Ultrastructural study of myxoma virus morphogenesis

Summary. Poxviruses are among the largest and most complex viruses known. Vaccinia virus, the prototype of the family Poxviridae, has been studied much more than myxoma virus. The aim of this work was to have a better knowledge about myxoma virus morphogenesis. The characterization of the main stages of MV morphogenesis was achieved by ultrastructural and immunological analysis. Specific antibodies were raised against M022L and M071L, two envelope proteins of extracellular enveloped virus and intracellular mature virus, respectively. The main stages of assembly were similar to those seen with other poxviruses, and the duration of the whole replication cycle was estimated to be around 16 h, longer than what was described for vaccinia virus. Morphological changes of infected cells were associated with the development of long cellular projections and enlarged microvilli. Intracellular enveloped viruses are associated with the cytoskeleton to move through the cell. Unlike earlier studies, as many cell-associated enveloped viruses as intracellular enveloped viruses were observed in relation with specialized microvilli, although these structures were rarely noticed. Finally, an unusual spreading process was observed, which uses cytoplasmic corridors.     

Interferon-induced prolonged biochemical response reduces hepatocarcinogenesis in hepatitis C virus infection

The aim of this study was to elucidate indicator of interferon (IFN) therapy for reducing hepatocellular carcinoma (HCC) in chronic hepatitis C patients without eradicating hepatitis C virus (HCV) RNA during IFN therapy. Inclusion criteria were biopsy-proven chronic hepatitis or liver cirrhosis, IFN period for more than 1.5 years and persistently positive HCV-RNA during IFN therapy. Two hundred thirty-six patients satisfied above criteria were treated with IFN for 1.5-5 years (median 1.8 years, mean 2 years). Mean age was 55.1 years and male was 145 (61%). Eighty-one (34%) patients had severe fibrosis of the liver. These patients were prospectively monitored about HCC after the termination of IFN therapy. We regarded biochemical response (BR) as normalization of serum aminotransferase and alpha-fetoprotein for more than 1 year during IFN therapy. Cumulative rate of development of HCC after the termination of IFN therapy was 9.1% at 5th year and 26.5% at 10th year. Cox proportional analysis showed that HCC development after the termination of IFN therapy occurred when histological staging was advanced (P < 0.0001) and BR was not achieved (P = 0.009), age was >60 years (P = 0.026). The relative risk of HCC development in patients with BR was 0.36 compared with patients without BR. The attainment of BR during IFN therapy is effective in reducing hepatocarcinogenesis for patients with chronic HCV infection.     

Occult hepatitis B virus and hepatitis C virus infections

Occult HBV infection is a well-recognised clinical entity characterised by the detection of HBV-DNA in serum and/or in liver in the absence of detectable hepatitis B surface antigen (HBsAg). Occult HBV infection has been described not only in patients who have resolved an acute or chronic HBV infection but also in patients without any serological markers of a past HBV infection. Occult HBV infection in patients with chronic HCV infection may induce more severe liver disease and lower response rate to interferon treatment. The existence of occult HCV infections has been also reported more recently. Occult HCV infection is characterised by the presence of HCV-RNA in liver and peripheral blood mononuclear cells in the absence of detectable serum HCV-RNA. Occult HCV infection may occur under two different clinical situations: in hepatitis C antibody-(anti-HCV) negative and serum HCV-RNA-negative patients with abnormal liver function tests and in anti-HCV-positive patients who have no detectable serum HCV-RNA and who have normal liver enzymes. The clinical relevance of occult HCV infections is still under investigation.     

Ultrastructural aspects of the dengue virus (flavivirus) particle morphogenesis

The pathway of dengue virus infection in both mosquito and Vero cells in culture has been described. However, a number of stages associated with dengue virus morphogenesis remain unclear. For this reason further study involving electron microscopic in situ hybridisation of viral RNA and immunolocalisation of envelope proteins was carried out. The data obtained support the hypothesis that both viral RNA and viral proteins assemble when anchored to the viral-induced smooth membrane structures which occur within the lumen of the rER. Following the formation of the nucleocapsid, virus particles acquire their envelopes inside the lumen of the rER and associated structures. Some virus particles only are transferred to the Golgi system for maturation and are delivered from the cell by exocytosis. Nevertheless, the majority of virus particles do not pass the Golgi system but instead remain enclosed in rER-derived vesicles, even after cell and syncytial lysis. The virus replication pathway is a cell line independent process.     

Risk factors for hepatitis C virus infection among Koreans according to the hepatitis C virus genotype

To investigate risk factors for HCV infection according to the genotype, we studied 178 patients positive for HCV-PCR and 226 controls that were negative for the anti-HCV antibody. One hundred and twenty five controls (community control) were recruited from spouses of HCV-PCR-positive patients and the other 101 from hospital visitors (hospital control). HCV genotyping was performed by PCR, and epidemiological data were obtained from all participants. The distribution of HCV genotypes was as follows -- 1a (0.6%), 1b (39.9%), 2a (38.2%), 2b (0%), 3 (1.1%), and unclassified (20.2%). By multivariate analysis, blood transfusion (OR 2.90) and endoscopy (OR 2.80) were found to be risk factors for HCV genotype 1b versus the community control. Similarly, blood transfusion (OR 3.17) was found to be risk factors for HCV genotype 1b versus the hospital control. Blood transfusion (OR 2.75) and endoscopy (OR 3.57) were risk factors for HCV genotype 2a versus the community control, and blood transfusion (OR 4.55) and endoscopy (OR 2.16) were those versus the hospital control. Our results suggest that the risk factors for HCV infection are similar among the different genotypes. Blood transfusion and endoscopy were found to be associated with HCV infection.     

Ultrastructural studies on the morphogenesis of the Densonucleosis virus (Parvovirus)

Morphogenesis of Densonucleosis virus (DNV), a Parvovirus, has been studied in its natural host, the larvae of Galleria mellonella. This infection is characterized by the formation of dense, DNA-positive inclusions in the hypertrophied nuclei of the infected cells. This type of lesion is observed in the majority of tissues, with the exception of the midgut.The first ultrastructural changes consist of the increase in the number of free ribosomes and of the formation of “microbody-like” structures arising from the accumulation of small, round bodies of 17–20 nm inside the vesicles.In the nucleus, the nucleolus undergoes hypertrophy which is accompanied by the segregation of its fibrillar and granular components. The development of the granular portion coincides with the synthesis of the double-stranded DNA of the replicative form inside the virogenic stroma. As the infection progresses, the granular portion of the nucleolus regresses, in favor of the fibrillar portion. On the other hand, DNV seems to stimulate the formation of intranuclear bodies associated with the virogenic stroma. The virions are assembled inside the virogenic stroma and may be in relation with the nucleolus and the intranuclear bodies. Each particle encapsidates a single-strand of the replicative form, giving rise to two distinct populations of mature virions. The replication of DNV shows certain similarities with that of other Parvoviruses, particularly of the AAV and H-1.     

Molecular basis for antibody cross-reactivity between the hepatitis C virus core protein and the host-derived GOR protein.

The presence of antibodies reactive to a recently cloned host-derived antigen GOR is highly correlated with the presence of antibodies to the hepatitis C virus (HCV), We explored the molecular basis for this observation, and address the following question: are antibodies reactive with GOR_19-27 (QKAKSNPNR) a result of a cross-reactivity triggered by the antigenic region at residues 9-17 of HCV core (RKTKRNTNR)? We compared the relative antibody avidity between antibodies reactive to both regions, and determined the residues essential for antibody binding using substitution peptide analogues. Of 96 sera assayed, 60 were found positive for anti-HCV, and of these 55 were found to react with HCV core. Twenty-nine sera were found reactive to the GOR peptide, and these were all reactive to HCV core. In most cases the relative antibody avidity of antibodies reactive to GOR was higher for the HCV core peptide. In 21 of the GOR-reactive sera we were able to determine the essential residues for antibody binding. The essential residues in > 50% of all tested sera coincided with the well conserved residues Lys¹⁰, Lys¹², Asn¹⁴, and Asn¹⁶. Also, reactivity to GOR was not related to any certain serotype of antibodies to HCV. Taken together, these findings explain at the molecular level the observed cross-reactivity between these two proteins, since sequence homology per se is not evidence for cross-reactivity.     

MICA and recovery from hepatitis C virus and hepatitis B virus infections

The polymorphic MHC class I chain-related A (MICA) gene encodes a ligand that has different binding affinities for the NKG2D activating receptor of CD8+ T cells and natural killer (NK) cells. We hypothesized that MICA heterogeneity would affect recovery from hepatitis C virus (HCV) and hepatitis B virus (HBV) infections. To test the hypothesis, we initially typed known MICA polymorphisms for 228 persons who cleared HCV infection and 442 persons with persistent hepatitis C matched on other factors affecting viral persistence. Although MICA(*)015 was detected more than two-fold more often in persons with viral clearance (odds ratio 0.36, 95% confidence interval=0.19, 0.80), it occurred in fewer than 5% of the study population. In a similar analysis of 442 persons with chronic hepatitis B and 768 matched controls who recovered, MICA(*)015 was detected in 2.0% of persons with chronic hepatitis B and only 0.9% of controls. No significant associations were detected with other MICA polymorphisms. While further investigation may reveal a structural basis of the MICA(*)015 associations, these data provide little support for the hypothesis that differential distribution of MICA alleles substantially affects recovery from HCV and HBV infections.     

Dendritic cells and hepatitis C virus

Hepatitis C virus (HCV) induces chronic persistent infection that can lead to the development of hepatocellular carcinoma. We have searched for the presence of HCV genomic RNA in cells from hematopoietic origin and have, among others, documented such sequences in B cells as well as dendritic cells (DC) derived from monocytes. The allostimulatory capacity of these latter cells was found altered in chronic patients while it appeared restored in long term responders to therapy.     

Ultrastructural and physicochemical characterization of the hepatitis C virus recovered from the serum of an agammaglobulinemic patient

Summary.  Hepatitis C virus (HCV) morphology and physicochemical properties remain unclear because HCV usually circulates in a complexed form in association with immunoglobulins. In the present work, we were interested in the characterization of HCV particles derived from the serum of an anti-HCV negative/HCV RNA positive agammaglobulinemic patient suffering from chronic type C hepatitis. Physicochemical properties of the virus particles were determined by serum centrifugation on a 10 60% isopycnic sucrose density gradient. HCV RNA quantified by bDNA was found in a major peak at density 1.13 g/ml and in a minor peak at densities 1.05 1.07 g/ml. By electron microscopy, 45 nm large core-like particles were found at the 1.13 g/ml density while 60 nm large virus-like particles similar to other members of the Flaviviridae family were visualized at the 1.06 1.07 g/ml densities. This confirms some studies reporting the low density of HCV as compared to other members of the Flaviviridae family. Received April 23, 1998 Accepted June 24, 1998     

Predicting factors of present hepatitis C virus infection among patients positive for the hepatitis C virus antibody

Background/Aims

To identify the predicting factors of present hepatitis C virus (HCV) infection among patients with positivity for antibodies to HCV (anti-HCV).

Methods

We analyzed patients who showed positive enzyme immunoassay (EIA) results and performed an HCV RNA test as a confirmatory test at Kyung Hee University Hospital at Gangdong from June 2006 to July 2012. The features distinguishing the groups with positive and negative HCV RNA results were reviewed.

Results

In total, 490 patients were included. The results of the HCV RNA test were positive and negative in 228 and 262 patients, respectively. The index value of anti-HCV, mean age, platelet counts, total bilirubin, prothrombin time international normalized ratio, albumin and alanine transaminase (ALT) levels differed significantly between the two groups. On multivariable analysis, an index value of anti-HCV >10 [odds ratio (OR)=397.27, P<0.001), ALT >40 IU/L (OR=3.64, P=0.001), and albumin <3.8 g/dL (OR=2.66, P=0.014) were related to present HCV infection.

Conclusions

Although EIA is not a quantitative test, considering the anti-HCV titer with ALT and albumin levels may be helpful in predicting present of HCV infection.     

Interferon-lambda and therapy for chronic hepatitis C virus infection

Interferon (IFN)-α, a type-I IFN, is widely used to treat chronic hepatitis C virus infection, but the broad expression of IFN-α receptors often leads to adverse reactions in many organs. Here, we examine IFN-λ, a type-III IFN, as a therapeutic alternative to IFN-α. Like IFN-α, IFN-λ also induces antiviral activity in hepatocytes, but might induce fewer adverse reactions because its receptor is largely restricted to cells of epithelial origin. We also discuss the recent discovery of single nucleotide polymorphisms (SNPs) near the human IFN-λ3 gene, IL28B, that correlate strongly with the ability to achieve a sustained virological response to therapy with pegylated IFN-α plus ribavirin in patients with chronic hepatitis C.     

Type-specific antibody for hepatitis C virus detected by use of NS-4 peptide and hepatitis C virus genome in Korea

Hepatitis C virus, often possessing mutant genes, have the features that allow them to avoid host's immunologic response and further cause chronic progressive infections. Therefore, it is essential for those patients infected of HCV to receive improved diagnostic procedures. And it is equally important to investigate the course of disease progression and the response to treatment. The goal of this study is to review the efficacy of the third generation immunoblot assay and standardized RT-PCR-Hybridization assay, and in contrast with genotype identification(genotyping), the followings were briefly evaluated for the efficacy of serotype identification(serotyping) by using NS-4 peptide in the observation of the course and in the treatment of patients with HCV hepatitis. 1. The true positive rate in 132 cases showing repeated positives with 3rd generation anti-HCV EIA are 81.8% by immunoblot assay and 75.8% by RT-PCR-Hybridization assay. 2. The 79.5% concordance of immunoblot and RT-PCR-Hybridization assay is shown. The negative results from immunoblot assay are also negative in RT-PCR-Hybridization assay. 3. Among 95 patients with HCV hepatitis patients in 95 cases, the serotype 1, 2 and 4 were 53.2%, 45.2%, and 1.6%, respectively. In 29 cases, the genotypes of patients with HCV showed 1b in 15 cases, 2a/2c in 8 cases, 2b in 2 cases and mixed type in 4 cases. 4. In comparison between serotype and genotype, they showed 75.9% concordance. But serotyping showed higher efficacy in experimental procedures and sampling conditions, with more convenience. Based on above evaluation and reference review, it is reasonable to check with 3rd generation immunoblot assay the samples producing repeated positive results from anti-HCV EIA. For more definitive diagnosis of HCV infection, it is appropriate to confirm and double-check with standardized RT-PCR-Hybridization assay. Lastly, it is strongly suggested that for observation of progression and for choice of interferon treatment, serotype identification(serotyping) is more useful in practice than genotype identification (genotyping).     

Ultrastructural studies on the replication and morphogenesis of Nairobi sheep disease virus,aNairovirus

Summary TheNairovirus Nairobi sheep disease virus (NSDV) affects sheep and goats causing severe hemorrhagic gastroenteritis and high mortality. Replication and morphogenesis of NSDV was determined by electron microscopic examination of ultra-thin sections of 143B and BHK-21 cells at varying times after infection. By 4h post-infection (p.i.) of 143B cells, virions budding from the luminal side of the bilayer membrane of smooth membrane vesicles were observed. Morphologically mature virus particles were electron-dense, spherical and of uniform size (100 nm diameter) and accumulated in smooth membrane vesicles associated with the Golgi complex. In BHK-21 clone 13 cells, mature virus particles in smooth membrane vesicles were present by 8h p.i. The morphogenesis of NSDV was restricted to the smooth membrane vesicles of Golgi complex, and budding of virus from other sites was not detected. Extracellular virus particles were observed by 10h p.i., before expression of cytopathic effects. The cytopathic effects were observed at 24h p.i. in 143B cells and at 36h p.i. in BHK-21 cells. The morphology and morphogenesis of NSDV in BHK-21 cells and in 143B cells resembles that of other members of the familyBunyaviridae.