Development of a Real-Time Cell Analysing (RTCA) method as a fast and accurate screen for the selection of chikungunya virus replication inhibitors

BACKGROUND: The xCELLigence real-time cell analysis (RTCA) system is an established electronic cell sensor array. This system uses microelectronic biosensor technology that is verified for real-time, label-free, dynamic and non-offensive monitoring of cellular features, including detection of viral cytopathic effect (CPE). Screening viral replication inhibitors based on presence of CPE has been applied for different viruses, including chikungunya virus (CHIKV). However, most CPE-based methods, including MTT and MTS assays, do not provide information on the initiation of CPE nor the changes in reaction rate of the virus propagation over time. Therefore, in this study we developed an RTCA method as an accurate and time-based screen for antiviral compounds against CHIKV. METHODS: CHIKV-infected Vero cells were used as an in vitro model to establish the suitability of the RTCA system as a quantitative analysis method based on the induction of CPE. We also performed an MTS assay as a CPE-based conventional method. Experimental assays were carried out to evaluate the optimal seeding density of the Vero cells, cytotoxicity of the tested compounds, titration of CHIKV and the antiviral activity of ribavirin, which has been reported as an effective compound against CHIKV in vitro replication. RESULTS: The optimal time point for viral inoculation was 18 h after seeding the cells. We determined that the maximum non-toxic dose (MNTD) of ribavirin was 200 μg/ml for Vero cells. Regarding the dynamic monitoring of Vero cell properties during antiviral assay, approximately 34 h post-infection, the normalised Cell Index (CI) values of CHIKV-infected Vero cells started to decrease, while the vehicle controls did not show any significant changes. We also successfully showed the dose dependent manner of ribavirin as an approved in vitro inhibitor for CHIKV through our RTCA experiment. CONCLUSION: RTCA technology could become the prevailing tool in antiviral research due to its accurate output and the opportunity to carry out quality control and technical optimisation

Dengue virus type 1 clade replacement in recurring homotypic outbreaks

BACKGROUND: Recurring dengue outbreaks occur in cyclical pattern in most endemic countries. The recurrences of dengue virus (DENV) infection predispose the population to increased risk of contracting the severe forms of dengue. Understanding the DENV evolutionary mechanism underlying the recurring dengue outbreaks has important implications for epidemic prediction and disease control. RESULTS: We used a set of viral envelope (E) gene to reconstruct the phylogeny of DENV-1 isolated between the periods of 1987–2011 in Malaysia. Phylogenetic analysis of DENV-1 E gene revealed that genotype I virus clade replacements were associated with the cyclical pattern of major DENV-1 outbreaks in Malaysia. A total of 9 non-conservative amino acid substitutions in the DENV-1 E gene consensus were identified; 4 in domain I, 3 in domain II and 2 in domain III. Selection pressure analyses did not reveal any positively selected codon site within the full length E gene sequences (1485 nt, 495 codons). A total of 183 (mean dN/dS = 0.0413) negatively selected sites were found within the Malaysian isolates; neither positive nor negative selection was noted for the remaining 312 codons. All the viruses were cross-neutralized by the respective patient sera suggesting no strong support for immunological advantage of any of the amino acid substitutions. CONCLUSION: DENV-1 clade replacement is associated with recurrences of major DENV-1 outbreaks in Malaysia. Our findings are consistent with those of other studies that the DENV-1 clade replacement is a stochastic event independent of positive selection

Nipah Virus Infection of Immature Dendritic Cells Increases Its Transendothelial Migration Across Human Brain Microvascular Endothelial Cells

Nipah virus (NiV) can infect multiple organs in humans with the central nervous system (CNS) being the most severely affected. Currently, it is not fully understood how NiV spreads throughout the body. NiV has been shown to infect certain leukocyte populations and we hypothesized that these infected cells could cross the blood-brain barrier (BBB), facilitating NiV entry into the CNS. Here, three leukocyte types, primary immature dendritic cells (iDC), primary monocytes (pMO), and monocytic cell line (THP-1), were evaluated for permissiveness to NiV. We found only iDC and THP-1 were permissive to NiV. Transendothelial migration of mock-infected and NiV-infected leukocytes was then evaluated using an in vitro BBB model established with human brain microvascular endothelial cells (HBMEC). There was approximately a threefold increase in migration of NiV-infected iDC across endothelial monolayer when compared to mock-infected iDC. In contrast, migration rates for pMO and THP-1 did not change upon NiV infection. Across TNF-α-treated endothelial monolayer, there was significant increase of almost twofold in migration of NiV-infected iDC and THP-1 over mock-infected cells. Immunofluorescence analysis showed the migrated NiV-infected leukocytes retained their ability to infect other cells. This study demonstrates for the first time that active NiV infection of iDC and THP-1 increased their transendothelial migration activity across HBMEC and activation of HBMEC by TNF-α further promoted migration. The findings suggest that NiV infection of leukocytes to disseminate the virus via the "Trojan horse" mechanism is a viable route of entry into the CNS

Emergence of the Asian lineage dengue virus type 3 genotype III in Malaysia

Background: Dengue virus type 3 genotype III (DENV3/III) is associated with increased number of severe infections when it emerged in the Americas and Asia. We had previously demonstrated that the DENV3/III was introduced into Malaysia in the late 2000s. We investigated the genetic diversity of DENV3/III strains recovered from Malaysia and examined their phylogenetic relationships against other DENV3/III strains isolated globally. Results: Phylogenetic analysis revealed at least four distinct DENV3/III lineages. Two of the lineages (DENV3/III-B and DENV3/III-C) are current actively circulating whereas the DENV3/III-A and DENV3/III-D were no longer recovered since the 1980s. Selection pressure analysis revealed strong evidence of positive selection on a number of amino acid sites in PrM, E, NS1, NS2a, NS2b, NS3, NS4a, and NS5. The Malaysian DENV3/III isolates recovered in the 1980s (MY.59538/1987) clustered into DENV3/III-B, which was the lineage with cosmopolitan distribution consisting of strains actively circulating in the Americas, Africa, and Asia. The Malaysian isolates recovered after the 2000s clustered within DENV3/III-C. This DENV3/III-C lineage displayed a more restricted geographical distribution and consisted of isolates recovered from Asia, denoted as the Asian lineage. Amino acid variation sites in NS5 (NS5-553I/M, NS5-629 T, and NS5-820E) differentiated the DENV3/III-C from other DENV3 viruses. The codon 629 of NS5 was identified as a positively selected site. While the NS5-698R was identified as unique to the genome of DENV3/III-C3. Phylogeographic results suggested that the recent Malaysian DENV3/III-C was likely to have been introduced from Singapore in 2008 and became endemic. From Malaysia, the virus subsequently spread into Taiwan and Thailand in the early part of the 2010s and later reintroduced into Singapore in 2013. Conclusions: Distinct clustering of the Malaysian old and new DENV3/III isolates suggests that the currently circulating DENV3/III in Malaysia did not descend directly from the strains recovered during the 1980s. Phylogenetic analyses and common genetic traits in the genome of the strains and those from the neighboring countries suggest that the Malaysian DENV3/III is likely to have been introduced from the neighboring regions. Malaysia, however, serves as one of the sources of the recent regional spread of DENV3/III-C3 within the Asia region

Body weight and survival rate of mice following lethal challenge with influenza virus.

<p>At 18 days following the last immunization, all mice in study group B were intranasally challenged with 10 LD₅₀ of H1N1/A/TN/1-560/2009-MA2 virus. (A) Body weight and (B) survival of mice orally immunized with PBS, LL-HA1/L/AcmA or HA1/L/AcmA, or subcutaneously immunized with HA1/L/AcmA-FA, was monitored daily for 14 days following virus challenge. The body weight of mice on the day of viral challenge was used as the baseline weight to determine the body weight changes post-viral challenge. Asterisks indicate statistically significant differences in comparison with the PBS-treated group (*p<0.05; **p<0.01; ***p<0.001).</p

Binding optimization of HA1/L/AcmA recombinant protein to <i>L</i>. <i>lactis</i>.

<p>(A) The percentage of <i>L</i>. <i>lactis</i> surface displaying HA1/L/AcmA recombinant protein after incubation with different amounts of HA1/L/AcmA recombinant protein. (B) The percentage of <i>L</i>. <i>lactis</i> surface displaying HA1/L/AcmA recombinant protein after incubation with HA1/L/AcmA recombinant protein for 1 h, 2 h, 3 h, and 4 h, respectively. (C) The percentage and mean fluorescence intensity (MFI) value of <i>L</i>. <i>lactis</i> surface displaying HA1/L/AcmA recombinant protein after incubation with HA1/L/AcmA recombinant protein in GM17 and PBS, respectively. The data represents mean ± standard deviation. Asterisk indicates statistically significant differences between groups (**P<0.01).</p

Body weight and survival rate of immunized mice upon lethal challenge with influenza virus.

<p>Body weight and survival rate of immunized mice upon lethal challenge with influenza virus.</p

Additional file 2: of Emergence of the Asian lineage dengue virus type 3 genotype III in Malaysia

Phylogeography of DENV3/III-C. (KML 1198 kb