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Mutagenic and clastogenic characterization of poststerilized poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer biosynthesized by Delftia acidovorans

Siew EL, Rajab NF, Osman AB, Sudesh K, Inayat-Hussain SH

J Biomed Mater Res A, 2009 Dec;91(3):786-94.
PMID: 19051306 DOI: 10.1002/jbm.a.32290

Polyhydroxyalkanoates (PHA) are naturally occurring biopolyesters that have great potential in the medical field. However, the leachables resulting from sterilization process of the biomaterials may exert toxic effect including genetic damage. Here, we demonstrate that although gamma-irradiation of poly(3-hydroxybutyrate-co-50 mol % 4-hydroxybutyrate) [P(3HB-co-4HB)] did not cause any change in the morphology by scanning electron microscopy, there was a significant degradation of this copolymer where the molecular weight was reduced by 37% after sterilization indicating the generation of leachables. Therefore, further investigation on the ability of the extract of this poststerilized copolymer to induce mutagenic effect was performed using Ames test (S. typhimurium strains TA1535 and TA1537) and umu test (S. typhimurium strain TA1535/pSK1002). Additionally, the capability of the extract to induce clastogenic effect was determined using Chinese hamster lung V79 fibroblast cells. Our results showed that with and without the presence of S9 metabolic activation, no mutagenic effects were observed in both Ames and umu tests when treated with P(3HB-co-4HB) extract. Similarly, treatment of P(3HB-co-4HB) extract in V79 fibroblast cells showed no significant production of micronuclei when compared with the positive control (Mitomycin C). Together, these results indicate that leachables of poststerilized P(3HB-co-4HB) cause no mutagenic and clastogenic effects.

Matched MeSH terms: Cricetinae
Fulltext Comparison of the pathogenicity of Nipah virus isolates from Bangladesh and Malaysia in the Syrian hamster

DeBuysscher BL, de Wit E, Munster VJ, Scott D, Feldmann H, Prescott J

PLoS Negl Trop Dis, 2013;7(1):e2024.
PMID: 23342177 DOI: 10.1371/journal.pntd.0002024

Nipah virus is a zoonotic pathogen that causes severe disease in humans. The mechanisms of pathogenesis are not well described. The first Nipah virus outbreak occurred in Malaysia, where human disease had a strong neurological component. Subsequent outbreaks have occurred in Bangladesh and India and transmission and disease processes in these outbreaks appear to be different from those of the Malaysian outbreak. Until this point, virtually all Nipah virus studies in vitro and in vivo, including vaccine and pathogenesis studies, have utilized a virus isolate from the original Malaysian outbreak (NiV-M). To investigate potential differences between NiV-M and a Nipah virus isolate from Bangladesh (NiV-B), we compared NiV-M and NiV-B infection in vitro and in vivo. In hamster kidney cells, NiV-M-infection resulted in extensive syncytia formation and cytopathic effects, whereas NiV-B-infection resulted in little to no morphological changes. In vivo, NiV-M-infected Syrian hamsters had accelerated virus replication, pathology and death when compared to NiV-B-infected animals. NiV-M infection also resulted in the activation of host immune response genes at an earlier time point. Pathogenicity was not only a result of direct effects of virus replication, but likely also had an immunopathogenic component. The differences observed between NiV-M and NiV-B pathogeneis in hamsters may relate to differences observed in human cases. Characterization of the hamster model for NiV-B infection allows for further research of the strain of Nipah virus responsible for the more recent outbreaks in humans. This model can be used to study NiV-B pathogenesis, transmission, and countermeasures that could be used to control outbreaks.

Matched MeSH terms: Cricetinae
Effects of administration of alpha-tocopherol and tocotrienols on serum lipids and liver HMG CoA reductase activity

Khor HT, Ng TT

Int J Food Sci Nutr, 2000;51 Suppl:S3-11.
PMID: 11271854

Male hamsters were fed on semi-synthetic diets containing commercial corn oil (CO), isolated corn oil triglycerides (COTG), COTG supplemented with 30 ppm of alpha-tocopherol (COTGTL) and COTG supplemented with 81 ppm of alpha-tocopherol (COTGTH) as the dietary lipid for 45 days. Male albino guinea pigs were fed on commercial chow pellets and treated with different dosages of tocopherol and tocotrienols intra-peritoneally for 6 consecutive days. Serum and liver were taken for analysis. Our results show that stripping corn oil of its unsaponifiable components resulted in COTG which yielded lower serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) and raised high-density lipoprotein cholesterol (HDL-C) and serum triglycerides (TG) levels. These results indicate that the COTG with its fatty acids are responsible for the hypocholesterolemic effect exhibited by corn oil. However, supplementing the COTG diet with alpha-tocopherol (alpha-T) at 30 ppm significantly raised the serum TC, LDL-C and TG levels, but did not alter the HDL-C level, indicating that alpha-T is hypercholesterolemic. Supplementing the COTG diet with alpha-T at 81 ppm raised the serum TC level but to a lesser extent as compared to that obtained with 30-ppm alpha-T supplementation. The increased TC, in this case, was reflected mainly by an increased in HDL-C level as the LDL-C level was unchanged. The TG level was also raised but to a lesser extent than that obtained with a lower alpha-T supplementation. The liver HMG CoA reductase (HMGCR) activity was exhibited (56%) by the COTG as compared to CO. Supplementation of alpha-T at 30 ppm to the COTG diet resulted in further inhibition (76%) of the liver HMGCR activity. On the contrary, supplementation of alpha-T at 81 ppm to COTG diet resulted in a highly stimulatory effect (131%) on the liver HMGCR activity. Short-term studies with guinea pigs treated intra-peritoneally with alpha-T showed that at low dosage (5 mg) the HMGCR activity was inhibited by 46% whereas increasing the dosage of alpha-T to 20 mg yielded lesser inhibition (18%) as compared to that of the control. Further increase in the dosage of alpha-T to 50 mg actually resulted in 90% stimulation of the liver HMGCR activity as compared to the control. These results clearly indicate that the effect of alpha-T on HMGCR activity was dose-dependent. Treatment of the guinea pigs with 10 mg of tocotrienols (T3) resulted in 48% inhibition of the liver HMGCR activity. However, treatment with a mixture of 5 mg of alpha-T with 10 mg of T3 resulted in lesser inhibition (13%) of the liver HMGCR activity as compared to that obtained with 10 mg of T3. The above results indicate that the alpha-T is hypercholesterolemic in the hamster and its effect on liver HMGCR is dose-dependent. T3 exhibited inhibitory effect on liver HMGCR and alpha-T attenuated the inhibitory effect of T3 on liver HMGCR.

Matched MeSH terms: Cricetinae
Fulltext Rapid detection of pathogenic leptospires by lyophilized reagent-based polymerase chain reaction

Lee SV, Tai ES, Mutalib AR, Khairani-Bejo S, Bahaman AR

Trop Biomed, 2011 Dec;28(3):497-505.
PMID: 22433877 MyJurnal

A simple and reliable tool for the early diagnosis of leptospirosis is urgently needed. We report the development of a lyophilized reagent-based polymerase chain reaction (PCR) assay targeting lipL32 gene, which is present only in pathogenic leptospires. To determine the effectiveness of the newly developed assay in the early diagnosis of leptospirosis, the sensitivity and specificity was evaluated. In simulated clinical samples, the assay was able to detect 10² and 10³ leptospires/ml in spiked urine and blood samples, respectively. In experimentally infected animals, leptospiral DNA could be detected in blood and lung samples as early as Day 1 post infection. This assay was also shown to be stable and remained sensitive for up to five months at ambient temperature. Hence, this lyophilized reagent-based PCR assay with high specificity, sensitivity and stability would provide a simple, rapid and reliable method in diagnosing acute leptospirosis, especially in the field of veterinary medicine.

Matched MeSH terms: Cricetinae
[The biological and hemagglutinating properties of Ghetah arbovirus]

Leont'eva NA, Fadeeva LL

Vopr. Virusol., 1969 Jul-Aug;14(4):464-8.
PMID: 4982330
Matched MeSH terms: Cricetinae
Antiviral activity of selected flavonoids against Chikungunya virus

Lani R, Hassandarvish P, Shu MH, Phoon WH, Chu JJ, Higgs S, et al.

Antiviral Res, 2016 Sep;133:50-61.
PMID: 27460167 DOI: 10.1016/j.antiviral.2016.07.009

This study focuses on the antiviral activity of selected flavonoids against the Chikungunya virus (CHIKV), a mosquito-transmitted virus that can cause incapacitating arthritis in infected individuals. Based on the results of screening on Vero cells, the tested compounds were evaluated further with various assays, including cytotoxicity assay, virus yield assay by quantitative reverse transcription polymerase chain reaction (qRT-PCR), virus RNA replication assay with a CHIKV replicon cell line, Western blotting, and quantitative immunofluorescence assay. Baicalein, fisetin, and quercetagetin displayed potent inhibition of CHIKV infection, with 50% inhibitory concentrations [IC50] of 1.891 μg/ml (6.997 μM), 8.444 μg/ml (29.5 μM), and 13.85 μg/ml (43.52 μM), respectively, and with minimal cytotoxicity. The time-of-addition studies and various antiviral assays demonstrated that baicalein and quercetagetin mainly inhibited CHIKV binding to the Vero cells and displayed potent activity against extracellular CHIKV particles. The qRT-PCR, immunofluorescence assay, and Western blot analyses indicated that each of these flavonoids affects CHIKV RNA production and viral protein expression. These data provide the first evidence of the intracellular anti-CHIKV activity of baicalein, fisetin, and quercetagetin.

Matched MeSH terms: Cricetinae
The hypolipidemic effects of Tamarindus indica fruit pulp extract in normal and diet-induced hypercholesterolemic hamsters are associated with altered levels of serum proteins

Lim CY, Junit SM, Aziz AA, Jayapalan JJ, Hashim OH

Electrophoresis, 2018 12;39(23):2965-2973.
PMID: 30280388 DOI: 10.1002/elps.201800258

The hypolipidemic effects of Tamarindus indica fruit pulp extract (Ti-FPE) have been earlier reported but the underlying molecular mechanisms are still uncertain. In this study, hamsters fed with Ti-FPE, both in the absence and presence of high-cholesterol diet, were shown to have significantly reduced levels of serum triglyceride, LDL-C and total cholesterol. The Ti-FPE-fed non-hypercholesterolemic hamsters also showed significant enhanced levels of serum apolipoprotein A1, antithrombin III, transferrin and vitamin D binding protein. In diet-induced hypercholesterolemic hamsters, apolipoprotein A1, antithrombin III and transferrin, which were relatively low in levels, became significantly enhanced when the hamsters were fed with Ti-FPE. These Ti-FPE-fed hypercholesterolemic hamsters also showed significant higher levels of serum vitamin D binding protein. When the different treated groups of hamsters were analyzed for the levels of the four serum proteins by ELISA, similar altered abundance were detected. Ingenuity Pathway Analysis of the Ti-FPE modulated serum proteins singled out "Lipid metabolism, molecular transport, small molecule biochemistry" as the top network. Our results suggest that the hypolipidemic effects of Ti-FPE are associated with alterations of serum proteins that are known to be cardioprotective and involved in the metabolism of lipids. The MS data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD010232.

Matched MeSH terms: Cricetinae
Establishment of a Nipah virus rescue system

Yoneda M, Guillaume V, Ikeda F, Sakuma Y, Sato H, Wild TF, et al.

Proc Natl Acad Sci U S A, 2006 Oct 31;103(44):16508-13.
PMID: 17053073

Nipah virus (NiV), a paramyxovirus, was first discovered in Malaysia in 1998 in an outbreak of infection in pigs and humans and incurred a high fatality rate in humans. Fruit bats, living in vast areas extending from India to the western Pacific, were identified as the natural reservoir of the virus. However, the mechanisms that resulted in severe pathogenicity in humans (up to 70% mortality) and that enabled crossing the species barrier were not known. In this study, we established a system that enabled the rescue of replicating NiVs from a cloned DNA by cotransfection of a constructed full-length cDNA clone and supporting plasmids coding virus nucleoprotein, phosphoprotein, and polymerase with the infection of the recombinant vaccinia virus, MVAGKT7, expressing T7 RNA polymerase. The rescued NiV (rNiV), by using the newly developed reverse genetics system, showed properties in vitro that were similar to the parent virus and retained the severe pathogenicity in a previously established animal model by experimental infection. A recombinant NiV was also developed, expressing enhanced green fluorescent protein (rNiV-EGFP). Using the virus, permissibility of NiV was compared with the presence of a known cellular receptor, ephrin B2, in a number of cell lines of different origins. Interestingly, two cell lines expressing ephrin B2 were not susceptible for rNiV-EGFP, indicating that additional factors are clearly required for full NiV replication. The reverse genetics for NiV will provide a powerful tool for the analysis of the molecular mechanisms of pathogenicity and cross-species infection.

Matched MeSH terms: Cricetinae
Fulltext Dengue virus infection-enhancing activity in serum samples with neutralizing activity as determined by using FcγR-expressing cells

Moi ML, Lim CK, Chua KB, Takasaki T, Kurane I

PLoS Negl Trop Dis, 2012;6(2):e1536.
PMID: 22389741 DOI: 10.1371/journal.pntd.0001536

Progress in dengue vaccine development has been hampered by limited understanding of protective immunity against dengue virus infection. Conventional neutralizing antibody titration assays that use FcγR-negative cells do not consider possible infection-enhancement activity. We reasoned that as FcγR-expressing cells are the major target cells of dengue virus, neutralizing antibody titration assays using FcγR-expressing cells that determine the sum of neutralizing and infection-enhancing activity, may better reflect the biological properties of antibodies in vivo.

Matched MeSH terms: Cricetinae
A rapid immune plaque assay for the detection of Hendra and Nipah viruses and anti-virus antibodies

Crameri G, Wang LF, Morrissy C, White J, Eaton BT

J Virol Methods, 2002 Jan;99(1-2):41-51.
PMID: 11684302

Rapid immune plaque assays have been developed to quantify biohazard level 4 agents Hendra and Nipah viruses and detect neutralising antibodies to both viruses. The methods rely on the fact that both viruses rapidly generate large syncytia in monolayers of Vero cells within 24 h and that monospecific antiserum to the Hendra virus phosphoprotein (P) detects that protein in both Hendra and Nipah virus-induced syncytia after methanol fixation of virus-infected cells. The P protein is a constituent of the ribonucleoprotein core of the viruses and a component of the viral RNA-dependent RNA polymerase and is made in significant amounts in infected cells. In the immune plaque assay, anti-P antibody is localised by an alkaline phosphatase-linked second antibody and the Western blot substrates 5-bromo-4-chloro-3-indolyl phosphate and p-nitro blue tetrazolium. A modification of the rapid immune plaque assay was also used to detect antibodies to Nipah virus in a panel of porcine field sera from Malaysia and the results showed good agreement between the immune plaque assay and a traditional serum neutralisation test. After methanol fixation, plates can be stored for up to 7 months and may be used in the immune plaque assay to complement the enzyme-linked immunosorbent assay screening of sera for antibodies to Nipah virus. At present, all enzyme-linked immunosorbent assay positive sera are subject to confirmatory serum neutralisation tests. Use of the immune plaque assay may reduce the number of sera requiring confirmatory neutralisation testing for Nipah virus antibodies under biohazard level 4 conditions by identifying those that generate false positive in the enzyme-linked immunosorbent assay.

Matched MeSH terms: Cricetinae
Fulltext Optimization of Bangladesh and Malaysian genotype recombinant reporter Nipah viruses for in vitro antiviral screening and in vivo disease modeling

Lo MK, Jain S, Davies KA, Sorvillo TE, Welch SR, Coleman-McCray JD, et al.

Antiviral Res, 2024 Nov;231:106013.
PMID: 39326503 DOI: 10.1016/j.antiviral.2024.106013

Nipah virus (NiV) causes near-annual outbreaks of fatal encephalitis and respiratory disease in South Asia with a high mortality rate (∼70%). Since there are no approved therapeutics for NiV disease in humans, the WHO has designated NiV and henipaviral diseases priority pathogens for research and development. We generated a new recombinant green fluorescent reporter NiV of the circulating Bangladesh genotype (rNiV-B-ZsG) and optimized it alongside our previously generated Malaysian genotype reporter counterpart (rNiV-M-ZsG) for antiviral screening in primary-like human respiratory cell types. Validating our platform for rNiV-B-ZsG with a synthetic compound library directed against viral RNA-dependent RNA polymerases, we identified a hit compound and confirmed its sub-micromolar activity against wild-type NiV, green fluorescent reporter, and the newly constructed bioluminescent red fluorescent double reporter (rNiV-B-BREP) NiV. We furthermore demonstrated that rNiV-B-ZsG and rNiV-B-BREP viruses showed pathogenicity comparable to wild-type NiV-B in the Syrian golden hamster model of disease, supporting additional use of these tools for both pathogenesis and advanced pre-clinical studies in vivo.

Matched MeSH terms: Cricetinae
Fulltext Recombinant measles virus vaccine expressing the Nipah virus glycoprotein protects against lethal Nipah virus challenge

Yoneda M, Georges-Courbot MC, Ikeda F, Ishii M, Nagata N, Jacquot F, et al.

PLoS One, 2013;8(3):e58414.
PMID: 23516477 DOI: 10.1371/journal.pone.0058414

Nipah virus (NiV) is a member of the genus Henipavirus, which emerged in Malaysia in 1998. In pigs, infection resulted in a predominantly non-lethal respiratory disease; however, infection in humans resulted in over 100 deaths. Nipah virus has continued to re-emerge in Bangladesh and India, and person-to-person transmission appeared in the outbreak. Although a number of NiV vaccine studies have been reported, there are currently no vaccines or treatments licensed for human use. In this study, we have developed a recombinant measles virus (rMV) vaccine expressing NiV envelope glycoproteins (rMV-HL-G and rMV-Ed-G). Vaccinated hamsters were completely protected against NiV challenge, while the mortality of unvaccinated control hamsters was 90%. We trialed our vaccine in a non-human primate model, African green monkeys. Upon intraperitoneal infection with NiV, monkeys showed several clinical signs of disease including severe depression, reduced ability to move and decreased food ingestion and died at 7 days post infection (dpi). Intranasal and oral inoculation induced similar clinical illness in monkeys, evident around 9 dpi, and resulted in a moribund stage around 14 dpi. Two monkeys immunized subcutaneously with rMV-Ed-G showed no clinical illness prior to euthanasia after challenge with NiV. Viral RNA was not detected in any organ samples collected from vaccinated monkeys, and no pathological changes were found upon histopathological examination. From our findings, we propose that rMV-NiV-G is an appropriate NiV vaccine candidate for use in humans.

Matched MeSH terms: Cricetinae
Fulltext Lethal Nipah virus infection induces rapid overexpression of CXCL10

Mathieu C, Guillaume V, Sabine A, Ong KC, Wong KT, Legras-Lachuer C, et al.

PLoS One, 2012;7(2):e32157.
PMID: 22393386 DOI: 10.1371/journal.pone.0032157

Nipah virus (NiV) is a recently emerged zoonotic Paramyxovirus that causes regular outbreaks in East Asia with mortality rate exceeding 75%. Major cellular targets of NiV infection are endothelial cells and neurons. To better understand virus-host interaction, we analyzed the transcriptome profile of NiV infection in primary human umbilical vein endothelial cells. We further assessed some of the obtained results by in vitro and in vivo methods in a hamster model and in brain samples from NiV-infected patients. We found that NiV infection strongly induces genes involved in interferon response in endothelial cells. Among the top ten upregulated genes, we identified the chemokine CXCL10 (interferon-induced protein 10, IP-10), an important chemoattractant involved in the generation of inflammatory immune response and neurotoxicity. In NiV-infected hamsters, which develop pathology similar to what is seen in humans, expression of CXCL10 mRNA was induced in different organs with kinetics that followed NiV replication. Finally, we showed intense staining for CXCL10 in the brain of patients who succumbed to lethal NiV infection during the outbreak in Malaysia, confirming induction of this chemokine in fatal human infections. This study sheds new light on NiV pathogenesis, indicating the role of CXCL10 during the course of infection and suggests that this chemokine may serve as a potential new marker for lethal NiV encephalitis.

Matched MeSH terms: Cricetinae
Genome structure of Sagiyama virus and its relatedness to other alphaviruses

Shirako Y, Yamaguchi Y

J Gen Virol, 2000 May;81(Pt 5):1353-60.
PMID: 10769079

Sagiyama virus (SAG) is a member of the genus Alphavirus in the family Togaviridae, isolated in Japan from mosquitoes in 1956. We determined the complete nucleotide sequence of the SAG genomic RNA from the original stock virus which formed a mixture of plaques with different sizes, and that from a full-length cDNA clone, pSAG2, infectious RNA transcripts from which formed uniform large plaques on BHK-21 cells. The SAG genome was 11698 nt in length exclusive of the 3' poly(A) tail. Between the complete nucleotide sequences of the full-length cDNA clone, pSAG2, and the consensus sequence from the original stock virus, there were nine amino acid differences; two each in nsP1, nsP2 and E1, and three in E2, some of which may be responsible for plaque phenotypic variants in the original virus stock. SAG was most closely related to Ross River virus among other alphaviruses fully sequenced, with amino acid sequence identities of 86% in the nonstructural proteins and of 83% in the structural proteins. The 3' terminal 280 nt region of SAG was 82% identical to that of Barmah Forest virus, which was otherwise not closely related to SAG. Comparison of the nucleotide sequence of SAG with partial nucleotide sequences of Getah virus (GET), which was originally isolated in Malaysia in 1955 and is closely related to SAG in serology and in biology, showed near identity between the two viruses, suggesting that SAG is a strain of GET.

Matched MeSH terms: Cricetinae
Regulation of G protein signaling by the 70kDa heat shock protein

Lim WK, Kanelakis KC, Neubig RR

Cell Signal, 2013 Feb;25(2):389-96.
PMID: 23153586 DOI: 10.1016/j.cellsig.2012.11.002

G protein-coupled receptors (GPCRs) transduce extracellular signals to the interior of the cell by activating membrane-bound guanine nucleotide-binding regulatory proteins (G proteins). An increasing number of proteins have been reported to bind to and regulate GPCRs. We report a novel regulation of the alpha(2A) adrenergic receptor (α(2A)-R) by the ubiquitous stress-inducible 70kDa heat shock protein, hsp70. Hsp70, but not hsp90, attenuated G protein-dependent high affinity agonist binding to the α(2A)-R in Sf9 membranes. Antagonist binding was unchanged, suggesting that hsp70 uncouples G proteins from the receptor. As hsp70 did not bind G proteins but complexed with the α(2A)-R in intact cells, a direct interaction with the receptor seems likely. In the presence of hsp70, α(2A)-R-catalyzed [(35)S]GTPγS binding was reduced by approximately 70%. In contrast, approximately 50-fold higher concentrations of hsp70 were required to reduce agonist binding to the stress-inducible 5-hydroxytryptamine(1A) receptor (5-HT(1A)-R). In heat-stressed CHO cells, the α(2A)-R was significantly uncoupled from G proteins, coincident with an increased localization of hsp70 at the membrane. The contrasting effect of hsp70 on the α(2A)-R compared to the 5-HT(1A)-R suggests that during stress, upregulation of hsp70 may attenuate signaling from specific GPCRs as part of the stress response to foster survival.

Matched MeSH terms: Cricetinae
Degradation of MicroRNA miR-466d-3p by Japanese Encephalitis Virus NS3 Facilitates Viral Replication and Interleukin-1β Expression

Jiang H, Bai L, Ji L, Bai Z, Su J, Qin T, et al.

J Virol, 2020 07 16;94(15).
PMID: 32461319 DOI: 10.1128/JVI.00294-20

Japanese encephalitis virus (JEV) infection alters microRNA (miRNA) expression in the central nervous system (CNS). However, the mechanism contributing to miRNA regulation in the CNS is not known. We discovered global degradation of mature miRNA in mouse brains and neuroblastoma (NA) cells after JEV infection. Integrative analysis of miRNAs and mRNAs suggested that several significantly downregulated miRNAs and their targeted mRNAs were clustered into an inflammation pathway. Transfection with miRNA 466d-3p (miR-466d-3p) decreased interleukin-1β (IL-1β) expression and inhibited JEV replication in NA cells. However, miR-466d-3p expression increased after JEV infection in the presence of cycloheximide, indicating that viral protein expression reduced miR-466d-3p expression. We generated all the JEV coding proteins and demonstrated NS3 helicase protein to be a potent miRNA suppressor. The NS3 proteins of Zika virus, West Nile virus, and dengue virus serotype 1 (DENV-1) and DENV-2 also decreased miR-466d-3p expression. Results from helicase-blocking assays and in vitro unwinding assays demonstrated that NS3 could unwind pre-miR-466d and induce miRNA dysfunction. Computational models and an RNA immunoprecipitation assay revealed arginine-rich domains of NS3 to be crucial for pre-miRNA binding and degradation of host miRNAs. Importantly, site-directed mutagenesis of conserved residues in NS3 revealed that R226G and R202W reduced the binding affinity and degradation of pre-miR-466d. These results expand the function of flavivirus helicases beyond unwinding duplex RNA to degrade pre-miRNAs. Hence, we revealed a new mechanism for NS3 in regulating miRNA pathways and promoting neuroinflammation.IMPORTANCE Host miRNAs have been reported to regulate JEV-induced inflammation in the CNS. We found that JEV infection could reduce expression of host miRNA. The helicase region of the NS3 protein bound specifically to miRNA precursors and could lead to incorrect unwinding of miRNA precursors, thereby reducing the expression of mature miRNAs. This observation led to two major findings. First, our results suggested that JEV NS3 protein induced miR-466d-3p degradation, which promoted IL-1β expression and JEV replication. Second, arginine molecules on NS3 were the main miRNA-binding sites, because we demonstrated that miRNA degradation was abolished if arginines at R226 and R202 were mutated. Our study provides new insights into the molecular mechanism of JEV and reveals several amino acid sites that could be mutated for a JEV vaccine.

Matched MeSH terms: Cricetinae
Fulltext Antigenic Variation of East/Central/South African and Asian Chikungunya Virus Genotypes in Neutralization by Immune Sera

Chua CL, Sam IC, Merits A, Chan YF

PLoS Negl Trop Dis, 2016 08;10(8):e0004960.
PMID: 27571254 DOI: 10.1371/journal.pntd.0004960

BACKGROUND: Chikungunya virus (CHIKV) is a re-emerging mosquito-borne virus which causes epidemics of fever, severe joint pain and rash. Between 2005 and 2010, the East/Central/South African (ECSA) genotype was responsible for global explosive outbreaks across India, the Indian Ocean and Southeast Asia. From late 2013, Asian genotype CHIKV has caused outbreaks in the Americas. The characteristics of cross-antibody efficacy and epitopes are poorly understood.
METHODOLOGY/PRINCIPAL FINDINGS: We characterized human immune sera collected during two independent outbreaks in Malaysia of the Asian genotype in 2006 and the ECSA genotype in 2008-2010. Neutralizing capacity was analyzed against representative clinical isolates as well as viruses rescued from infectious clones of ECSA and Asian CHIKV. Using whole virus antigen and recombinant E1 and E2 envelope glycoproteins, we further investigated antibody binding sites, epitopes, and antibody titers. Both ECSA and Asian sera demonstrated stronger neutralizing capacity against the ECSA genotype, which corresponded to strong epitope-antibody interaction. ECSA serum targeted conformational epitope sites in the E1-E2 glycoprotein, and E1-E211K, E2-I2T, E2-H5N, E2-G118S and E2-S194G are key amino acids that enhance cross-neutralizing efficacy. As for Asian serum, the antibodies targeting E2 glycoprotein correlated with neutralizing efficacy, and I2T, H5N, G118S and S194G altered and improved the neutralization profile. Rabbit polyclonal antibody against the N-terminal linear neutralizing epitope from the ECSA sequence has reduced binding capacity and neutralization efficacy against Asian CHIKV. These findings imply that the choice of vaccine strain may impact cross-protection against different genotypes.
CONCLUSION/SIGNIFICANCE: Immune serum from humans infected with CHIKV of either ECSA or Asian genotypes showed differences in binding and neutralization characteristics. These findings have implications for the continued outbreaks of co-circulating CHIKV genotypes and effective design of vaccines and diagnostic serological assays.

Matched MeSH terms: Cricetinae
Fulltext Identifying Early Target Cells of Nipah Virus Infection in Syrian Hamsters

Baseler L, Scott DP, Saturday G, Horne E, Rosenke R, Thomas T, et al.

PLoS Negl Trop Dis, 2016 Nov;10(11):e0005120.
PMID: 27812087 DOI: 10.1371/journal.pntd.0005120

BACKGROUND: Nipah virus causes respiratory and neurologic disease with case fatality rates up to 100% in individual outbreaks. End stage lesions have been described in the respiratory and nervous systems, vasculature and often lymphoid organs in fatal human cases; however, the initial target organs of Nipah virus infection have not been identified. Here, we detected the initial target tissues and cells of Nipah virus and tracked virus dissemination during the early phase of infection in Syrian hamsters inoculated with a Nipah virus isolate from Malaysia (NiV-M) or Bangladesh (NiV-B).
METHODOLOGY/PRINCIPAL FINDINGS: Syrian hamsters were euthanized between 4 and 48 hours post intranasal inoculation and tissues were collected and analyzed for the presence of viral RNA, viral antigen and infectious virus. Virus replication was first detected at 8 hours post inoculation (hpi). Nipah virus initially targeted type I pneumocytes, bronchiolar respiratory epithelium and alveolar macrophages in the lung and respiratory and olfactory epithelium lining the nasal turbinates. By 16 hpi, virus disseminated to epithelial cells lining the larynx and trachea. Although the pattern of viral dissemination was similar for both virus isolates, the rate of spread was slower for NiV-B. Infectious virus was not detected in the nervous system or blood and widespread vascular infection and lesions within lymphoid organs were not observed, even at 48 hpi.
CONCLUSIONS/SIGNIFICANCE: Nipah virus initially targets the respiratory system. Virus replication in the brain and infection of blood vessels in non-respiratory tissues does not occur during the early phase of infection. However, virus replicates early in olfactory epithelium and may serve as the first step towards nervous system dissemination, suggesting that development of vaccines that block virus dissemination or treatments that can access the brain and spinal cord and directly inhibit virus replication may be necessary for preventing central nervous system pathology.

Matched MeSH terms: Cricetinae
The antioxidant effect of the Malaysian Gelam honey on pancreatic hamster cells cultured under hyperglycemic conditions

Batumalaie K, Qvist R, Yusof KM, Ismail IS, Sekaran SD

Clin Exp Med, 2014 May;14(2):185-95.
PMID: 23584372 DOI: 10.1007/s10238-013-0236-7

Type 2 diabetes consists of progressive hyperglycemia, insulin resistance, and pancreatic β-cell failure which could result from glucose toxicity, inflammatory cytokines, and oxidative stress. In the present study, we investigate the effect of pretreatment with Gelam honey (Melaleuca spp.) and the individual flavonoid components chrysin, luteolin, and quercetin, on the production of reactive oxygen species (ROS), cell viability, lipid peroxidation, and insulin content in hamster pancreatic cells (HIT-T15 cells), cultured under normal and hyperglycemic conditions. Phenolic extracts from a local Malaysian species of Gelam honey (Melaleuca spp.) were prepared using the standard extraction methods. HIT-T15 cells were cultured in 5 % CO2 and then preincubated with Gelam honey extracts (20, 40, 60, and 80 μg/ml) as well as some of its flavonoid components chrysin, luteolin, and quercetin (20, 40, 60, and 80 μM), prior to stimulation by 20 and 50 mM of glucose. The antioxidative effects were measured in these cultured cells at different concentrations and time point by DCFH-DA assay. Pretreatment of cells with Gelam honey extract or the flavonoid components prior to culturing in 20 or 50 mM glucose showed a significant decrease in the production of ROS, glucose-induced lipid peroxidation, and a significant increase in insulin content and the viability of cells cultured under hyperglycemic condition. Our results show the in vitro antioxidative property of the Gelam honey and the flavonoids on the β-cells from hamsters and its cytoprotective effect against hyperglycemia.

Matched MeSH terms: Cricetinae
Fulltext A golden hamster model for human acute Nipah virus infection

Wong KT, Grosjean I, Brisson C, Blanquier B, Fevre-Montange M, Bernard A, et al.

Am J Pathol, 2003 Nov;163(5):2127-37.
PMID: 14578210 DOI: 10.1016/S0002-9440(10)63569-9

A predominantly pig-to-human zoonotic infection caused by the novel Nipah virus emerged recently to cause severe morbidity and mortality in both animals and man. Human autopsy studies showed the pathogenesis to be related to systemic vasculitis that led to widespread thrombotic occlusion and microinfarction in most major organs especially in the central nervous system. There was also evidence of extravascular parenchymal infection, particularly near damaged vessels (Wong KT, Shieh WJ, Kumar S, Norain K, Abdullah W, Guarner J, Goldsmith CS, Chua KB, Lam SK, Tan CT, Goh KJ, Chong HT, Jusoh R, Rollin PE, Ksiazek TG, Zaki SR, Nipah Virus Pathology Working Group: Nipah virus infection: Pathology and pathogenesis of an emerging paramyxoviral zoonosis. Am J Pathol 2002, 161:2153-2167). We describe here a golden hamster (Mesocricetus auratus) model that appears to reproduce the pathology and pathogenesis of acute human Nipah infection. Hamsters infected by intranasal or intraperitoneal routes died within 9 to 29 days or 5 to 9 days, respectively. Pathological lesions were most severe and extensive in the hamster brain. Vasculitis, thrombosis, and more rarely, multinucleated endothelial syncytia, were found in blood vessels of multiple organs. Viral antigen and RNA were localized in both vascular and extravascular tissues including neurons, lung, kidney, and spleen, as demonstrated by immunohistochemistry and in situ hybridization, respectively. Paramyxoviral-type nucleocapsids were identified in neurons and in vessel walls. At the terminal stage of infection, virus and/or viral RNA could be recovered from most solid organs and urine, but not from serum. The golden hamster is proposed as a suitable model for further studies including pathogenesis studies, anti-viral drug testing, and vaccine development against acute Nipah infection.

Matched MeSH terms: Cricetinae

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