Filariae-Retrovirus Co-infection in Mice is Associated with Suppressed Virus-Specific IgG Immune Response and Higher Viral Loads.

Worldwide more than 2 billion people are infected with helminths, predominantly in developing countries. Co-infections with viruses such as human immunodeficiency virus (HIV) are common due to the geographical overlap of these pathogens. Helminth and viral infections induce antagonistic cytokine responses in their hosts. Helminths shift the immune system to a type 2-dominated immune response, while viral infections skew the cytokine response towards a type 1 immune response. Moreover, chronic helminth infections are often associated with a generalized suppression of the immune system leading to prolonged parasite survival, and also to a reduced defence against unrelated pathogens. To test whether helminths affect the outcome of a viral infection we set up a filarial/retrovirus co-infection model in C57BL/6 mice. Although Friend virus (FV) infection altered the L. sigmodontis-specific immunoglobulin response towards a type I associated IgG2 isotype in co-infected mice, control of L. sigmodontis infection was not affected by a FV-superinfection. However, reciprocal control of FV infection was clearly impaired by concurrent L. sigmodontis infection. Spleen weight as an indicator of pathology and viral loads in spleen, lymph nodes (LN) and bone marrow (BM) were increased in L. sigmodontis/FV-co-infected mice compared to only FV-infected mice. Numbers of FV-specific CD8+ T cells as well as cytokine production by CD4+ and CD8+ cells were alike in co-infected and FV-infected mice. Increased viral loads in co-infected mice were associated with reduced titres of neutralising FV-specific IgG2b and IgG2c antibodies. In summary our findings suggest that helminth infection interfered with the control of retroviral infection by dampening the virus-specific neutralising antibody response.

Inhibition of catecholamine degradation ameliorates while chemical sympathectomy aggravates the severity of acute Friend retrovirus infection in mice.

Several lines of evidence indicate that the sympathetic nervous system (SNS) might be involved in the pathogenesis and progression of retroviral infections. However, experimental data are scarce and findings inconsistent. Here, we investigated the role of the SNS during acute infection with Friend virus (FV), a pathogenic murine retrovirus that causes polyclonal proliferation of erythroid precursor cells and splenomegaly in adult mice. Experimental animals were infected with FV complex, and viral load, spleen weight, and splenic noradrenaline (NA) concentration was analyzed until 25 days post infection. Results show that FV infection caused a massive but transient depletion in splenic NA during the acute phase of the disease. At the peak of the virus-induced splenomegaly, splenic NA concentration was reduced by about 90% compared to naïve uninfected mice. Concurrently, expression of the catecholamine degrading enzymes monoamine oxidase A (MAO-A) and catechol-O-methyltransferase (COMT) was significantly upregulated in immune cells of the spleen. Pharmacological inhibition of MAO-A and COMT by the selective inhibitors clorgyline and 3,5-dinitrocatechol, respectively, efficiently blocked NA degradation and significantly reduced viral load and virus-induced splenomegaly. In contrast, chemical sympathectomy prior to FV inoculation aggravated the acute infection and extended the duration of the disease. Together these findings demonstrate that catecholamine availability at the site of viral replication is an important factor affecting the course of retroviral infections.

Brainstem 1H nuclear magnetic resonance (NMR) spectroscopy: marker of demyelination and repair in spinal cord.

Measuring in vivo spinal cord injury and repair remains elusive. Using magnetic resonance spectroscopy (MRS) we examined brainstem N-acetyl-aspartate (NAA) as a surrogate for spinal cord injury in two mouse strains with different reparative phenotypes following virus-induced demyelination. Swiss Jim Lambert (SJL) and Friend Virus B (FVB) mice progressively demyelinate with axonal loss. FVB mice demyelinate similarly but eventually remyelinate coincident with functional recovery. Brainstem NAA levels drop in both but recover in FVB mice. Chronically infected SJL mice lost 30.5% of spinal cord axons compared to FVB mice (7.3%). In remyelination-enhancing or axon-preserving clinical trials, brainstem MRS may be a viable endpoint to represent overall spinal cord dysfunction.

Development of a real-time quantitative reverse transcriptase PCR assay for detection of the Friend leukemia virus load in murine plasma.

Friend leukemia virus (FLV), a murine retrovirus, has been used as a model for elucidation of human immunodeficiency virus (HIV) immunopathogenesis and evaluation of anti-HIV drug effects for several decades. However, no method for direct detection of the plasma viral load has yet been reported. In this study, a TaqMan real-time quantitative reverse transcriptase PCR (qRT-PCR) assay was established for the rapid detection and quantitation of FLV. Measurement of the absolute FLV load was achieved through synthesis of a standard RNA from within the FLV envelope gene for generation of a standard curve. The assay allows quantitation over a range from 20 to 2 x 10(8) RNA copies per reaction in a two-step real-time quantitative reverse transcriptase PCR protocol. The relationships between the initially injected FLV dose and the plasma FLV load and spleen index were explored. Following this, the in vivo effects of zidovudine, adefovir dipivoxil, and entecavir on mice infected with FLV were evaluated. The results showed that the plasma FLV load was not proportional to the spleen index over the same FLV injection dosage series, although a trend was observed. When evaluated using plasma viral load, high dose (15 mg/(kg d)) adefovir dipivoxil was capable of significant inhibition of FLV replication in mice. The qRT-PCR assay described here allows specific, sensitive and direct detection of FLV and may also provide more precise measurement of FLV load.

Evaluation of the Friend Virus model for the development of improved adenovirus-vectored anti-retroviral vaccination strategies.

We evaluated the suitability of the Friend Virus (FV) model for the development of improved adenovirus vectors for anti-retroviral vaccination using two types of adenovirus vectors, encoding F-MuLV Env and Gag, which differed only in their fiber genes (Ad5 and Ad5F35). Genetically FV-resistant C57BL/6 mice and highly susceptible CB6F1 hybrid mice were vaccinated by either homologous or heterologous prime-boost regimen. After FV challenge, viral loads in the spleens of C57BL/6 mice were reduced approximately 250-fold and were below the detection threshold in >50% of the mice. Vaccination outcome was critically influenced by the route of vector administration. In CB6F1 mice, vaccination resulted in reduced viremia, delayed onset of splenomegaly, and induction of FV-specific T cells as assessed by tetramer staining. Heterologous prime-boost vaccination resulted in significantly higher neutralizing antibody titers, translating into improved immune protection, in contrast to coexpression of cytokines. Our results suggest that the FV model can provide insight into the development of improved adenovirus vectors for HIV-1 vaccination.

Effects of type I interferons on Friend retrovirus infection.

The type I interferon (IFN) response plays an important role in the control of many viral infections. However, since there is no rodent animal model for human immunodeficiency virus, the antiviral effect of IFN-alpha and IFN-beta in retroviral infections is not well characterized. In the current study we have used the Friend virus (FV) model to determine the activity of type I interferons against a murine retrovirus. After FV infection of mice, IFN-alpha and IFN-beta could be measured between 12 and 48 h in the serum. The important role of type I IFN in the early immune defense against FV became evident when mice deficient in IFN type I receptor (IFNAR(-/-)) or IFN-beta (IFN-beta(-/-)) were infected. The levels of FV infection in plasma and in spleen were higher in both strains of knockout mice than in C57BL/6 wild-type mice. This difference was induced by an antiviral effect of IFN-alpha and IFN-beta and was most likely mediated by antiviral enzymes as well as by an effect of these IFNs on T-cell responses. Interestingly, the lack of IFNAR and IFN-beta enhanced viral loads during acute and chronic FV infection. Exogenous IFN-alpha could be used therapeutically to reduce FV replication during acute but not chronic infection. These findings indicate that type I IFN plays an important role in the immediate antiviral defense against Friend retrovirus infection.

Dietary copper restriction-induced changes in myocardial gene expression and the effect of copper repletion.

Dietary copper (Cu) restriction leads to cardiac hypertrophy and failure in mice, and Cu repletion (CuR) reverses the hypertrophy and prevents the transition to heart failure. The present study was undertaken to determine changes in myocardial gene expression involved in Cu deficient (CuD) cardiomyopathy and its reversal by CuR. Analysis was performed on three groups of mice: 4-week-old CuD mice that exhibited signs of cardiac failure, their age-matched copper-adequate (CuA) controls, and the CuD mice that were re-fed adequate Cu for 2 weeks. Total RNA was isolated from hearts and subjected to cDNA micro-array and real-time reverse transcription-polymerase chain reaction analysis. Dietary CuD caused a decrease in cardiac mRNA of beta-MHC, L-type Ca(2+) channel, K-dependent NCX, MMP-2, -8, and -13, NF-kappaB, and VEGF. The mRNA levels of ET-1, TGF-beta, TNF-alpha, and procollagen-I-alpha1 and III-alpha1 were increased in the CuD cardiac tissue. Copper repletion resulted in cardiac mRNA levels of most of the genes examined returning to control levels, although the K-dependent NCX and MMP-2 values did not reach those of the CuA control. In addition, CuR caused an increase in beta-MHC, L-type Ca(2+)channel, MMP-13 to levels surpassing those of CuA control, and a decrease in ET-1, and TNF-alpha mRNA levels. In summary, changes in gene expression of elements involved in contractility, Ca(2+) cycling, and inflammation and fibrosis may account for the altered cardiac function found in CuD mice. The return to normal cardiac function by CuR may be a result of the favorable regression in gene expression of these critical components in myocardial tissue.

Unique three-repeat sequences containing FVa, LVb/C4, and CORE motifs in LTR-U3 of Friend murine leukemia virus clone A8 accelerate the induction of thymoma in rat.

Friend murine leukemia virus (Fr-MLV) clone A8 causes thymoma 7 weeks postinfection in rats with a more rapid progression than clone 57. The U3 region of A8-LTR contains a unique structure of enhancer motifs consisting of three repeats of a 38-bp sequence containing FVa, LVb/C4, and CORE motifs. Replacement or deletion of the 38-bp sequence in the A8-U3 resulted in a marked reduction in tumorigenicity. Furthermore, the virus with 57-U3 gained high tumorigenicity after construction of the three 38-bp repeats in the U3 region. These findings indicated that the repeats of the 38-bp sequence of A8-LTR are essential for the rapid induction of thymoma. Interestingly, the repeat of the 38-bp sequence did not accelerate the amount of integrated viral DNA in the thymus during the early phase of infection, although it contributed to higher production of infectious virus. Thus, it was demonstrated that the ability to induce thymoma, which correlates with virus titer in the thymus, is not determined by the rate of viral DNA integration into the host genome.

Leukemia-related transcription factor TEL accelerates differentiation of Friend erythroleukemia cells.

TEL belongs to a member of the ETS family transcription factors that represses transcription of target genes such as FLI-1. Although TEL is essential for establishing hematopoiesis in neonatal bone marrow, its role in erythroid lineage is not understood. To investigate a role for TEL in erythroid differentiation, we introduced TEL into mouse erythroleukemia (MEL) cells. Overexpressing wild-type-TEL in MEL cells enhanced differentiation induced by hexamethylene bisacetamide or dimethylsulfoxide, as judged by the increased levels of erythroid-specific delta-aminolevulinate synthase and beta-globin mRNAs. TEL bound to a corepressor mSin3A through the helix-loop-helix domain. A TEL mutant lacking this domain still bound to the ETS binding site, but lost its transrepressional effect. This mutant completely blocked erythroid differentiation in MEL cells. Moreover, it showed dominant-negative effects over TEL-mediated transcriptional repression and acceleration of erythroid differentiation. Endogenous TEL mRNA was found to increase during the first 3 days in differentiating MEL cells and drastically decrease thereafter. All these data suggest that TEL might play some role in erythroid cell differentiation.

Temporal effects of gamma interferon deficiency on the course of Friend retrovirus infection in mice.

The current studies demonstrate complex and seemingly contradictory effects by gamma interferon (IFN-gamma) on Friend virus (FV) infection. Both temporal and tissue-specific effects were observed. During the first week of infection, IFN-gamma-deficiency caused increased levels of FV infection in multiple tissues. Surprisingly, however, by 2 weeks postinfection, IFN-gamma-deficient mice had significantly lower levels of infection in both the spleen and bone marrow compared to wild-type mice. The rapid reduction of virus in the IFN-gamma-deficient mice correlated with a more rapid virus-neutralizing antibody response than was observed in the wild-type mice. Furthermore, the virus-neutralizing antibody response in wild-type mice could be accelerated by ablation of their IFN-gamma response. Although the IFN-gamma-deficient mice developed an accelerated virus-neutralizing antibody response, they did not class-switch to immunoglobulin G class immunoglobulins nor could they maintain long-term virus-neutralizing antibody titers. Eventually, all of the IFN-gamma-deficient mice failed to keep persistent virus in check and developed fatal FV-induced erythroleukemia.

Identification of genetic determinants that regulate tumorigenicity of Friend murine leukemia virus in rats.

A neuropathogenic variant of Friend murine leukemia virus (FrMLV), clone A8, has been shown to cause thymoma and infiltration of leukemic cells to organs at 7-8 weeks post-infection in rats with a more rapid progression than clone 57. We have previously reported that the determinant for induction of aggressive leukemia in rats is located in the ClaI-AatII fragment containing the long terminal repeat (LTR) and the 5' half of the 5' leader sequence of A8 virus. Further studies of chimeric viruses restricted the determinant for the induction of thymoma to only the 0.6-kb ClaI-KpnI fragment of A8. This fragment contains a 0.1 kb region of the 3' terminus of the env gene, the intergenic region, the U3, and the 5' half of the R region in the LTR. Major differences in the fragment between A8 and 57 viruses were found in the U3 region, especially in the enhancer motifs. These results indicate that the enhancer region of A8-LTR contributes to the manifestation of thymoma with rapid progression in rats.

Distribution of ecotropic retrovirus receptor protein in rat brains detected by immunohistochemistry.

Friend murine leukaemia virus (FrMLV) FrC6 clone A8 causes spongiform degeneration in the central nervous system (CNS) of newborn but not 3-week-old rats. To assess whether expression of the ecotropic MLV receptor (CAT-1) in the CNS correlates with the pathogenicity of the A8 virus, we generated an anti-CAT-1 antibody raised against a synthetic peptide that corresponds to the carboxyl-terminal amino acid sequence of CAT-1. In the CNS of newborn and 3 to 4-week-old rats, a strong immunoreactivity against the antibody was detected in most of the endothelial cells. However, almost no expression of CAT-1 was detected in the CNS of 21-week-old rats. In newborn rats, many parenchymal cells in the brain as well as the vascular wall expressed CAT-1 antigen. These findings suggest that retrovirus receptor-bearing glial cells contribute to the neuropathogenesis of MLV, including clone A8, which induces spongiosis in rats only when inoculated into newborns.

Role of interleukin-4 (IL-4), IL-12, and gamma interferon in primary and vaccine-primed immune responses to Friend retrovirus infection.

The immunological resistance of a host to viral infections may be strongly influenced by cytokines such as interleukin-12 (IL-12) and gamma interferon (IFN-gamma), which promote T helper type 1 responses, and IL-4, which promotes T helper type 2 responses. We studied the role of these cytokines during primary and secondary immune responses against Friend retrovirus infections in mice. IL-4- and IL-12-deficient mice were comparable to wild-type B6 mice in the ability to control acute and persistent Friend virus infections. In contrast, more than one-third of the IFN-gamma-deficient mice were unable to maintain long-term control of Friend virus and developed gross splenomegaly with high virus loads. Immunization with a live attenuated vaccine virus prior to challenge protected all three types of cytokine-deficient mice from viremia and high levels of spleen virus despite the finding that the vaccinated IFN-gamma-deficient mice were unable to class switch from immunoglobulin M (IgM) to IgG virus-neutralizing antibodies. The results indicate that IFN-gamma plays an important role during primary immune responses against Friend virus but is dispensable during vaccine-primed secondary responses.

Immunity to retroviral infection: the Friend virus model.

Friend virus infection of adult immunocompetent mice is a well established model for studying genetic resistance to infection by an immunosuppressive retrovirus. This paper reviews both the genetics of immune resistance and the types of immune responses required for recovery from infection. Specific major histocompatibility complex (MHC) class I and II alleles are necessary for recovery, as is a non-MHC gene, Rfv-3, which controls virus-specific antibody responses. In concordance with these genetic requirements are immunological requirements for cytotoxic T lymphocyte, T helper, and antibody responses, each of which provides essential nonoverlapping functions. The complexity of responses necessary for recovery from Friend virus infection has implications for both immunotherapies and vaccines. For example, it is shown that successful passive antibody therapy is dependent on MHC type because of the requirement for T cell responses. For vaccines, successful immunization requires priming of both T cell and B cell responses. In vivo depletion experiments demonstrate different requirements for CD8(+) T cells depending on the vaccine used. The implications of these studies for human retroviral diseases are discussed.

Factors affecting induction of neurological disorders in mice by paralysis-inducing Friend-related PVC viruses.

Our previous studies showed that the passage of the Friend virus complex through rats generated variant MuLVs, designated PVC111, PVC211, PVC321 and PVC441, that induced neurological disorders associated with tremor and paralysis. In this study, we tested the pathogenicity of four different PVC viruses in mice. Although histopathological studies revealed spongiform degeneration in the spinal cords of NFS mice infected with each PVC virus, only PVC441 frequently induced tremor and paralysis. After a long latency, all of these viruses induced leukemia associated with severe anemia. Further studies with PVC441 revealed dose- and age-dependence for tremor induction. In contrast to NFS mice, BALB/c, DBA/2 and C57BL/6 mice infected with PVC441 virus showed no neurological symptoms, although the virus could be isolated from the tissues of central nervous system. Despite the absence of neurological symptoms, a high degree of neuronal degeneration in the lumbar spinal cord was found in PVC441-infected BALB/c mice. A low degree of neuronal degeneration was found in PVC441-infected DBA/2 or C57BL/6 mice. Genetic crosses of these resistant mice with susceptible NFS mice indicated that resistance to tremor induction by PVC441 was dominant in all mouse strains and suggested that various host genes may control the susceptibility of mice to tremor induction by PVC441 virus.

Characterization of a neurologic disease induced by a polytropic murine retrovirus: evidence for differential targeting of ecotropic and polytropic viruses in the brain.

A variety of ecotropic murine leukemia viruses cause neurodegenerative disease. We describe here the clinical and histopathological features of a neurologic disease induced by a polytropic murine leukemia virus, FMCF98. Clinical disease was dominated by hyperexcitability and ataxia, and the histopathology was characterized primarily by astrocytosis and astrocytic degeneration. The viral envelope gene harbored the determinants of neurovirulence, since the chimeric virus Fr98E, which contained the envelope gene of FMCF98 on a background of the nonneurovirulent virus FB29, caused a similar disease. The disease caused by Fr98E differed from that induced by the coisogenic neurovirulent ecotropic virus FrCasE in clinical presentation, histopathology, and distribution of virus in the central nervous system. Since Fr98E contains a polytropic envelope gene and FrCasE contains an ecotropic envelope gene, these phenotypic differences appeared to be determined by envelope sequences and may reflect differences in virus receptor usage in the central nervous system.

Murine leukemia virus-induced neurodegeneration of rats: enhancement of neuropathogenicity correlates with enhanced viral tropism for macrophages, microglia, and brain vascular cells.

A highly neuropathogenic retrovirus, NT40, was generated by serially passaging an infectious molecular clone of Friend murine leukemia virus, FB29, through F344 Fisher rats. NT40 induced severe neurological signs such as reflex abnormalities and ataxia within 4-6 weeks following neonatal inoculation. FB29 led to only very mild neurological dysfunctions with longer incubation periods. Pathological alterations were characterized by mild (FB29) to extensive (NT40) noninflammatory spongiform degeneration, mainly of brain-stem areas. Infectious center assays revealed that viral titers in brain tissues of NT40-infected rats were 100-fold higher than those of FB29-infected animals. Employing immunohistochemistry, in situ hybridization, and flow cytometry, NT40 was found to infect many endothelial cells of brain blood vessels and microglia, whereas FB29 infected only microglia and those to a lower extent. However, when isolated from adult diseased rats, microglial cells turned out in both cases to be nonproductively infected with either FB29 or NT40. Of peripheral organs, we found enhanced levels of NT40 in peritoneal macrophages but not in spleen, thymus, or serum when compared to FB29. Altogether these data suggest that an expanded cellular tropism within the CNS and elevated viral titers in macrophages and microglia correlated with enhancement of neuropathogenicity.

Characterization of monoclonal antibodies recognizing neurotropic Friend murine leukemia virus.

We isolated a replication-competent, neurotropic retrovirus (FrC6 virus) and its molecular clone A8 from the NB-tropic Friend murine leukemia virus (FLV) complex. For detection and characterization of the FrC6 and A8 viruses, monoclonal antibodies (MAbs) against the FLV complex were established. Thirty MAbs, each of which reacted with the FLV-producing cell line, were tested for potential neutralizing activities; only two MAbs inhibited the proliferation of the A8 virus. These two MAbs were ineffective or had very weak neutralizing activities toward the non-neurotropic FLV strain clone 57 virus. Further characterization of MAbs by immunoprecipitation revealed that 4 MAbs recognized the envelope protein of the A8 virus. Two of these 4 MAbs recognized the surface glycoprotein gp70, requiring the conformational epitope of the virus for this recognition, while the other two MAbs, which were reactive with the transmembrane protein p15E, were conformation-independent. Both of the MAbs against gp70 distinguished neuropathogenic and non-neuropathogenic viruses to some extent, through neutralizing activity or binding activity detected by immunoprecipitation, whereas the two MAbs against p15E reacted with the viruses in a similar manner. Furthermore, one of the MAbs distinguished the viral antigen in the wall of the vacuolation that composes the spongiotic lesion induced by FrC6 viral infection of the brain.

Accumulation of unintegrated viral DNA in rat glial cells infected with neurotropic Friend murine leukemia virus.

FrC6 virus isolated from Friend murine leukemia virus (FLV) as a neurotropic virus clone induced a high-degree of accumulation of unintegrated viral DNA during infection of rat glial cells by 72 h post-infection. When anti-FLV neutralizing antibody, dextran sulfate, or 3'-azido-3'-deoxythymidine (AZT) was added to the culture within 24 h after infection, the accumulation of unintegrated viral DNA was inhibited. However, after 30-36 h post-infection, addition of anti-FLV antibody or dextran sulfate scarcely inhibited the accumulation of the unintegrated viral DNA, while addition of AZT at 30-36 h post-infection still reduced the amount of unintegrated viral DNA. Our results demonstrate that after 30-36 h post-infection, when second-round infection had already taken place, further superinfection with cell-free virus to glial cells was not required for the accumulation of unintegrated viral DNA. Possible mechanisms are discussed.

A low oncogenic variant of Friend murine leukemia virus with strong immunosuppressive properties.

Friend leukemia complex (FLC) is known to induce immunosuppression but the use of FLC in studies of immune cells function is disadvantageous since the immunosuppression always is accompanied by an acute erythroleukemia. To obtain immunosuppressive variants of FLC with reduced leukemogenic potential, we isolated T-helper cells from FLC infected mice, and passed lysates of the cells to recipient uninfected mice. A group of these mice developed a condition distinct from the disease induced by FLC. A viral stock prepared from these mice, designated Fd-MIV for friend derived murine immunodeficiency virus, induced a profound suppression of the primary antibody response without acute transformation in adult NMRI mice. Terminally a wasting disease with weight loss, atrophy of the thymus and lymph nodes and renal disease was observed in some mice. Analysis of viral DNA and RNA from infected NIH 3T3 cells showed that Fd-MIV contained at least two viral components, a 8.4 kb friend murine leukemia virus (F-MuLV) and a 7.4 kb mink cell focus (MCF)/xenotropic virus related genome. The 7.4 kb genome was not detected in Fd-MIV infected, immunocompromised mice indicating that the 8.4 kb genome might be responsible for the disease.