Comparison of serum hepatitis B virus replication markers in patients with chronic hepatitis B: studies on HBeAg/Anti-HBe system, viral dna polymerase and HBV-DNA

The detection of HBV-DNA in serum by molecular hybridization is the most sensitive and specific marker of replication and infectivity of hepatitis B virus and currently is proposed as a routine diagnostic technique in the follow-up of HBV - related diseases. Comparing different techniques already described, we found that direct spotting of serum samples on nitrocellulose membranes under vacuum filtration, followed by denaturing and neutralizing washes is more practical, simple, sensible and reproducible. DNA polymerase assay using phosphonoformic acid as specific viral inhibitor has shown 86.8% of concordance with HBV-DNA detection, and so, it is an useful alternative in the follow-up of hepatitis B chronic patients. We found 19.2% HBeAg positive samples with no other markers of viral replication and no anti-HBe positive sample had detectable HBV-DNA. Discordance between the 2 systems have been extensively described, and we confirm this for the first time in our country. Molecular biological techniques are essential to determine the replication status of chronic hepatitis B patients.

Ultrastructural aspects of virus replication in one fatal case and several other isolates from a dengue type 2 outbreak in Rio de Janeiro

Dengue virus replication in mosquito cell cultures was observed by electron microscopy in one fatal and 40 classical isolates from a dengue type 2 outbreak in Rio de Janeiro and compared with the prototype New Guinea C strain. All the Brazilian isolates presented, beside the classical structured dengue virus particles, fuzzy coated virus-like particles, never observed in thereferencial New Guinea C virus strain. more numerous DEN-2 virus particles, fuzzy coated virus-like particles, defective virus particles and smooth membrane structures inside the rough endoplasmic reticulum characterized the unique fatal isolate examined.

Virucidal activity of Colombian Lippia essential oils on dengue virus replication in vitro

The inhibitory effect of Lippia alba and Lippia citriodora essential oils on dengue virus serotypes replication in vitro was investigated. The cytotoxicity (CC50) was evaluated by the MTT assay and the mode of viral inhibitory effect was investigated with a plaque reduction assay. The virus was treated with the essential oil for 2 h at 37ºC before cell adsorption and experiments were conducted to evaluate inhibition of untreated-virus replication in the presence of oil. Antiviral activity was defined as the concentration of essential oil that caused 50% reduction of the virus plaque number (IC50). L. alba oil resulted in less cytotoxicity than L. citriodora oil (CC50: 139.5 vs. 57.6 μg/mL). Virus plaque reduction for all four dengue serotypes was observed by treatment of the virus before adsorption on cell. The IC50 values for L. alba oil were between 0.4-32.6 μg/mL and between 1.9-33.7 μg/mL for L. citriodora oil. No viral inhibitory effect was observed by addition of the essential oil after virus adsorption. The inhibitory effect of the essential oil seems to cause direct virus inactivation before adsorption on host cell.

Inhibition of Mayaro virus replication by prostaglandin A1 and B2 in Vero cells

The effect of prostaglandins (PGA1 and PGB2) on the replication of Mayaro virus was studied in Vero cells. PGA1 and PGB2 antiviral activity was found to be dose-dependent. However, while 10 µg/ml PGB2 inhibited virus yield by 60%, at the same dose PGA1 suppressed virus replication by more than 90%. SDS-PAGE analysis of [35S]-methionine-labelled proteins showed that PGA1 did not alter cellular protein synthesis. In infected cells, PGA1 slightly inhibited the synthesis of protein C, while drastically inhibiting the synthesis of glycoproteins E1 and E2.

Brazilian dengue virus type 1 replication in mosquito cell cultures

The development of dengue viruses type 1 obtained from accute human sera and inoculated into mosquito cell cultures, was observed by standard transmission electron microscopy and cytochemical staining. It follows the trans-type mechanism already estabilished of other dengue types. Directed passage of single virus particles across the cell membrane seems to be a pathway of entry and exit in dengue-1 infected cells. The nature of numerous electron translucent vesicles and tubules, produced simmultaneously during virus replication inside the rough endoplasmic reticulum, was analyzed by cytochemical tests. The largest amount of virus particles was produced inside cell syncytia.

Studies on the replication of Mayaro virus grown in interferon treated cells

Mayaro virus grown in interferon treated infected cells has been characterized with regard to its ability to replicate in vertebrate (TC7) and invertebrate (Aedes albopictus) cells. Virus purified from interferon treated TC7 cells adsorbs and penetrates to the same extent as the control virus. During infection, these virus particles caused inhibition of host protein synthesis and synthesized the same spectrum of viral proteins as normal virus. This population however, was apparently more sensitive to interferon treatment. Electron microscopy of TC7 cells showed the presence of numerous aberrant virus particles budding from the plasma membrane.

Prostaglandin A1 Inhibits Replication of Classical Swine Fever Virus

Prostaglandins (Pgs) have been shown to inhibit the replication of several DNA and RNA viruses. Here we report the effect of prostaglandin (PgA1) on the multiplication of a positive strand RNA virus, Classical Swine Fever Virus (CSFV) in PK15 cells. PgA1 was found to inhibit the multiplication of CSFV. At a concentration of 5 µg/ml, which was nontoxic to the cells, PgA1 inhibitis virus production in 99%. In PgA1 treated cells the size and number of characteristic Classical Swine Fever focus decreased in amount.

Detection of replication-defective hepatitis A virus based on the correlation between real-time polymerase chain reaction and ELISA in situ results

ELISA in situ can be used to titrate hepatitis A virus (HAV) particles and real-time polymerase chain reaction (RT-PCR) has been shown to be a fast method to quantify the HAV genome. Precise quantification of viral concentration is necessary to distinguish between infectious and non-infectious particles. The purpose of this study was to compare cell culture and RT-PCR quantification results and determine whether HAV genome quantification can be correlated with infectivity. For this purpose, three stocks of undiluted, five-fold diluted and 10-fold diluted HAV were prepared to inoculate cells in a 96-well plate. Monolayers were then incubated for seven, 10 and 14 days and the correlation between the ELISA in situ and RT-PCR results was evaluated. At 10 days post-incubation, the highest viral load was observed in all stocks of HAV via RT-PCR (10(5) copies/mL) (p = 0.0002), while ELISA revealed the highest quantity of particles after 14 days (optical density = 0.24, p < 0.001). At seven days post-infection, there was a significant statistical correlation between the results of the two methods, indicating equivalents titres of particles and HAV genome during this period of infection. The results reported here indicate that the duration of growth of HAV in cell culture must be taken into account to correlate genome quantification with infectivity.

Peroral susceptibility of Aedes albifasciatus and Culex pipiens complex mosquitoes (Diptera: Culicidae) from Argentina to western equine encephalitis virus

The transmission cycle of western equine encephalitis (WEE) virus in South America is unknown. A WEE virus strain was isolated from Aedes albifasciatus in Argentina during the WEE epizootic of 1982-83. Also, Culex pipiens from Argentina was reported to be able to transmit WEE virus experimentally, but other results indicate that Cx. pipiens from the USA is refractory to this virus. We determined the susceptibility of Argentina strains of Ae. albifasciatus and Culex pipiens complex mosquites to infection by WEE virus by the oral route. Adult females were fed on chicks infected with a WEE virus strain isolated in Cordoba Province, Argentina, or were fed on a blood/virus suspension. Each mosquito ingested between 10(1.6) to 10(6.4) vero cell plaque-forming units of virus. Each of 28 Ae. albifasciatus was positive for virus from the fourth day postfeeding, and there was evidence for virus replication. In contrast, 0/44 Cx. p. quinquefasciatus and only 1/15 Cx. p. pipiens was positive. Aedes albifasciatus is susceptible to infection by WEE virus and should be considered a potential vector of this virus in Argentina. Both subspecies of Cx. pipiens are refractory to peroral infection by WEE virus and probably do not play a role in the WEE virus cycle in Argentina.

RC-IAL cell line: sensitivity of rubella virus grow

OBJECTIVE: The rapid growth of the rubella virus in RC-IAL² with development of cytopathic effect, in response to rubella virus infection, is described. For purposes of comparison, the rubella virus RA-27/3 strain was titered simultaneously in the RC-IAL, Vero, SIRC and RK13 cell lines. METHODS: Rubella virus RA-27/3 strain are inoculated in the RC-IAL cell line (rabbit Kidney, Institute Adolfo Lutz). Plates containing 1.5x10(5) cells/ml of RC-IAL line were inoculated with 0.1ml s RA-27/3 strain virus containing 1x 10(4)TCID50/0.1ml. A 25% cytopathic effect was observed after 48 hours and 100% after 96 hours. The results obtained were compared to those observed with the SIRC, Vero and RK13 cell lines. Rubella virus was detected by immunohistochemistry. RESULTS: With the results, it was possible to conclude that the RC-IAL cell line is a very good substrate for culturing rubella virus. The cells inoculated with rubella virus were examined by phase contrast microscopy and showed the characteristic rounded, bipolar and multipolar cells. The CPE in RC-IAL was observed in the first 48 hours and the curve of the increased infectivity was practically the same as observed in other cell lines. CONCLUSIONS: These findings are important since this is one the few cell lines described in the literature with a cytopathic effect. So it can be used for antigen preparation and serological testing for the diagnosis of specific rubella antibodies.

Replication of dengue viruses in mosquito cell cultures: a model from ultrastructural observations

Mosquito cell cultures infected with human sera from dengue-1 and dengue-2 outbreaks, started in Rio de Janeiro by 1986 and 1990 respectively, were examined by electron microscopy at different times post the infection of cell cultures. More information was obtained about cell penetration of virus particles in the presence or not of antibodies, their pathway inside the cells, replication mode and exit. Infectiveness of the virus at those different stages can only be attributed to the particles appearing inside the trans-Golgi vesicles; most of all newly formed virus particles remain inside the RER-derived cell vesicles or inside lysosomes, even during cell lysis. Groups of larges particles, 65-75 nm in diameter at dengue-2 infections, persist during cell passage. The large amounts of smooth membrane structures, as vesicles or tabules inside the RER are attributed to a cell response to viral infection.

In situ hybridization of hepatitis C virus RNA in liver cells of an experimentally infected rhesus macaque

The liver tissue of a rhesus macaque inoculated with hepatitis C virus (HCV) has been analyzed for the presence of HCV RNA using the technique of in situ hybridization, both at light and electron microscopy levels. The animal was inoculated by the intrasplenic route using a HCV infected autogenic hepatocyte transplant. The serum sample used to infect the hepatocyte cells was characterized by polymerase chain reaction technique and shown to be positive for HCV RNA, genotype 3 with 10(7) RNA copies/ml. In situ hybridization was performed using a complementary negative strand probe made with the specific primer. We were able to detect and localize viral RNA in altered membranes of the rough endoplasmic reticulum of infected liver cells, showing evidence of virus replication in vivo.

Coat protein and RNAs of Cole latent virus are not present within chloroplasts of Chenopodium quinoa-infected cells

Cole latent virus (CoLV), genus Carlavirus, was studied by electron microscopy and biochemical approaches with respect both to the ultrastructure of the Chenopodium quinoa infected cells and to its association with chloroplasts. The CoLV was observed to be present as scattered particles interspersed with membranous vesicles and ribosomes or as dense masses of virus particles. These virus particles reacted by immunolabelling with a polyclonal antibody to CoLV. Morphologically, chloroplasts, mitochondria and nuclei appeared to be unaltered by virus infection and virus particles were not detected in these organelles. However, virus particle aggregates were frequently associated with the outer membrane of chloroplasts and occasionally with peroxisomes. Chloroplasts were purified by Percoll gradient, and the coat protein and virus-associated RNAs were extracted and analyzed by Western and Northern blots respectively. Coat protein and CoLV-associated RNAs were not detected within this organelle. The results presented in this work indicate that the association CoLV/chloroplasts, observed in the ultrastructural studies, might be a casual event in the host cell, and that the virus does not replicate inside the organelle.

Sugarcane yellow leaf virus infection leads to alterations in photosynthetic efficiency and carbohydrate accumulation in sugarcane leaves

Infection by Sugarcane yellow leaf virus (ScYLV) causes severe leaf symptoms in sugarcane (Saccharum spp.) hybrids, which indicate alterations in its photosynthetic apparatus. To gain an overview of the physiological status of infected plants, we evaluated chlorophyll a fluorescence and gas exchange assays, correlating the results with leaf metabolic surveys, i.e., photosynthetic pigments and carbohydrate contents. When compared to healthy plants, infected plants showed a reduction in potential quantum efficiency for photochemistry of photosystem (PSII) and alterations in the filling up of the plastoquinone (PQ) pool. They also showed reduction in the CO2 net exchange rates, probably as a consequence of impaired quantum yield. In addition, reductions were found in the contents of photosynthetic leaf pigments and in the ratio chlorophyll a/chlorophyll b (chla/chlb). Carbohydrate content in the leaves was increased as a secondary effect of the ScYLV infection. This article discusses the relation of virus replication and host defense responses with general alterations in the photosynthetic apparatus and in the metabolism of infected plants.