Generation of a Live-Attenuated Strain of Chikungunya Virus from an Indian Isolate for Vaccine Development.

Chikungunya virus (CHIKV) re-emergence in the last decade has resulted in explosive epidemics. Along with the classical symptoms of fever and debilitating arthralgia, there were occurrences of unusual clinical presentations such as neurovirulence and mortality. These generated a renewed global interest to develop prophylactic vaccines. Here, using the classical approach of virus attenuation, we developed an attenuated CHIKV strain (RGCB355/KL08-p75) for the purpose. Repeated passaging (75 times) of a local clinical isolate of ECSA lineage virus in U-87 MG human astrocytoma cells, an interferon-response-deficient cell line, resulted in efficient adaptation and attenuation. While experimental infection of 3-day old CHIKV-susceptible BALB/c pups with the parent strain RGCB355/KL08-p4 resulted in death of all the animals, there was 100% survival in mice infected with the attenuated p75. In adult, immunocompetent, CHIKV-non-susceptible C57BL/6 mice, inoculation with p75 induced high antibody response without any signs of disease. Both p4 and p75 strains are uniformly lethal to interferon-response-deficient AG129 mice. Passive protection studies in AG129 mice using immune serum against p75 resulted in complete survival. Whole-genome sequencing identified novel mutations that might be responsible for virus attenuation. Our results establish the usefulness of RGCB355/KL08-p75 as a strain for vaccine development against chikungunya.

Interferon regulated gene (IRG) expression-signature in a mouse model of chikungunya virus neurovirulence.

Interferon regulated genes (IRGs) are critical in controlling virus infections. Here, we analyzed the expression profile of IRGs in the brain tissue in a mouse model of chikungunya virus (CHIKV) neurovirulence. Neurovirulence is one of the newer complications identified in disease caused by re-emerging strains of CHIKV, an alphavirus with positive-strand RNA in the Togaviridae family. In microarray analysis, we identified significant upregulation of 269 genes, out of which a predominant percentage (76%) was IRGs. The highly modulated IRGs included Ifit1, Ifi44, Ddx60, Usp18, Stat1, Rtp4, Mnda, Gbp3, Gbp4, Gbp7, Oasl2, Oas1g, Ly6a, Igtp, and Gbp10, along with many others exhibiting lesser changes in expression levels. We found that these IRG mRNA transcripts are modulated in parallel across CHIKV-infected mouse brain tissues, human neuronal cell line IMR-32 and hepatic cell line Huh-7. The genes identified to be highly modulated both in mouse brain and human neuronal cells were Ifit1, Ifi44, Ddx60, Usp18, and Mnda. In Huh-7 cells, however, only two IRGs (Gbp4 and Gbp7) showed a similar level of upregulation. Concordant modulation of IRGs in both mice and human cells indicates that they might play important roles in regulating CHIKV replication in the central nervous system (CNS). The induction of several IRGs in CNS during infection underscores the robustness of IRG-mediated innate immune response in CHIKV restriction. Further studies on these IRGs would help in evolving possibilities for their targeting in host-directed therapeutic interventions against CHIKV.

Nucleophosmin (NPM1)/B23 in the Proteome of Human Astrocytic Cells Restricts Chikungunya Virus Replication.

Chikungunya virus (CHIKV), a positive-stranded RNA virus, can cause neurological complications by infecting the major parenchymal cells of the brain such as neurons and astrocytes. A proteomic analysis of CHIKV-infected human astrocytic cell line U-87 MG revealed tight functional associations among the modulated proteins. The predominant cellular pathways involved were of transcription-translation machinery, cytoskeletol reorganization, apoptosis, ubiquitination, and metabolism. In the proteome, we could also identify a few proteins that are reported to be involved in host-virus interactions. One such protein, Nucleophosmin (NPM1)/B23, a nucleolar protein, showed enhanced cytoplasmic aggregation in CHIKV-infected cells. NPM1 aggregation was predominantly localized in areas wherein CHIKV antigen could be detected. Furthermore, we observed that inhibition of this aggregation using a specific NPM1 oligomerization inhibitor, NSC348884, caused a significant dose-dependent enhancement in virus replication. There was a marked increase in the amount of intracellular viral RNA, and ∼105-fold increase in progeny virions in infected cells. Our proteomic analysis provides a comprehensive spectrum of host proteins modulated in response to CHIKV infection in astrocytic cells. Our results also show that NPM1/B23, a multifunctional chaperone, plays a critical role in restricting CHIKV replication and is a possible target for antiviral strategies.

Utility of a rapid immunochromatographic strip test in detecting canine parvovirus infection compared with polymerase chain reaction.

AIM: The present study was undertaken to detect the presence of canine parvovirus (CPV) in fecal samples of diarrheic dogs by conventional polymerase chain reaction (PCR) and immunochromatographic (IC) strip test and to compare the diagnostic potential of these tests. MATERIALS AND METHODS: A total of 50 fecal samples collected from diarrheic dogs suspected for CPV infection were subjected to PCR using CPV-555 primer amplifying the gene coding for the VP1 protein. These samples were also tested by IC strip test using a commercial rapid Ag test kit. The results were statistically analyzed using McNemar test. RESULTS: A total of 22 samples (44%) were detected as positive by PCR, which yielded a specific amplicon of 583 bp. In IC strip test, 18 (36%) samples were found to be positive. The sensitivity of the test as compared to PCR was found to be 72.22% and specificity was 92.86%. Positive predictive value and negative predictive value of IC strip test was found to be 88.89% and 81.25%, respectively. Statistical analysis of the results of PCR and IC assay using McNemar test revealed no significant difference (p>0.05). CONCLUSION: The IC strip test could be employed as a rapid field level diagnostic tool for the diagnosis of canine parvoviral diarrhea.