Early Phase Of Acute Infection Research Articles

Nonsteroidal Anti-Inflammatory Drugs in Viral Infections Disease, Specially COVID-19.

During the current SARS-CoV-2 (COVID-19) pandemic, some reports were presented based on those nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroids may exacerbate symptoms in COVID-19 patients. According to this, we aimed to collate information available in published articles to identify any evidence behind these statements with the aim of helping clinicians on how best to treat patients. We could not find published conclusive evidence for or against the use of NSAIDs in COVID-19 patients. Meanwhile, there appeared to be some evidence that corticosteroids may be beneficial if utilized in the early acute phase of infection, however, conflicting WHO (World Health Organization) evidence surrounding corticosteroid use in certain viral infections means this evidence is not conclusive. Given the current availability of literature, caution should be exercised until further evidence emerges surrounding the use of NSAIDs and corticosteroids in COVID-19 patients. However, the availability of reliable information for clinicians and patients is paramount.

Transcriptome profiling and differential expression analysis of the immune-related genes during the early phase of acute infection with Aeromonas hydrophila in the Chinese sucker (Myxocyprinus asiaticus)

Chinese sucker (Myxocyprinus asiaticus), a unique species of Catostomidae in Asia and China's second-class protected animal species, has important value both in research and economy field. Moreover, Chinese sucker are susceptible to infection by Aeromonas hydrophila. However, until now the information related to the immune system against A. hydrophila of Chinese sucker is unclear. Therefore, to understand the molecular basis underlying the host immune response to A. hydrophila infection, Illumina HiSeq™ 2000 was used to analyse the spleen transcriptome of infected Chinese sucker for the first time. De nova assembly of paired-end reads yielded 693,660 unigenes. Comparative analysis of the expression profile between bacteria challenge fish and control fish identified a total of 1390 differentially expressed genes (padj<0.05 and |log2FoldChange| > 1), including 907 up-regulated genes and 483 down-regulated genes. A significant enrichment analysis of these DEGs in spleen revealed major immune-related pathways, including Toll-like receptor, T cell receptor, complement and coagulation cascades, MAPK and NF-kB. The selected DEGs were validated using qPCR. Moreover, the expression level of nineteen immune-related genes were detected at 4, 12 and 24 h post infection. The results showed that a various types of molecules were involved in the antibacterial immune response, including pattern recognition receptors (TLR2, TLR4, TLR6 and PGRP5), interleukins (IL1B, IL6, IL10, IL11 and IL12), chemokines (CXCL1, CXCL2 and CXCL8), activity regulation molecules of immune cells (CD68, CD200, CD247, CSF3 and CSF3R), and complement components (C6, C7). Altogether, the results obtained on immune-related genes may allow for a better understanding of immunity in Chinese sucker to A. hydrophila, carrying out detailed functional analysis of these genes and developing strategies for efficient immune protection against infections in Chinese sucker, which will be great significance for healthy breeding and resource conservation of Chinese sucker.

COVID-19 and treatment with NSAIDs and corticosteroids: should we be limiting their use in the clinical setting?

Given the current SARS-CoV-2 (COVID-19) pandemic, the availability of reliable information for clinicians and patients is paramount. There have been a number of reports stating that non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids may exacerbate symptoms in COVID-19 patients. Therefore, this review aimed to collate information available in published articles to identify any evidence behind these claims with the aim of advising clinicians on how best to treat patients. This review found no published evidence for or against the use of NSAIDs in COVID-19 patients. Meanwhile, there appeared to be some evidence that corticosteroids may be beneficial if utilised in the early acute phase of infection, however, conflicting evidence from the World Health Organisation surrounding corticosteroid use in certain viral infections means this evidence is not conclusive. Given the current availability of literature, caution should be exercised until further evidence emerges surrounding the use of NSAIDs and corticosteroids in COVID-19 patients.

Inhibition of type I interferon signaling abrogates early Mycobacterium bovis infection

BackgroundMycobacterium bovis (M. bovis) is the principal causative agent of bovine tuberculosis; however, it may also cause serious infection in human being. Type I IFN is a key factor in reducing viral multiplication and modulating host immune response against viral infection. However, the regulatory pathways of Type I IFN signaling during M. bovis infection are not yet fully explored. Here, we investigate the role of Type I IFN signaling in the pathogenesis of M. bovis infection in mice.MethodsC57BL/6 mice were treated with IFNAR1-blocking antibody or Isotype control 24 h before M. bovis infection. After 21 and 84 days of infection, mice were sacrificed and the role of Type I IFN signaling in the pathogenesis of M. bovis was investigated. ELISA and qRT-PCR were performed to detect the expression of Type I IFNs and related genes. Lung lesions induced by M. bovis were assessed by histopathological examination. Viable bacterial count was determined by CFU assay.ResultsWe observed an abundant expression of Type I IFNs in the serum and lung tissues of M. bovis infected mice. In vivo blockade of Type I IFN signaling reduced the recruitment of neutrophils to the lung tissue, mediated the activation of macrophages leading to an increased pro-inflammatory profile and regulated the inflammatory cytokine production. However, no impact was observed on T cell activation and recruitment in the early acute phase of infection. Additionally, blocking of type I IFN signaling reduced bacterial burden in the infected mice as compared to untreated infected mice.ConclusionsAltogether, our results reveal that Type I IFN mediates a balance between M. bovis-mediated inflammatory reaction and host defense mechanism. Thus, modulating Type I IFN signaling could be exploited as a therapeutic strategy against a large repertoire of inflammatory disorders including tuberculosis.

Distribution pattern of bovine viral diarrhoea virus type 1 genome in lymphoid tissues of experimentally infected sheep.

In this study, cellular localization and the distribution pattern of BVDV genome in lymphoid tissues during the course of experimental acute BVDV-1 infection of sheep was investigated. Tonsils, mesenteric lymph nodes (MLN) and spleen were collected on 3, 6, 9, 12 and 15days post infection (dpi) from twenty 4-month-old lambs, experimentally inoculated intra-nasally with 5 × 10(5) TCID50 of a non-cytopathic (ncp) BVDV-1 isolate, Ind-17555. Tissues collected from ten mock-infected lambs served as controls. In situ hybridization (ISH) was carried out in paraformaldehyde fixed paraffin embedded tissue sections using digoxigenin labelled riboprobe targeting 5'-UTR of BVDV-1. BVDV genome was detected at all the intervals from 3 dpi to 15 dpi in the lymphoid tissues with variations between the intervals and also amongst the infected sheep. During the early phase of acute infection, presence of viral genome was more in tonsils than MLN and spleen, whereas the distribution was higher in MLN during later stages. BVDV-1 genome positive cells included lymphocytes, macrophages, plasma cells, reticular cells and sometimes crypt epithelial cells. Genome distribution was frequently observed in the lymphoid follicles of tonsils, MLN and spleen, besides the crypt epithelium in tonsils, paracortex and medullary sinus and cords of MLN. Most abundant and widespread distribution of BVDV-1 genome was observed on 6 dpi while there was a reduction in number and intensity of positive signals by 15 dpi in most of the infected animals. This is the first attempt made to study the localisation of BVDV-1 in lymphoid tissues of acutely infected sheep by in situ hybridization. The results show that the kinetics of BVDV-1 distribution in lymphoid tissues of experimentally infected non-pregnant sheep follows almost a similar pattern to that demonstrated in BVDV infected cattle.

Lack of variant specific CD8+ T-cell response against mutant and pre-existing variants leads to outgrowth of particular clones in acute hepatitis C

BackgroundCTL escape mutations have been described during acute hepatitis C in patients who developed chronic disease later on. Our aim was to investigate the mutual relationship between HCV specific CD8+ T cells and evolution of the viral sequence during early acute HCV infection.ResultsWe sequenced multiple clones of NS3 1406 epitope in 4 HLA-A*02 patients with acute hepatitis C genotype 1b infection. Pentamers specific for the variants were used to monitor the corresponding CD8+ T cell response. We observed outgrowth of mutations, which induced only a weak and thus potentially insufficient CD8+ T cell response. In one patient we observed outgrowth of variant epitopes with similarities to a different genotype rather than de novo mutations most probably due to a lack of responsiveness to these likely pre-existing variants. We could show that in acute hepatitis C CTL escape mutations occur much earlier than demonstrated in previous studies.ConclusionsThe adaption of the virus to a new host is characterized by a high and rapid variability in epitopes under CD8+ T cell immune pressure. This adaption takes place during the very early phase of acute infection and strikingly some sequences were reduced below the limit of detection at some time points but were detected at high frequency again at later time points. Independent of the observed variability, HCV-specific CD8+ T cell responses decline and no adaption to different or new antigens during the course of infection could be detected.

CD4+T Cells Are Not Essential for Control of Early AcuteCryptosporidium parvumInfection in Neonatal Mice

Cryptosporidiosis is an important diarrheal disease of humans and neonatal livestock caused by Cryptosporidium spp. that infect epithelial cells. Recovery from Cryptosporidium parvum infection in adult hosts involves CD4(+) T cells with a strong Th1 component, but mechanisms of immunity in neonates are not well characterized. In the present investigation with newborn mice, similar acute patterns of infection were obtained in C57BL/6 wild-type (WT) and T and B cell-deficient Rag2(-/-) mice. In comparison with uninfected controls, the proportion of intestinal CD4(+) or CD8(+) T cells did not increase in infected WT mice during recovery from infection. Furthermore, infection in neonatal WT mice depleted of CD4(+) T cells was not exacerbated. Ten weeks after WT and Rag2(-/-) mice had been infected as neonates, no patent infections could be detected. Treatment at this stage with the immunosuppressive drug dexamethasone produced patent infections in Rag2(-/-) mice but not WT mice. Expression of inflammatory markers, including gamma interferon (IFN-γ) and interleukin-12p40 (IL-12p40), was higher in neonatal WT mice than in Rag2(-/-) mice around the peak of infection, but IL-10 expression was also higher in WT mice. These results suggest that although CD4(+) T cells may be important for elimination of C. parvum, these cells are dispensable for controlling the early acute phase of infection in neonates.

IL-17 Produced during Trypanosoma cruzi Infection Plays a Central Role in Regulating Parasite-Induced Myocarditis

BackgroundChagas disease is a neglected disease caused by the intracellular parasite Trypanosoma cruzi. Around 30% of the infected patients develop chronic cardiomyopathy or megasyndromes, which are high-cost morbid conditions. Immune response against myocardial self-antigens and exacerbated Th1 cytokine production has been associated with the pathogenesis of the disease. As IL-17 is involved in the pathogenesis of several autoimmune, inflammatory and infectious diseases, we investigated its role during the infection with T. cruzi.Methodology/Principal FindingsFirst, we detected significant amounts of CD4, CD8 and NK cells producing IL-17 after incubating live parasites with spleen cells from normal BALB/c mice. IL-17 is also produced in vivo by CD4+, CD8+ and NK cells from BALB/c mice on the early acute phase of infection. Treatment of infected mice with anti-mouse IL-17 mAb resulted in increased myocarditis, premature mortality, and decreased parasite load in the heart. IL-17 neutralization resulted in increased production of IL-12, IFN-γ and TNF-α and enhanced specific type 1 chemokine and chemokine receptors expression. Moreover, the results showed that IL-17 regulates T-bet, RORγt and STAT-3 expression in the heart, showing that IL-17 controls the differentiation of Th1 cells in infected mice.Conclusion/SignificanceThese results show that IL-17 controls the resistance to T. cruzi infection in mice regulating the Th1 cells differentiation, cytokine and chemokine production and control parasite-induced myocarditis, regulating the influx of inflammatory cells to the heart tissue. Correlations between the levels of IL-17, the extent of myocardial destruction, and the evolution of cardiac disease could identify a clinical marker of disease progression and may help in the design of alternative therapies for the control of chronic morbidity of chagasic patients.

Cutting Edge: Programmed Death-1 Up-Regulation Is Involved in the Attrition of Cytomegalovirus-Specific CD8+ T Cells in Acute Self-Limited Hepatitis B Virus Infection

Attrition of heterologous virus-specific CD8(+) T cells has been demonstrated in murine viral infection; however, little is known regarding this phenomenon in human viral infections. In this study, we observed that CMV-specific CD8(+) T cells displayed numerical decline and functional impairment in the early phase of acute infection, whereas programmed death-1 (PD-1) expression was significantly up-regulated by these CMV-specific CD8(+) T cells. This early PD-1 up-regulation was found to be closely associated with the increased apoptotic sensitivity of CMV-specific CD8(+) T cells. The in vitro addition of anti-PD-1 further enhanced the spontaneous apoptosis of CMV-specific CD8(+) T cells; however, blockade of the PD-1-mediated pathway with anti-PD-L1 significantly restored the CMV-specific CD8(+) T cell proliferation and IFN-gamma production. Thus, PD-1 plays a crucial role in the attrition of CMV-specific CD8(+) T cells in acute hepatitis B virus infection, which in turn, influences the preexisting homeostatic virus-specific CD8(+) T cell pool.

Quantitative Analysis of Virulent Duck Enteritis Virus Loads in Experimentally Infected Ducklings

To better understand the pathogenesis of duck virus enteritis (DVE), the levels of viral DNA in various tissues of ducklings during acute stage of virulent duck enteritis virus (DEV) infection were investigated by using quantitative real-time polymerase chain reaction. The results show that the viral levels of DEV in systemic organs have a close correlation with the progression of disease. The rapid dissemination and active replication of virulent DEV in multiple systemic organs at the early phase of acute infection accelerate the progression of disease. The levels of viral DNA increase sharply soon after developed clinical signs of disease, and the extent of increase and the magnitude of DEV DNA load in various tissues of ducklings after the exhibition of clinical signs may be a critical determinant of the outcome of DEV infection. The relatively high levels of DEV in bursa and small intestine tissues of dead ducklings most likely reflect the abundance of target epithelial and lymphoid cells in these tissues, which therefore play a key role in the pathogenesis of acute DVE and manifest as severe tissue lesions on the bursa and small intestine.

Acute HIV infection: it takes more than guts

To understand the cellular and molecular mechanisms of acute HIV pathogenesis. Recent studies have given us new insights into the mechanisms of acute HIV pathogenesis by demonstrating the 'systemic' destruction of the CD4 memory T cell compartment. This destruction occurs well before the emergence of a strong and broad immune response, highlighting the failure of the immune response to contain early viral infection and destruction. However, recent data also suggest that very few founder populations of cells are infected early, at the portal of entry, making them ideal targets for vaccine-induced immune responses that may aid in the effective control of early infection and transmission. HIV causes a massive destruction of memory CD4 T cells during the early acute phase of infection. This destruction proceeds largely in the absence of emerging antiviral immune responses, and severely disables the ability of the immune system to generate secondary immune responses. Early preservation of the memory CD4 compartment by shifting emphasis of antiretroviral therapeutic strategies to early treatment, and development of vaccines that can induce strong and broad immune responses, will be critical to prevent the destructive effects of early HIV infection.

Immunopathogenesis of Hepatitis C Virus Infection: Multifaceted Strategies Subverting Innate and Adaptive Immunity

Hepatitis C virus (HCV) infection is one of the major causes of chronic liver disease worldwide. The critical role of innate as well as adaptive immunity has been reported in HCV persistence and liver injury. In the early phase of acute infection, HCV continues to replicate in the liver, suggesting the HCV capability of inhibiting innate immunity. The sustained, vigorous and multiepitope-specific CD4+ and CD8+ T cell responses are essential for spontaneous HCV clearance. HCV-specific CD8+ T cells are primary elements for HCV clearance by inducing hepatocyte apoptosis, in which Fas/CD95 is fundamentally involved. However, once HCV persistency develops, HCV utilizes multifaceted arms to subvert various immune effectors. During IFNalpha-based therapy, the enhancement of HCV-specific CD4+ T cell response followed by HCV eradication has been reported, however, it remains obscure whether the therapeutic HCV clearance is able to restore the durable immune competency to HCV. Further investigation is still warranted to establish the means to direct HCV-specific immune responses in the desired way.

Chemokine Production and Leukocyte Recruitment to the Lungs of Paracoccidioides brasiliensis-Infected Mice Is Modulated by Interferon-γ

Chemokines and chemokine receptors play a role in cell recruitment during granulomatous inflammatory reactions. Here, we evaluated the expression of chemokines and chemokine receptors and their regulation by IFN-gamma in the course of Paracoccidioides brasiliensis (Pb) infection in mice. We found an association between KC and MIP-1alpha (CCL3) production and neutrophil infiltration in the lungs of Pb-infected mice during the early acute phase of infection. High levels of RANTES/CCL5, MCP-1/CCL2, IP-10/CXCL10, and Mig/CXCL9 simultaneously with mononuclear cell infiltration in the lungs was found. In the absence of IFN-gamma (GKO mice) we observed increased production of KC and MIP-1alpha and chronic neutrophilia. Moreover, we found a change in the chemokine receptor profiles expressed by wild-type (WT) versus GKO animals. Increased expression of CXCR3 and CCR5, and low levels of CCR3 and CCR4 were observed in the lungs of Pb-infected WT mice, whereas the opposite effect was observed in the lungs of GKO mice. Consistent with these results, infected cells from WT mice preferentially migrated in response to IP-10 (CXCR3 ligand), while those from GKO mice migrated in response to eotaxin/CCL11 (CCR3 ligand). These results suggest that IFN-gamma modulates the expression of chemokines and chemokine receptors as well as the kind of cells that infiltrate the lungs of Pb-infected mice.

The hepatologist in the haematologists' camp.

The hepatologist in the haematologists' camp.

Stage-dependent role of nitric oxide in control of Trypanosoma cruzi infection.

Trypanosoma cruzi, the causative agent of Chagas' disease, is known to be susceptible to nitric oxide (NO)-dependent killing by gamma interferon-activated macrophages. Mice deficient for inducible nitric oxide synthase (iNOS) are highly susceptible to T. cruzi, and inhibition of iNOS from the beginning of infection was reported to lead to an increase in trypomastigotes in the blood and to high mortality. In the present study, we investigated whether NO production is essential for the control of T. cruzi in all phases of the infection. BALB/c mice were treated at different time intervals after T. cruzi infection with an iNOS inhibitor, aminoguanidine or L-N6-(1-iminoethyl)-lysine (L-NIL). Treatment initiated with the beginning of the infection resulted in 100% mortality by day 16 postinfection (p.i.). If treatment was started later during the acute phase at the peak of parasitemia (day 20 p.i.), all the mice survived. Parasitemia was cleared and tissue amastigotes became undetectable in these mice even in the presence of the iNOS inhibitor L-NIL. Inhibition of iNOS in the chronic phase of the infection, i.e., from day 60 to day 120 p.i., with L-NIL did not result in a reappearance of parasitemia. These data suggest that while NO is essential for T. cruzi control in the early phase of acute infection, it is dispensable in the late acute and chronic phase, revealing a fundamental difference in control mechanisms compared to those in infections by other members of the order Kinetoplastida, e.g., Leishmania major.

The role of IL-12 in experimental Trypanosoma cruzi infection.

Host resistance to Trypanosoma cruzi infection is dependent on both natural and acquired immune responses. During the early acute phase of infection in mice, natural killer (NK) cell-derived IFN-gamma is involved in controlling intracellular parasite replication, mainly through the induction of nitric oxide biosynthesis by activated macrophages. We have shown that IL-12, a powerful inducer of IFN-gamma production by NK cells, is synthesized soon after trypomastigote-macrophage interaction. The role of IL-12 in the control of T. cruzi infection in vivo was determined by treating infected mice with anti-IL-12 monoclonal antibody (mAb) and analyzing both parasitemia and mortality during the acute phase of infection. The anti-IL-12 mAb-treated mice had higher levels of parasitemia and mortality compared to control mice. Also, treatment of infected mice with mAb specific for IFN-gamma or TNF-alpha inhibited the protective effect of exogenous IL-12. On the other hand, TGF-beta and IL-10 produced by infected macrophages inhibited the induction and effects of IL-12. Therefore, while IL-12, TNF-alpha and IFN-gamma correlate with resistance to T. cruzi infection, TGF-beta and IL-10 promote susceptibility. These results provide support for a role of innate immunity in the control of T. cruzi infection. In addition to its protective role, IL-12 may also be involved in the modulation of T. cruzi-induced myocarditis, since treatment of infected mice with IL-12 or anti-IL-12 mAb leads to an enhanced or decreased inflammatory infiltrate in the heart, respectively. Understanding the role of the cytokines produced during the acute phase of T. cruzi infection and their involvement in protection and pathogenesis would be essential to devise new vaccines or therapies.

Role of T-Cell Subsets in Acute and Persistent E-55 +Murine Leukemia Virus Infection in Susceptible Progressor and Resistant Long-Term Nonprogressor Mouse Strains

Previous studies from this laboratory have demonstrated that E-55 +MuLV-infected BALB/c-H-2 k(BALB.K) mice progress to develop thymic lymphoma about 7 months after infection whereas infected C57BL/10-H-2 k(B10.BR) mice are long-term nonprogressors that fail to develop disease even after 2 years of infection. Both resistant long-term nonprogressor (B10.BR) and progressor (BALB.K) mice generate an early immune response that results in a dramatic decrease in the number of virus-infected cells. Despite this early immune response, mice from both strains become persistently infected. However, resistant B10.BR mice also demonstrate a late T-cell-mediated response that may be causally related to long-term nonprogression whereas susceptible BALB.K mice fail to demonstrate this late T-cell response. In the present studies, the T-cell subsets involved in the effective early immune response in both B10.BR and BALB.K mice as well as the late T-cell response in B10.BR mice were determined by in vivoantibody-mediated depletion. Results from these studies demonstrate that during the early acute phase of infection, elimination of CD4 +T cells ablated the ability of both BALB.K and B10.BR mice to decrease the burden of virus-infected cells. However, elimination of CD8 +T cells ablated this result in BALB.K but not B10.BR mice. Thus, despite the fact that both immunocompetent B10.BR and BALB.K mice are able to decrease the number of virus-infected cells during the early acute phase of infection, there is a difference in the T-cell subsets that mediate this effect in these strains of mice. In addition, characterization of the late immune response that keeps virus at very low levels during the persistent stage of virus infection in resistant B10.BR mice demonstrated that simultaneous elimination of both CD4 +and CD8 +T cells allowed the emergence of virus-infected cells whereas the elimination of either subset alone showed no effect compared to untreated control mice that are immunologically intact. Since B10.BR and BALB.K are identical with respect to their H-2 k-haplotypes, it appears that the differences between these strains with respect to the generation of effective early and late anti-virus immune responses are regulated by a non- H-2-linked gene(s).

Differentiation of primary from secondary anti-EBNA-1-negative cases by determination of avidity of VCA-IgG

background: Serological techniques are used to determine Epstein Barr virus (EBV) etiology of a constellation of signs or symptoms related to lymphadenopathy, fever, respiratory tract infection, mononucleosis, hepatitis, thrombocytopenia or neurological disorder. Anti-Epstein Barr Nuclear antigen (EBNA)-1 is regularly negative during the first 3–4 weeks after the onset of clinical symptoms indicating acute EBV infection (primary anti-EBNA-1-negative). It may, however, also be negative in immunocompromised convalescent individuals (secondary anti-EBNA-1-negative) such as tumor patients, HIV-positive patients and transplant recipients. Objectives: The aim of this study was to determine the frequency of secondary anti-EBNA-1-negative cases and to find a way to distinguish them from primary anti-EBNA-1-negative cases using anticomplementary immunofluorescence (ACIF) and enzyme immunoassay (EIA). Study design: All sera sent to our institute for EBV serology during one year were routinely tested for Viral Capsid antibody (VCA)-IgM, VCA-IgG and anti-EBNA-1. Results: VCA-IgG-positive/anti-EBNA-1-negative cases (13.5% of total VCA-IgG-positive) comprised 55% primary and 45% secondary anti-EBNA-1-negative cases. Detection of secondary anti-EBNA-1-negative cases was independent of the method used, i.e., ACIF or EIA. VCA-IgG retained its high avidity in secondary anti-EBNA-1-negative cases, whereas primary anti-EBNA-1-negative cases taken during the early phase of acute infection showed low avidity of VCA-IgG. Conclusion: Determination of the avidity of VCA-IgG routinely and in concert with standard serodiagnosis (VCA-IgG, VCA-IgM, anti-EBNA-1) can enable the differentiation of primary and secondary anti-EBNA-1-negative cases.

Influence of genes within the MHC on mortality and brain cyst development in mice infected with Toxoplasma gondii: kinetics of immune regulation in BALB H‐2 congenic mice

Previous work has shown that genes within the major histocompatibility complex (MHC) of the mouse influence resistance and susceptibility to Toxoplasma gondii infection. Initial studies presented here using B10 H-2 congenic and recombinant haplotype mice inoculated via the oral route with the low virulence Beverley strain of T. gondii confirm the D region localization of MHC-linked control of brain cyst number. All B10 mice were, however, exquisitely sensitive to minor changes in virulence of the parasite inoculum resulting in high mortality during the early acute phase of infection. Further experiments examining mortality and brain cyst number in BALB MHC congenic mice inoculated via different routes indicated that the BALB background would provide a more favourable genetic environment in which to analyse kinetics of MHC controlled immune regulation following infection via the natural (oral) route. In studies comparing d and k haplotype mice a dramatic inverse relationship between splenic CD4:CD8 T cell ratios and brain cyst number was observed, particularly in the strain (BALB/K; H-2k) most susceptible to high brain cyst numbers and subsequent toxoplasmic encephalitis. Of particular interest was the observation that splenomegaly and the relative increase in the splenic CD8 T cell population preceded and accompanied the very dramatic and rapid increase in brain cyst formation. The results suggest that the too rapid development of a potent anti-parasite response in the viscera may drive the parasite to encyst in the brain.

The clinical significance of molecular variation within the hepatitis B virus genome

Although HBV has a circular DNA genome that is partially double stranded, it replicates by means of an RNA intermediate. The process is catalyzed by a translation product of the polymerase open reading frame that has reverse transcriptase activity. The enzyme is found in association with the virion and achieves a high rate of nucleotide misincorporation during transcription because such enzymes lack proofreading activity. The virus is remarkable for its efficient use of nucleic acid because the genome is only 3.2 kb long and yet it encodes four groups of proteins and their regulatory elements (1). This is achieved in some regions by reading the sequence through different frames to direct the synthesis of distinct proteins from the same genetic material. Only a few of the mutations that occur during the normal replication cycle therefore permit the entry of a new virus to the pool for natural selection. Nevertheless, patients with chronic hepatitis have been shown to have viruses with different sequences cocirculating, and some regions of the genome are poorly conserved between different isolates (2). The existence of patients with evidence of HBV replication but no evidence of liver disease, both in the early phase of acute infection (3) and in some chronic infections (4), has led to the suggestion that the virus is not directly cytopathic but that it is the immune response to infected liver cells that causes the hepatitis (5). The development of polymerase chain reaction (PCR) sequencing techniques has allowed us to determine whether sequence variation occurs under host selection and offers an explanation for the variable outcome of HBV infection.