Kinetic analysis of pathogenicity and tissue tropism of gyrovirus 3 in experimentally infected chickens.

Gyrovirus 3 (GyV3), the third novel emerging species of the genus Gyrovirus of the Anelloviridae family, has been described in multiple hosts. Epidemiologically, there are suggestions that GyV3 is associated with diarrhea/proventriculitis, however, no direct causal evidence exists between GyV3 infection and specific clinical diseases. Herein, we infected special pathogen-free (SPF) chickens with GyV3, and then assessed the pathogenicity and tissue tropism. The results revealed that GyV3 induced persistent infection characterized by diarrhea, aplastic anemia, immunosuppression, and persistent systemic lymphocytic inflammation. Clinically, the infected chickens presented ruffled feathers, diarrhea, anemia, and weight loss. Aplastic anemia was characterized by progressive depletion of hematopoietic cells in the bone marrow, immunosuppression was associated with atrophy of the thymus, spleen, and bursa of Fabricious, progressive lymphocytic inflammations were characterized by proventriculitis, adrenalitis, pancreatitis, hepatitis, nephritis, and bronchitis. Viral loads of GyV3 in tissues exhibited "M", "N", "W" or "V" type dynamic changes. The highest level of viral loads was reported in bone marrow at 7dpi, followed by the adrenal gland at 2 dpi, the sciatic nerve at 7 dpi, and bile at 35 dpi. The bone marrow and kidney demonstrate the strongest immunostaining of GyV3-VP1 antigen and were suggested as the target tissues of GyV3. Collectively, GyV3 is an immunosuppressive pathogenic virus that targets the bone marrow and kidney in chickens. Exploring the pathogenicity and tissue tropism of GyV3 will guide the basic understanding of the biology of GyV3 and its pathogenesis in chickens.

Cross-species pathogenicity of gyrovirus 3 in experimentally infected chickens and mice.

Gyrovirus 3 (GyV3) has been identified in humans and other hosts, suggesting its cross-species pathogenicity, which poses an increased public health risk. In the current study, we established chicken and mouse models of GyV3 infection. We found that GyV3 induced persistent infections, characterized by viremia, aplastic anemia, immunosuppression, and systematic lymphocytic inflammation, in both species. Kinetic viral loads and antigen expression demonstrated rapid viral replication and broad tissue tropism of GyV3 in both models. The highest viral loads and the strongest antigen immunostaining were present in bone marrow and cerebrum in both chickens and mice, indicating that these are target tissues for GyV3. Genetic diversity analysis of VP1 in infected chickens and mice showed that GyV3 adapts to new hosts via rapid evolution of the hypervariable region of the gene encoding the structural protein VP1. Overall, our results indicate that GyV3 is a cross-species pathogenic virus; therefore, more attention needs to be paid to high levels of GyV3-induced neurotropism and aplastic anemia as a public health risk.

Aplastic Anemia Frequency and Management in Pediatric Liver Transplantations Due to Non-A-E Hepatitis.

BACKGROUND: Hepatitis-associated aplastic anemia (HAAA) is a rare complication that presented with bone marrow failure after acute hepatitis. HAAA usually occurs in adolescent men within 1-6 months following hepatitis. Most of HAAA's etiology has non-A-E viral hepatitis. METHODS: Our retrospective study included patients with acute fulminant hepatitis who had been treated in Ege University Pediatric Gastroenterology, Hepatology and Nutrition Department and Izmir Kent Hospital Clinical, laboratory, and epidemiological data of the patients were collected from the files. RESULTS: In this study, 499 children underwent liver transplantation (LT) in two pediatric transplantation centers. Sixty-eight (13.6%) out of 499 patients, underwent liver transplantation due to fulminant hepatic failure (FHF). Therefore, a total of 64 patients (34 girls, 30 boys) with a diagnosis of FHF have included in the study. Thirty-two (50.0%) of 64 FHF were due to non-A-E hepatitis and 4 out of the 64 patients (6.2%) with FHF developed HAAA. All of the patients received prednisolone as immunosuppression treatment after LT. Three patients were also given Tacrolimus and 1 received an additional mycophenolate mofetil. One of the patients was given prednisolone and cyclosporine treatment without tacrolimus. Bone marrow transplantation was performed in 1 patient (25.0%). Two of the patients received immunosuppressive treatment including rabbit-derived anti-thymocyte globulin, cyclosporine, and initially prednisolone. CONCLUSION: In children who underwent liver transplantation for non-A-E FHF are at high risk to develop aplastic anemia. The clinicians should be alert after orthotropic liver transplantation patient could develop aplastic anemia and early treatment with immunosuppressive therapies result in a more successful outcome.

The Impact of Hepatitis on Clinical Outcomes for Pediatric Patients with Aplastic Anemia.

OBJECTIVES: To assess the prognostic role of hepatitis in pediatric patients with aplastic anemia and the incidence of hepatitis B among patients with hepatitis-associated aplastic anemia in an area with a previously high prevalence of hepatitis B after nationwide hepatitis B vaccination for 30 years. STUDY DESIGN: Pediatric patients (n = 78) with aplastic anemia were enrolled in this study, including 9 with hepatitis-associated aplastic anemia. We collected the clinical characteristics, etiologies of the aplastic anemia, hepatitis B virus serology and serum hepatitis B viral load, response to the treatments, and survival outcome from the participants. We applied univariate and multivariate Cox regression analysis to evaluate the correlations between clinical features and survival outcome. Survival analysis was done using Cox regression model and Kaplan-Meier curves. RESULTS: Patients with hepatitis-associated aplastic anemia were related to significantly worse survival prognosis when compared with patients with non-hepatitis-associated aplastic anemia, and hepatitis-associated aplastic anemia was the only independent prognostic factor to predict a poor survival outcome in our patients with aplastic anemia by multivariable analysis. In none of the total 78 patients was aplastic anemia related to hepatitis B virus infection. CONCLUSIONS: Patients with hepatitis-associated aplastic anemia had a significantly worse prognosis when compared with patients whose aplastic anemia was not hepatitis-associated. This study demonstrates the potential benefit of hepatitis B vaccination in decreasing the incidence of hepatitis-associated aplastic anemia in children.

Optimal pre-emptive cytomegalovirus therapy threshold in a patient population with high prevalence of seropositive status.

INTRODUCTION: Preemptive therapy (PET) for cytomegalovirus (CMV) reactivation post allogeneic hematopoietic stem cell transplantation (SCT) was shown to decrease the incidence of CMV disease. However, the optimal PET threshold is elusive. PURPOSE OF THE STUDY: To examine the efficacy of PET initiation at a viral threshold of 1000 copies/mL (1560 IU/mL) in a patient population with high prevalence of CMV seropositive status. PATIENTS AND METHODS: A single center retrospective review of patients that underwent allogeneic SCT was done. RESULTS: A total of 195 allogeneic SCT recipients were included with median follow up of 18.1 (0.7-95.6) months. A total of 178 (91 %) of patients had a positive CMV PCR with median days to initial reactivation post SCT of 17 (1-1187); 129 patients had peak CMV titer < 1000 copies/mL (low titer) whereas the remaining 49 patients had a peak titer ≥ 1000 copies/mL (high titer). 120 (93 %) of patients with low titers cleared spontaneously with median time to clearance of 40 days (4-188). One patient in the high titer group developed CMV disease. At multivariable analysis; age at SCT HR 1.02 (1.004-1.04; 0.017), malignant vs. benign condition HR 9.4 (2.47-61; 0.0005) and cGVHD HR 0.37 (0.2-0.65; 0.0005) were significant for OS. CONCLUSIONS: CMV reactivation post SCT was very common in patients with high prevalence of seropositive status. A PET threshold of 1000 copies/mL (1560 IU/mL) appears desirable as it was associated with spontaneous clearance in over 90 % of patients while minimizing treatment related toxicity. Validation of these observations is warranted.

Hepatitis-associated Aplastic Anaemia in Children.

BACKGROUND: Children with idiopathic acute liver failure (IALF) are at a high risk of developing life-threatening bone marrow failure (BMF). The aim of the study was to describe the development, therapy and prognosis of this hepatitis-associated aplastic anaemia (HAAA) in comparison to isolated acquired aplastic anaemia. RESULTS: We retrospectively found 18 patients (9 female) of HAAA between 1984 and 2017 with an age of 1.4-16.4 years. Fifteen of them fulfilled the SAA criteria, 3 had a bone marrow hypoplasia. Eleven of these children received liver transplantation (LTx) (these were 11 of 42 (26%) children receiving LTx for IALF), 6 patients recovered without LTx. The first signs of BMF, thrombocytopaenia and leucocytopaenia, occurred before LTx in all cases. During the follow-up period 8 patients reached haematological remission, 6 received haematopoietic stem cell transplantation (HSCT). Seven children died in a median of 304 days after the first symptoms mostly because of bleedings and infections. To date, extensive investigations failed to detect a genetically, viral or immunological aetiology. No AA was diagnosed in the 41 patients receiving liver transplants during the same period for ALF of known aetiology. As a comparison group, we collected the data of patients with isolated SAA. 73% achieved a remission after Immunosuppressive therapy (IST) without HSCT, and none of them died during the follow-up period. CONCLUSION: Blood counts should be examined early and regularly (0-22 days after onset) in patients with IALF. Aggressive treatment with LTx, IST and HSCT appears to improve the prognosis.

T-lymphocyte subsets and Th1/Th2 cytokines in convalescent patients with Epstein-Barr virus-associated aplastic anemia.

Objective: The aim of this study was to analyze T-lymphocyte subsets and Th1/Th2 cytokines in convalescent patients with Epstein-Barr virus (EBV)-associated aplastic anemia (AA).Methods: Sixty AA patients were enrolled, who were in remission following immunosuppressive therapy, including 34 EBV-negative cases and 26 EBV-positive cases. Their complete blood count (CBC), T-lymphocyte subsets, Th1/Th2 cytokines were analyzed. The correlation between EBV-DNA and T-lymphocyte subsets was evaluated, as well as the relationship between EBV-DNA and Th1/Th2 cytokines. The presence of EBV-DNA in peripheral blood mononuclear cells (PBMCs) was also assessed in 60 normal controls.Results: EBV-DNA was detected in 26/60 (43.33%) patients and 21/60 (35.00%) controls. EBV-DNA copy number in AA patients was higher than in controls (Z = -2.138, P = 0.033). The percentage of CD3+CD4+ T-lymphocytes and the ratio of CD4+/CD8+ T-lymphocytes in the EBV-negative group were higher than in the EBV-positive group (P = 0.001 and 0.001, respectively). EBV was positively correlated with CD3+CD8+ T-lymphocyte percentages (Pearson R: 0.496, P = 0.009). Moreover, EBV was positively correlated with IL-10 and IFN-γ levels (Pearson R: 0.559, P = 0.002 and Pearson R: 0.621, P = 0.001, respectively).Conclusions: EBV-DNA copy number in AA patients was higher than in normal controls. Both AA and EBV infection may cause changes in the levels of T-lymphocyte subsets. We recommend monitoring the changes in the immune function and EBV infection simultaneously in AA patients, especially following immunosuppressive therapy.

Risk Factors and Predictive Scoring System For Post-Transplant Lymphoproliferative Disorder after Hematopoietic Stem Cell Transplantation.

We analyzed data from 64,539 consecutive patients in the Japanese national transplant registry, including 40,195 after allogeneic hematopoietic stem cell transplantation (HSCT), 24,215 after autologous HSCT and 129 after syngeneic HSCT, of whom 299 developed Epstein-Barr virus-positive post-transplant lymphoproliferative disorder (PTLD). The probability of developing PTLD at 2 years post-HSCT was .79% after allogeneic transplantation, .78% after syngeneic transplantation, and .11% after autologous transplantation. The following variables were identified as risk factors after allogeneic HSCT in multivariate analysis: antithymocyte globulin (ATG) use in a conditioning regimen, ATG use for acute graft-versus-host disease (GVHD) treatment, donor other than an HLA-matched related donor, aplastic anemia, second or subsequent allogeneic HSCT, the most recent year of transplantation, and acute GVHD. The probability at 2 years increased particularly after 2009 (1.24%) than before 2009 (.45%). To stratify the risk of PTLD before allogeneic HSCT, we developed a novel 5-point scoring system based on 3 pretransplant risk factors: ATG use in a conditioning regimen (high dose, 2 points; low dose, 1 point), donor type (HLA-mismatched related donor, 1 point; unrelated donor, 1 point; cord blood, 2 points), and aplastic anemia (1 point). Patients were classified into 4 risk groups according to the summed points: low risk (0 or 1 point), intermediate risk (2 points), high risk (3 points), and very high risk (4 or 5 points) groups, with probabilities at 2 years of .3%, 1.3%, 4.6%, and 11.5%, respectively. Our scoring system is useful for predicting patients at high risk for PTLD. Careful observation and close monitoring of Epstein-Barr virus reactivation are warranted for these high-risk patients.

Clinical heterogeneity of human parvovirus B19 infection following adult liver transplantation.

Severe aplastic anemia and its secondary comorbidities associated with human parvovirus B19 infection is a rare and sometimes refractory complication following liver transplantation.We retrospectively reviewed data for 217 adult liver transplant recipients from donations after death in China March 2013 through May 2017, 5 patients with human parvovirus B19 infectious diseases were teased out, and diagnoses were made from positive serological marker, bone marrow aspiration, and genome assay, other hemolytic causes were excluded. Severe aplastic anemia and its comorbidities were confirmed, combination of immunoglobulin and blood transfusion as well as immunosuppressant switch was employed for 5 recipients.Four male and 1 female recipients were diagnosed with human parvovirus B19 infections based on clinical presentations, bone marrow aspiration, and nested PCR, age ranged from 47 to 62 years, the onset time from liver transplantation varied from 29 to 415 days, anemia improved in 5 patients, 2 deaths occurred due to parvovirus-related morbidities, 1 patient died from de novo carcinoma of the tongue 2 years later and unrelated to parvovirus, 2 other recipients are still alive.Human parvovirus B19 infectious disease is a rare but clinically significant infection whose comorbidities will bring about more attentions.

The Pathophysiology of Acquired Aplastic Anemia: Current Concepts Revisited.

Idiopathic acquired aplastic anemia is a rare, life-threatening bone marrow failure syndrome characterized by cytopenias and hypocellular bone marrow. The pathophysiology is unknown; the most favored model is of a dysregulated immune system leading to autoreactive T-cell destruction of hematopoietic stem and progenitor cells in a genetically susceptible host. The authors review the literature and propose that the major driver of acquired aplastic anemia is a combination of hematopoietic stem and progenitor cells intrinsic defects and an inappropriately activated immune response in the setting of a viral infection. Alterations in bone marrow microenvironment may also contribute to the disease process.

Epstein Barr Virus Infection Affects Function of Cytotoxic T Lymphocytes in Patients with Severe Aplastic Anemia.

Severe aplastic anemia (SAA) is characterized by pancytopenia and failure of hematopoietic function and is caused by excessive functioning of cytotoxic T lymphocytes (CTLs). EBNA-1, a nucleoprotein of the Epstein Barr virus (EBV), can influence the proliferation and function of lymphocytes. We therefore tested the number of EBV copies in the CD8+ T cells of 27 patients with SAA and 10 healthy control subjects and observed the influences of EBNA-1 upon the CD8+ T cells of patients with SAA. The results showed that more EBV copies were found in the CD8+ T cells of patients with untreated SAA than in patients with SAA in remission or in the healthy control subjects. Their copy number was positively correlated with the expression of granzyme B and perforin, the secretion level of interferon-γ in CD8+ T cells, and the viability of CD8+ T cells, whereas no correlation was seen between the copy number and the interleukin 4 secretion level or the apoptosis rate. Meanwhile, the expression of granzyme B and perforin was reduced after EBNA-1 gene knockdown, whereas the interferon-γ secretion level and cell viability declined. Therefore, we infer that EBV infection may be a factor in the activation of CTLs and in damaging the bone marrow hematopoietic function of patients with SAA.

Acute hepatitis as a prequel to very severe aplastic anemia.

Severe aplastic anemia is a rare and potentially life-threatening disease of the bone marrow often requiring allogeneic hematopoietic stem cell transplantation. Pathogenesis of the disease can vary and often remains enigmatic. Occasionally, severe aplastic anemia is associated with prior severe acute hepatitis. Differential diagnosis of acute non-viral hepatitis challenges the physician as pathogenesis remains unclear.We here present a case of a young patient presenting with acute hepatitis followed by severe aplastic anemia successfully treated with allogeneic hematopoietic stem cell transplantation. Due to immunosuppressive treatment with azathioprine for acute hepatitis of putative autoimmune pathogenesis and coincident infection with parvovirus B19, diagnosis of the sequential disease of acute hepatitis followed by severe aplastic anemia was complicated. We discuss the caveats and present a review of the literature.

Parvovirus B19 Is Associated with a Significant Decrease in Hemoglobin Level among Children <5 Years of Age with Anemia in Northwestern Tanzania.

Parvovirus B19 (B19) can cause transient aplastic crisis and lead to acute severe anemia. This study investigated the relationship between B19 and anemia among children <5 years old in the city of Mwanza, Tanzania. An enzyme immunoassay was used to detect B19 IgM- and IgG-specific antibodies among children with various categories of anemia according to the World Health Organization (WHO) guidelines. A total of 265 children with median age of 28.5 months (interquartile range 18-39.5) were investigated. Eighty-six children (32.5%) had severe anemia. B19-specific IgM and IgG antibodies were detected in 24 (9%) and 46 (17.4%) children, respectively. Low hemoglobin (Hb) level (p = 0.031), Plasmodium falciparum infection (p = 0.001) and residing in rural areas (p = 0.025) independently predicted B19 IgM seropositivity. Acute B19 infection decreased Hb level by 1.1 g/dl (p = 0.003). In malaria endemic areas, acute B19 infections should be considered among children with severe anemia from rural areas.

Resolution of acute hepatitis B-associated aplastic anaemia with antiviral therapy.

A previously healthy 44-year-old woman presented with 3 days of worsening petechial rash, epistaxis and fatigue. Admission labs revealed pancytopenia, low reticulocyte index and elevated liver enzymes. Bone marrow biopsy demonstrated a profoundly hypocellular bone marrow without dysplasia and additional testing demonstrated an acute hepatitis B infection. In the context of an acute hepatitis B infection, elevated liver enzymes and aplastic anaemia, our patient was diagnosed with severe hepatitis-associated aplastic anaemia due to an acute hepatitis B infection. She was initiated on antiviral therapy with tenofovir and briefly received immunosuppressive therapy with a robust sustained improvement in her blood counts. Acute hepatitis B-associated aplastic anaemia is an exceptionally rare presentation of aplastic anaemia. We present acute hepatitis B-associated aplastic anaemia that resolved with antiviral therapy, which to our knowledge is the second such case reported in the literature and the first using tenofovir.

Aplasia medular transitória associada a infecção por parvovírus B19 / Transient aplastic crisis caused by Parvovirus B19 infection

O parvovírus B19 é um eritrovírus humano com tropismo para as células progenitoras da medula óssea, sendo responsável por um grande espectro de manifestações clínicas, desde infecções assintomáticas até crises aplásicas graves. Os autores apresentam o caso de uma mulher de 40 anos, com história de anemia ferropênica por menorragias, que desenvolveu quadro clínico com febre, cefaleias, petéquias e, osteriormente, exantema nas pernas, associado à hipoplasia medular com redução transitória da contagem de todas as linhagens celulares hematológicas. A suspeita de infecção aguda por parvovírus B19 foi confirmada pela detecção de anticorpos IgM antiparvovírus B19 no sangue periférico, por meio de teste imunoenzimático (ELISA). Os achados do mielograma no 5o dia após a admissão, apesar de haver ainda tão só recuperação parcial das linhagens celulares hematológicas no sangue periférico, revelaram linhagens celulares medulares normais. A paciente teve recuperação espontânea, apenas com terapêutica de suporte.

Parvovirus B19 is a human erythrovirus with tropism for erythroid progenitor cells. It is responsible for a wide range of clinical manifestations, from asymptomatic infections to severe aplastic crises. The authors present the case of a 40 year-old female patient with history of iron-deficiency anemia from menorrhagia who presented with fever, headache, petechiae,and later,rash on lower limbs, associated with medullary hypoplasia and transient decrease of all hematologic cell lines.The suspicion of acute Parvovirus B19 infection was confirmed by the detection of anti-Parvovirus B19 IgM antibodies through Enzyme-Linked Immunosorbent Assay (ELISA). Although there was only partial recovery of the hematologic cell lineage in peripheral blood, findings on myelogram 5 days after admission showed normal hematopoietic cell lines. The patient had spontaneous recovery only with supportive treatment.

Ileal mass-like lesion induced by Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in a patient with aplastic anemia.

Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening hyperinflammatory syndrome characterized by activated macrophages engulfing erythrocytes, leukocytes, platelets, and their precursor cells in bone marrow, liver, spleen, or lymph nodes. We report a case of Epstein-Barr virus (EBV)-associated HLH unusually presenting as an ileal mass. A 23-year-old man presented initially with persistent fever unresponsive to antibiotics and pancytopenia. A bone marrow aspiration and biopsy were used to diagnose the patient with aplastic anemia and HLH. A relatively well-defined low-density mass was radiologically noted in the terminal ileum, along with enlarged lymph nodes, and was suspected to be malignant lymphoma or an abscess. The ileocecectomy specimen revealed a transmural hemorrhagic infarction with numerous activated macrophages phagocytosing erythrocytes, plasma cells, and lymphocytes, and he was diagnosed with EBV-associated HLH. The patient received an allo-unrelated peripheral blood stem-cell transplantation and expired due to graft-versus-host disease following liver failure. The present case is very unique, in that EBV-associated HLH presented with an unusual ileal mass resulting from hemorrhagic infarction in a patient with aplastic anemia, suggesting variability in the biological behavior of EBV-associated disease.