Morphology of hepatitis C and hepatitis B virus particles as detected by immunogold electron microscopy

We performed indirect immunogold electron microscopy (EM) for immunological identification and characterization of hepatitis C virus (HCV). To clarify the morphology of HCV, an indirect immunogold EM of two plasma samples from patients with high HCV RNA titers was carried out using antibodies specific for the putative HCV envelope protein (E) 1. Spherical virus particles 55–65 nm in diameter with delicate spike projections were detected in the 1.14–1.16 g/ml fractions after sucrose density gradient centrifugation. Polyclonal and monoclonal antibodies to the putative HCV E1 specifically recognized these particles. In addition, immunogold EM of the samples was also performed to uncover the morphology of HCV core particles. Spherical particles 33–40 nm in diameter (average, 37 nm) were detected in the 1.22- to 1.25-g/ml fractions by conventional EM after sucrose density gradient centrifugation. Immunogold EM using rabbit polyclonal antibody (RR8) specific for the putative HCV core protein and colloidal gold-labeled goat antirabbit IgG showed binding of the gold particles with RR8. Some of the HCV core particles showed icosahedric morphology. Optical rotation technique showed that the HCV core particles exhibit sixfold symmetry and that the length of the regular hexagon side is approximately 20 nm, suggesting that they have an icosahedric structure. Further, the detection limit of the indirect immunogold EM was evaluated in 11 plasma samples from chronic hepatitis B patients with different degrees of hepatitis B virus (HBV) DNA titers using antihepatitis B surface antigen antibody. The study showed that the detection limit of virus using this method is 10⁷ virions/ml.     

Unidentified virus-like particles are detected in plasmas with elevated ALT levels: are they significant of etiological agent(s) of non-B,non-C hepatitis?

GB virus C (GBV-C) and hepatitis G virus (HGV) have been proposed as new viruses etiologically implicated in non-B, non-C hepatitis, but the morphology of these particular virus particles is still unknown, and most cases of non-A to E hepatitis do not relate to their infections. We tried to visualize virus-like particles (VLPs) in plasma samples from hepatitis B surface antigen- and antibody to hepatitis C virus (HCV)-negative blood donors with elevated alanine aminotransferase (ALT), and examined the association of the virus-like particles and the genomes of parenterally transmissible GBV-C/HGV. Twenty-three plasma samples, 13 with elevated ALT levels and 10 with normal ALT values, from blood donors without infections of hepatitis B virus (HBV) and HCV, were subjected to a 20%–60% sucrose density gradient centrifugation, and virus-like particles were observed by electron microscopy. GBV-C/HGV RNAs in the plasmas were tested. Virus-like particles were found in the fractions with densities of 1.15–1.16 g/ml from 12 of 13 (92.3%) plasmas with elevated ALT levels and 1 of 10 (10%) normal controls. The ultrastructural morphology of visualized VLPs was pleomorphic in size and appearance; the majority of the VLPs were 50- to 80-nm spherical particles with a 35- to 45-nm inner core and 9- to 12-nm-long surface spikelike projections. Rodlike VLPs 50–70 nm in diameter with a length of 110–160 nm were also observed in the same samples. The incidence of detection of the circulating VLPs was significantly (P < 0.001) related to elevated ALT levels, but GBV-C/HGV RNAs were detected in none of the plasmas containing the virus-like particles. Spherical VLPs are detected in HBV- and HCV-negative plasmas significantly correlated with the elevation of ALT, suggesting that they are implicated in non-B, non-C hepatitis.     

Effects of bafilomycin A1 on Japanese encephalitis virus in C6/36 mosquito cells

Summary.  Involvement of intracellular acidic compartments in the early phase of Japanese encephalitis (JE) virus infection of C6/36 mosquito cells was examined by bafilomycin A1, a specific inhibitor of vacuolar type H+-ATPase (V-ATPase). Dose dependent reduction of viral envelope protein (E) produced into the infected culture fluid was observed by pretreating the cells with 0.25 to 1.0 μM bafilomycin Al. In synchronized infection, cell surface-bound virions were internalized immediately by heating at 31 °C, followed by the release of nucleocapsid into the cytosol within a short lag period. Subcellular distribution of infecting ²H-uridine-labeled viral RNA (V-RNA) and its RNase sensitivity were analyzed by fractionation in Percoll density gradient centrifugation. At a 10 min chasing period, an RNase resistant V-RNA peak was found in fractions with a mean density of 1.05g/ml corresponding to the endosome, while an RNase sensitive V-RNA peak was detected at density range of 1.052–1.054 g/ml corresponding to the ribosome in C6/36 cell homogenate. The results indicate that JE virus infection in C6/36 cells proceeded through the endocytic pathway involving intracellular acidic compartments which was affected by bafilomycin A1. Received December 19, 1997 Accepted March 19, 1998     

Characterisation of a virus from Australia that is closely related to papaya mosaic potexvirus

Summary.  We have isolated a previously undescribed potexvirus from Alternanthera pungens (Amaranthaceae) in southern Queensland, Australia. This virus was shown to have a moderately wide experimental host range, infecting plants in nine of the twelve families tested. Using specific antibodies, a plate trapped antigen ELISA was developed, allowing detection of virions down to 0.8 μg/ml of leaf extract. Virions averaged 554 nm long and had a capsid protein with a M_r of 23.1 × 10³. A portion of the genome containing the capsid protein ORF and 3′ untranslated region was cloned and sequenced. From both serological and amino acid sequence comparisons, the virus was shown to be closely related to papaya mosaic potexvirus (PMV). To determine the taxonomic status of the virus, we assessed variation in the amino acid sequence of capsid proteins of distinct species within the potexvirus genus, as well as variation between strains of the same virus. When the core region of the capsid proteins were compared, distinct species had a maximum of 62.2% sequence identity, whereas strains had a minimum of 88.8% identity. By comparison, the core region of the capsid proteins of the Alternanthera virus and PMV had 79.8  identity. We have concluded that the Alternanthera virus is a different species from PMV, and its relationship with PMV resembles that of potyvirus subgroup members. Accepted August 3, 1998 Received May 22, 1998     

Hepatitis C virus particles of different density in the blood of chronically infected immunocompetent and immunodeficient patients: Implications for virus clearance by antibody

The purpose of this study was to analyse the influence of the humoral immune response on the generation and clearance of hepatitis C virus (HCV) RNA containing particles in the blood of chronically infected patients. Blood samples were fractionated by sequential flotation ultracentrifugation and HCV RNA was recovered in three fractions: low density of < 1.063 g/ml, intermediate density of 1.063-1.21 g/ml, and high density of > 1.21 g/ml. Serum low-density lipoproteins co-fractionated with the low-density particles, and high-density lipoproteins co-fractionated with the intermediate-density particles. Immunoglobulins were found exclusively in the high-density fractions. In patients with congenital immunodeficiencies, with no or low serum antibodies to the virus, mean HCV RNA titres were equal in each fraction, at approximately 10(5) IU/ml. In antibody-positive, immunocompetent patients, however, virus titres in the low-density fraction and those in the high-density fraction were reduced or absent in most patients, suggesting that virus particles in these fractions are subject to antibody-mediated clearance. Particles of intermediate density were approximately equal in titre in both patient groups, suggesting that these particles are neither generated by, nor cleared, as a result of the humoral immune response. Immunoprecipitation experiments indicated that particles of intermediate density were not complexed with either high-density lipoprotein or immunoglobulins. Elucidation of the mechanisms by which these particles are generated and maintained in the blood may provide valuable insight into the mechanism of virus persistence.     

Narcissus symptomless virus: a new carlavirus of daffodils

Summary. A filamentous virus, with particles 600–650 nm long, was purified from Narcissus pseudonarcissus (daffodil) in Hangzhou and an antiserum prepared. After mechanical inoculation, the virus could be detected serologically in Narcissus species but not in some commonly used virus indicators. Infection was symptomless. The complete sequence of the genomic RNA (8281 nt) showed six predicted ORFs typical of carlaviruses. Pairwise comparisons of gene sequences and phylogenetic analysis demonstrated that the new virus should be classified as a carlavirus but that it was not closely related to members of any current species. We propose the name Narcissus symptomless virus (NSV).     

Enveloped particles in the serum of chronic hepatitis C patients

HCV particles were isolated from the plasma of chronically infected patients. The virus was analysed by sucrose density gradient centrifugation. The fractions were tested for viral RNA, core antigen and envelope proteins by using a monoclonal antibody directed against the natural E1E2 complex (D32.10). Two populations of particles containing RNA plus core antigen were separated: the first with a density of 1.06-1.08 g/ml did not contain the envelope proteins; the second with a density between 1.17 and 1.21 g/ml expressed both E1 and E2 glycoproteins. Electron microscopy of the enveloped population after immunoprecipitation with D32.10 showed spherical particles with a rather featureless surface and with a diameter around 40 nm. Immuno-gold staining gave evidence that the E1E2 complex was indeed positioned at the surface of these particles.     

Synergism in replication of cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) RNA in orchid protoplasts

Summary.  An in vitro orchid protoplast isolation method to study replication kinetics of CymMV and ORSV was developed. This method allows the isolation of viable and raphid-free petal protoplasts from an orchid hybrid, {\it Dendrobium} Sonia (Dendrobium Caesar × Dendrobium Tomie Drake). The optimum field strength for both viral RNA to achieve good efficiency of electroporation was 750 V/cm and the optimum viral RNA concentration required was 1 μg and 4 μg per 2 × 10⁶ protoplasts for CymMV and ORSV, respectively. Autoradiographs of Northern blots depicting the viral genomic and subgenomic RNA in the extracts, referred to as the “Replication Footprint Profiles” (RFP) of specific CymMV/ORSV virus were prepared at different time intervals. Viral RNA synthesis reached a maximum at 18 h for CymMV and 24 h for ORSV. When CymMV and ORSV viral RNA were electroporated into the protoplasts simultaneously, detection signals of both the positive and negative strand viral RNA increased as compared to the singly infected protoplasts. Thus, synergism in replication of CymMV and ORSV was observed in orchid protoplasts. Received November 20, 1997 Accepted February 25, 1998     

Molecular characterization and interviral relationships of a flexuous filamentous virus causing mosaic disease of sugarcane (Saccharum officinarum L.) in India

Summary.  A virus isolate causing mosaic disease of commercial sugarcane was purified to homogeneity. Electron microscopy revealed flexuous filamentous virus particles of ca 890 × 15 nm. The virus isolate reacted positively with heterologous antiserum to narcissus latent virus form UK, but failed to react with potyvirus group specific antiserum. N-terminal sequencing of the intact coat protein (CP) and the tryptic peptides indicated that the virus was probably a potyvirus but distinct from several reported potyviruses. Comparison of the 3′-terminal 1084 nucleotide sequence of the RNA genome of this virus revealed 93.6% sequence identity in the coat protein coding region with the recently described sugarcane streak mosaic virus (Pakistani isolate). The molecular weight of the coat protein (40 kDa) was higher than that deduced from the amino acid sequence (34 kDa). The apparent increase in size was shown to be due to glycosylation of the coat protein which has not been reported thus far in the family, Potyviridae. This is the first report on the molecular characterization of a virus causing mosaic disease of sugarcane in India and the results demonstrate that the virus is a strain of sugarcane streak mosaic virus, a member of the Tritimovirus genus of the Potyviridae. We have named it sugarcane streak mosaic virus – Andhra Pradesh isolate (SCSMV-AP). Received October 14, 1997 Accepted August 7, 1998     

Binding of human lipoproteins (low, very low, high density lipoproteins) to recombinant envelope proteins of hepatitis C virus

Heterogeneities in the density of hepatitis C virus (HCV)-RNA-carrying material from human sera (1.03–1.20 g/ml) are partially due to the binding of lipoproteins [low density (LDL), very low density (VLDL), high density (HDL) lipoproteins] and immunoglobulins. In this study we demonstrate the binding of recombinant HCV envelope protein (E1/E2) to human LDL, VLDL and HDL on a molecular basis. The binding of lipoproteins was restricted to the middle part of the E1 gene product (amino acids 222–336) and the C-terminal part of the E2 protein (amino acids 523–809). Lipoproteins did not bind to recombinant HCV core protein. Received: 22 December 1999     

Establishment of infectious HCV virion-producing cells with newly designed full-genome replicon RNA

Hepatitis C virus (HCV) replicon systems enable in-depth analysis of the life cycle of HCV. However, the previously reported full-genome replicon system is unable to produce authentic virions. On the basis of these results, we constructed newly designed full-genomic replicon RNA, which is composed of the intact 5'-terminal-half RNA extending to the NS2 region flanked by an extra selection marker gene. Huh-7 cells harboring this full-genomic RNA proliferated well under G418 selection and secreted virion-like particles into the supernatant. These particles, which were round and 50?nm in diameter when analyzed by electron microscopy, had a buoyant density of 1.08?g/mL that shifted to 1.19?g/mL after NP-40 treatment; these figures match the putative densities of intact virions and nucleocapsids without envelope. The particles also showed infectivity in a colony-forming assay. This system may offer another option for investigating the life cycle of HCV.     

Identification and characterisation of tomato torrado virus,a new plant picorna-like virus from tomato

Summary. A new virus was isolated from tomato plants from the Murcia region in Spain which showed symptoms of ‘torrado disease’; very distinct necrotic, almost burn-like symptoms on leaves of infected plants. The virus particles are isometric with a diameter of approximately 28 nm. The viral genome consists of two (+)ssRNA molecules of 7793 (RNA1) and 5389 nts (RNA2). RNA1 contains one open reading frame (ORF) encoding a predicted polyprotein of 241 kDa that shows conserved regions with motifs typical for a protease-cofactor, a helicase, a protease and an RNA-dependent RNA polymerase. RNA2 contains two, partially overlapping ORFs potentially encoding proteins of 20 and 134 kDa. These viral RNAs are encapsidated by three proteins with estimated sizes of 35, 26 and 23 kDa. Direct protein sequencing mapped these coat proteins to ORF2 on RNA2. Phylogenetic analyses of nucleotide and derived amino acid sequences showed that the virus is related to but distinct from viruses belonging to the genera Sequivirus, Sadwavirus and Cheravirus. This new virus, for which the name tomato torrado virus is proposed, most likely represents a member of a new plant virus genus.     

Complete nucleotide sequence of the genome organization of RNA2 of patchouli mild mosaic virus, a new favavirus

Summary.  The complete nucleotide sequence and the genome organization of the RNA 2 of a patchouli mild mosaic virus (PaMMV) was determined. The sequence consists of 3591 nucleotides and contains a single long open reading frame sufficient to code for 118K protein. Three proteins of 52 K, 44 K and 22 K could be encoded by the PaMMV RNA 2 genome. Our analysis of the N-terminal sequences of two species of coat protein (CP) allowed precise location of the CP cistrons within the polyprotein. 44 K and 22 K proteins are the coat proteins. The positions of the cleavage sites are Gln/Ala between 44 K and 22 K coat proteins and Gln/Gly between 52 K and 44 K proteins. Comparison of PaMMV RNA 2 with comoviral and nepoviral RNA 2 showed no sequence similarity. These results as well as previous serological studies strongly suggest that PaMMV is a member in the genus Fabavirus. Received May 29, 1998 Aaccepted July 20, 1998     

Virus-like particles of calicivirus as epitope carriers

Summary.  The VP60 of rabbit haemorrhagic disease virus (RHDV), when expressed in baculovirus, self-assembles into virus-like particles (VLP) which are antigenically and immunogenically indistinguishable from native virions. When the N-terminal 30 amino acid residues of VP60 were deleted and substituted by a well characterized six residue epitope from bluetongue virus capsid protein VP7 (Btag), the fusion protein retained its ability to self-assemble into VLPs. However, the size of these particles was only 27 nm, compared to 40 nm of VLPs derived from native VP60. The antigenicity of both VP60 and the Btag was retained as evident from ELISA and Western blot analyses. When Btag was fused at the C-terminus of VP60 without deletion, the fusion proteins formed VLPs of 40 nm in size and also retained their antigenicity, but the Btag antigenicity appeared weak at this fusion site. Received December 9, 1998/Accepted March 19, 1999     

Neolignans inhibit Trypanosoma cruzi infection of its triatomine insect vector, Rhodnius prolixus

Two neolignans, burchellin and nordihydroguaiaretic acid (NDGA), were toxic only to Trypanosoma cruzi clone Dm28c maintained in brain heart infusion (BHI) medium at a concentration of 100 μg/ml, not 10 μg/ml. When Rhodnius prolixus was fed with epimastigotes of T. cruzi and treated simultaneously with a single dose of burchellin or NDGA at 10 μg/ml of blood meal the number of parasites in the gut decreased. Whereas burchellin was only partially active, NDGA drastically reduced the number of epimastigotes and metacyclic trypomastigotes of T. cruzi in the excreta (urine plus feces). When the insect larvae were pretreated with burchellin or NDGA at 20 days before the infection with T. cruzi a significant reduction in the number of parasites in the gut occurred. However, when both compounds were applied at 20 days after the establishment of T. cruzi infection, although burchellin significantly reduced the gut infection, neither compound could abolish the infection entirely within the subsequent 15 days. Received: 15 June 1998 / Accepted: 15 August 1998     

Complete nucleotide sequences and genome characterization of double-stranded RNA 1 and RNA 2 in the Raphanus sativus-root cv. Yipinghong

Summary. Four distinct double-stranded (ds) RNA bands were extracted from leaves of Raphanus sativus-root cv. Yipinghong with yellowing at the leaf edge in China. Purified viral particles of 28–30 nm in diameter contained dsRNA segments with the same number and mobility as these extracted directly from radish leaves. The two major dsRNA segments, namely RasR 1 and RasR 2, were 1866 and 1791 bp in length, respectively. Computer analysis predicted that they both contained a single open reading frame (ORF) on their plus-stranded RNA, putatively encoding a RNA dependent RNA polymerase and a capsid protein similar to that encoded by members of the family Partitiviridae. In addition, both RasR 1 and RasR 2 were highly conserved at the 5′ untranslated regions (UTR) and had an adenosine-uracil rich stretch at the 3′ UTR, with an identical terminal motif (5′-AAAAUAAAACC-3′). Taken together, these results suggest that the two major dsRNA segments constitute the genome of a partitivirus infecting radish.     

Characterization of the large (L) RNA of peanut bud necrosis tospovirus

Summary.  The nucleocapsids purified from peanut plants systemically infected with peanut bud necrosis virus (PBNV), a member of the genus Tospovirus, contained both viral(v) and viral complementary(vc) sense L RNAs. Defective forms of L RNA containing ‘core polymerase region’ were observed. The full length L RNA of PBNV was sequenced using overlapping cDNA clones. The 8911 nucleotide L RNA contains a single open reading frame (ORF) in the vc strand, and encodes a protein of 330 kDa. At the 5′ and 3′ termini of the v sense RNA there were 247 and 32 nt untranslated regions, respectively, containing an 18 nt complementary sequence with one mismatch. Comparisons of the predicted amino acid sequence of the L protein of PBNV with other members of Bunyaviridae suggest that the L protein of PBNV is a viral polymerase. The L protein had highest identity in the ‘core-polymerase domain’ with the corresponding regions of other tospoviruses, tomato spotted wilt virus and impatiens necrotic spot virus. Received October 17, 1997 Accepted June 16, 1998