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Rabies virus (RABV) is the causative agent of rabies, a lethal neurological disease in mammals. RABV strains can be classified into fixed strains (laboratory strains) and street strains (field/clinical strains), which have different properties including cell tropism and neuroinvasiveness. RABV Toyohashi strain is a street strain isolated in Japan from an imported case which had been bitten by rabid dog in the Philippines. In order to facilitate molecular studies of RABV, we established a reverse genetics (RG) system for the study of the Toyohashi strain. The recombinant virus was obtained from a cDNA clone of Toyohashi strain and exhibited similar growth efficiency as the original virus in cultured cell lines. Both the original and recombinant strains showed similar pathogenicity with high neuroinvasiveness in mice, and the infected mice developed a long and inconsistent incubation period, which is characteristic of street strains. We also generated a recombinant Toyohashi strain expressing viral phosphoprotein (P protein) fused with the fluorescent protein mCherry, and tracked the intracellular dynamics of the viral P protein using live-cell imaging. The presented reverse genetics system for Toyohashi strain will be a useful tool to explore the fundamental molecular mechanisms of the replication of RABV street strains. [ABSTRACT FROM AUTHOR]

Emerging and reemerging tick-borne orthonairovirus infections (family Nairoviridae), which are genetically distinct from Crimean-Congo hemorrhagic fever virus, have been recently reported in East Asia. Here, we have established a mouse infection model using type-I/II interferon receptor-knockout mice (AG129) both for a better understanding the pathogenesis of these infections and validation of antiviral agents using Yezo virus (YEZV), a novel virus causing febrile illnesses associated with tick bites in Japan and China. YEZV-inoculated AG129 mice developed hepatitis with body weight loss and died by 6 days post infection. Blood biochemistry tests showed elevated liver enzyme levels, similar to YEZV-infected human patients. AG129 mice treated with favipiravir survived lethal YEZV infection, demonstrating the anti-YEZV effect of this drug. The present mouse model will help us better understand the pathogenicity of the emerging tick-borne orthonairoviruses and the development of specific antiviral agents for their treatment. Author summary: Establishment of animal models are pivotal in the study of newly emerging infectious diseases. In this report, we show that mice lacking interferon responses can serve as a lethal animal model for emerging tick-borne orthonairovirus infections. Yezo virus is a tick-borne orthonairovirus that is genetically distinct from previously reported human pathogenic orthonairoviruses such as Crimean-Congo Hemorrhagic fever virus, and has recently been reported to cause acute febrile illnesses in humans in Japan and China. Type-I/II interferon receptor-knockout mice (AG129 mice) infected with Yezo virus developed acute disease with elevated liver enzyme levels, including AST and ALT, similar to human patients. Our pathological and virological analyses in this mouse model revealed that Yezo virus mainly replicates in the liver and spleen and causes fatal hepatitis in AG129 mice. Furthermore, we could demonstrate the antiviral effect of favipiravir for Yezo virus infection. Thus, this animal model will facilitate studies of emerging tick-borne orthonairovirus infections which will allow a better understanding of disease pathogenesis and will permit the screening of potential antiviral agents. [ABSTRACT FROM AUTHOR]

The most conserved fusion loop (FL) domain present in the flavivirus envelope protein has been reported as a dominant epitope for cross-reactive antibodies to mosquito-borne flaviviruses (MBFVs). As a result, establishing accurate serodiagnosis for MBFV infections has been difficult as anti-FL antibodies are induced by both natural infection and following vaccination. In this study, we modified the most conserved FL domain to overcome this cross-reactivity. We showed that the FL domain of lineage I insect-specific flavivirus (ISFV) has differences in antigenicity from those of MBFVs and lineage II ISFV and determined the key amino acid residues (G106, L107, or F108), which contribute to the antigenic difference. These mutations were subsequently introduced into subviral particles (SVPs) of dengue virus type 2 (DENV2), Zika virus (ZIKV), Japanese encephalitis virus (JEV), and West Nile virus (WNV). In indirect enzyme-linked immunosorbent assays (ELISAs), these SVP mutants when used as antigens reduced the binding of cross-reactive IgG and total Ig induced by infection of ZIKV, JEV, and WNV in mice and enabled the sensitive detection of virus-specific antibodies. Furthermore, immunization of ZIKV or JEV SVP mutants provoked the production of antibodies with lower cross-reactivity to heterologous MBFV antigens compared to immunization with the wild-type SVPs in mice. This study highlights the effectiveness of introducing mutations in the FL domain in MBFV SVPs with lineage I ISFV-derived amino acids to produce SVP antigens with low cross-reactivity and demonstrates an improvement in the accuracy of indirect ELISA-based serodiagnosis for MBFV infections. Key points: • The FL domain of Lineage I ISFV has a different antigenicity from that of MBFVs. • Mutated SVPs reduce the binding of cross-reactive antibodies in indirect ELISAs. • Inoculation of mutated SVPs induces antibodies with low cross-reactivity. [ABSTRACT FROM AUTHOR]

Mosquitoes interact with various organisms in the environment, and female mosquitoes in particular serve as vectors that directly transmit a number of microorganisms to humans and animals by blood-sucking. Comprehensive analysis of mosquito-borne viruses has led to the understanding of the existence of diverse viral species and to the identification of zoonotic arboviruses responsible for significant outbreaks and epidemics. In the present study on mosquito-borne bunyaviruses we employed a broad-spectrum RT-PCR approach and identified eighteen different additional species in the Phenuiviridae family and also a number of related but unclassified bunyaviruses in mosquitoes collected in Zambia. The entire RNA genome segments of the newly identified viruses were further analyzed by RNA sequencing with a ribonuclease R (RNase R) treatment to reduce host-derived RNAs and enrich viral RNAs, taking advantage of the dsRNA panhandle structure of the bunyavirus genome. All three or four genome segments were identified in eight bunyavirus species. Furthermore, L segments of three different novel viruses related to the Leishbunyaviridae were found in mosquitoes together with genes from the suspected host, the Crithidia parasite. In summary, our virus detection approach using a combination of broad-spectrum RT-PCR and RNA sequencing analysis with a simple virus enrichment method allowed the discovery of novel bunyaviruses. The diversity of bunyaviruses is still expanding and studies on this will allow a better understanding of the ecology of hematophagous mosquitoes. [ABSTRACT FROM AUTHOR]

Mosquitoes interact with various organisms in the environment, and female mosquitoes in particular serve as vectors that directly transmit a number of microorganisms to humans and animals by blood-sucking. Comprehensive analysis of mosquito-borne viruses has led to the understanding of the existence of diverse viral species and to the identification of zoonotic arboviruses responsible for significant outbreaks and epidemics. In the present study on mosquito-borne bunyaviruses we employed a broad-spectrum RT-PCR approach and identified eighteen different additional species in the Phenuiviridae family and also a number of related but unclassified bunyaviruses in mosquitoes collected in Zambia. The entire RNA genome segments of the newly identified viruses were further analyzed by RNA sequencing with a ribonuclease R (RNase R) treatment to reduce host-derived RNAs and enrich viral RNAs, taking advantage of the dsRNA panhandle structure of the bunyavirus genome. All three or four genome segments were identified in eight bunyavirus species. Furthermore, L segments of three different novel viruses related to the Leishbunyaviridae were found in mosquitoes together with genes from the suspected host, the Crithidia parasite. In summary, our virus detection approach using a combination of broad-spectrum RT-PCR and RNA sequencing analysis with a simple virus enrichment method allowed the discovery of novel bunyaviruses. The diversity of bunyaviruses is still expanding and studies on this will allow a better understanding of the ecology of hematophagous mosquitoes. [ABSTRACT FROM AUTHOR]

A novel multiplex loop-mediated isothermal amplification (LAMP) method combined with DNA chromatography was developed for the simultaneous detection of three important respiratory disease-causing viruses: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus. Amplification was performed at a constant temperature, and a positive result was confirmed by a visible colored band. An in-house drying protocol with trehalose was used to prepare the dried format multiplex LAMP test. Using this dried multiplex LAMP test, the analytical sensitivity was determined to be 100 copies for each viral target and 100–1000 copies for the simultaneous detection of mixed targets. The multiplex LAMP system was validated using clinical COVID-19 specimens and compared with the real-time qRT-PCR method as a reference test. The determined sensitivity of the multiplex LAMP system for SARS-CoV-2 was 71% (95% CI: 0.62–0.79) for cycle threshold (Ct) ≤ 35 samples and 61% (95% CI: 0.53–0.69) for Ct ≤40 samples. The specificity was 99% (95%CI: 0.92–1.00) for Ct ≤35 samples and 100% (95%CI: 0.92–1.00) for the Ct ≤40 samples. The developed simple, rapid, low-cost, and laboratory-free multiplex LAMP system for the two major important respiratory viral diseases, COVID-19 and influenza, is a promising field-deployable diagnosis tool for the possible future 'twindemic, ' especially in resource-limited settings. [ABSTRACT FROM AUTHOR]

In parallel with vaccination, oral antiviral agents are highly anticipated to act as countermeasures for the treatment of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Oral antiviral medication demands not only high antiviral activity but also target specificity, favorable oral bioavailability, and high metabolic stability. Although a large number of compounds have been identified as potential inhibitors of SARS-CoV-2 infection in vitro, few have proven to be effective in vivo. Here, we show that oral administration of S-217622 (ensitrelvir), an inhibitor of SARS-CoV-2 main protease (Mpro; also known as 3C-like protease), decreases viral load and ameliorates disease severity in SARS-CoV-2–infected hamsters. S-217622 inhibited viral proliferation at low nanomolar to submicromolar concentrations in cells. Oral administration of S-217622 demonstrated favorable pharmacokinetic properties and accelerated recovery from acute SARS-CoV-2 infection in hamster recipients. Moreover, S-217622 exerted antiviral activity against SARS-CoV-2 variants of concern, including the highly pathogenic Delta variant and the recently emerged Omicron BA.5 and BA.2.75 variants. Overall, our study provides evidence that S-217622, an antiviral agent that is under evaluation in a phase 3 clinical trial (clinical trial registration no. jRCT2031210350), has remarkable antiviral potency and efficacy against SARS-CoV-2 and is a prospective oral therapeutic option for COVID-19. A worthwhile antiviral: Despite the success of vaccines and early antivirals in combating the ongoing SARS-CoV-2 pandemic, there remains room for improvement. Here, Sasaki et al. tested the ability of a main protease (Mpro) inhibitor, S-217622 (ensitrelvir), to protect hamsters from disease after SARS-CoV-2 infection. The authors found that S-217622 retained activity against the Mpro of multiple strains of the virus and reduced disease severity in hamsters when administered orally as a prophylactic or as a therapeutic. Hamsters treated prophylactically with S-217622 did not spread virus to cohoused hamsters. These results support further development of S-217622, which is now under evaluation in a phase 3 clinical trial. [ABSTRACT FROM AUTHOR]

Nucleic acid test (NAT), most typically quantitative PCR, is one of the standard methods for species specific flavivirus diagnosis. Semi-comprehensive NATs such as pan-flavivirus PCR which covers genus Flavivirus are also available; however, further specification by sequencing is required for species level differentiation. In this study, a semi-comprehensive detection system that allows species differentiation of flaviviruses was developed by integration of the pan-flavivirus PCR and Nanopore sequencing. In addition, a multiplexing method was established by adding index sequences through the PCR with a streamlined bioinformatics pipeline. This enables defining cut-off values for observed read counts. In the laboratory setting, this approach allowed the detection of up to nine different flaviviruses. Using clinical samples collected in Vietnam and Brazil, seven different flaviviruses were also detected. When compared to a commercial NAT, the sensitivity and specificity of our system were 66.7% and 95.4%, respectively. Conversely, when compared to our system, the sensitivity and specificity of the commercial NAT were 57.1% and 96.9%, respectively. In addition, Nanopore sequencing detected more positive samples (n = 8) compared to the commercial NAT (n = 6). Collectively, our study has established a semi-comprehensive sequencing-based diagnostic system for the detection of flaviviruses at extremely affordable costs, considerable sensitivity, and only requires simple experimental methods. [ABSTRACT FROM AUTHOR]

Although the spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has resulted in a worldwide pandemic, there are currently no virus-specific drugs that are fully effective against SARS-CoV-2. Only a limited number of human-derived cells are capable of supporting SARS-CoV-2 replication and the infectivity of SARS-CoV-2 in these cells remains poor. In contrast, monkey-derived Vero cells are highly susceptibility to infection with SARS-CoV-2, although they are not suitable for the study of antiviral effects by small molecules due to their limited capacity to metabolize drugs compared to human-derived cells. In this study, our goal was to generate a virus-susceptible human cell line that would be useful for the identification and testing of candidate drugs. Towards this end, we stably transfected human lung-derived MRC5 cells with a lentiviral vector encoding angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV-2. Our results revealed that SARS-CoV-2 replicates efficiently in MRC5/ACE2 cells. Furthermore, viral RNA replication and progeny virus production were significantly reduced in response to administration of the replication inhibitor, remdesivir, in MRC5/ACE2 cells compared with Vero cells. We conclude that the MRC5/ACE2 cells will be important in developing specific anti-viral therapeutics and will assist in vaccine development to combat SARS-CoV-2 infections. [ABSTRACT FROM AUTHOR]

Tick-borne flaviviruses (TBFVs) infect mammalian hosts through tick bites and can cause various serious illnesses, such as encephalitis and hemorrhagic fevers, both in humans and animals. Despite their importance to public health, there is limited epidemiological information on TBFV infection in Africa. Herein, we report that a novel flavivirus, Mpulungu flavivirus (MPFV), was discovered in a Rhipicephalus muhsamae tick in Zambia. MPFV was found to be genetically related to Ngoye virus detected in ticks in Senegal, and these viruses formed a unique lineage in the genus Flavivirus. Analyses of dinucleotide contents of flaviviruses indicated that MPFV was similar to those of other TBFVs with a typical vertebrate genome signature, suggesting that MPFV may infect vertebrate hosts. Bioinformatic analyses of the secondary structures in the 3′-untranslated regions (UTRs) revealed that MPFV exhibited unique exoribonuclease-resistant RNA (xrRNA) structures. Utilizing biochemical approaches, we clarified that two xrRNA structures of MPFV in the 3′-UTR could prevent exoribonuclease activity. In summary, our findings provide new information regarding the geographical distribution of TBFV and xrRNA structures in the 3′-UTR of flaviviruses. [ABSTRACT FROM AUTHOR]

The spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains. Author summary: SARS-CoV-2 uses its spike (S) protein to enter target cells. Unlike other similar coronaviruses, the nascent S protein has a polybasic cleavage motif and is cleaved by the host protease. We have identified SARS-CoV-2 variants with mutations at the cleavage motif of S protein (S gene mutants) which undergo inefficient proteolytic cleavage, generate smaller plaques, and infect fewer cell lines. Notably, S gene mutants emerged rapidly through SARS-CoV-2 propagation in Vero cells. Since Vero cells are commonly used for SARS-CoV-2 propagation, it is a very real possibility that researchers have performed experiments, screened antivirals, and developed vaccines using SARS-CoV-2 S gene mutants without realizing. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) causes persistent outbreaks in endemic and non‐endemic regions in Zambia. However, the epidemiology of the disease is poorly understood, particularly during the inter‐epidemic periods. We conducted surveillance for ASF in asymptomatic domestic pigs and soft ticks in selected Zambian provinces. While serum samples (n = 1,134) were collected from crossbred pigs from all study sites between 2014 and 2017, whole blood (n = 300) was collected from both crossbred and indigenous pigs in Eastern Province (EP) in 2017. Soft ticks were collected from Mosi‐oa‐Tunya National Park in Southern Province (SP) in 2019. Sera were screened for antibodies against ASF by ELISA while genome detection in whole blood and soft ticks was conducted by PCR. Ticks were identified morphologically and by phylogenetic analysis of the 16S rRNA gene. Seroprevalence was highest in EP (50.9%, 95% CI [47.0–54.9]) compared to significantly lower rates in SP (2.9%, 95% CI [1.6–5.1]). No antibodies to ASFV were detected in Lusaka Province. In EP, the prevalence of ASFV genome was 11.7% (35/300), significantly higher (OR = 6.2, 95% CI [2.4–16.6]) in indigenous pigs compared to crossbred pigs. The pooled prevalence of ASFV genome in ticks was 11.0%, 95% CI [8.5–13.9]. Free‐range husbandry system was the only factor that was significantly associated with seropositive (p <.0001, OR = 39.3) and PCR‐positive results (p <.001, OR = 5.7). Phylogenetically, based on the p72 gene, ASFV from Ornithodoros moubata ticks detected in this study belonged to genotype I, but they separated into two distinct clusters. Besides confirming ASF endemicity in EP and the presence of ASFV‐infected ticks in SP, these results provide evidence for exposure of domestic pigs to ASFV in non‐endemic regions during the inter‐epidemic period. [ABSTRACT FROM AUTHOR]

Background: The objectives of this study were to investigate the relationships between polymorphisms at the interferon lambda (IFNL) locus and CD4+:CD8+ ratio normalisation in people living with HIV (PLWH) on effective antiretroviral therapy (ART); and to examine whether these polymorphisms influence the composition of T lymphocyte compartments in long-term treated HIV-1 infection. Methods: A cross-sectional study in PLWH enrolled into the Mater Immunology study. We performed IFNL genotyping on stored samples and evaluated the association of IFNL single-nucleotide polymorphisms (rs368234815 and rs12979860) with CD4+:CD8+ ratio normalization (> 1) and expanded CD4+ and CD8+ T-cell subsets; CD45RO+CD62L+ (central-memory), CD45RO+ CD62L−(effector-memory) and CD45RO−CD62L+ (naïve), using logistic and linear regression models, respectively. Results: 190 ambulatory PLWH recruited to the main study, 143 were included in the analysis (38 had no stored DNA and 9 no T-lymphocyte subpopulation). Of 143 included, the median age (IQR) was 45(39–48) years, 64% were male and 66% were of Caucasian ethnicity. Heterosexual-contact (36%), injecting drug-use (33%) and men who have sex with men (24%) were the most presented HIV-transmission risk groups. The majority of subjects (90.2%) were on ART with 79% of the cohort having an undetectable HIV-RNA (< 40 copies/ml) and the time since ART initiation was 7.5 (3.7–10.4) year. rs368234815 and rs12979860 displayed similar allelic frequencies, with minor alleles ΔG and T representing 39% and 42%, respectively, of circulating alleles. rs368234815 ΔG/ΔG minor homozygotes were significantly associated with increased odds for attaining a normalised CD4+:CD8+ ratio compared to rs368234815 T/T major homozygotes in PLWH virologically suppressed on effective ART (OR = 3.11; 95% CI [1.01:9.56]). rs368234815 ΔG/ΔG homozygosity was also significantly associated with lower levels of CD4+ effector memory T-cells (regression coefficient: − 7.1%, p = 0.04) and CD8+ naïve T-cell subsets were significantly higher in HIV-1 mono-infected PLWH with rs368234815 ΔG/ΔG (regression coefficient: + 7.2%, p = 0.04). Conclusions: In virally-suppressed, long-term ART-treated PLWH, rs368234815 ΔG/ΔG homozygotes were more likely to have attained normalisation of their CD4+:CD8+ ratio, displayed lower CD4+ effector memory and higher naive CD8+ T-cells. Further studies are needed to replicate our findings in other, larger and more diverse cohorts and to determine the impact of IFNL genetic-variation on CD4+:CD8+ ratio normalisation and clinical outcomes in PLWH. [ABSTRACT FROM AUTHOR]

Dropped head syndrome (DHS) has been rarely observed in amyotrophic lateral sclerosis (ALS), and the neuropathological findings of this condition have almost never been described. The identification of transactivation response DNA‐binding protein 43 kDa (TDP‐43), which binds to RNA/DNA has provided a new method for studying ALS and frontotemporal lobar degeneration (FTLD). Post‐mortem examination of an adult sudden death case of a 71‐year‐old patient who complained of DHS exhibited severe loss of anterior motor neurons in the cervical cord (C4‐6). Loss of nerve fibers of the anterior roots was striking compared with posterior roots, together with marked neurogenic atrophy of posterior muscles semispinalis cervicis. Bunina bodies were found in large neurons of Betz giant cells, but not in the motor neurons of spinal cords, or neurons of bulbar regions. Phosphorylated TDP‐43 (p‐TDP‐43)‐positive structures were detected in the residual neurons of the cervical, thoracic and lumber cords, hypoglossal nucleus, cerebellar dentate nucleus and parahippocampal cortex, together with ubiquitin‐positive inclusions. Phosphorylated Tau positive structures in neuronal cytoplasm were found in the amygdala, entorhinal cortex and parahippocampal cortex, some of which co‐expressed p‐TDP‐43. The medial zone of cervical cords may be the first onset site, and that is the cause of head drop in the early stage of ALS. In spite of detailed examination, the direct cause of sudden death was not verified. This autopsy report revealed the relation of DHS which is a rare clinical manifestation of ALS, and neuropathological findings. [ABSTRACT FROM AUTHOR]

Japanese encephalitis virus (JEV) exerts a profound burden of viral encephalitis. We have investigated the differentially expressed transcripts in the neuronal transcriptome during JEV infection by RNA sequencing (RNA-Seq) of virus-infected SH-SY5Y human neuroblastoma cells. Gene ontology analysis revealed significant enrichment from two main pathways: endoplasmic reticulum (ER)-nucleus signaling (P value: 5.75E−18; false discovery rate [FDR] 3.11E−15) and the ER unfolded protein response (P value: 7.58E−18; FDR 3.11E−15). qPCR validation showed significant upregulation and differential expression (P < 0.01) of ER stress-signaling transcripts (SESN2, TRIB3, DDIT3, DDIT4, XBP1, and ATF4) at 24 h post-infection for both low (LN) and high (HN) neurovirulence JEV strains. Immunoblot analysis following JEV infection of SH-SY5Y cells showed an increase in levels of SESN2 protein following JEV infection. Similarly, Zika virus (MR766) infection of SH-SY5Y showed a titer-dependent increase in ER stress-signaling transcripts; however, this was absent or diminished for DDIT4 and ATF4, respectively, suggestive of differences in the induction of stress-response transcripts between flaviviruses. Interestingly, SLC7A11 and SLC3A2 mRNA were also both deregulated in JEV-infected SH-SY5Y cells and encode the two constituent subunits of the plasma membrane xCT amino acid antiporter that relieves oxidative stress by export of glutamate and import of cystine. Infection of SH-SY5Y and HEK293T cells by the JEV HN strain Sw/Mie/40/2004 lead to significant upregulation of the SLC7A11 mRNA to levels comparable to DDIT3. Our findings suggest upregulation of antioxidants including SESN2 and, also, the xCT antiporter occurs to counteract the oxidative stress elicited by JEV infection. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) circulation occurs between non-human primates (NHPs) in a sylvatic transmission cycle. To investigate evidence of flavivirus infection in NHPs in Zambia, we performed a plaque reduction neutralization test (PRNT) to quantify neutralizing antibodies. PRNT revealed that sera from NHPs (African green monkeys and baboons) exhibited neutralizing activity against ZIKV (34.4%; 33/96), whereas a PRNT for yellow fever virus using NHP sera showed no neutralization activity. ZIKV genomic RNA was not detected in splenic tissues from NHPs, suggesting that the presence of anti-ZIKV neutralizing antibodies represented resolved infections. Our evidence suggests that ZIKV is maintained in NHP reservoirs in Zambia. [ABSTRACT FROM AUTHOR]

Detection and sequencing of chikungunya virus (CHIKV) genome was performed using a combination of a modified reverse transcription loop-mediated isothermal amplification (RT-LAMP) method and a MinION sequencer. We developed the protocol for drying all the reagents for the RT-LAMP in a single reaction tube. Using this system, the CHIKV genome was effectively amplified under isothermal conditions, and used as a template for MinION sequencing with a laptop computer. Our in-house RT-LAMP method and MinION sequencing system were also validated with RNAs and serum samples from recent outbreaks of CHIKV patients in Brazil. The obtained sequence data confirmed the CHIKV outbreaks and identified the genotype. In summary, our established inexpensive on-site genome detection and sequencing system is applicable for both diagnosis of CHIKV infected patients and genotyping of the CHIKV virus in future outbreak in remote areas. [ABSTRACT FROM AUTHOR]

A 24 year‐old female presented with a mass lesion in the right temporal lobe. This case was difficult to diagnose using histological and immunological methods and therefore molecular analyses were applied to provide a definitive diagnosis. The tumor was well‐demarcated, partially cystic, and irregularly‐enhanced on gadolinium‐enhanced T1‐weighted magnetic resonance images. Pathologically, a large part of the tumor consisted of cells with fine cytoplasmic processes on a myxoid and mucinous background. Cells formed a microcystic structure around the mucinous tissue. Numerous eosinophilic granular bodies, but not Rosenthal fibers, were present. The solid and compact regions of the tumor were composed of fasciculation of dense fibrous glial tissues and occasional multinucleated giant cells. Tumor cells and their fragmented cytoplasmic processes were positively stained with GFAP, while eosinophilic granular bodies were both positive and negative. Xanthomatous changes were not detected and the reticulin fibers were restricted to vascular tissues. The MIB1 index was scored as approximately 10%. In molecular analyses of BRAF, the KIAA1549‐BRAF (K16‐B9) fusion gene was detected in all tumor regions, whereas BRAF V600E mutation was not detected by either conventional Sanger sequencing or the Eprobe‐PCR method. Based on the results of the molecular analyses, this case was diagnosed as pilocytic astrocytoma. [ABSTRACT FROM AUTHOR]

Rabies virus (RABV) is the causative agent of fatal neurological disease. Cellular attachment is the initial and essential step for viral infections. Although extensive studies have demonstrated that RABV uses various target cell molecules to mediate infection, no specific molecule has been identified as an attachment factor for RABV infection. Here we demonstrate that cellular heparan sulfate (HS) supports RABV adhesion and subsequent entry into target cells. Enzymatic removal of HS reduced cellular susceptibility to RABV infection, and heparin, a highly sulfated form of HS, blocked viral adhesion and infection. The direct binding between RABV glycoprotein and heparin was demonstrated, and this interaction was shown to require HS N- and 6-O-sulfation. We also revealed that basic amino acids in the ectodomain of RABV glycoprotein serve as major determinants for the RABV-HS interaction. Collectively, our study highlights a previously undescribed role of HS as an attachment factor for RABV infection. [ABSTRACT FROM AUTHOR]

Globally, hepatitis C virus (HCV) is one of the major causes of hepatocellular carcinoma and liver cirrhosis. For clinical decision making, genetic variation in the interferon-λ ( IFNL) cluster has been utilised as a baseline predictor of natural and interferon-based treatment-induced viral clearance. In Vietnam, where HCV genotypes 1 (g1) and g6 predominate, no prior studies have been conducted investigating associations of IFNL3/4 polymorphisms with spontaneous clearance (SC) or HCV viral load (VL) in chronic infection. In this study, we have investigated the host genetic variations in IFNL loci to determine the association of IFNL3/4 polymorphisms with HCV SC and baseline VLs in a Vietnamese HCV-seropositive cohort. The majority of the cohort harboured major homozygous polymorphisms in IFNL3/4 cluster (i.e. rs12979860-CC: 82.7%; rs8099917-TT: 84.8% and rs368234815-TT/TT: 85.5%) and the SC rates in these groups were 15.8%, 16.3% and 15.7%, respectively. In the minor allele groups, the resolution rates were lower (12% in rs12979860 non-CC, 9.1% in rs8099917 non-TT and 9.5% in rs368234815 non-TT/TT). Furthermore, in individuals harbouring minor alleles, females achieved higher SC rates than males. HCV g6-infected rs12979860 major homozygous individuals had significantly higher viral loads than individuals with minor alleles (CC: 6.56 log IU/ml vs. non-CC: 5.66 log IU/ml; P = 0.021). The association between IFNL3/4 genotypes with elevated HCV VL observed in HCV g6-infected individuals may have implications for the progression of liver disease in Southeast Asian countries where this viral genotype predominates and therefore warrants further studies. [ABSTRACT FROM AUTHOR]

Enteroviruses (EVs) are associated with a broad spectrum of clinical presentation, including aseptic meningitis (AM), encephalitis, hand, foot and mouth disease, acute flaccid paralysis, and acute flaccid myelitis. Epidemics occur sporadically and are associated with increased cases of AM in children. The present study describes the seroepidemiological analysis of circulating EVs in Ireland from 2005 to 2014 and phylogenetic characterization of echovirus 30 (E-30), enterovirus A71 (EV-A71), and enterovirus D68 (EV-D68). EV VP1 genotyping was applied to viral isolates and clinical samples, including cerebrospinal fluid (CSF), and those isolates that remained untypeable by neutralising anti-sera. An increase in AM cases from 2010 to 2014 was associated with an E-30 genogroup variant VII and sequences clustered phylogenetically with those detected in AM outbreaks in France and Italy. EV-D68 viral RNA was not detected in CSF samples and no neurological involvement was reported. Three EV-A71 positive CSF samples were identified in patients presenting with AM. A phylogenetic analysis of respiratory-associated EV-D68 and EV-A71 cases in circulation was performed to determine baseline epidemiological data. EV-D68 segregated with clades B and B(1) and EV-A71 clustered as subgenogroup C2. The EV VP1 genotyping method was more sensitive than neutralising anti-sera methods by virus culture and importantly demonstrated concordance between EV genotypes in faecal and CSF samples which should facilitate EV screening by less invasive sampling approaches in AM presentations. [ABSTRACT FROM AUTHOR]

We introduce here MATtrack, an open source MATLAB-based computational platform developed to process multi-Tiff files produced by a photo-conversion time lapse protocol for live cell fluorescent microscopy. MATtrack automatically performs a series of steps required for image processing, including extraction and import of numerical values from Multi-Tiff files, red/green image classification using gating parameters, noise filtering, background extraction, contrast stretching and temporal smoothing. MATtrack also integrates a series of algorithms for quantitative image analysis enabling the construction of mean and standard deviation images, clustering and classification of subcellular regions and injection point approximation. In addition, MATtrack features a simple user interface, which enables monitoring of Fluorescent Signal Intensity in multiple Regions of Interest, over time. The latter encapsulates a region growing method to automatically delineate the contours of Regions of Interest selected by the user, and performs background and regional Average Fluorescence Tracking, and automatic plotting. Finally, MATtrack computes convenient visualization and exploration tools including a migration map, which provides an overview of the protein intracellular trajectories and accumulation areas. In conclusion, MATtrack is an open source MATLAB-based software package tailored to facilitate the analysis and visualization of large data files derived from real-time live cell fluorescent microscopy using photoconvertible proteins. It is flexible, user friendly, compatible with Windows, Mac, and Linux, and a wide range of data acquisition software. MATtrack is freely available for download at eleceng.dit.ie/courtney/MATtrack.zip. [ABSTRACT FROM AUTHOR]

Host genetic factors influence treatment responses to antiviral therapy in chronic hepatitis C virus (HCV) infection. We retrospectively investigated associations between host genetic markers and treatment-induced virologic responses to dual therapy with interferon-α and ribavirin in chronically infected HCV genotype 1 (g1)- and genotype 3 (g3)-infected individuals. A total of 171 patients (89 HCV g1 and 82 HCV g3 infected) were investigated for genetic markers influencing treatment-induced sustained virologic response (SVR). Overall, SVR was observed for 46/89 (52%) HCV g1- and 57/82 (70%) HCV g3-infected patients. Of the 4 interleukin 28B ( IL28B) single-nucleotide polymorphisms (SNPs), rs12979860 was the host genetic marker most significantly associated with failure to achieve an SVR in HCV g1-infected individuals [ P=3.83×10−4; odds ratio (OR)=5.61; confidence interval (CI)=2.07-15.18] and gave a positive predictive value for treatment failure of 81.3% for minor homozygotes (TT). Using additive ( P=3.54×10−4) and dominant models ( P=3.83×10−4), a dosage effect of the T allele was observed, with the dominance term not significant for this SNP. Logistic regression showed an association between HLA-C1/C1 and rapid virologic response in HCV g1 infections with an OR relative to the heterozygote of 10.0 (95% CI: 1.6-62.5, P=0.014). HLA-C2 homozygosity was a significant predictor of nonresponse to treatment in HCV g1-infected individuals ( P=0.023). [ABSTRACT FROM AUTHOR]

Understanding the dynamic properties of cellular proteins in live cells and in real time is essential to delineate their function. In this context, we introduce the Fluorescence Recovery After Photobleaching-Photoactivation unit (Andor) combined with the Nikon Eclipse Ti E Spinning Disk (Andor) confocal microscope as an advantageous and robust platform to exploit the properties of the Dendra2 photoconvertible fluorescent protein (Evrogen) and analyse protein subcellular trafficking in living cells. A major advantage of the spinning disk confocal is the rapid acquisition speed, enabling high temporal resolution of cellular processes. Furthermore, photoconversion and imaging are less invasive on the spinning disk confocal as the cell exposition to illumination power is reduced, thereby minimizing photobleaching and increasing cell viability. We have tested this commercially available platform using experimental settings adapted to track the migration of fast trafficking proteins such as UBC9, Fibrillarin and have successfully characterized their differential motion between subnuclear structures. We describe here step-by-step procedures, with emphasis on cellular imaging parameters, to successfully perform the dynamic imaging and photoconversion of Dendra2-fused proteins at high spatial and temporal resolutions necessary to characterize the trafficking pathways of proteins. [ABSTRACT FROM AUTHOR]

Studies have identified solid organ transplant recipients who remain asymptomatic despite maintaining CHL. Factors which determine the CHL state remain poorly understood but are likely to involve immunological control of the viral infection. We monitored expression of PD-1, a marker of T-cell exhaustion and viral persistence, on CD8 T cells in patients who resolved EBV infection as determined by undetectable EBV DNA ( REI) and CHL patients. PD-1 expression on CD8 T cells was increased in the first year post-transplant irrespective of EBV outcome, and most CD8 T cells continued to express PD-1 for up to three yr post-transplant. Although all patient groups showed similar frequencies of EBV-specific CD8+ T cells, PD-1 expression on these cells increased in the post-transplant groups compared with the pretransplant patients. Functional studies of EBV-specific CD8+ T cells stimulated with BZLF or LMP2 peptide pools revealed monofunctional IFN-γ responses. Our results indicate that PD-1 expression on CD8 T cells post-transplant may result from factors other than antigenic stimulation. [ABSTRACT FROM AUTHOR]

Viroporins, which are encoded by a wide range of animal viruses, oligomerize in host cell membranes and form hydrophilic pores that can disrupt a number of physiological properties of the cell. Little is known about the relationship between host cell proteins and viroporin activity. The human JC polyomavirus (JCV) is the causative agent of progressive multifocal leukoencephalopathy. The JCV-encoded agnoprotein, which is essential for viral replication, has been shown to act as a viroporin. Here we demonstrate that the JCV agnoprotein specifically interacts with adaptor protein complex 3 through its δ subunit. This interaction interrupts adaptor protein complex 3–mediated vesicular trafficking with suppression of the targeting of the protein to the lysosomal degradation pathway and instead permits the transport of agnoprotein to the cell surface with resulting membrane permeabilization. The findings demonstrate a previously undescribed paradigm in virus–host interactions allowing the host to regulate viroporin activity and suggest that the viroporins of other viruses may also be highly regulated by specific interactions with host cell proteins. [ABSTRACT FROM AUTHOR]

The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is a genetically diverse pathogen infecting approximately 2-3% of the world's population. Herein, we describe results of a large, multicentre serological and molecular epidemiological study cataloguing the prevalence and genetic diversity of HCV in five regions of Vietnam; Ha Noi, Hai Phong, Da Nang, Khanh Hoa and Can Tho. Individuals (n = 8654) with varying risk factors for infection were analysed for the presence of HCV Ab/Ag and, in a subset of positive specimens, for HCV RNA levels (n = 475) and genotype (n = 282). In lower risk individuals, including voluntary blood donors, military recruits and pregnant women, the prevalence of infection was 0.5% (n = 26/5250). Prevalence rates were significantly higher (p<0.001) in intravenous drug users (IDUs; 55.6%, n = 556/1000), dialysis patients (26.6%, n = 153/575) commercial sex workers (CSWs; 8.7%, n = 87/1000), and recipients of multiple blood transfusions (6.0%, n = 32/529). The prevalence of HCV in dialysis patients varied but remained high in all regions (11-43%) and was associated with the receipt of blood transfusions [OR: 2.08 (1.85-2.34), p = 0.001], time from first transfusion [OR: 1.07 (1.01 -1.13), p = 0.023], duration of dialysis [OR: 1.31 (1.19-1.43), p<0.001] and male gender [OR: 1.60 (1.06-2.41), p = 0.026]. Phylogenetic analysis revealed high genetic diversity, particularly amongst dialysis and multi-transfused patients, identifying subtypes 1a (33%), 1b (27%), 2a (0.4%), 3a (0.7%), 3b (1.1%), 6a (18.8%), 6e (6.0%), 6h (4.6%), 6l (6.4%) and 2 clusters of novel genotype 6 variants (2.1 %). HCV genotype 1 predominated in Vietnam (60%, n = 169/282) but the proportion of infections attributable to genotype 1 varied between regions and risk groups and, in the Southern part of Vietnam, genotype 6 viruses dominated in dialysis and multi-transfused patients (73.9%). This study confirms a high prevalence of HCV infection in Vietnamese IDUs and, notably, reveals high levels of HCV infection associated with dialysis and blood transfusion. [ABSTRACT FROM AUTHOR]

Recombination in Hepatitis C virus (HCV) is considered to be rare. In this study, we performed a phylogenetic analysis of 1278 full-length HCV genome sequences to identify potential recombination events. Nine inter-genotype recombinants were identified, all of which have been previously reported. This confirms the rarity of inter-genotype HCV recombinants. The analysis also identified five inter-subtype recombinants, four of which are documented for the first time (EU246930, EU246931, EU246932, and EU246937). Specifically, the latter represent four different novel recombination types (6a/6o, 6e/ 6o, 6e/6h, and 6n/6o), and this was well supported by seven independent methods embedded in RDP. The breakpoints of the four novel HCV recombinants are located within the NS5B coding region and were different from all previously reported breakpoints. While the locations of the breakpoints identified by RDP were not identical, they are very close. Our study suggests that while recombination in HCV is rare, this warrants further investigation. [ABSTRACT FROM AUTHOR]

Hepatitis B (HBV) infection is endemic in Viet Nam, with up to 8.4 million individuals estimated to be chronically infected. We describe results of a large, multicentre seroepidemiological and molecular study of the prevalence of HBV infection and blood-borne viral coinfections in Viet Nam. Individuals with varying risk factors for infection (n = 8654) were recruited from five centres; Ha Noi, Hai Phong, Da Nang, Khanh Hoa and Can Tho. A mean prevalence rate of 10.7% was observed and levels of HBsAg were significantly higher in injecting drug users (IDUs) (17.4%, n = 174/1000) and dialysis patients (14.3%, n = 82/575) than in lower-risk groups (9.4%; p<0.001). Coinfection with HIV was seen in 28% of HBV-infected IDUs (n = 49/ 174) and 15.2% of commercial sex workers (CSWs; n = 15/99). HCV infection was present in 89.8% of the HBV-HIV coinfected IDUs (n = 44/49) and 40% of HBV-HIV coinfected CSWs (n = 16/40). Anti-HDV was detected in 10.7% (n = 34/318) of HBsAg positive individuals. Phylogenetic analysis of HBV S gene (n = 187) showed a predominance of genotype B4 (82.6%); genotypes C1 (14.6%), B2 (2.7%) and C5 (0.5%) were also identified. The precore mutation G1896A was identified in 35% of all specimens, and was more frequently observed in genotype B (41%) than genotype C (3%; p<0.0001). In the immunodominant 'a' region of the surface gene, point mutations were identified in 31% (n = 58/187) of sequences, and 2.2% (n = 4/187) and 5.3% (n = 10/187) specimens contained the major vaccine escape mutations G145A/R and P120L/Q/S/T, respectively. 368 HBsAg positive individuals were genotyped for the IL28B SNP rs12979860 and no significant association between the IL28B SNP and clearance of HBsAg, HBV viral load or HBeAg was observed. This study confirms the high prevalence of HBV infection in Viet Nam and also highlights the significant levels of blood-borne virus coinfections, which have important implications for hepatitis-related morbidity and development of effective management strategies. [ABSTRACT FROM AUTHOR]

Background: In contrast with human T-cell leukemia virus type 1 (HTLV-1) that causes ATL (adult T-cell leukemia), HTLV-2 has not been causally linked to malignant disease. The minus strand of the HTLV genomes encode the regulatory proteins HTLV-1 bZIP factor (HBZ) for HTLV-1 and antisense protein of HTLV-2 (APH-2) for HTLV-2. Unlike the viral proteins Tax1 and Tax2, both HBZ and APH-2 are constitutively expressed in infected cells suggesting that they may play important roles in the pathogenesis of these viruses. To date, very little is known about the function of APH-2 except that it inhibits Tax2-mediated transcription of HTLV-2 genes. In the present study, we investigated the role of APH-2 in basal and Tax2B-mediated activation of the AP-1 pathway. Results: We demonstrate that, unlike HBZ, APH-2 stimulates basal AP-1 transcription by interacting with c-Jun and JunB through its non-conventional bZIP domain. In addition, when Tax2 and APH-2 are co-expressed, they physically interact in vivo and in vitro and APH-2 acts as an inhibitor of Tax2-mediated activation of AP-1 transcription. Conclusions: This report is the first to document that HTLV-2 can modulate the AP-1 pathway. Altogether our results reveal that, in contrast with HBZ, APH-2 regulates AP-1 activity in a Tax2-dependant manner. As the AP-1 pathway is involved in numerous cellular functions susceptible to affect the life cycle of the virus, these distinct biological properties between HBZ and APH-2 may contribute to the differential pathogenic potential of HTLV-1 and HTLV-2. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is capable of disrupting different facets of lipid metabolism and lipids have been shown to play a crucial role in the viral life cycle. The aim of this study was to examine the effect HCV infection has on the hepatocyte metabolome. Huh-7.5 cells were infected using virus produced by the HCV J6/JFH1 cell culture system and cells were harvested 24, 48, and 72-hours following infection. Metabolic profiling was performed using a non-targeted multiple platform methodology combining ultrahigh performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/ MS2) and gas chromatography/mass spectrometry (GC/MS). There was a significant increase in a number of metabolites involved in nucleotide synthesis and RNA replication during early HCV infection. NAD levels were also significantly increased along with several amino acids. A number of lipid metabolic pathways were disrupted by HCV infection, resulting in an increase in cholesterol and sphingolipid levels, altered phospholipid metabolism and a possible disruption in mitochondrial fatty acid transport. Fluctuations in 59-methylthioadenosine levels were also noted, along with alterations in the glutathione synthesis pathway. These results highlight a number of previously unreported metabolic interactions and give a more in depth insight into the effect HCV has on host cell biochemical processes. [ABSTRACT FROM AUTHOR]

Human adenovirus (HAdV) serotype 14 is rarely identified. However, an emerging variant, termed HAdV14p1, recently has been described in the United States in association with outbreaks of acute respiratory disease with high rates of illness and death. We retrospectively analyzed specimens confirmed positive for HAdV by immunofluorescence, virus culture, or real-time PCR during July 1, 2009-July 31, 2010, and describe 9 cases of HAdV14p1 infection with characteristic mutations in the fiber and E1A genes that are phylogenetically indistinguishable from the viruses previously detected in the United States. Three patients died; 2 were immunocompromised, and 1 was an immunocompetent adult. We propose that surveillance should be increased for HAdV-14p1 and recommend that this virus be considered in the differential diagnosis of sudden-onset acute respiratory disease, particularly fatal infections, for which an etiology is not clear. [ABSTRACT FROM AUTHOR]

Background: The HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition of its Nuclear Localisation Signal (NLS) by multiple import factors, which include transportin and importin β. However, it remains unclear which nuclear import pathway(s) predominate in vivo, and the cellular environment that modulates Rev nucleocytoplasmic shuttling remains to be characterised. Results: In our study, we have identified the cellular protein HIC (Human I-mfa domain-Containing protein) as a novel interactor of HIV-1 Rev. We demonstrate that HIC selectively interferes with Rev NLS interaction with importin β and impedes its nuclear import and function, but does not affect Rev nuclear import mediated by transportin. Hence, the molecular determinants mediating Rev-NLS recognition by importin β and transportin appear to be distinct. Furthermore, we have employed HIC and M9 M, a peptide specifically designed to inhibit the transportinmediated nuclear import pathway, to characterise Rev nuclear import pathways within different cellular environments. Remarkably, we could show that in 293T, HeLa, COS7, Jurkat, U937, THP-1 and CEM cells, Rev nuclear import is cell type specific and alternatively mediated by transportin or importin β, in a mutually exclusive fashion. Conclusions: Rev cytoplasmic sequestration by HIC may represent a novel mechanism for the control of Rev function. These studies highlight that the multivalent nature of the Rev NLS for different import receptors enables Rev to adapt its nuclear trafficking strategy. [ABSTRACT FROM AUTHOR]

Virus infections can result in a range of cellular injuries and commonly this involves both the plasma and intracellular membranes, resulting in enhanced permeability. Viroporins are a group of proteins that interact with plasma membranes modifying permeability and can promote the release of viral particles. While these proteins are not essential for virus replication, their activity certainly promotes virus growth. Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease resulting from lytic infection of oligodendrocytes by the polyomavirus JC virus (JCV). The genome of JCV encodes six major proteins including a small auxiliary protein known as agnoprotein. Studies on other polyomavirus agnoproteins have suggested that the protein may contribute to viral propagation at various stages in the replication cycle, including transcription, translation, processing of late viral proteins, assembly of virions, and viral propagation. Previous studies from our and other laboratories have indicated that JCV agnoprotein plays an important, although as yet incompletely understood role in the propagation of JCV. Here, we demonstrate that agnoprotein possesses properties commonly associated with viroporins. Our findings demonstrate that: (i) A deletion mutant of agnoprotein is defective in virion release and viral propagation; (ii) Agnoprotein localizes to the ER early in infection, but is also found at the plasma membrane late in infection; (iii) Agnoprotein is an integral membrane protein and forms homo-oligomers; (iv) Agnoprotein enhances permeability of cells to the translation inhibitor hygromycin B; (v) Agnoprotein induces the influx of extracellular Ca2+; (vi) The basic residues at amino acid positions 8 and 9 of agnoprotein key are determinants of the viroporin activity. The viroporin-like properties of agnoprotein result in increased membrane permeability and alterations in intracellular Ca2+ homeostasis leading to membrane dysfunction and enhancement of virus release. [ABSTRACT FROM AUTHOR]

The oligomerization of HIV-1 Gag and Gag-Pol proteins, which are assembled at the plasma membrane, leads to viral budding. The budding generally places the viral components under non-reducing conditions. Here the effects of non-reducing conditions on Gag structures and viral RNA protection were examined. Using different reducing conditions and SDS-PAGE, it was shown that oligomerized Gag possesses intermolecular covalent bonds under non-reducing conditions. In addition, it was demonstrated that the mature viral core contains a large amount of covalent bonded Gag multimers, as does the immature core. Viral genomic RNA becomes sensitive to ribonuclease in reducing conditions. These results suggest that, under non-reducing conditions, covalent bonded Gag multimers are formed within the viral particles and play a role in protection of the viral genome. [ABSTRACT FROM AUTHOR]

Natalizumab therapy in multiple sclerosis has been associated with JC polyomavirus–induced progressive multifocal leucoencephalopathy. We hypothesized that natalizumab may also lead to reactivation of BK, a related human polyomavirus capable of causing morbidity in immunosuppressed groups. Patients with relapsing remitting multiple sclerosis treated with natalizumab were prospectively monitored for reactivation of BK virus in blood and urine samples, and for evidence of associated renal dysfunction. In this cohort, JC and BK DNA in blood and urine; cytomegalovirus (CMV) DNA in blood and urine; CD4 and CD8 T-lymphocyte counts and ratios in peripheral blood; and renal function were monitored at regular intervals. BK subtyping and noncoding control region sequencing was performed on samples demonstrating reactivation. Prior to commencement of natalizumab therapy, 3 of 36 patients with multiple sclerosis (8.3%) had BK viruria and BK reactivation occurred in 12 of 54 patients (22.2%). BK viruria was transient in 7, continuous in 2 patients, and persistent viruria was associated with transient viremia. Concomitant JC and CMV viral loads were undetectable. CD4:CD8 ratios fluctuated, but absolute CD4 counts did not fall below normal limits. In four of seven patients with BK virus reactivation, transient reductions in CD4 counts were observed at onset of BK viruria: these resolved in three of four patients on resuppression of BK replication. No renal dysfunction was observed in the cohort. BK virus reactivation can occur during natalizumab therapy; however, the significance in the absence of renal dysfunction is unclear. We propose regular monitoring for BK reactivation or at least for evidence of renal dysfunction in patients receiving natalizumab. [ABSTRACT FROM AUTHOR]

The molecular characterization of measles virus (MeV) is a valuable epidemiological tool to monitor virus transmission and to discriminate between imported and endemic infection. There has been significant immigration into Ireland in recent years and many individuals originate from regions of high measles incidence. Ireland has had a number of outbreaks of MeV which appear attributable to sub-optimal vaccine uptake and possibly imported strains as new genotypes have been identified in recent years. To ascertain any significant changes in circulating measles genotypes we investigated 65 confirmed measles cases between the years 2002 and 2007. The laboratory diagnosis of measles was confirmed by detection of measles-specific IgM in oral fluid in conjunction with a real-time polymerase chain reaction assay targeting the MeV hemagglutinin gene. Phylogenetic analysis based on the 3′ hypervariable region of the nucleoprotein gene was performed and three genotypes, all within measles clade D, were found to be circulating during this time period. In 2002 and 2003, genotype D8 (n = 2) was observed whereas genotype D7 was dominant in 2003 (n = 31). A distinct change in the circulating MeV genotype and increased genetic diversity was observed between 2004 and 2007. All cases were within genotype D4 (n = 32) but were phylogenetically distinct from each other. These data provide important epidemiologic baseline information on MeV in Ireland and facilitates detailed examination of measles transmission. J. Med. Virol. 81:125-129, 2009. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Human metapneumovirus (hMPV) is a cause of respiratory illness ranging from wheezing to bronchiolitis and pneumonia in children. A quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay was developed for the detection of all four main genetic lineages of hMPV and employed to validate an indirect immunofluorescence (IF) assay to detect hMPV positive specimens. The IF assay detected 24 positives from a screen of 625 randomly selected pediatric respiratory specimens collected (3.8% prevalence). From this cohort of 625 specimens, 229 were also tested by real-time RT-PCR assay. This included the 24 IF positive specimens and 205 randomly selected specimens from both study periods. In addition to confirming all the IF positives, the real-time assay detected an additional six hMPV positive specimens giving rise to a combined prevalence of 4.8%. Phylogenetic analysis showed that hMPV subtypes A2b and B2 to be the most prevalent genotypes circulating in our population and surprisingly no hMPV subgroups A1 or B1 were detected during this study period. Based on this phylogenetic analysis, we propose the existence of sub-clusters of hMPV genotype B2 present in our population which we term subtypes B2a and B2b. The mean log 10 copies/ml of quantitative RT-PCR determinations from these 30 hMPV positive respiratory specimens was 6.35 (range = 4.44-8.15). Statistical analysis of quantitative RT-PCR determinations of viral load from these 30 respiratory specimens suggests that hMPV genotype B specimens have a higher viral load than hMPV genotype A isolates ( P < 0.03). J. Med. Virol. 80:510-516, 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Coinfection with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) is associated with an accelerated course of HCV infection and a faster progression to severe liver disease. We have investigated whether the development of liver disease in coinfected patients is associated with specific chemokine and cytokine production. Four cohorts—HCV/HIV-coinfected patients, HCV-monoinfected patients, HIV-monoinfected patients, and healthy control subjects—were studied. Serum levels of the 10-kDa interferon—y—in—ducible protein (IP-10) were higher in all 3 groups of infected patients than in control subjects (P< .0001). HCV/HIV-coinfected patients had significantly higher IP-10 levels than monoinfected patients. In HCV-monoinfected patients, liver fibrosis scores and liver enzyme levels were positively correlated with IP-10 levels. Elevated IP-10 levels are associated with and may contribute to liver damage in both HCV-mono-infected and HCV/HIV-coinfected patients. [ABSTRACT FROM AUTHOR]

This paper examines data from the Cape Fear region of North Carolina, an area at elevated exposure to hurricanes and catastrophic risk. The findings support an earlier documented pattern of price declinations with successive hurricane landfalls. The findings also reveal a tempering of this trend in the years after the last major strike in 1999. A test statistic is constructed for the timing and intensity of the real estate market reaction to perceptions of catastrophic risk. A Chow test is used to frame market responses to repeated hurricane landfalls in the study area. The test reveals a structural shift in the housing market in the periods following the last two in a series of four hurricanes. Home prices recover and market stability returns in the years following the last storm. The findings are important to varied stakeholders in the coastal real estate market. [ABSTRACT FROM AUTHOR]