Application of real-time RT-PCR in vector surveillance and assessment of replication kinetics of an emerging novel ECSA genotype of Chikungunya virus in Aedes aegypti

Chikungunya has emerged as one of the most important arboviral infection of global significance. Expansion of Chikungunya virus endemic areas can be ascribed to naive population, increasing vector population and adaptability of virus to new vector. In this study, a SYBR Green I based quantitative RT-PCR assay was developed. The assay was found to be 10-fold more sensitive than conventional RT-PCR and no cross reactivity was observed with related alphaviruses and flaviviruses. The detection efficiency of the assay was impervious to mosquitoes of different pool sizes. Vector surveillance has resulted in detection of CHIKV RNA in Aedes aegypti, confirming its vectorial potential for CHIKV in northern India. The assessment of the assay was further carried out by studying the competence of Indian Ae. aegypti for CHIKV, which revealed 100% infection rate and dissemination rate with 60% transmission rate. The replication kinetics of CHIKV in different anatomical sites of Ae. aegypti revealed highest titre at day 6 post infection in midgut and at day 10 post infection in saliva, legs and wings. The implementation of the assay in detecting lower viral load makes it a remarkable tool for surveillance of virus activity in mosquitoes.     

Domain-III FG loop of the dengue virus type 2 envelope protein is important for infection of mammalian cells and Aedes aegypti mosquitoes

The FG extended loop in domain III of the dengue virus type 2 (DENV2) envelope protein is postulated to be a molecular determinant for host cell infectivity. To determine the contribution of the FG loop to virus infectivity, an infectious cDNA clone of DENV2 was manipulated by deleting amino acids in the loop (VEPGΔ) to mimic tick-borne flaviviruses or by substituting these AAs with RGD or RGDK/S to mimic motifs present in other mosquito-borne flaviviruses. We found the FG loop to be dispensable for infection of C6/36 cells but critical for infection of Aedes aegypti mosquito midguts and mammalian cells. All the FG loop mutants were able to bind to and enter mammalian cells but replication of VEPGΔ in Vero cells at 37 °C was delayed until acquisition of secondary mutations. Reduced binding of DENV2 type-specific monoclonal antibody 3H5 to mutant viruses confirmed the FG loop motif as its target epitope.     

A trade-off in replication in mosquito versus mammalian systems conferred by a point mutation in the NS4B protein of dengue virus type 4

An acceptable live-attenuated dengue virus vaccine candidate should have low potential for transmission by mosquitoes. We have identified and characterized a mutation in dengue virus type 4 (DEN4) that decreases the ability of the virus to infect mosquitoes. A panel of 1248 mutagenized virus clones generated previously by chemical mutagenesis was screened for decreased replication in mosquito C6/36 cells but efficient replication in simian Vero cells. One virus met these criteria and contained a single coding mutation: a C-to-U mutation at nucleotide 7129 resulting in a Pro-to-Leu change in amino acid 101 of the nonstructural 4B gene (NS4B P101L). This mutation results in decreased replication in C6/36 cells relative to wild-type DEN4, decreased infectivity for mosquitoes, enhanced replication in Vero and human HuH-7 cells, and enhanced replication in SCID mice implanted with HuH-7 cells (SCID-HuH-7 mice). A recombinant DEN4 virus (rDEN4) bearing this mutation exhibited the same set of phenotypes. Addition of the NS4B P101L mutation to rDEN4 bearing a 30 nucleotide deletion (Delta30) decreased the ability of the double-mutant virus to infect mosquitoes but increased its ability to replicate in SCID-HuH-7 mice. Although the NS4B P101L mutation decreases infectivity of DEN4 for mosquitoes, its ability to enhance replication in SCID-HuH-7 mice suggests that it might not be advantageous to include this specific mutation in an rDEN4 vaccine. The opposing effects of the NS4B P101L mutation in mosquito and vertebrate systems suggest that the NS4B protein is involved in maintaining the balance between efficient replication in the mosquito vector and the human host.     

Genetic variations of SOCS1 are associated with chronic hepatitis B virus infection

Suppressor of cytokine signaling (SOCS)-1 is involved in viral infection through regulation of both innate and adaptive immunity. The SOCS1 gene polymorphisms may affect the outcome of viral infection. The relationship between SOCS1 polymorphisms and hepatitis B virus (HBV) infection has not yet been explored. This study genotyped SOCS1 rs243327 and rs33932899 polymorphisms in 477 patients with chronic HBV infection, 93 HBV infection resolvers and 215 healthy controls. In statistical analysis, p-values less than 0.05 in multiple comparisons were corrected by Bonferroni method and presented as p_c. The results showed that the allele T-containing genotypes (CT + TT) of rs243327 were higher in HBV patients than resolvers and lower in resolvers than healthy controls although the difference was not significant. The allele T of rs243327 was significantly lower in resolvers than controls (p = 0.033). The genotype GC and allele C of rs33932899 were significantly less frequent in HBV patients than controls (p_c < 0.001 and p < 0.001, respectively). The haplotype T/G of rs243327/rs33932899 was significantly more frequent in HBV patients than resolvers (p_c < 0.001) or controls (p_c = 0.009). These data indicate that SOCS1 polymorphisms might affect the susceptibility and outcome of HBV infection.     

A novel coding-region RNA element modulates infectious dengue virus particle production in both mammalian and mosquito cells and regulates viral replication in Aedes aegypti mosquitoes

Dengue virus (DENV) is an enveloped flavivirus with a positive-sense RNA genome transmitted by Aedes mosquitoes, causing the most important arthropod-borne viral disease affecting humans. Relatively few cis-acting RNA regulatory elements have been described in the DENV coding-region. Here, by introducing silent mutations into a DENV-2 infectious clone, we identify the conserved capsid-coding region 1 (CCR1), an RNA sequence element that regulates viral replication in mammalian cells and to a greater extent in Ae. albopictus mosquito cells. These defects were confirmed in vivo, resulting in decreased replication in Ae. aegypti mosquito bodies and dissemination to the salivary glands. Furthermore, CCR1 does not regulate translation, RNA synthesis or virion retention but likely modulates assembly, as mutations resulted in the release of non-infectious viral particles from both cell types. Understanding the role of CCR1 could help characterize the poorly-defined stage of assembly in the DENV life cycle and uncover novel anti-viral targets.     

两株登革2型病毒感染BALB/C小鼠特异性抗体产生动态的比较

目的：两株登革2型病毒(Dengue Type 2 virus，DV2)初次和再次感染BALB／C小鼠建立动物感染模型，对其产生特异性抗体进行动态观察，探讨感染DV2NGC株和DV2临床分离株(B株)引起的体液免疫应答的差异。方法：两株DV2毒株模拟自然感染途径，经皮下多点注射建立BALB／C小鼠感染动物模型；采用间接ELISA法检测感染动物血浆中抗DV2特异性IgM类抗体、IgG类抗体，并同时分离病毒观察病毒血症期的变化。结果：不同DV2毒株初次、再次感染BALB／C小鼠后诱导特异性Ig产生的类别存在差异，且Dv2B株再次感染动物后表现为病毒血症期相对延长。结论：两株DV2诱导特异性抗体产生的动态与病毒血症期不同。     

Genetic, biochemical, and structural characterization of a new densovirus isolated from a chronically infected Aedes albopictus C6/36 cell line

We report the isolation, sequencing, biochemical, and structural characterization of a previously undescribed virus in a chronically infected Aedes albopictus C6/36 cell line. This virus is identified as a new densovirus under the Densovirinae subfamily of the Parvoviridae based on its biological and morphologic properties as well as sequence homologies, and is tentatively designated A. albopictus C6/36 cell densovirus (C6/36 DNV). Analysis of the 4094 nt of the C6/36 DNV genome revealed that the plus strand had three large open reading frames (ORFs): a left ORF, a right ORF, and a mid-ORF (within the left ORF), whose potential coding capacities are 91.0, 40.8, and 41.2 kDa, respectively. The left ORF likely encodes the nonstructural protein NS-1, which contains NTP-binding and helicase domains. The right ORF likely encodes structural proteins, VP1 and VP2. Our analyses revealed that C6/36 DNV has a similar genomic organization and shares very high homology in nucleotide sequence and amino acid sequences with Aedes aegypti densovirus (AaeDNV) and A. albopictus densovirus (AalDNV), members of the genus Brevidensovirus of the Densovirinae. Similar to other densoviruses, C6/36 DNV has a different genomic organization and no recognizable sequence homology with viruses in the Parvovirinae. The three-dimensional (3D) reconstruction of the C6/36 DNV at 15.6-A resolution by electron cryomicroscopy (cryoEM) revealed distinctive outer surface features not previously seen in other parvoviruses, indicating structural divergence of densoviruses, in addition to its genomic differences, while the inner surface of the C6/36 DNV capsid exhibits features that are conserved among parvoviruses.     

MASP2 gene polymorphism is associated with susceptibility to hepatitis C virus infection

Hepatitis C virus (HCV) has become a major public health issue and is prevalent in most countries. We examined several MASP2 functional polymorphisms in 104 Brazilian patients with moderate and severe chronic hepatitis C using the primers set to amplify the region encoding the first domain (CUB1), a critical region for the formation of functional mannan-binding lectin (MBL)/MBL-associated serine proteases (MASP)-2 complexes, and the fifth domain (CCP2), which is essential for C4 cleavage of the MASP2 gene. We identified five single nucleotide polymorphisms in patients and controls: p. R99Q, p. D120G, p.P126L, p.D371Y, and p.V377A. Our results show that the p.D371Y variant (c.1111 G > T) is associated with susceptibility to HCV infection (p = 0.003, odds ratio = 6.33, 95% confidence interval = 1.85-21.70). Considered as a dominant function for the T allele, this variant is associated with high plasma levels of the MASP-2 in hepatitis C patients (p < 0.001). However, further functional investigations are necessary to understand the degree of involvement between MASP2 and the HCV susceptibility.     

Euprosterna elaeasa virus genome sequence and evolution of the Tetraviridae family: Emergence of bipartite genomes and conservation of the VPg signal with the dsRNA Birnaviridae family

The Tetraviridae is a family of non-enveloped positive-stranded RNA insect viruses that is defined by the T = 4 symmetry of virions. We report the complete Euprosterna elaeasa virus (EeV) genome sequence of 5698 nt with no poly(A) tail and two overlapping open reading frames, encoding the replicase and capsid precursor, with ∼67% amino acid identity to Thosea asigna virus (TaV). The N-terminally positioned 17 kDa protein is released from the capsid precursor by a NPGP motif. EeV has 40 nm non-enveloped isometric particles composed of 58 and 7 kDa proteins. The 3′-end of TaV/EeV is predicted to form a conserved pseudoknot. Replicases of TaV and EeV include a newly delineated VPg signal mediating the protein priming of RNA synthesis in dsRNA Birnaviridae. Results of rooted phylogenetic analysis of replicase and capsid proteins are presented to implicate recombination between monopartite tetraviruses, involving autonomization of a sgRNA, in the emergence of bipartite tetraviruses. They are also used to revise the Tetraviridae taxonomy.     

深圳市首例本地登革热3型病例的病原学检测与分析

目的对2016年深圳市报告的首例本地疑似登革热病例查明病因.分离鉴定病原体,从分子水平分析分离株的生物学特征.方法对疑似患者血清标本采用ELISA、胶体金免疫层析法和荧光RT-PCR方法分别检测登革病毒抗体、NS1抗原和病毒核酸,并用C6/36细胞分离登革病毒.采用RT-PCR方法扩增病毒PrM/M-E基因后进行序列测定,并与不同国家和地区的登革毒株进行同源性比较和进化树分析.结果从患者血清标本中检测到登革病毒IgM抗体、NS1抗原和登革3型病毒核酸,并成功分离到登革3型病毒,将其命名为DENV3-SZ1648.深圳市登革3型病毒分离株SZ1648与登革3型国际标准株H87株、国内外流行株80-2、GWL-25株在PrM/M-E基因上核苷酸同源性分别为92.0％、91.8％和90.3％,而与登革1、2、4型国际标准株HAWAII、NGC、H241同源性分别为68.7％、64.2％和63.2％.进化树显示SZ1648株与2007年印度尼西亚分离株MKS-0098亲缘关系最近,在进化树的同一分支上,和85-159株(Indonedia 1985)、2167株(Tahiti 1989)、29472株(Fiji1992)等同属基因Ⅰ亚型.患者发病前1个月在深圳居住,无输血史、无外出史.结论从病原学、血清学和分子生物学特征上均证实该本地病例是由登革3型病毒引起,这也是深圳市首次报道存在本地登革3型病毒的疫情,该毒株最有可能来源于印度尼西亚.     

Characterization of biofilms formed by Candida parapsilosis, C. metapsilosis, and C. orthopsilosis

Infections due to Candida parapsilosis have been associated with the ability of this fungus to form biofilms on indwelling medical devices. Recently, C. parapsilosis isolates were reclassified into 3 genetically non-identical classes: C. parapsilosis, C. orthopsilosis, and C. metapsilosis. Little information is available regarding the ability of these newly reclassified species to form biofilms on biomedical substrates. In this study, we characterized biofilm formation by 10 clinical isolates each of C. parapsilosis, C. orthopsilosis, and C. metapsilosis. Biofilms were allowed to form on silicone elastomer discs to early (6 h) or mature (48 h) phases and quantified by tetrazolium (XTT) and dry weight assays. Surface topography and three-dimensional architecture of the biofilms were visualized using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM), respectively. Metabolic activity assay revealed strain-dependent biofilm forming ability of the 3 species tested, while biomass determination revealed that all 3 species formed equivalent biofilms (P>0.05 for all comparisons). SEM analyses of representative isolates of these species showed biofilms with clusters of yeast cells adherent to the catheter surface. Additionally, confocal microscopy analyses showed the presence of cells embedded in biofilms ranging in thickness between 62 and 85 μm. These results demonstrate that similar to C. parapsilosis, the 2 newly identified Candida species (C. orthopsilosis and C. metapsilosis) were able to form biofilms.     

Functional characterization of ex vivo blood myeloid and plasmacytoid dendritic cells after infection with dengue virus

Myeloid and plasmacytoid dendritic cells (mDC and pDC) are naturally distinctive subsets. We exposed both subsets to dengue virus (DV) in vitro and investigated their functional characteristics. High levels of DV replication in mDC were found to correlate with DC-SIGN expression. Production of inflammatory cytokines by mDC increased gradually after DV-infection, which was dependent on DV replication. Co-stimulatory markers were upregulated on mDC upon DV-infection. On the contrary, lower levels of DV-replication were observed in pDC, but the cytokine production in pDC was quicker and stronger. This cytokine response was not dependent on viral replication, but dependent on cell endosomal activity and TLR7, and could be also induced by purified DV genome RNA. These results clearly suggested functional differences between mDC and pDC in response to DV infection. Additionally, the TLR7-mediated recognition of DV RNA may be involved in pDC functional activation.     

Chemokines expression during Leptospira interrogans serovar Copenhageni infection in resistant BALB/c and susceptible C3H/HeJ mice

The role of innate immune responses in protection against leptospirosis remains unclear. We examined the expression of the chemokines CCL2/JE (MCP-1), CCL3/MIP-1α (MIP-1α) and CXCL1/KC (IL-8) regarding resistance and susceptibility to leptospirosis in experimental mice models BALB/c and C3H/HeJ, respectively. A virulent strain of Leptospira interrogans serovar Copenhageni was used in this study. Twenty-five animals of each mouse strain of C3H/HeJ and BALB/c, were infected intraperitoneally with 10⁶ cells. Five un-infected animals of each strain were kept as control. Mortality of C3H/HeJ mouse was observed while BALB/c mice were asymptomatic. The presence of leptospire DNA in tissues of infected animals was demonstrated by PCR. Chemokines were measured in serum, spleen, liver, kidney and lung of both strains of animals using immunoenzymatic assay (ELISA). Elevations in the levels of chemokines MCP-1 and IL-8 occurred in all organs and sera of C3H/HeJ and BALB/c infected mice. The levels of MIP-1α were lower when compared to MCP-1 and IL-8 in all analyzed organs, with a slight increase in liver and kidney. Our results indicate that the expression of inflammatory mediators can vary greatly, depending on the tissue and mouse strains. It is possible that the resistance to Leptospira can be partially correlated to the increase of MIP-1α observed in BALB/c mice, while an increasing and a sustained expression of MCP-1 and IL-8 in the lungs of C3H/HeJ mice can be correlated to the severity and progression of leptospirosis.     

Clinical and genetic analysis of MAPT, GRN, and C9orf72 genes in Korean patients with frontotemporal dementia

The hexanucleotide repeat expansion (GGGGCC) in chromosome 9 open-reading frame 72 (C9orf72) and mutations in the microtubule-associated protein tau (MAPT) and progranulin (GRN) genes are known to be associated with the main causes of familial or sporadic amyotrophic lateral sclerosis and frontotemporal dementia (FTD) in Western populations. These genetic abnormalities have rarely been studied in Asian FTD populations. We investigated the frequencies of mutations in MAPT and GRN and the C9orf72 abnormal expansion in 75 Korean FTD patients. Two novel missense variants of unknown significance in the MAPT and GRN were detected in each gene. However, neither abnormal C9orf72 expansion nor pathogenic MAPT or GRN mutation was found. Our findings indicate that MAPT, GRN, and C9orf72 mutations are rare causes of FTD in Korean patients.     

Isolation and characterization of Solenopsis invicta virus 3, a new positive-strand RNA virus infecting the red imported fire ant, Solenopsis invicta

We report the discovery of a new virus from the red imported fire ant, Solenopsis invicta. Solenopsis invicta virus 3 (SINV-3) represents the third virus discovered from this ant species using the metagenomics approach. The single (positive)-strand RNA, monopartite, bicistronic genome of SINV-3 was sequenced in entirety (GenBank accession number FJ528584), comprised of 10,386 nucleotides, and polyadenylated at the 3′ terminus. This genome size was confirmed by Northern analysis. The genome revealed 2 large open reading frames (ORFs) in the sense orientation with an untranslated region (UTR) at each end and between the two ORFs. The 5′ proximal ORF (ORF 1) encoded a predicted protein of 299.1 kDa (2580 amino acids). The 3′ proximal ORF (ORF 2) encoded a predicted protein of 73.2 kDa (651 amino acids). RNA-dependent RNA polymerase (RdRp), helicase, and protease domains were recognized in ORF 1. SDS-PAGE separation of purified SINV-3 particles yielded 2 bands (ostensibly capsid proteins) with a combined molecular mass of 77.3 kDa which was similar to the mass predicted by ORF 2 (73.2 kDa). Phylogenetic analysis of the conserved amino acid sequences containing domains I to VIII of the RdRp from dicistroviruses, iflaviruses, plant small RNA viruses, picornaviruses, and 4 unassigned positive-strand RNA viruses revealed a trichotomous phenogram with SINV-3 and Kelp fly virus comprising a unique cluster. Electron microscopic examination of negatively stained samples of SINV-3 revealed isometric particles with apparent projections and a diameter of 27.3 ± 1.3 nm. SINV-3 was successfully transmitted to uninfected workers by feeding. The minus (replicative) strand of SINV-3 was detected in worker ants indicating replication of the virus. The possibility of using SINV-3 as a microbial control agent for fire ants is discussed.     

Wolbachia endosymbionts and human disease control

Most human filarial nematode parasites and arthropods are hosts for a bacterial endosymbiont, Wolbachia. In filaria, Wolbachia are required for normal development, fertility and survival, whereas in arthropods, they are largely parasitic and can influence development and reproduction, but are generally not required for host survival. Due to their obligate nature in filarial parasites, Wolbachia have been a target for drug discovery initiatives using several approaches including diversity and focused library screening and genomic sequence analysis. In vitro and in vivo anti-Wolbachia antibiotic treatments have been shown to have adulticidal activity, a long sought goal of filarial parasite drug discovery. In mosquitoes, it has been shown that the presence of Wolbachia can inhibit the transmission of certain viruses, such as Dengue, Chikungunya, Yellow Fever, West Nile, as well as the infectivity of the malaria-causing protozoan, Plasmodium and filarial nematodes. Furthermore, Wolbachia can cause a form of conditional sterility that can be used to suppress populations of mosquitoes and additional medically important insects. Thus Wolbachia, a pandemic endosymbiont offers great potential for elimination of a wide-variety of devastating human diseases.     

Lack of evidence for an interaction between Buchnera GroEL and Banana bunchy top virus (Nanoviridae)

Circulative plant viruses such as luteovirids and geminiviruses have been shown to bind to GroEL proteins produced by endosymbiotic bacteria harboured within hemipteran vectors. These interactions seem to prevent the degradation of the viral particles in the aphid's haemocoel. Similarly to luteovirids and geminiviruses, Banana bunchy top virus (BBTV), a member of the Nanoviridae family, is transmitted in a persistent, circulative manner and can be detected in the haemolymph of the aphid vector, Pentalonia nigronervosa. To date, it is not known if BBTV can interact with GroEL. In this study, we localised and inferred the phylogeny of a Buchnera aphidicola endosymbiont inhabiting P. nigronervosa. Furthermore, we predicted the 3D structure of Buchnera GroEL and detected the protein in the haemolymph of P. nigronervosa. Interactions were tested using 3 different assays: immunocapture PCR, dot blot, and far-western blot assays; however, none of them showed evidence of a BBTV–GroEL interaction. We concluded that it was unlikely that BBTV interacted with Buchnera GroEL either in vitro or in vivo and we discuss possible alternatives by which BBTV viral particles are able to avoid the process of degradation in the aphid haemocoel.     

Mutations in UBQLN2 and SIGMAR1 genes are rare in Korean patients with amyotrophic lateral sclerosis

Mutations in the UBQLN2 and SIGMAR1 genes were recently identified in X-linked dominant amyotrophic lateral sclerosis and/or frontotemporal dementia (ALS and/or FTD) and FTD and/or motor neuron disease, respectively. Subsequent studies, however, found that UBQLN2 mutations were rare, and the pathogenicity of SIGMAR1 mutation in FTD and/or motor neuron disease was controversial. In the present study, we analyzed mutations in the UBQLN2 and SIGMAR1 genes in a Korean cohort of 258 patients with familial ALS (n = 9) or sporadic (sALS; n = 258) ALS. One novel UBQLN2 variant (p.D314E) was observed in 2 patients with sALS and 5 of 727 controls indicating that this variant might be a rare polymorphism rather than a disease-causing mutation. A novel SIGMAR1 gene variant in the 3′-untranslated region (c.*58T>C) was found in 1 sALS and was absent in 727 control samples. Taken together, our data suggest that causative mutations in the UBQLN2 and SIGMAR1 genes are rare in Korean patients with either familial or sporadic ALS.     

Mutational analysis of GIGYF2, ATP13A2 and GBA genes in Brazilian patients with early-onset Parkinson's disease

In the last decade, several genes have been linked to Parkinson's disease (PD), including GIGYF2, ATP13A2 and GBA. To explore whether mutations in these genes contribute to development of PD in the Brazilian population, we screened 110 patients with early-onset PD. No clearly pathogenic mutations were identified in ATP13A2 and GIGYF2. In contrast, we identified a significantly higher frequency of known pathogenic mutations in GBA gene among the PD cases (6/110 = 5.4%) when compared to the control group (0/155) (P = 0.0047). Our results strongly support an association between GBA gene mutations and an increased risk of PD. Mutations in GIGYF2 and ATP13A2 do not seem to represent a risk factor to the development of PD in the Brazilian population. Considering the scarcity of studies on GIGYF2, ATP13A2 and GBA mutation frequency in Latin American countries, we present significant data about the contribution of these genes to PD susceptibility.