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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]

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]

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]

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]

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]

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]

Adult T-cell leukemia-lymphoma (ATLL) is a group of T-cell malignancies caused by infection with human T-lymphotropic virus type I (HTLV-I). Although the pathogenesis of ATLL remains incompletely understood, the viral regulatory protein Tax is centrally involved in cellular transformation. Here we describe the generation of HTLV-I Tax transgenic mice using the Lck proximal promoter to restrict transgene expression to developing thymocytes. After prolonged latency periods, transgenic mice developed diffuse large-cell lymphomas and leukemia with clinical, pathological and immunological features characteristic of acute ATLL. Transgenic mice were functionally immunocompromised and they developed opportunistic infections. Fulminant disease also developed rapidly in SCID mice after engraftment of lymphomatous cells from transgenic mice. Flow cytometry showed that the cells were CD4− and CD8−, but CD44+, CD25+ and cytoplasmic CD3+. This phenotype is indicative of a thymus-derived pre–T-cell phenotype, and disease development was associated with the constitutive activation of NF-κB. Our model accurately reproduces human disease and will provide a tool for analysis of the molecular events in transformation and for the development of new therapeutics. [ABSTRACT FROM AUTHOR]

The human polyomavirus JC virus (JCV) is ubiquitous and can be shed in the urine of more than 40% of the healthy population. Amplification and sequencing of JCV from urine has allowed a distinctive map of the distribution of JCV genotypes worldwide. To define the frequency of JCV urinary excretion and genotype distribution in Ireland, urines from 121 healthy individuals and from 94 immunocompromised individuals (human immunodeficiency virus [HIV]-positive patients and rheumatoid arthritis patients) were collected. JCV DNA was detected by polymerase-chain reaction (PCR) with subsequent nucleotide sequencing of a fragment of the major capsid protein (VP1). JCV was detected in 20.7% of healthy individuals and was found significantly more often in the urine of HIV-positive patients (54.2%; P [ABSTRACT FROM AUTHOR]

Human T-lymphotropic virus type 1 (HTLV-1) infection is associated with the clonal expansion and transformation of mature T lymphocytes. While the mechanisms involved are incompletely understood the viral regulatory protein Tax plays a central role in these processes. Recent studies employing genomic and proteomic approaches have demonstrated the marked complexity of gene deregulation associated with Tax expression and confirmed the remarkable pleiotropism of this protein as evidenced by the numerous Tax–cellular protein interactions in infected cells. In this review, we summarize the role of Tax in the deregulation of selected cellular-signaling pathways. Specifically, this has focused on the influence and interaction of Tax with the AP-1 and NF-AT transcription factors, PDZ domain-containing proteins, Rho-GTPases, and the Janus kinase/signal transducer and activator of transcription and transforming growth factor-β-signaling pathways. In addition to identifying the deregulation of events within these pathways, attempts have been made to highlight differences between HTLV-1 and -2, which may relate to differences in their pathogenic properties.Oncogene (2005) 24, 5965–5975. doi:10.1038/sj.onc.1208975 [ABSTRACT FROM AUTHOR]

To understand the mechanisms involved in the pathogenesis of human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), three in vivo phenomena which have been observed in the peripheral blood of patients and differing from that in asymptomatic HTLV-I carriers must be taken into consideration: (a) the presence of increased HTLV-I viral load, (b) a higher immune responsiveness against HTLV-I antigens, and (c) biased nucleotide substitutions in the HTLV-I pX region which indicate a decreased selection pressure for viral amino acid changes. We now propose a hypothesis which focuses on the in vivo dynamics of HTLV-I infected lymphocyte migration and which incorporates these features. In addition, the hypothesis assumes the existence of a deviation in immune surveillance for HTLV-I in the central nervous system (CNS) in spite of the presence of frequent specific immune effectors. We suggest that in the active phase of HAM/TSP, accompanied with or following autoaggressive interactions between infected lymphocytes and immunocompetent cells in the CNS, there is a consequential reflux of the infected lymphocytes to the peripheral blood. The reflux of infected cells would be expected to provide peripheral blood with tissue-derived HTLV-I proviruses which have been indulged and propagated in an immune-privileged site. This process would result in and account for the observed increase in viral load and the substitution bias in HTLV-I sequences in the peripheral blood. [ABSTRACT FROM AUTHOR]

The mechanisms involved in the restriction of the cellular tropism of HIV-1 to cells of primate origin remain to be clearly defined. However, a number of studies have shown that this is determined not only at the level of the cellular receptor(s) or virus entry, but at a number of additional and later stages in virus replication. We have recently reported that the reverse transcription of HIV-1 RNA is markedly enhanced by the association of the gag encoded nucleocapsid p15 protein and cellular topoisomerase I. In the present study we have investigated if the recruitment of cellular topoisomerase 1 during virus replication is important in determining the cellular tropism of HIV-1. Employing a stable murine cell line, L929, expressing both human CD4 and topoisomerase I, it could be demonstrated that effective proviral DNA synthesis occurred following infection. In contrast in cells expressing only human CD4, proviral DNA synthesis was not detected. In addition we have co-expressed fusin, a protein known to act as an accessory factor as the virus entry stage in infection of T cell tropic HIV-1, to support viral entry completely. However no progeny virus could be detected after HIV-1 infection. These results suggest that reverse transcription in vivo is critically dependent on the presence of cellular topoisomerase I, and support the view that involvement of this enzyme is important in HIV-1 replication. Moreover the findings suggest that other factors which remained to be identified, are involved in restricting HIV-1 replication in non-primate cells. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor about the origin and the patterns of dissemination of the HTLV-I virus strain. Discovery of the Cosmopolitan HTLV-I Cosmopolitan subtype in African ethnic populations; Use of molecular clock calculations on an 1,500 year-old Andean mummy to prove the virus' origins in South America; Mummy's possesion of a contemporary strain.

The human T lymphotropic viruses type I (HTLV-I) and type II (HTLV-II) are closely related retroviruses which are associated with the development of lymphoproliferative disorders. In vitro studies have demonstrated that the virus regulatory protein Tax plays a central role in cell transformation and proliferation, and that this appears to be due to the ability of the protein to disregulate both cell cycle controls and the transcription of a number of cellular genes.