seroprevalence and genetic diversity: Investigation of recent dengue outbreaks in Nepal

Aim: To determine the virus-host response of DENV serotypes co-circulating in Nepal.

Principal investigator: Dr. Anurag Adhikari

Ongoing Research on virus-host response

This study aims to assess antibody’s capacity to neutralize the dengue virus titer by employing Foci Reduction Neutralizing Test. For this purpose, Vero E6 cells are grown and maintained in BSL-2 laboratory. Vero E6 cells were infected with Dengue virus 1-4 with different MOI in order to facilitate virus propagation (Figure1- 2). After super infection, the supernatant of virus culture was concentrated and collected which underwent through PCR for virus detection (Figure 3). To determine virus titer, we performed foci forming assay which utilizes crystal violet staining in order to spot the foci formed in cells monolayers resulting due to cytopathic effect of dengue virus on cells (Figure 4). C6/36 cell lines are being maintained in BSL-2 laboratory (Figure 5) and our further plan involves utilization of C6/36 cells for dengue virus propagation.

Figure 1: Illustration of dengue virus infection on Vero E6 cells.

Figure 1 : Illustration of dengue virus infection on Vero E6 cells.

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Figure 2: represents (A) Vero E6 cells monolayer at 20x objective lens magnification after 24 hours of infection with DENV1 MOI 0.2, (B) DENV2 MOI 0.1, (C) DENV3 MOI 0.1, and (D) DENV4 MOI 0.1.

 

Figure 3: Gel electrophoresis-PCR for virus detection after harvesting virus from supernatant.

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Figure 4: represents the Foci Forming assay in which    5 x 10^4 Vero E6 cells present in each well were infected with (A) DENV1 MOI 2, (B) DENV2 MOI 1, (C) DENV3 MOI 1, (D) DENV4 MOI 1, and (E) represents mock infected Vero E6 cells.

 

 

 

 

 

 

 

 

 

 

Figure 5: C6/36 observation of P4 at 20x and 10x at 10 days of seeding.

Figure 5: C6/36 observation of P4 at 10x after 13 days of seeding.

 

B) Immune response against Human Immunodeficiency Virus infection

Aim: Understanding the humoral and cell-mediated immune response against the Human Immunodeficiency Virus infection.

Principal Investigator: Anurag Adhikari

Details on the humoral immunity are explained in the KaVA-Program [see  https://kribs.org.np/kava-program/ ]

 

Recent findings and ongoing research in HIV cell-mediated immune response

Within the HIV cell-mediated study, we have optimized the peripheral blood mononuclear cells (PBMC) isolation and long-term liquid nitrogen-based cryopreservation at our laboratory. These isolated and stored PBMCs from HIV-infected individuals and controls have been documented for their pre-long term storage absolute cell count and viability (Figure 1-2).  To understand the extent of the recovery of the functional stimulatory property of these cryopreserved cells, we developed and optimized the PBMC stimulation assay and studied their capacity to produce Interferon-Gamma cytokine (Figure 3). [Figures 1-3 are unpublished data-manuscript on preparation-please cite original source or contact adhikari.a[at]kribs.org.np before sharing].

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 3: Representative image of spot forming unit (SFU) [LEFT]. SFU/million PBMC for HIV versus healthy donor PBMC in stimulated (PMA) and non stimulated ELISpot assay [RIGHT].

C) Orientia tsutsugamushi infection and associated immune response in human

Principal Investigator: Anurag Adhikari

Initial results and outcomes: Summary (Unpublished paper, under preparation)

Phylogenetic analysis of the partial genome of Orientia tsutsugamushi 47kDa gene (GenBank accession: OL770337-OL770352) showed a close genetic relation with Karp-UK strain (among the 87.5% of the patients), as well with CRF93-Thailand strain (6.25% of the patients), and Karp-Thailand strain (6.25% of the patients). Similarly, phylogenetic analysis of Orientia tsutsugamushi 56kDa gene (GenBank accession: OL770323-OL770336) showed close relation with Gilliam-Bangladesh strain (among the 35.7% of the patients), Karp-Bangladesh strain (28.6% of the patients), Gilliam-UK strain (14.3% of the patients), Shimokoshi-Taiwan strain (14.3% of the patients), and with Vietnam strain (7.14% of the patients). We also show that the patient-derived groEL gene of Anaplasma (GenBank accession: OL770355 -OL770356) was closely related to the D-GB-gro-8-South Korea strain (5.5% of typhus suspected patients), and the rpoB gene of Leptospira (GenBank accession: OL770353-OL770354) was closely related to Linhai 56609-China strain (5.5% of typhus suspected patients).

This study shows that the Karp, Gilliam, and Shimokoshi strains of Orientia tsutsugamushi are circulating in southwest Nepal. Additionally, this study identifies the co-presence of Anaplasma, and Leptospira in the febrile symptomatic patient. Further genotype and serotype screening study for these bacteria among febrile patients is a current need so as to identify the tick-borne bacterial diversity and its associated disease burden among the Nepalese population.

Figure 1:. Phylogenetic tree of Orientia tsutsugamushi 56kDa type-specific antigen. The phylogenetic tree was inferred using the neighbor-joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The Genbank accession numbers for the O. tsutsugamushi strains retrieved are indicated and Accession numbers of published sequences: (OL770323-OL770336).

 

Figure 2: Phylogenetic tree of Orientia tsutsugamushi 47kDa type-specific antigen. The phylogenetic tree was inferred using the neighbor-joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The Genbank accession numbers for the O. tsutsugamushi strains retrieved are indicated and Accession numbers of published sequences: (OL770337-OL770352).

 

Figure 3: Phylogenetic tree of Leptospira rpoB gene. The phylogenetic tree was inferred using the neighbor-joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The Genbank accession numbers for the O. tsutsugamushi strains retrieved are indicated and Accession numbers of published sequences: (OL770353-OL770354)

Figure 4: Phylogenetic tree of Anaplasma groEl gene.The phylogenetic tree was inferred using the neighbor-joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The Genbank accession numbers for the O. tsutsugamushi strains retrieved are indicated and Accession numbers of published sequences: (OL770355 -OL770356).

GenBank sequence accession links

Orientia tsutsugamushi 47kDa gene (GenBank accession of 16 sequences: OL770337-OL770352)

https://www.ncbi.nlm.nih.gov/nuccore/?term=OL770337%3AOL770352%5Bpacc%5D 

 

Orientia tsutsugamushi 56kDa gene (GenBank accession of 14 sequences: OL770323-OL770336)

https://www.ncbi.nlm.nih.gov/nuccore/?term=OL770323%3AOL770336%5Bpacc%5D

 

Anaplasma, (GenBank accession of 2 sequences: OL770355 -OL770356)

https://www.ncbi.nlm.nih.gov/nuccore/?term=OL770355%3AOL770356%5Bpacc%5D

 

Leptospira, (GenBank accession of 2 sequences: OL770353-OL770354)

https://www.ncbi.nlm.nih.gov/nuccore/?term=OL770353%3AOL770354%5Bpacc%5D