Insights into the Host Range, Genetic Diversity, and Geographical Distribution of Jingmenviruses.

Jingmenvirus is a recently identified group of segmented RNA viruses phylogenetically linked with unsegmented Flaviviridae viruses. Primarily identified in various tick genera originating in China, Jingmenvirus geographical distribution has rapidly expanded to cover Africa, South America, Caribbean, and Europe. The identification of Jingmen-related viruses in various mammals, including febrile humans, opens the possibility that Jingmenviruses may be novel tick-borne arboviruses. In this study, we aimed at increasing knowledge of the host range, genetic diversity, and geographical distribution of Jingmenviruses by reporting for the first time the identification of Jingmenviruses associated with Rhipicephalus microplus ticks originating in the French Antilles (Guadeloupe and Martinique islands), with Amblyomma testudinarium ticks in Lao PDR, and with Ixodes ricinus ticks in metropolitan France, and from urine of Pteropus lylei bats in Cambodia. Analyses of the relationships between the different Jingmenvirus genomes resulted in the identification of three main phylogenic subclades, each of them containing both tick-borne and mammal-borne strains, reinforcing the idea that Jingmenviruses may be considered as tick-borne arboviruses. Finally, we estimated the prevalence of Jingmenvirus-like infection using luciferase immunoprecipitation assay screening (LIPS) of asymptomatic humans and cattle highly exposed to tick bites. Among 70 French human, 153 Laotian human, and 200 Caribbean cattle sera tested, only one French human serum was found (slightly) positive, suggesting that the prevalence of Jingmenvirus human and cattle infections in these areas is probably low.IMPORTANCE Several arboviruses emerging as new pathogens for humans and domestic animals have recently raised public health concern and increased interest in the study of their host range and in detection of spillover events. Recently, a new group of segmented Flaviviridae-related viruses, the Jingmenviruses, has been identified worldwide in many invertebrate and vertebrate hosts, pointing out the issue of whether they belong to the arbovirus group. The study presented here combined whole-genome sequencing of three tick-borne Jingmenviruses and one bat-borne Jingmenvirus with comprehensive phylogenetic analyses and high-throughput serological screening of human and cattle populations exposed to these viruses to contribute to the knowledge of Jingmenvirus host range, geographical distribution, and mammalian exposure.

Potential for Co-Infection of a Mosquito-Specific Flavivirus, Nhumirim Virus, to Block West Nile Virus Transmission in Mosquitoes.

Nhumirim virus (NHUV) is an insect-specific virus that phylogenetically affiliates with dual-host mosquito-borne flaviviruses. Previous in vitro co-infection experiments demonstrated prior or concurrent infection of Aedes albopictus C6/36 mosquito cells with NHUV resulted in a 10,000-fold reduction in viral production of West Nile virus (WNV). This interference between WNV and NHUV was observed herein in an additional Ae. albopictus mosquito cell line, C7-10. A WNV 2K peptide (V9M) mutant capable of superinfection with a pre-established WNV infection demonstrated a comparable level of interference from NHUV as the parental WNV strain in C6/36 and C7-10 cells. Culex quinquefasciatus and Culex pipiens mosquitoes intrathoracically inoculated with NHUV and WNV, or solely with WNV as a control, were allowed to extrinsically incubate the viruses up to nine and 14 days, respectively, and transmissibility and replication of WNV was determined. The proportion of Cx. quinquefasciatus mosquitoes capable of transmitting WNV was significantly lower for the WNV/NHUV group than the WNV control at seven and nine days post inoculation (dpi), while no differences were observed in the Cx. pipiens inoculation group. By dpi nine, a 40% reduction in transmissibility in mosquitoes from the dual inoculation group was observed compared to the WNV-only control. These data indicate the potential that infection of some Culex spp. vectors with NHUV could serve as a barrier for efficient transmissibility of flaviviruses associated with human disease.

Hepatitis C-like viruses are produced in cells from rabbit and hare DNA.

Hepatitis C virus (HCV), a major causative agent of acute and chronic liver disease, belongs to the Flaviviridæ family and contains a single-strand positive-sense RNA genome, which upon virus entry and uncoating, functions as mRNAs and thus can be directly translated into proteins by host cell machinery. To date the HCV origin remains unclear and HCV life cycle and pathogenesis are not enlightened processes due to the absence of HCV efficient cell cultures systems or animals models. Here we show that rabbit and hare HCV-like viruses, RHCV and HHCV respectively, are formed after the inoculation of genomic DNA in Madin-Darby bovine kidney cell line cultures. RHCV is closely related to the HCV-1a/HCV-1b genotypes and HHCV is more closely related to the HCV-1b genotype. These findings could contribute to the understanding of HCV origin as well as clarify the virus life cycle, pathogenesis, evolution and diversity.

Development of an algorithm for production of inactivated arbovirus antigens in cell culture.

Arboviruses are medically important pathogens that cause human disease ranging from a mild fever to encephalitis. Laboratory diagnosis is essential to differentiate arbovirus infections from other pathogens with similar clinical manifestations. The Arboviral Diseases Branch (ADB) reference laboratory at the CDC Division of Vector-Borne Diseases (DVBD) produces reference antigens used in serological assays such as the virus-specific immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Antigen production in cell culture has largely replaced the use of suckling mice; however, the methods are not directly transferable. The development of a cell culture antigen production algorithm for nine arboviruses from the three main arbovirus families, Flaviviridae, Togaviridae, and Bunyaviridae, is described here. Virus cell culture growth and harvest conditions were optimized, inactivation methods were evaluated, and concentration procedures were compared for each virus. Antigen performance was evaluated by the MAC-ELISA at each step of the procedure. The antigen production algorithm is a framework for standardization of methodology and quality control; however, a single antigen production protocol was not applicable to all arboviruses and needed to be optimized for each virus.

Preliminary findings on Bagaza virus (Flavivirus: Flaviviridae) growth kinetics, transmission potential & transovarial transmission in three species of mosquitoes.

BACKGROUND & OBJECTIVES: Bagaza virus (BAGV), a flavivirus synonymous with Israel turkey meningoencephalitis virus, has been found to circulate in India. BAGV has recently been held responsible for inducing febrile illness in humans and causing unusually high mortality to wild birds in Spain. A study was therefore, undertaken to determine its replication kinetics in certain mosquitoes and to determine vector competence and potential of the mosquitoes to transmit BAGV experimentally. METHODS: Aedes aegypti, Culex tritaeniorhynchus and Cx quinquefasciatus mosquitoes were inoculated with BAGV; samples were harvested every day and titrated in BHK-21 cell line. Vector competence and experimental transmission were determined by examining the saliva of infected mosquitoes for virus and induction of sickness in suckling mice, respectively. RESULTS: Cx. tritaeniorhynchus and Ae. aegypti mosquitoes yielded 5 log10 and 4.67 log10 TCID50/ml of virus on day 3 post-infection (PI), respectively while Cx. quinquefasciatus yielded a titre of 4 log10 TCID50/ml on day 4 PI. BAGV was detected in saliva of all the infected mosquitoes demonstrating their vector competence. Experimental transmission of BAGV to infant mice as well as transovarial transmission was demonstrated by Cx. tritaeniorhynchus but not by Ae. aegypti and Cx. quinquefasciatus mosquitoes. INTERPRETATION & CONCLUSIONS: Replication of BAGV to high titres and dissemination to saliva in three most prevalent mosquitoes in India is of immense public health importance. Though no major outbreak involving man has been reported yet, BAGV has a potential to cause outbreaks in future.

Viral arthritides.

Viral infections may manifest as acute or chronic arthritis. Joint involvement arises from either direct infection of the joint, through an immunological response directed towards the virus or autoimmunity. Epidemiological clues to the diagnosis include geographic location and exposure to vector-borne, blood-borne or sexually transmitted viruses. Although not always possible, it is important to diagnose the pathogenic virus, usually by serology, nucleic acid tests or rarely, viral culture. In general, viral arthritides are self-limiting and treatment is targeted at symptomatic relief. This article focuses on the causes, clinical features, diagnosis and treatment of viral arthritides.

Architects of assembly: roles of Flaviviridae non-structural proteins in virion morphogenesis.

Viruses of the Flaviviridae family, including hepatitis C, dengue and bovine viral diarrhoea, are responsible for considerable morbidity and mortality worldwide. Recent advances in our understanding of virion assembly have uncovered commonalities among distantly related members of this family. We discuss the emerging hypothesis that physical virion components are not alone in forming the infectious particle, but that non-structural proteins are intimately involved in orchestrating morphogenesis. Pinpointing the roles of Flaviviridae proteins in virion production could reveal new avenues for antiviral therapeutics.

Genetic and phenotypic characterization of the newly described insect flavivirus, Kamiti River virus.

We have described in the accompanying paper by Sang, et al., ([57], Arch Virol 2003, in press) the isolation and identification of a new flavivirus, Kamiti River virus (KRV), from Ae. macintoshi mosquitoes that were collected as larvae and pupae from flooded dambos in Central Province, Kenya. Among known flaviviruses, KRV was shown to be most similar to, but genetically and phenotypically distinct from, Cell fusing agent virus (CFAV). KRV was provisionally identified as an insect-only flavivirus that fails to replicate in vertebrate cells or in mice. We report here the further characterization of KRV. Growth in cell culture was compared to that of CFAV; although growth kinetics were similar, KRV did not cause the cell fusion that is characteristic of CFAV infection. The KRV genome was found to be 11,375 nucleotides in length, containing a single open reading frame encoding 10 viral proteins. Likely polyprotein cleavage sites were identified, which were most similar to those of CFAV and were comparable to those of other flaviviruses. Sequence identity with other flaviviruses was low; maximum identity was with CFAV. Possible terminal secondary structures for the 5' and 3' non-coding regions (NCR) were similar to those predicted for other flaviviruses. Whereas CFAV was isolated from insect cells in the laboratory, the isolation of KRV demonstrates the presence of an insect-only flavivirus in nature and raises questions regarding potential interactions between this virus and other mosquito-borne viruses in competent vector populations. Additionally, this virus will be an important tool in future studies to determine markers associated with flavivirus host specificity.

Effect of coinfection with GB virus C on survival among patients with HIV infection.

BACKGROUND: Previous studies have suggested that people with human immunodeficiency virus (HIV) infection who are coinfected with GB virus C (GBV-C, or hepatitis G virus) have delayed progression of HIV disease. GBV-C is related to hepatitis C virus but does not appear to cause liver disease. METHODS: We examined the effect of coinfection with GBV-C on the survival of patients with HIV infection. We also evaluated cultures of peripheral-blood mononuclear cells infected with both viruses to determine whether GBV-C infection alters replication in vitro. RESULTS: Of 362 HIV-infected patients, 144 (39.8 percent) had GBV-C viremia in two tests. Forty-one of the patients with GBV-C viremia (28.5 percent) died during the follow-up period, as compared with 123 of the 218 patients who tested negative for GBV-C RNA (56.4 percent; P<0.001). The mean duration of follow-up for the entire cohort was 4.1 years. In a Cox regression analysis adjusted for HIV treatment, baseline CD4+ T-cell count, age, sex, race, and mode of transmission of HIV, the mortality rate among the 218 HIV-infected patients without GBV-C coinfection was significantly higher than that among the 144 patients with GBV-C coinfection (relative risk, 3.7; 95 percent confidence interval, 2.5 to 5.4). HIV replication, as measured by the detection of p24 antigen in culture supernatants, was reproducibly inhibited in cultures of peripheral-blood mononuclear cells by GBV-C coinfection. Coinfection did not alter the surface expression of HIV cellular receptors on peripheral-blood mononuclear cells, as determined by flow cytometry. CONCLUSIONS: GBV-C infection is common in people with HIV infection and is associated with significantly improved survival.

Hepatitis G virus biology, epidemiology, and clinical manifestations: Implications for blood safety.

Hepatitis G virus (HGV), also called GBV-C, is a single positive-standard RNA virus belonging to the Flaviviridae family. In 50% to 75% of infections, HGV is cleared with plasma RNA disappearing as anti-E2 becomes detectable; in other cases, HGV infection becomes chronic. The prevalence of HGV RNA in blood donors ranges from 1% to 4%, and the rate of anti-E2, indicating resolved infection, ranges from 3% to 14%. HGV is transmitted by transfusion of blood components and has been transmitted by nonvirally inactivated factor VIII concentrate. Despite extensive study, HGV has not been identified as a causative agent of any type of liver disease or any other known clinical condition. Molecular biology data show a lack of hepatotropism; preliminary data indicate that the site of HGV replication may be in mononuclear cells in bone marrow or spleen but not in peripheral blood or lymph nodes. The combined clinical and laboratory data strongly support the contention that HGV is not a hepatotropic virus and that this virus was inappropriately named hepatitis G. Because the data do not indicate any pathologic effects of HGV, it is not appropriate to screen the blood supply for HGV RNA.

Review of the epidemiology, molecular characterization and tropism of the hepatitis G virus/GBV-C.

The hepatitis G virus and GBV-C are recently discovered variants of the same virus belonging to the family Flaviviridae (HGV/GBV-C). Although initially thought to be a hepatitis virus, it has been shown to have no association with liver disease. This paper reviews the data relating to the discovery, global prevalence, natural history, disease association, molecular features, replication and tissue tropism of HGV/GBV-C.

Toward a surrogate model for hepatitis C virus: An infectious molecular clone of the GB virus-B hepatitis agent.

GB virus-B (GBV-B) is a member of the Flaviviridae family of viruses. This RNA virus infects tamarins, but its natural host is not known. GBV-B has special interest because it is the virus that is most closely related to hepatitis C virus (HCV), an important human pathogen. In the present study, we identified a previously unrecognized sequence at the 3' end of the GBV-B genome. This new 3' terminal sequence can form several predicted stem-loop structures as is typical for other members of the Flaviviridae family. We constructed molecular clones and showed that the new 3' UTR sequence was critical for in vivo infectivity. After intrahepatic transfection of two tamarins with RNA transcripts of the full-length GBV-B clone, we detected high viral titers from Week 1 postinoculation with peak titers of approximately 10(8) genome equivalents/ml. The viremic pattern of GBV-B infection in the transfected animals was the same as in animals inoculated intravenously with the virus pool used as the cloning source. The sequence of the recombinant virus was recovered from one of the tamarins and shown to be identical to that of the infectious clone. The development of severe hepatitis in both tamarins infected with the recombinant GBV-B virus provides formal proof that GBV-B is a true hepatitis virus.

Association of antibody to GB virus C (hepatitis G virus) with viral clearance and protection from reinfection.

GB virus C (GBV-C) RNA and envelope antibody were assessed in a median of 4 samples collected over 6.5 years among injection drug users (IDUs). A marker of GBV-C infection was detected in 110 (94.8%) of 116 IDUs. GBV-C RNA was detected at all visits in 32, was never detected in 70, was acquired in 7, and was cleared in 8. The odds of detecting anti-GBV-C were 103-fold higher in participants without detectable RNA (64 of 70) than in IDUs with persistent RNA (3 of 32; P < 10(-7)). Anti-GBV-C was detected in all 8 instances of RNA clearance. GBV-C RNA never reappeared once it was cleared, and there were no new GBV-C infections among 61 anti-GBV-C-positive IDUs observed for 382 person-years, though all had ongoing drug use. Studies using RNA testing alone may significantly underestimate the occurrence of GBV-C infection. Anti-GBV-C is highly associated with viral clearance and protection from reinfection.

Immunofluorescence studies on the replication of some arboviruses in nucleated and enucleated cells.

Porcine stable kidney (PS) or Vero cells infected with either flavi-(Japanese encephalitis--JE, West Nile--WN, and Dengue--DEN-2) or alphaviruses (Chikungunya--CHIK and Sindbis--SIN) were stained in indirect fluorescent antibody (FA) assay with anti-JE virus monoclonal (MoAb) Hx-3 (flavivirus cross-reactive) and polyclonal (immune PF) antibodies. By 48 hr post infection (p.i.), 15 to 20% of the three flaviviruses and CHIK virus infected cells, which revealed positive cytoplasmic immunofluorescence (IF), showed intranuclear IF. By 24 hr p.i., the intranuclear IF was not observed or became diminished. The enucleation of cells by cytochalasin B treatment prior to the infection with any of the three flaviviruses resulted in the loss of IF compared with the cells enucleated after the infection (18 hr p.i.) whereas SIN or CHIK virus-infected cells reacted similarly by the either method. These findings indicate an essential role of the nucleus in the replication of the flaviviruses only and while replicating in the infected cells, flaviviruses and CHIK virus might express viral specific proteins in the cell nuclei.