CD4+ and CD8+ T cells are required to prevent SARS-CoV-2 persistence in the nasal compartment.

SARS-CoV-2 is the causative agent of COVID-19 and continues to pose a significant public health threat throughout the world. Following SARS-CoV-2 infection, virus-specific CD4+ and CD8+ T cells are rapidly generated to form effector and memory cells and persist in the blood for several months. However, the contribution of T cells in controlling SARS-CoV-2 infection within the respiratory tract are not well understood. Using C57BL/6 mice infected with a naturally occurring SARS-CoV-2 variant (B.1.351), we evaluated the role of T cells in the upper and lower respiratory tract. Following infection, SARS-CoV-2-specific CD4+ and CD8+ T cells are recruited to the respiratory tract and a vast proportion secrete the cytotoxic molecule Granzyme B. Using antibodies to deplete T cells prior to infection, we found that CD4+ and CD8+ T cells play distinct roles in the upper and lower respiratory tract. In the lungs, T cells play a minimal role in viral control with viral clearance occurring in the absence of both CD4+ and CD8+ T cells through 28 days post-infection. In the nasal compartment, depletion of both CD4+ and CD8+ T cells, but not individually, results in persistent and culturable virus replicating in the nasal compartment through 28 days post-infection. Using in situ hybridization, we found that SARS-CoV-2 infection persisted in the nasal epithelial layer of tandem CD4+ and CD8+ T cell-depleted mice. Sequence analysis of virus isolates from persistently infected mice revealed mutations spanning across the genome, including a deletion in ORF6. Overall, our findings highlight the importance of T cells in controlling virus replication within the respiratory tract during SARS-CoV-2 infection.

Concerted synergy between viral-specific IgG and CD8 + T cells is critical for clearance of an HCV-related rodent hepacivirus.

BACKGROUND AND AIMS: Evidence assessing the role of B cells and their antibodies, or lack thereof, in the spontaneous resolution of acute HCV infection is conflicting. Utilization of a strictly hepatotropic, HCV-related rodent hepacivirus (RHV) model circumvents many of the challenges facing the field in characterizing the immunological correlates of dichotomous infection outcomes. This study seeks to elucidate the importance of B cells in the clearance of acute RHV infection. APPROACH AND RESULTS: µMT mice were infected i.v. with RHV and found to develop chronic infection for over a year. Wild-type (WT) mice depleted of B cells also exhibited persistent viremia that resolved only upon B cell resurgence. The persistent infection developed by B1-8i and AID cre/cre mice revealed that antigen-specific, class-switched B cells or their antibodies were crucial for viral resolution. Virus-specific CD8 + and CD4 + T cells were characterized in these mice using newly developed major histocompatibility complex class I and II tetramers and ex vivo peptide stimulation. Immunoglobulin G (IgG) was purified from the serum of RHV- or lymphocytic choriomeningitis virus Armstrong-infected mice after viral clearance and passively transferred to AID cre/cre recipients, revealing viral clearance only in αRHV IgG recipients. Further, the transfer of αRHV IgG into B cell-depleted recipients also induced viral resolution. This ability of RHV-specific IgG to induce viral clearance was found to require the concomitant presence of CD8 + T cells. CONCLUSIONS: Our findings demonstrate a cooperative interdependence between immunoglobulins and the T cell compartment that is required for RHV resolution. Thus, HCV vaccine regimens should aim to simultaneously elicit robust HCV-specific antibody and T cell responses for optimal protective efficacy.

Intrinsic p53 Activation Restricts Gammaherpesvirus-Driven Germinal Center B Cell Expansion during Latency Establishment.

Gammaherpesviruses (GHV) are DNA tumor viruses that establish lifelong latent infections in lymphocytes. For viruses such as Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68), this is accomplished through a viral gene-expression program that promotes cellular proliferation and differentiation, especially of germinal center (GC) B cells. Intrinsic host mechanisms that control virus-driven cellular expansion are incompletely defined. Using a small-animal model of GHV pathogenesis, we demonstrate in vivo that tumor suppressor p53 is activated specifically in B cells that are latently infected by MHV68. In the absence of p53, the early expansion of MHV68 latency was greatly increased, especially in GC B cells, a cell-type whose proliferation was conversely restricted by p53. We identify the B cell-specific latency gene M2, a viral promoter of GC B cell differentiation, as a viral protein sufficient to elicit a p53-dependent anti-proliferative response caused by Src-family kinase activation. We further demonstrate that EBV-encoded latent membrane protein 1 (LMP1) similarly triggers a p53 response in primary B cells. Our data highlight a model in which GHV latency gene-expression programs that promote B cell proliferation and differentiation to facilitate viral colonization of the host trigger aberrant cellular proliferation that is controlled by p53. IMPORTANCE: Gammaherpesviruses cause lifelong infections of their hosts, commonly referred to as latency, that can lead to cancer. Latency establishment benefits from the functions of viral proteins that augment and amplify B cell activation, proliferation, and differentiation signals. In uninfected cells, off-schedule cellular differentiation would typically trigger anti-proliferative responses by effector proteins known as tumor suppressors. However, tumor suppressor responses to gammaherpesvirus manipulation of cellular processes remain understudied, especially those that occur during latency establishment in a living organism. Here we identify p53, a tumor suppressor commonly mutated in cancer, as a host factor that limits virus-driven B cell proliferation and differentiation, and thus, viral colonization of a host. We demonstrate that p53 activation occurs in response to viral latency proteins that induce B cell activation. This work informs a gap in our understanding of intrinsic cellular defense mechanisms that restrict lifelong GHV infection.

The monarch butterfly in Mexico: a conservation model.

Each fall, millions of monarch butterflies (Danaus plexippus L.) travel from Canada and the United States to overwinter in Mexico and California. In 2022, the IUCN listed migratory monarchs as endangered because of their population decline. The main accepted drivers are widespread use of herbicides, effects of climate, and land use change that causes habitat loss. We analyzed the main actions taken to officially protect the overwintering sites and the migration phenomenon with the establishment of the Monarch Butterfly Biosphere Reserve in 2000. The conservation of the monarch overwintering sites in Mexico is an example of continuous work from their discovery to the present. We highlight the importance of monitoring the areas covered by overwintering monarchs in Mexico. These colonies represent the largest concentrations of monarch populations in the world. In the last 10 years, the average area covered by monarchs was 2.72 ( ± 0.47 SE) hectares.

Soluble ORF2 protein enhances HEV replication and induces long-lasting antibody response and protective immunity in vivo.

BACKGROUND AND AIMS: The HEV is a small positive-sense RNA virus that encodes a cytoplasmic form of the capsid protein (ORF2c), essential for virion structure, and a secreted glycosylated form (ORF2s) that accumulates at high titer in serum and can mask neutralizing epitopes. We explored the contribution of ORF2s to HEV replication and its role in generating antibodies against ORF2 in a nonhuman primate model. APPROACH AND RESULTS: We used a recombinant HEV genotype 3 variant that does not express ORF2s due to the introduction of stop codons (ORF2s mut ). Rhesus macaques (RMs) were given intrahepatic injections of infectious wildtype HEV (ORF2s wt ) RNA or a variant lacking ORF2s expression (ORF2s mut ). The replication of the ORF2s mut virus was delayed by ~2 weeks compared with ORF2s wt , and peak titers were nearly tenfold lower. Reversions of the 3 mutations that blocked ORF2s expression were not detected in the ORF2s mut genomes, indicating genetic stability. However, serum antibodies against ORF2 were transiently detected in RMs infected with ORF2s mut , whereas they were long-lasting in RMs infected with ORF2s wt . Moreover, RMs infected with ORF2s mut were more susceptible to reinfection, as evidenced by the viral RNA detected in fecal samples and the expansion of HEV-specific CD8 + T cells. CONCLUSIONS: These findings indicate that ORF2s may be dispensable for viral replication in vivo but is required for long-lived antibody-mediated responses that protect against HEV re-exposure.

Differential immune transcriptomic profiles between vaccinated and resolved HCV reinfected subjects.

Successive episodes of hepatitis C virus (HCV) infection represent a unique natural rechallenge experiment to define correlates of long-term protective immunity and inform vaccine development. We applied a systems immunology approach to characterize longitudinal changes in the peripheral blood transcriptomic signatures in eight subjects who spontaneously resolved two successive HCV infections. Furthermore, we compared these signatures with those induced by an HCV T cell-based vaccine regimen. We identified a plasma cell transcriptomic signature during early acute HCV reinfection. This signature was absent in primary infection and following HCV vaccine boost. Spontaneous resolution of HCV reinfection was associated with rapid expansion of glycoprotein E2-specifc memory B cells in three subjects and transient increase in E2-specific neutralizing antibodies in six subjects. Concurrently, there was an increase in the breadth and magnitude of HCV-specific T cells in 7 out of 8 subjects. These results suggest a cooperative role for both antibodies and T cells in clearance of HCV reinfection and support the development of next generation HCV vaccines targeting these two arms of the immune system.

Prospecting the Resilience of Several Spanish Ancient Varieties of Red Grape under Climate Change Scenarios.

BACKGROUND: Climate change results in warmer air temperatures and an uncertain amount and distribution of annual precipitations, which will directly impact rainfed crops, such as the grapevine. Traditionally, ancient autochthones grapevine varieties have been substituted by modern ones with higher productivity. However, this homogenization of genotypes reduces the genetic diversity of vineyards which could make their ability to adapt to challenges imposed by future climate conditions difficult. Therefore, this work aimed to assess the response of four ancient grapevine varieties to high temperatures under different water availabilities, focusing on plant water relations, grape technological and phenolic maturity, and the antioxidant capacity of the must. METHODS: The study was conducted on fruit-bearing cuttings grown in pots in temperature-gradient greenhouses. A two-factorial design was established where two temperature regimes, ambient and elevated (ambient + 4 °C), were combined with two water regimes, full irrigation and post-veraison deficit irrigation, during fruit ripening. RESULTS: There were significant differences among the ancient varieties regarding plant water relations and fruit quality. CONCLUSION: This research underlines the importance of evaluating the behavior of ancient grapevine varieties that could offer good options for the adaptation of viticulture to future climate conditions.

Serum neutralization activity declines but memory B cells persist after cure of chronic hepatitis C.

The increasing incidence of hepatitis C virus (HCV) infections underscores the need for an effective vaccine. Successful vaccines to other viruses generally depend on a long-lasting humoral response. However, data on the half-life of HCV-specific responses are lacking. Here we study archived sera and mononuclear cells that were prospectively collected up to 18 years after cure of chronic HCV infection to determine the role of HCV antigen in maintaining neutralizing antibody and B cell responses. We show that HCV-neutralizing activity decreases rapidly in potency and breadth after curative treatment. In contrast, HCV-specific memory B cells persist, and display a restored resting phenotype, normalized chemokine receptor expression and preserved ability to differentiate into antibody-secreting cells. The short half-life of HCV-neutralizing activity is consistent with a lack of long-lived plasma cells. The persistence of HCV-specific memory B cells and the reduced inflammation after cure provide an opportunity for vaccination to induce protective immunity against re-infection.

Brief Research Report: Anti-SARS-CoV-2 Immunity in Long Lasting Responders to Cancer Immunotherapy Through mRNA-Based COVID-19 Vaccination.

Cancer patients (CPs) have been identified as particularly vulnerable to SARS-CoV-2 infection, and therefore are a priority group for receiving COVID-19 vaccination. From the patients with advanced solid tumors, about 20% respond very efficiently to immunotherapy with anti-PD1/PD-L1 antibodies and achieve long lasting cancer responses. It is unclear whether an efficient cancer-specific immune response may also correlate with an efficient response upon COVID-19 vaccination. Here, we explored the antiviral immune response to the mRNA-based COVID-19 vaccine BNT162b2 in a group of 11 long-lasting cancer immunotherapy responders. We analysed the development of SARS-CoV-2-specific IgG serum antibodies, virus neutralizing capacities and T cell responses. Control groups included patients treated with adjuvant cancer immunotherapy (IMT, cohort B), CPs not treated with immunotherapy (no-IMT, cohort C) and healthy controls (cohort A). The median ELISA IgG titers significantly increased after the prime-boost COVID vaccine regimen in all cohorts (Cohort A: pre-vaccine = 900 (100-2700), 3 weeks (w) post-boost = 24300 (2700-72900); Cohort B: pre-vaccine = 300 (100-2700), 3 w post-boost = 8100 (300-72900); Cohort C: pre-vaccine = 500 (100-2700), 3 w post-boost = 24300 (300-72900)). However, at the 3 w post-prime time-point, only the healthy control group showed a statistically significant increase in antibody levels (Cohort A = 8100 (900-8100); Cohort B = 900 (300-8100); Cohort C = 900 (300-8100)) (P < 0.05). Strikingly, while all healthy controls generated high-level antibody responses after the complete prime-boost regimen (Cohort A = 15/15 (100%), not all CPs behaved alike [Cohort B= 12/14 (84'6%); Cohort C= 5/6 (83%)]. Their responses, including those of the long-lasting immunotherapy responders, were more variable (Cohort A: 3 w post-boost (median nAb titers = 95.32 (84.09-96.93), median Spike-specific IFN-γ response = 64 (24-150); Cohort B: 3 w post-boost (median nAb titers = 85.62 (8.22-97.19), median Spike-specific IFN-γ response (28 (1-372); Cohort C: 3 w post-boost (median nAb titers = 95.87 (11.8-97.3), median Spike-specific IFN-γ response = 67 (20-84)). Two long-lasting cancer responders did not respond properly to the prime-boost vaccination and did not generate S-specific IgGs, neutralizing antibodies or virus-specific T cells, although their cancer immune control persisted for years. Thus, although mRNA-based vaccines can induce both antibody and T cell responses in CPs, the immune response to COVID vaccination is independent of the capacity to develop an efficient anti-cancer immune response to anti PD-1/PD-L1 antibodies.

Neutralization and receptor use of infectious culture-derived rat hepacivirus as a model for HCV.

BACKGROUND AND AIMS: Lack of tractable immunocompetent animal models amenable to robust experimental challenge impedes vaccine efforts for HCV. Infection with rodent hepacivirus from Rattus norvegicus (RHV-rn1) in rats shares HCV-defining characteristics, including liver tropism, chronicity, and pathology. RHV in vitro cultivation would facilitate genetic studies on particle production, host factor interactions, and evaluation of antibody neutralization guiding HCV vaccine approaches. APPROACH AND RESULTS: We report an infectious reverse genetic cell culture system for RHV-rn1 using highly permissive rat hepatoma cells and adaptive mutations in the E2, NS4B, and NS5A viral proteins. Cell culture-derived RHV-rn1 particles (RHVcc) share hallmark biophysical characteristics of HCV and are infectious in mice and rats. Culture adaptive mutations attenuated RHVcc in immunocompetent rats, and the mutations reverted following prolonged infection, but not in severe combined immunodeficiency (SCID) mice, suggesting that adaptive immune pressure is a primary driver of reversion. Accordingly, sera from RHVcc-infected SCID mice or the early acute phase of immunocompetent mice and rats were infectious in culture. We further established an in vitro RHVcc neutralization assay, and observed neutralizing activity of rat sera specifically from the chronic phase of infection. Finally, we found that scavenger receptor class B type I promoted RHV-rn1 entry in vitro and in vivo. CONCLUSIONS: The RHV-rn1 infectious cell culture system enables studies of humoral immune responses against hepacivirus infection. Moreover, recapitulation of the entire RHV-rn1 infectious cycle in cell culture will facilitate reverse genetic studies and the exploration of tropism and virus-host interactions.

Changes in climate drive recent monarch butterfly dynamics.

Declines in the abundance and diversity of insects pose a substantial threat to terrestrial ecosystems worldwide. Yet, identifying the causes of these declines has proved difficult, even for well-studied species like monarch butterflies, whose eastern North American population has decreased markedly over the last three decades. Three hypotheses have been proposed to explain the changes observed in the eastern monarch population: loss of milkweed host plants from increased herbicide use, mortality during autumn migration and/or early-winter resettlement and changes in breeding-season climate. Here, we use a hierarchical modelling approach, combining data from >18,000 systematic surveys to evaluate support for each of these hypotheses over a 25-yr period. Between 2004 and 2018, breeding-season weather was nearly seven times more important than other factors in explaining variation in summer population size, which was positively associated with the size of the subsequent overwintering population. Although data limitations prevent definitive evaluation of the factors governing population size between 1994 and 2003 (the period of the steepest monarch decline coinciding with a widespread increase in herbicide use), breeding-season weather was similarly identified as an important driver of monarch population size. If observed changes in spring and summer climate continue, portions of the current breeding range may become inhospitable for monarchs. Our results highlight the increasingly important contribution of a changing climate to insect declines.

Resolution of hepatitis E virus infection in CD8+ T cell-depleted rhesus macaques.

BACKGROUND & AIMS: HEV is a significant cause of acute hepatitis globally. Some genotypes establish persistent infection when immunity is impaired. Adaptive immune mechanisms that mediate resolution of infection have not been identified. Herein, the requirement for CD8+ T cells to control HEV infection was assessed in rhesus macaques, a model of acute and persistent HEV infection in humans. METHODS: Rhesus macaques were untreated or treated with depleting anti-CD8α monoclonal antibodies before challenge with an HEV genotype (gt)3 isolate derived from a chronically infected human patient. HEV replication, alanine aminotransferase, anti-capsid antibody and HEV-specific CD4+ and CD8+ T cell responses were assessed after infection. RESULTS: HEV control in untreated macaques coincided with the onset of a neutralizing IgG response against the ORF2 capsid and liver infiltration of functional HEV-specific CD4+ and CD8+ T cells. Virus control was delayed by 1 week in CD8+ T cell-depleted macaques. Infection resolved with onset of a neutralizing IgG antibody response and a much more robust expansion of CD4+ T cells with antiviral effector function. CONCLUSIONS: Liver infiltration of functional CD8+ T cells coincident with HEV clearance in untreated rhesus macaques, and a 1-week delay in HEV clearance in CD8+ T cell-depleted rhesus macaques, support a role for this subset in timely control of virus replication. Resolution of infection in the absence of CD8+ T cells nonetheless indicates that neutralizing antibodies and/or CD4+ T cells may act autonomously to inhibit HEV replication. HEV susceptibility to multiple adaptive effector mechanisms may explain why persistence occurs only with generalized immune suppression. The findings also suggest that neutralizing antibodies and/or CD4+ T cells should be considered as a component of immunotherapy for chronic infection. LAY SUMMARY: The hepatitis E virus (HEV) is a major cause of liver disease globally. Some genetic types (genotypes) of HEV persist in the body if immunity is impaired. Our objective was to identify immune responses that promote clearance of HEV. Findings indicate that HEV may be susceptible to multiple arms of the immune response that can act independently to terminate infection. They also provide a pathway to assess immune therapies for chronic HEV infection.

Early T follicular helper cell activity accelerates hepatitis C virus-specific B cell expansion.

Early appearance of neutralizing antibodies during acute hepatitis C virus (HCV) infection is associated with spontaneous viral clearance. However, the longitudinal changes in antigen-specific memory B cell (MBCs) associated with divergent HCV infection outcomes remain undefined. We characterized longitudinal changes in E2 glycoprotein-specific MBCs from subjects who either spontaneously resolved acute HCV infection or progressed to chronic infection, using single-cell RNA-seq and functional assays. HCV-specific antibodies in plasma from chronically infected subjects recognized multiple E2 genotypes, while those from spontaneous resolvers exhibited variable cross-reactivity to heterotypic E2. E2-specific MBCs from spontaneous resolvers peaked early after infection (4-6 months), following expansion of activated circulating T follicular helper cells (cTfh) expressing interleukin 21. In contrast, E2-specific MBCs from chronically infected subjects, enriched in VH1-69, expanded during persistent infection (> 1 year), in the absence of significantly activated cTfh expansion. Early E2-specific MBCs from spontaneous resolvers produced monoclonal antibodies (mAbs) with fewer somatic hypermutations and lower E2 binding but similar neutralization as mAbs from late E2-specific MBCs of chronically infected subjects. These findings indicate that early cTfh activity accelerates expansion of E2-specific MBCs during acute infection, which might contribute to spontaneous clearance of HCV.

Immune system control of hepatitis C virus infection.

Hepatitis C virus (HCV) remains a global public health problem even though more than 95% of infections can be cured by treatment with direct-acting antiviral agents. Resolution of viremia post antiviral therapy does not lead to protective immunity and therefore reinfections can occur. Immune cell detection of HCV activates signaling pathways that produce interferons and trigger the innate immune response against the virus, preventing HCV replication and spread. Cells in the innate immune system, including natural killer, dendritic, and Kupffer cells, interact with infected hepatocytes and present viral antigens to T and B cells where their effector responses contribute to infection outcome. Despite the immune activation, HCV can evade the host response and establish persistent infection. Plans to understand the correlates of protection and strategies to activate proper innate and adaptive immune responses are needed for development of an effective prophylactic vaccine that stimulates protective immunity and limits HCV transmission.

Multiscale seasonal factors drive the size of winter monarch colonies.

Monarch butterflies in eastern North America have declined by 84% on Mexican wintering grounds since the observed peak in 1996. However, coarse-scale population indices from northern US breeding grounds do not show a consistent downward trend. This discrepancy has led to speculation that autumn migration may be a critical limiting period. We address this hypothesis by examining the role of multiscale processes impacting monarchs during autumn, assessed using arrival abundances at all known winter colony sites over a 12-y period (2004-2015). We quantified effects of continental-scale (climate, landscape greenness, and disease) and local-scale (colony habitat quality) drivers of spatiotemporal trends in winter colony sizes. We also included effects of peak summer and migratory population indices. Our results demonstrate that higher summer abundance on northern breeding grounds led to larger winter colonies as did greener autumns, a proxy for increased nectar availability in southern US floral corridors. Colony sizes were also positively correlated with the amount of local dense forest cover and whether they were located within the Monarch Butterfly Biosphere Reserve, but were not influenced by disease rates. Although we demonstrate a demographic link between summer and fine-scale winter population sizes, we also reveal that conditions experienced during, and at the culmination of, autumn migration impact annual dynamics. Monarchs face a growing threat if floral resources and winter habitat availability diminish under climate change. Our study tackles a long-standing gap in the monarch's annual cycle and highlights the importance of evaluating migratory conditions to understand mechanisms governing long-term population trends.

Conditional mutagenesis in vivo reveals cell type- and infection stage-specific requirements for LANA in chronic MHV68 infection.

Gammaherpesvirus (GHV) pathogenesis is a complex process that involves productive viral replication, dissemination to tissues that harbor lifelong latent infection, and reactivation from latency back into a productive replication cycle. Traditional loss-of-function mutagenesis approaches in mice using murine gammaherpesvirus 68 (MHV68), a model that allows for examination of GHV pathogenesis in vivo, have been invaluable for defining requirements for specific viral gene products in GHV infection. But these approaches are insufficient to fully reveal how viral gene products contribute when the encoded protein facilitates multiple processes in the infectious cycle and when these functions vary over time and from one host tissue to another. To address this complexity, we developed an MHV68 genetic platform that enables cell-type-specific and inducible viral gene deletion in vivo. We employed this system to re-evaluate functions of the MHV68 latency-associated nuclear antigen (mLANA), a protein with roles in both viral replication and latency. Cre-mediated deletion in mice of loxP-flanked ORF73 demonstrated the necessity of mLANA in B cells for MHV68 latency establishment. Impaired latency during the transition from draining lymph nodes to blood following mLANA deletion also was observed, supporting the hypothesis that B cells are a major conduit for viral dissemination. Ablation of mLANA in infected germinal center (GC) B cells severely impaired viral latency, indicating the importance of viral passage through the GC for latency establishment. Finally, induced ablation of mLANA during latency resulted in complete loss of affected viral genomes, indicating that mLANA is critically important for maintenance of viral genomes during stable latency. Collectively, these experiments provide new insights into LANA homolog functions in GHV colonization of the host and highlight the potential of a new MHV68 genetic platform to foster a more complete understanding of viral gene functions at discrete stages of GHV pathogenesis.

Murine Gammaherpesvirus 68 Expressing Kaposi Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen (LANA) Reveals both Functional Conservation and Divergence in LANA Homologs.

Latency-associated nuclear antigen (LANA) is a multifunctional protein encoded by members of the Rhadinovirus genus of gammaherpesviruses. Studies using murine gammaherpesvirus 68 (MHV68) demonstrated that LANA is important for acute replication, latency establishment, and reactivation in vivo Despite structural similarities in their DNA-binding domains (DBDs), LANA homologs from Kaposi sarcoma-associated herpesvirus (KSHV) and MHV68 exhibit considerable sequence divergence. We sought to determine if KSHV and MHV68 LANA homologs are functionally interchangeable. We generated an MHV68 virus that encodes KSHV LANA (kLANA) in place of MHV68 LANA (mLANA) and evaluated the virus's capacity to replicate, establish and maintain latency, and reactivate. kLANA knock-in (KLKI) MHV68 was replication competent in vitro and in vivo but exhibited slower growth kinetics and lower titers than wild-type (WT) MHV68. Following inoculation of mice, KLKI MHV68 established and maintained latency in splenocytes and peritoneal cells but did not reactivate efficiently ex vivo kLANA repressed the MHV68 promoter for ORF50, the gene that encodes the major lytic transactivator protein RTA, while mLANA did not, suggesting a likely mechanism for the KLKI MHV68 phenotypes. Bypassing this repression by providing MHV68 RTA in trans rescued KLKI MHV68 replication in tissue culture and enabled detection of KLKI MHV68 reactivation ex vivo These data demonstrate that kLANA and mLANA are functionally interchangeable for establishment and maintenance of latency and suggest that repression of lytic replication by kLANA, as previously shown with KSHV, is a kLANA-specific function that is transferable to MHV68.IMPORTANCE Kaposi sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) are members of the Rhadinovirus genus of gammaherpesviruses. These viruses establish lifelong infections that place their respective human and murine hosts at risk for cancer. Latency-associated nuclear antigen (LANA) is a conserved Rhadinovirus protein that is necessary for long-term chronic infection by these viruses. To better understand the conserved functions performed by LANA homologs, we generated a recombinant MHV68 virus that encodes the KSHV LANA protein in place of the MHV68 LANA homolog. We determined that the KSHV LANA protein is capable of supporting MHV68 latency in a mouse model of chronic infection but also functions to repress viral replication. This work describes an in vivo model system for defining evolutionarily conserved and divergent functions of LANA homologs in Rhadinovirus infection and disease.

Population Genetics of Overwintering Monarch Butterflies, Danaus plexippus (Linnaeus), from Central Mexico Inferred from Mitochondrial DNA and Microsatellite Markers.

Population genetic variation and demographic history in Danaus plexippus (L.), from Mexico were assessed based on analyses of mitochondrial cytochrome c oxidase subunit I (COI; 658 bp) and subunit II (COII; 503 bp) gene segments and 7 microsatellite loci. The sample of 133 individuals included both migratory monarchs, mainly from 4 overwintering sites within the Monarch Butterfly Biosphere Reserve (MBBR) in central Mexico (states of Michoacán and México), and a nonmigratory population from Irapuato, Guanajuato. Haplotype (h) and nucleotide (π) diversities were relatively low, averaging 0.466 and 0.00073, respectively, for COI, and 0.629 and 0.00245 for COII. Analysis of molecular variance of the COI data set, which included additional GenBank sequences from a nonmigratory Costa Rican population, showed significant population structure between Mexican migratory monarchs and nonmigratory monarchs from both Mexico and Costa Rica, suggesting limited gene flow between the 2 behaviorally distinct groups. Interestingly, while the COI haplotype frequencies of the nonmigratory populations differed from the migratory, they were similar to each other, despite the great physical distance between them. Microsatellite analyses, however, suggested a lack of structure between the 2 groups, possibly owing to the number of significant deviations from Hardy-Weinberg equilibrium resulting from heterzoygote deficiencies found for most of the loci. Estimates of demographic history of the combined migratory MBBR monarch population, based on the mismatch distribution and Bayesian skyline analyses of the concatenated COI and COII data set (n = 89) suggested a population expansion dating to the late Pleistocene (~35000-40000 years before present) followed by a stable effective female population size (Nef) of about 6 million over the last 10000 years.

Desensibilización rápida al ácido acetilsalicílico de pacientes con cardiopatía isquémica: experiencia de un centro / Rapid desensitization to aspirin in patients with coronary artery disease: A single center experience

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