Tamarin virus

Hepatocyte cultures from tamarin grown on glass coverslips were infected with the day 16 inoculum on day 3 postplating and were harvested 4 days after infection. Immunofluorescence staining for NS3 protein. Hepatocytes grown on glass coverslips were inoculated with GBV-B 3 days postplating and harvested 3 days postinfection unless noted otherwise. To further demonstrate the specificity of the staining reaction, we performed blocking experiments in which the antiserum was adsorbed with either GST or NS3 lacking GST prior to being used in staining.

Unadsorbed antiserum again yielded a bright cytoplasmic staining Fig. In contrast, adsorption of the antiserum with purified NS3 completely eliminated the specific fluorescent staining Fig. Hepatocytes were inoculated with GBV-B and harvested 3 days p. Lower-magnification photographs of the cultures revealed that a high percentage of cells were infected in some fields Fig.

This represents efficient infection, since as described above, the standardized inoculum used in these experiments represented a multiplicity of infection of 0. To evaluate the spread of infection over time, cultures were inoculated with a fold-lower multiplicity of infection and were harvested at various times over a 3-week period.

Despite the extensive spread of infection over a 3-week period, some cells appeared to be resistant to infection. These cells may have lost hepatocyte differentiated functions required for infection, or they may have been protected by the induction of interferon in infected cells. The past decade has witnessed rapid advances in our understanding of HCV epidemiology, pathogenesis, and molecular biology of replication. The advances in molecular biology of replication have depended on the use of model systems to examine internal ribosome entry site function, polyprotein processing, the functions of individual proteins, and their interactions with the host cell.

The primary limiting factor in advances at this time is the lack of an adequate culture system. The development of infectious cDNA clones of HCV have provided a new means to develop tissue culture systems for genetic analysis of replication 3 , 11 , 13 , 29 — 31 ; however, this potential has not yet been realized.

A single system for the selection of cells that maintain high levels of replication of an HCV replicon expressing the nonstructural proteins has been described 20 , but widespread application of this technology has not been achieved. A number of viruses can serve as potential surrogates for HCV, including bovine viral diarrhea virus, a member of the pestivirus family.

This study significantly advances the GBV-B model through the development of a tissue culture system with high levels of replication and efficient in vitro infection.

Our previous studies with HCV replication in primary chimpanzee hepatocytes provided some of the most convincing evidence for in vitro replication of HCV; however, this system was limited in many aspects 16 , The level of replication rarely exceeded the quantity of virus present in inoculum, and the virus remained primarily cell associated. The GBV-B system described in this study was not dependent on strand-specific assays, since cell-associated viral RNA increased as much as fold in comparison to the input inoculum Fig.

In addition, the expression of NS3 could readily be detected by immunofluorescence. The high levels of GBV-B replication coupled with quantitative, real-time TaqMan RT-PCR provide an opportunity to optimize this system to maintain the high levels of replication observed in the first 2 weeks and potentially to define the limiting factors required for replication.

Use of the serum-free medium will permit studies on the impact of alterations in the levels of growth factors and hormones on replication as well as the effects of various cytokines. The recent development of infectious cDNA clones of GBV-B 6 ; Stanley Lemon, personal communication also suggests that this system will be useful for genetic analysis of replication.

Unlike HCV, it can be anticipated that transfection of primary tamarin hepatocytes with synthetic RNA from an infectious clone will lead to high levels of replication.

Chimeric viruses would certainly improve the utility of the system for the screening of HCV antiviral drugs, if such clones are infectious. The failure to develop chimeric clones between different genotypes of HCV 30 suggests that replacement of limited domains containing catalytic motifs would more likely be infectious than those containing global replacements.

In contrast, the ability of the dengue virus NS5 protein to provide function in trans to Kunjin virus replicons lacking a portion of the NS5 domain 12 suggests that such replacements are feasible. In addition, the high level of homology of GBV-B with HCV within certain motifs suggests that hybrid clones may not be required for the testing of some antiviral compounds in this system.

In addition, preliminary data indicate that both interferon and ribavirin are capable of suppressing GBV-B replication in this culture system unpublished data. The GBV-B tamarin model will also provide a valuable animal model in which to examine pathogenesis, antiviral therapy, and immune clearance of the virus.

Viral clearance occurred in both animals within 12 to 14 weeks. The ALT levels were significantly elevated within 2 weeks p. Whether the high levels of GBV-B replication are to some degree cytopathic or whether the early rise in ALT was due to an innate or specific immune response to infected cells was not determined in these studies. No overt cytopathic effect was observed in vitro in infected hepatocytes.

This does not exclude a low level of cytopathic effect or a slow but accelerated death of infected cells, since primary cultures of hepatocytes always contain some dying cells. The spread of the infection over 21 days to yield cultures that are uniformly positive for NS3 by immunofluorescence argues against a significant cytopathic effect, but detailed studies on the induction of apoptotic markers should be conducted before it is concluded that no cytopathic consequences of viral infection exist.

Seroconversion for anti-NS3 antibodies did not occur until 8 weeks p. Similar infection profiles for GBV-B have been reported previously This rapid viral clearance, rapid loss of antibody profile is very similar to those that we have recently described for chimpanzees inoculated with HCV 2.

Whether similar profiles occur in HCV-infected humans is difficult to determine, since the time of infection is rarely known in humans, and all viral markers are lost within months of infection. However, a number of studies support a higher level of viral clearance in humans than previously suspected 4 , 8 , At this time, chronic infections with GBV-B have not been reported.

The high levels of replication could contribute to a more intense immune response that favors viral clearance. Alternatively, there may be other intrinsic differences between the viruses and their hosts that lead to viral clearance.

If GBV-B is transmitted in nature in tamarins primarily through a parenteral route with low levels of sexual and maternal transmission, as is observed with HCV in humans, it is difficult to imagine maintenance of the virus in a population without some chronic infections, unless other animals serve as a reservoir for the virus. This of course assumes that tamarins are indeed the natural host for GBV-B. At this time too few animals have been followed long term with sensitive RT-PCR assays to exclude the possibility for a low percentage of chronic infections.

The potential exists for the induction of chronic infections through the use of transient immunosuppression with cyclosporin or FK Chronically infected animals would certainly be useful in a number of studies including the evaluation of antiviral drugs.

Perhaps one of the most promising potential applications of the tamarin model and the tissue culture system described in this report is development of an in vitro neutralization assay and an animal model for evaluating HCV vaccine approaches. We observed protective immunity in animals that had cleared the infection and were rechallenged, although a very transient low-level viremia was observed.

Whether this protection is due to a humoral or cellular immune response remains to be determined. Even if protection was provided by cellular immunity in these animals, the in vitro culture system provides an opportunity to rapidly evaluate the neutralizing potential of serum from small animals immunized with various envelope protein configurations.

National Center for Biotechnology Information , U. Journal List J Virol v. J Virol. Brasky , 2 and Robert E. Kathleen M. Robert E. Author information Article notes Copyright and License information Disclaimer. Loop , San Antonio, TX Phone: Fax: E-mail: gro. Received May 2; Accepted Sep This article has been cited by other articles in PMC.

Abstract GB virus-B GBV-B causes an acute hepatitis in tamarins characterized by increased alanine transaminase levels that quickly return to normal as the virus is cleared. Hepatitis GB inoculum. Hepatocyte cultures. Course of GBV-B infection in tamarins.

Open in a separate window. GBV-B rechallenge of previously infected tamarins. GBV-B replication in in vivo-infected hepatocytes.

Susceptibility of normal tamarin hepatocytes for in vitro GBV-B infection. Estimation of doubling time for GBV-B. Time of maximum susceptibility for in vitro infection. Titration of inoculum. Infectivity of tissue culture derived GBV-B. Cultures were harvested day 7 postinfection.

ND, not determined. Immunofluorescence of in vitro-infected hepatocytes. The incidence of transfusion-associated hepatitis G virus infection and its relation to liver disease. Dr Shriram Raghavan from Jananom Pvt Ltd, a science startup in Kovaipudur, Tamil Nadu believes that the local diet could be a reason why the state has seen fewer deaths due to Covid Dr Arunansu Talukdar, another panelist and professor general medicine at Kolkata Medical College, agreed with Dr Raghavan and added that taking supplements rich in zinc, vitamins A, C, B6, iron etc can help in boosting immunity and offering a stronger defence against the infection.

He cited example of rural communities in Bengal and Assam who eat lot of green leafy vegetables and green chillies — rich in anti-oxidants — in their daily diet and exhibit stronger immunity against infectious diseases. Read also: Phased lifting of lockdown step in right direction, says Dale Fisher.

Facebook Twitter Linkedin EMail. Looking for Something? Start a Conversation. Sudoku Play Now. Word Search Play Now. Antiviral activity of the tamarind seed lectin has been shown by techniques such as luciferase-based assay, plaque reduction assay and real-time PCR.

The research group at IIT Roorkee has filed a patent for an antiviral composition containing this tamarind antiviral protein. The researchers are now developing tamarind-based therapeutic agents for chikungunya. Click here for more Education News. Latest Exams School Campus.

IIT Roorkee Chikungunya.