Cell-associated Strain Research Articles

P30: Investigation of HIV-1 drug-resistant variants in plasma bulk and cell-associated strains ultra-deep sequencing from blood donors in Recife, Brazil

P30: Investigation of HIV-1 drug-resistant variants in plasma bulk and cell-associated strains ultra-deep sequencing from blood donors in Recife, Brazil

Genotypic resistance in plasma and peripheral blood lymphocytes in a group of naive HIV-1 patients

Background Since HIV infection cannot be completely eliminated due to the establishment of latently infected CD4+ T cell reservoirs, there is an urgent need for a clearer understanding of comparative resistance profiles between plasma and PBMC in HIV-1 patients. Objectives To assess the prevalence of mutations associated with drug resistance and to compare cell free and cell-associated strains. Study design Genotypic resistance analysis on viral DNA and plasma was performed in 31 therapy naive patients with chronic infection to check reverse transcriptase (RT) and protease resistance associated mutations before beginning antiretroviral therapy. Results Direct sequencing of DNA provirus disclosed key mutations (such as G190A/S, V106A, K103N and T215F) to RT inhibitors more frequently (7 patients out 31) than in plasma RNA (2 out of 31). In addition, major mutations (D30N, M46I, I50V, I84V) associated with drug resistance in the PR region were only found in PBMCs. Conclusions Despite the small number of patients, our results show a different resistance profile between plasma and PBMC compartments and may yield additional information for first-line antiretroviral regimens. Further investigations on larger series followed-up for a longer period of time are required to obtain in-depth information on the meaning of the mutations detectable in plasma and/or in PBMCs.

Identification of interferon-resistant subpopulations in several strains of measles virus: positive selection by growth of the virus in brain tissue

Subacute sclerosing panencephalitis (SSPE) is a chronic and usually fatal central nervous system disease caused by a persistent infection with measles virus. The pathogenic mechanisms of the disease are poorly understood, but restricted expression of viral antigens within the infected tissue appears to be involved. We have previously proposed that interferon (IFN) plays a role in the pathogenesis of SSPE by interacting with viral subpopulations that are relatively resistant to IFN-mediated inhibition. Such IFN-resistant viral subpopulations have now been identified in six independent strains of measles virus, two derived from patients with measles and four derived from patients with SSPE. By means of a replicative-plating procedure, these IFN-resistant viruses were found to be heterogeneous with respect to their growth in the presence of high levels of IFN. One viral form replicates fully, with complete destruction of the infected-cell culture, whereas the other form induces a restricted, self-limited form of cytopathic effect, similar to that seen with cell-associated strains of measles virus isolated from SSPE patients. Passage of a virus stock containing both of these viral forms through the central nervous system tissue of newborn hamsters strongly selects for the viral form associated with the self-limiting type of cytopathic effect. The presence of this form of IFN-resistant virus coupled with chronic production of IFN within the central nervous system may account for viral persistence in SSPE patients.

Transmission of measles virus encephalitis to ferrets by intracardiac inoculation of a cell-associated SSPE virus strain.

Ferret fibroblasts infected with a cell-associated strain of subacute sclerosing panencephalitis virus were inoculated into the hearts of ferrets in order to study whether the virus can spread from the blood to the brain in this animal model. Five of 21 inoculated ferrets developed encephalitis 5-7 days later and were sacrificed. Sick animals showed inflammatory lesions in the brain, both perivascular cuffings and infiltration of inflammatory cells in the choroid plexus and meninges. Virus was isolated in cell cultures from various parts of the brain and virus antigen was found by immunostaining, particularly in the cortex. Virus was not detected in inflammatory cells by immunostaining but in situ hybridization with a cDNA probe demonstrated measles virus RNA in neurons and glia cells surrounding perivascular inflammatory cuffings and in a lymph node of one ferret. Ferrets inoculated into the heart with cell-associated SSPE virus seem to be a suitable animal model to study how the virus spreads from the blood to the brain.

Identification of a nonproductive, cell-associated form of measles virus by its resistance to inhibition by recombinant human interferon.

Subacute sclerosing panencephalitis (SSPE) is a fatal disease in children and young adults that is caused by persistent infection of the central nervous system (CNS) by a nonproductive, cell-associated form of measles virus. Using an experimental model for SSPE (LEC viral strain in newborn hamsters), we have shown previously that establishment of such CNS infections involves selective elimination from the CNS of productively infected cells by host defensive mechanisms, coupled with the selective sparing of cells carrying nonproductive viral forms. That interferon (IFN) may play a role in this process was suggested by the disappearance of productively infected cells from the CNS tissues prior to the appearance of antiviral antibodies and by the demonstration of cell-associated, IFN-resistant viral variants in the virus stocks that were used. Results of this study support these conclusions by showing that similar IFN-resistant viral variants are present in the HBS strain of SSPE-derived measles virus and that these variants, in the presence of IFN, have properties that are similar to those of naturally occurring cell-associated strains of SSPE viruses, e.g., DR, IP3, and Biken. These IFN-resistant forms of HBS virus were isolated and were shown to maintain their resistance to inhibition by IFN after cloning. However, on removal of IFN, they reverted to productive forms similar to the parental HBS virus. The potential role of such viral forms in the pathogenesis of SSPE is discussed.

Suppression of Natural Killer Cell Activity and T Cell Proliferation by Fresh Isolates of Human Cytomegalovirus

Human cytomegalovirus (HCMV) infections are commonly associated with immunosuppression. A direct effect of this virus on lymphocyte functions in vitro, however, has not been shown. We have been investigating the effects of low-passage, fresh isolates of HCMV (cell-free and cell-associated) on natural killer cell (NK) activity and T cell proliferation. Cell-associated, low-passage isolates of HCMV markedly depressed NK activity. Suppression of NK activity was clearly manifested only after seven days of culture and could not be correlated with titer of virus or cell viability. Addition of interferon-alpha (IFN-alpha) but not interleukin-2 (IL-2) partially reconstituted the response, whereas depletion of infected monocytes prevented inhibition of NK-mediated lysis. The effects of cell-free and cell-associated isolates of HCMV on T cell proliferation differed in several respects from suppression of NK activity. Both cell-associated and cell-free isolates of HCMV completely abrogated antigen-specific and mitogen responses. This effect was apparent after only three days of culture with virus and was not reversed by either IFN or IL-2. Cell-associated strain AD169 also induced suppression but to a lesser extent. From observations reported here and other data, we suggest that HCMV can cause direct suppression of lymphocyte functions.

Round cell variant of measles virus: Neurovirulence and pathogenesis of acute encephalitis in newborn hamsters

A subacute sclerosing panencephalitis (SSPE) strain of measles virus has been previously shown to be composed of two interrelated but separable viral variants. One of these, the syncytiagenic or S variant, resembles defective, cell-associated strains of measles virus; while the other, the round cell inducing or RC variant, induces a highly productive infection in cell culture. It is now reported that the S variant is more neurovirulent in newborn hamsters than the RC variant and that viral replication in infected CNS tissues occurs in two phases. Early in the infection cell-free virus, composed primarily of the RC variant, is produced. Later, coincident with the appearance of antiviral antibody, cell-free virus rapidly disappears, leaving behind only cell-associated virus which resembles the S variant. Quantification of viral antigen expression in the infected tissues suggests that this change in the state of infection is not associated with antigenic modulation, but rather is the result of preferential elimination of RC variant infected cells.

Demonstration of immunoglobulin in brains of ferrets inoculated with an SSPE strain of measles virus: use of protein A conjugated to horseradish peroxidase.

Measles virus-specific immunoglobulin G (IgG) has been found in the brains of patients with subacute sclerosing panencephalitis (SSPE), a slowly progressing central nervous system (CNS) disease affecting children. IgG/albumin ratios indicate that the antibodies are probably synthesized in the CNS. In a ferret model system, protein A conjugated to horseradish peroxidase (PrAPx) was used to localize Ig's in brains of animals inoculated with a cell associated strain of SSPE. Ig's were found in plasma cells in various stages of antibody production both in perivascular inflammatory lesions and scattered throughout the cerebral cortex. These findings offer corroborative evidence that the Ig found in SSPE ferret brain and CSF is actively synthesized within the CNS. Antibody was also demonstrated in glial and neuronal cell bodies and processes and in postsynaptic profiles. These are the same sites where measles virus antigens are most frequently found and suggests the possibility of immune complex formation.

Induction of acute myoclonic encephalopathy in hamsters by subacute sclerosing panencephalitis virus.

Myoclonus is a characteristic neurological sign of subacute sclerosing panencephalitis (SSPE). Attempts were made to induce myoclonus in a large proportion of hamsters with a cell-associated strain of SSPE virus (the Biken strain) and thereby to establish an experimental model for study of the mechanism of development of this condition. When injected intracerebrally, Biken virus induced myoclonus within two to 14 days in 84% of the three- to nine-week-old hamsters tested. Electroencephalographic traces showed a periodic and synchronous discharge consisting of high-voltage slow waves and spikes that appeared coincidentally with myoclonus. Neurons in the cortex and thalamus of the affected animals had severely degenerated cytoplasm. Inflammatory changes, such as perivascular cuffing or infiltration of mononuclear cells, were not detected. Staining with immunoperoxidase revealed measles viral antigens in the cytoplasm and dendrites of the affected neurons. SSPE virus with the same properties as the parent virus was recovered from brain cells of sick animals by cocultivation with Vero cells.

Presence of oligoclonal immunoglobulin G bands and lack of matrix protein antibodies in cerebrospinal fluids and sera of ferrets with measles virus encephalitis

Young adult ferrets were immunized with measles vaccine and 5 to 6 weeks later inoculated intracerebrally with Vero cells persistently infected with cell-associated strain D.R. of measles virus isolated from a patient with subacute sclerosing panencephalitis. Of nine ferrets which survived the infection for 3 weeks or longer, five showed neurological signs. At the time of death they had widespread inflammation in their brains, and cell-associated virus was isolated from three ferrets sacrificed from 5 weeks to 7 months after inoculation. Four ferrets did not develop clinical signs, but two of these had mild inflammation in the brain 7 months and 2 1/2 years after inoculation, respectively. Cerebrospinal fluids drawn by cisternal puncture from infected ferrets at the time of sacrifice had neutralizing titers against measles virus similar to the titers found in sera, but antibody against the measles virus matrix protein was not detectable. Cerebrospinal fluid showed increased immunoglobulin G (IgG) and had distinct measles virus-specific oligoclonal IgG bands. The intensity of the bands correlated with the neutralizing titers of the fluids. These results confirm and extend earlier findings and indicate that persistent measles virus infection in ferrets is similar to human subacute sclerosing panencephalitis and can be used to study certain aspects of persistent brain infections leading to subacute encephalitis.

Presence of antibody in virus-infected brain cells of SSPE ferrets

Measles virus specific immunoglobulin G (IgG) has been found in the brains of patients with subacute sclerosing panencephalitis (SSPE), a slowly progressing disease of the central nervous system (CNS) in children. IgG/albumin ratios indicate that the antibodies are synthesized within the CNS. Using the ferret as an animal model to study the disease, we have been attempting to localize the Ig's in the brains of animals inoculated with a cell associated strain of SSPE. In an earlier report, preliminary results using Protein A conjugated to horseradish peroxidase (PrAPx) (Dynatech Diagnostics Inc., South Windham, ME.) to detect antibodies revealed the presence of immunoglobulin mainly in antibody-producing plasma cells in inflammatory lesions and not in infected brain cells.In the present experiment we studied the brain of an SSPE ferret with neutralizing antibody titers of 1:1024 in serum and 1:512 in CSF at time of sacrifice 7 months after i.c. inoculation with SSPE measles virus-infected cells. The animal was perfused with saline and portions of the brain and spinal cord were immersed in periodate-lysine-paraformaldehyde (P-L-P) fixative. The ferret was not perfused with fixative because parts of the brain were used for virus isolation.

Immunoperoxidase staining of simple nuclear bodies in sclerosing panencephalitis (SSPE) by antiserum to Measles nucleocapsids.

Nuclear bodies have been found with great consistency in brain tissue from patients with subacute sclerosing panencephalitis (SSPE). There has been controversy as to the significance of these structures and their relationship, if any, to the disease. In this study, a non-productive, cell-associated strain of SSPE measles virus grown in tissue culture on Vero cells showed the continued presence of nuclear bodies, frequently in close association with the continued presence of nuclear bodies, frequently in close association with the SSPE measles virus nucleocapsids. Immunoperoxidase (I-P) staining with specific antiserum against measles virus nucleocapsids was done. The resultant positive labeling of the nuclear bodies demonstrates that they contain measles virus antigen and suggests that the nuclear bodies play a role in the development of the measles virus nucleocapsids of SSPE.