RESUMO
BACKGROUND: Valganciclovir prophylaxis against cytomegalovirus (CMV) is recommended for solid organ transplant recipients, but is associated with drawbacks, including expense and leukopenia. Our center adopted a strategy of serial assessment with a CMV-specific T cell immunity panel (CMV-TCIP) and cessation of valganciclovir prophylaxis upon demonstration of adequate CD4+ responses in kidney transplant patients at high risk of CMV disease. METHODS: We retrospectively reviewed adult recipients of a kidney or pancreas transplant between August 2019 and July 2021 undergoing serial CMV-TCIP monitoring. Included patients were considered high risk for CMV, defined by donor positive (D+)/recipient negative (R-) CMV IgG serostatus, or recipient positive (R+) patients who received induction with a lymphocyte-depleting agent. Prophylaxis was discontinued after a patient's first CMV-specific CD4+ T cell value of ≥0.20%. Risk of clinically significant CMV infection (csCMVi) in those who underwent early discontinuation of CMV prophylaxis and predictors of CMV T cell immunity were analyzed. RESULTS: Of 54 included patients, 22 stopped prophylaxis early due to CMV-specific CD4+ T cell immunity at a median of 4.7 (IQR: 3.8-5.4) months after transplant. No instances of csCMVi were observed in the 22 patients who had prophylaxis discontinued early, of whom 19/22 were CMV R+ and 3/22 were CMV D+/R-. Donor/recipient CMV serostatus was predictive of immunity (p <.001). CONCLUSION: Early discontinuation of valganciclovir prophylaxis in patients with CMV CD4+ T cellular immunity appears safe and potentially beneficial in this preliminary series, especially in R+ patients. Further study is warranted, given that truncated prophylaxis may yield patient-level benefits.
Assuntos
Antivirais , Infecções por Citomegalovirus , Citomegalovirus , Transplante de Rim , Valganciclovir , Humanos , Infecções por Citomegalovirus/prevenção & controle , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Transplante de Rim/efeitos adversos , Masculino , Pessoa de Meia-Idade , Feminino , Estudos Retrospectivos , Antivirais/uso terapêutico , Antivirais/administração & dosagem , Valganciclovir/uso terapêutico , Valganciclovir/administração & dosagem , Citomegalovirus/imunologia , Adulto , Linfócitos T CD4-Positivos/imunologia , Transplantados , Idoso , Linfócitos T/imunologia , Fatores de RiscoRESUMO
The role of viral diversity in the pathogenesis of BK polyomavirus (BKPyV)-associated disease is poorly understood. Here, we report near full-length BKPyV genome sequences from two allogeneic hematopoietic cell transplant recipients infected with BKPyV genotype II, which is uncommon in the USA.
RESUMO
Cytomegalovirus (CMV) is a significant human pathogen, especially for immunocompromised patients, often treated with one or more antiviral drugs. Although the prevalence of resistance is low, the impact of drug resistant CMV infections on patient outcomes is high and genotypic testing is recommended when resistance is suspected. To assess the prevalence of CMV drug resistance mutations among samples submitted for genotypic testing, 2750 patient sample results were analyzed. Testing was performed by sequencing for ganciclovir (GCV), cidofovir (CDV), foscarnet (FOS), maribavir (MBV) and/or letermovir (LMV) resistance conferring mutations. Of the 2750 patient samples, 826 (30.04%) had resistance to one or more anti-CMV drug. Resistance mutations were most common in UL97, with 27.64% and 9.96% of samples having GCV and MBV mutations, respectively. Resistance mutations in UL54 were less common, with 6.11%, 5.98% and 1.76% of samples having GCV, CDV and FOS mutations, respectively. For LMV, resistance mutations in UL56 were present in 7.17% of samples, with mutations at codon 325 representing 80.95% of the observed LMV resistance mutations. Resistance to two drugs was identified in 215 samples and to 3 or more drugs in 35 samples. While a high prevalence of CMV resistance mutations was identified, this must be taken in the context of healthcare providers submitting samples from patients with suspected resistant CMV strains. For these patients, rapid monitoring for resistance allows treatment modifications based on objective results rather than empiric drug selection, which is particularly relevant given the presence of mutations conferring resistance to more than one drug.
Assuntos
Citomegalovirus , Transplantados , Humanos , Citomegalovirus/genética , Prevalência , DNA Polimerase Dirigida por DNA/genética , Proteínas Virais/genética , Antivirais/farmacologia , Antivirais/uso terapêutico , Ganciclovir/farmacologia , Ganciclovir/uso terapêutico , Foscarnet/farmacologia , Foscarnet/uso terapêutico , Cidofovir , Farmacorresistência Viral/genética , Mutação , BenzimidazóisRESUMO
BK polyomavirus (BKPyV) infection is common after hematopoietic stem cell transplantation (HSCT) and is associated with the development of hemorrhagic cystitis (HC). The role that BKPyV plays in the pathogenesis of HC is not well characterized. We investigated the impact of BKPyV diversity on the development of HC using a previously established cohort of pediatric HSCT patients. There were 147 urine samples with quantifiable BKPyV at month 1 after HSCT; 137 (93.2%) were amplified using our in-house polymerase chain reaction approach and sent for next-generation sequencing. Subtype Ia was most frequent (61.3%), followed by subtype Ib1 (31.4%). The median viral load of subtype Ia samples was higher than for subtype Ib1 at month 1. Across the protein coding regions, APOBEC-induced mutations and signature patterns associated with HC were identified. This is the largest sequencing study of a single cohort of HSCT patients, providing a vast resource of sequence data for future analyses.
Assuntos
Vírus BK , Cistite , Transplante de Células-Tronco Hematopoéticas , Infecções por Polyomavirus , Infecções Tumorais por Vírus , Humanos , Criança , Vírus BK/genética , Hemorragia/complicações , Transplante de Células-Tronco Hematopoéticas/efeitos adversosRESUMO
Symptomatic BK polyomavirus (BKPyV) infections are common and relevant in immunocompromised patients. Here, we present full-length BKPyV genomes from samples from patients who received hematopoietic cell transplants in the United States. These individuals had non-subtype I BKPyV, as determined by amplification, next-generation sequencing, and phylogenetic analysis.
RESUMO
BK polyomavirus (BKPyV) is a ubiquitous pathogen that typically results in asymptomatic infection. However, in immunocompromised individuals, BKPyV viral shedding in the urine can reach 109 copies per mL. These high viral levels within urine provide ideal samples for next-generation sequencing to accurately determine BKPyV genotype and identify mutations associated with pathogenesis. Sequencing data obtained can be further analyzed to better understand and characterize the genetic diversity present in BKPyV. Here, methods are described for the successful extraction of viral DNA from urine and the subsequent amplification methods to prepare a sample for next-generation sequencing.
Assuntos
Vírus BK , Infecções por Polyomavirus , Infecções Tumorais por Vírus , Vírus BK/genética , DNA Viral/genética , Humanos , Hospedeiro Imunocomprometido , Infecções por Polyomavirus/diagnóstico , Infecções Tumorais por Vírus/diagnóstico , Eliminação de Partículas ViraisRESUMO
BACKGROUND: The Eurofins GeneScan Technologies' VIRSeek SARS-CoV-2 Mplex kit is a RT (reverse transcription) real-time polymerase chain reaction (RT-qPCR) assay for the detection of two targets on the N-gene (nucleocapsid) of SARS-CoV-2. An extraction control, that allows monitoring of the extraction procedure and PCR inhibition, is included. OBJECTIVE: In silico analysis and wet testing showed inclusivity and exclusivity of the assay. The complete workflow starting from surface swabbing (VIRSeek PATHOSwab kit), RNA extraction (VIRSeek RNAExtractor), RT-PCR (VIRSeek SARS-CoV-2 Mplex), and evaluation with FastFinder was validated in comparison to the CDC method for detection of SARS-CoV-2 on stainless steel. METHOD: In silico analysis was performed by using the MFOLD online program. The matrix study was performed for stainless steel inoculated with SARS-CoV-2 isolated from the first documented US case of a traveler from Wuhan, China. RESULTS: For inclusivity, 15 764 sequences were analyzed and all mismatches (0.37% of the sequences had single mismatches) were considered non-critical. Cross reactivity for closely related viruses and background organisms was performed, resulting in correct exclusion of all. No significant differences were observed for the probability of detection (POD) study when comparing to the CDC method. CONCLUSIONS: Results of the inclusivity and exclusivity study show that the assay is specific for detection of SARS-CoV-2. The POD study showed no statistically significant difference compared to the CDC reference method, results were identical for the uninoculated and the high level. For the fractional recovery level, the candidate method detected 9/17 samples leading to a POD of 0.47, the reference method detected 11/20 samples leading to a POD of 0.55. HIGHLIGHT: The complete workflow starting from swabbing of the surface (VIRSeek PATHOSwab kit), RNA extraction (VIRSeek RNAExtractor), RT-PCR (VIRSeek SARS CoV-2 Mplex) and evaluation with FastFinder was validated in comparison to the US Centers for Disease Control and Prevention method for detection of SARS-CoV-2 on Stainless Steel.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Aço InoxidávelRESUMO
BK polyomavirus (BKPyV) infection can lead to nephropathy and hemorrhagic cystitis (HC). We evaluated BKPyV genotypes in two individuals after hematopoietic cell transplant (HCT). The first case developed HC and was infected with genotype Ib-2, while the second did not develop HC and was infected with genotype Ia.
RESUMO
SARS-CoV-2 has infected millions worldwide. The virus is novel, and currently there is no approved treatment. Convalescent plasma may offer a treatment option. We evaluated trends of IgM/IgG antibodies/plasma viral load in donors and recipients of convalescent plasma. 114/139 (82 %) donors had positive IgG antibodies. 46/114 donors tested positive a second time by NP swab. Among those retested, the median IgG declined (p < 0.01) between tests. 25/139 donors with confirmed SARS-CoV-2 were negative for IgG antibodies. This suggests that having had the infection does not necessarily convey immunity, or there is a short duration of immunity associated with a decline in antibodies. Plasma viral load obtained on 35/39 plasma recipients showed 22 (62.9 %) had non-detectable levels on average 14.5 days from positive test versus 6.2 days in those with detectable levels (p < 0.01). There was a relationship between IgG and viral load. IgG was higher in those with non-detectable viral loads. There was no relationship between viral load and blood type (p = 0.87) or death (0.80). Recipients with detectable viral load had lower IgG levels; there was no relationship between viral load, blood type or death.
Assuntos
Anticorpos Antivirais/administração & dosagem , COVID-19/sangue , COVID-19/terapia , SARS-CoV-2 , Adulto , Idoso , Feminino , Humanos , Imunização Passiva , Imunoglobulina G/administração & dosagem , Imunoglobulina M/administração & dosagem , Masculino , Pessoa de Meia-Idade , Soroterapia para COVID-19RESUMO
Individuals with HIV infection and two apolipoprotein L1 gene (APOL1) risk variants frequently develop nephropathy. Here we tested whether non-HIV viral infections influence nephropathy risk via interactions with APOL1 by assessing APOL1 genotypes and presence of urine JC and BK polyoma virus and plasma HHV6 and CMV by quantitative polymerase chain reaction. We analyzed 300 samples from unrelated and related first-degree relatives of African Americans with nondiabetic nephropathy using linear and nonlinear mixed models to account for familial relationships. The four groups evaluated were APOL1 zero/one versus two risk alleles, with or without nephropathy. Urine JCV and BKV were detected in 90 and 29 patients, respectively, whereas HHV6 and CMV were rare. Adjusting for family age at nephropathy, gender, and ancestry, presence of JCV genomic DNA in urine and APOL1 risk alleles were significantly negatively associated with elevated serum cystatin C, albuminuria (albumin-to-creatinine ratio over 30 mg/g), and kidney disease defined as an eGFR under 60 ml/min per 1.73 m(2) and/or albuminuria in an additive (APOL1 plus JCV) model. BK viruria was not associated with kidney disease. Thus, African Americans at increased risk for APOL1-associated nephropathy (two APOL1 risk variants) with JC viruria had a lower prevalence of kidney disease, suggesting that JCV interaction with APOL1 genotype may influence kidney disease risk.
Assuntos
Apolipoproteínas/genética , Negro ou Afro-Americano/genética , Vírus JC/isolamento & purificação , Nefropatias/genética , Nefropatias/virologia , Lipoproteínas HDL/genética , Infecções por Polyomavirus/virologia , Infecções Tumorais por Vírus/virologia , Adulto , Idoso , Albuminúria/etnologia , Albuminúria/genética , Albuminúria/virologia , Apolipoproteína L1 , Distribuição de Qui-Quadrado , Cistatina C/sangue , DNA Viral/urina , Feminino , Interação Gene-Ambiente , Predisposição Genética para Doença , Taxa de Filtração Glomerular , Humanos , Vírus JC/genética , Nefropatias/sangue , Nefropatias/etnologia , Nefropatias/fisiopatologia , Nefropatias/prevenção & controle , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Dinâmica não Linear , North Carolina/epidemiologia , Fenótipo , Infecções por Polyomavirus/etnologia , Prevalência , Fatores de Risco , Infecções Tumorais por Vírus/etnologiaRESUMO
BACKGROUND: The sensitivity of blood cultures for diagnosing invasive candidiasis (IC) is poor. METHODS: We performed a validated Candida real-time polymerase chain reaction (PCR) and the Fungitell 1,3-ß-D-glucan (BDG) assay on blood samples collected from prospectively identified patients with IC (n = 55) and hospitalized controls (n = 73). Patients with IC had candidemia (n = 17), deep-seated candidiasis (n = 33), or both (n = 5). Controls had mucosal candidiasis (n = 5), Candida colonization (n = 48), or no known Candida colonization (n = 20). RESULTS: PCR using plasma or sera was more sensitive than whole blood for diagnosing IC (P = .008). Plasma or sera PCR was more sensitive than BDG in diagnosing IC (80% vs 56%; P = .03), with comparable specificity (70% vs 73%; P = .31). The tests were similar in diagnosing candidemia (59% vs 68%; P = .77), but PCR was more sensitive for deep-seated candidiasis (89% vs 53%; P = .004). PCR and BDG were more sensitive than blood cultures among patients with deep-seated candidiasis (88% and 62% vs 17%; P = .0005 and .003, respectively). PCR and culture identified the same Candida species in 82% of patients. The sensitivity of blood cultures combined with PCR or BDG among patients with IC was 98% and 79%, respectively. CONCLUSIONS: Candida PCR and, to a lesser extent, BDG testing significantly enhanced the ability of blood cultures to diagnose IC.
Assuntos
Candida/isolamento & purificação , Candidíase Invasiva/diagnóstico , DNA Fúngico/sangue , Reação em Cadeia da Polimerase em Tempo Real/métodos , beta-Glucanas/sangue , Candida/química , Candida/genética , Candidemia/sangue , Candidemia/diagnóstico , Candidemia/microbiologia , Candidíase/sangue , Candidíase/diagnóstico , Candidíase/microbiologia , Candidíase Invasiva/sangue , Candidíase Invasiva/microbiologia , DNA Fúngico/genética , Humanos , Estudos Prospectivos , Proteoglicanas , Kit de Reagentes para Diagnóstico , Sensibilidade e EspecificidadeRESUMO
Diagnosis of invasive pulmonary aspergillosis (IPA) remains a major challenge to clinical microbiology laboratories. We developed rapid and sensitive quantitative PCR (qPCR) assays for genus- and species-specific identification of Aspergillus infections by use of TaqMan technology. In order to validate these assays and understand their potential diagnostic utility, we then performed a blinded study of bronchoalveolar lavage (BAL) fluid specimens from well-characterized models of IPA with the four medically important species. A set of real-time qPCR primers and probes was developed by utilizing unique ITS1 regions for genus- and species-specific detection of the four most common medically important Aspergillus species (Aspergillus fumigatus, A. flavus, A. niger, and A. terreus). Pan-Aspergillus and species-specific qPCRs with BAL fluid were more sensitive than culture for detection of IPA caused by A. fumigatus in untreated (P < 0.0007) and treated (P ≤ 0.008) animals, respectively. For infections caused by A. terreus and A. niger, culture and PCR amplification from BAL fluid yielded similar sensitivities for untreated and treated animals. Pan-Aspergillus PCR was more sensitive than culture for detection of A. flavus in treated animals (P = 0.002). BAL fluid pan-Aspergillus and species-specific PCRs were comparable in sensitivity to BAL fluid galactomannan (GM) assay. The copy numbers from the qPCR assays correlated with quantitative cultures to determine the pulmonary residual fungal burdens in lung tissue. Pan-Aspergillus and species-specific qPCR assays may improve the rapid and accurate identification of IPA in immunocompromised patients.
Assuntos
Aspergillus/isolamento & purificação , Aspergilose Pulmonar Invasiva/diagnóstico , Aspergilose Pulmonar Invasiva/microbiologia , Técnicas de Diagnóstico Molecular/métodos , Micologia/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Aspergillus/genética , Carga Bacteriana , Líquido da Lavagem Broncoalveolar/microbiologia , Primers do DNA/genética , DNA Espaçador Ribossômico/genética , Modelos Animais de Doenças , Feminino , Sondas de Oligonucleotídeos/genética , Coelhos , Sensibilidade e Especificidade , Fatores de TempoRESUMO
BACKGROUND: Early diagnosis and treatment of invasive pulmonary aspergillosis (IPA) improves outcome. METHODS: We compared the performance of publicly available pan-Aspergillus, Aspergillus fumigatus-, and Aspergillus terreus-specific real-time polymerase chain reaction (PCR) assays with the Platelia galactomannan (GM) assay in 150 bronchoalveolar lavage (BAL) samples from lung transplant recipients (16 proven/probable IPA, 26 Aspergillus colonization, 11 non-Aspergillus mold colonization, and 97 negative controls). RESULTS: The sensitivity and specificity of pan-Aspergillus PCR (optimal quantification cycle [Cq], ≤35.0 by receiver operating characteristic analysis) and GM (≥.5) for diagnosing IPA were 100% (95% confidence interval, 79%-100%) and 88% (79%-92%), and 93% (68%-100%) and 89% (82%-93%), respectively. The sensitivity and specificity of A. fumigatus-specific PCR were 85% (55%-89%) and 96% (91%-98%), respectively. A. terreus-specific PCR was positive for the 1 patient with IPA due to this species; specificity was 99% (148 of 149 samples). Aspergillus PCR identified 1 patient with IPA not diagnosed by GM. For BAL samples associated with Aspergillus colonization, the specificity of GM (92%) was higher than that of pan-Aspergillus PCR (50%; P = .003). Among negative control samples, the specificity of pan-Aspergillus PCR (97%) was higher than that of BAL GM (88%; P = .03). Positive results for both BAL PCR and GM testing improved the specificity to 97% with minimal detriment to sensitivity (93%). CONCLUSIONS: A recently developed pan-Aspergillus PCR assay and GM testing of BAL fluid may facilitate the diagnosis of IPA after lung transplantation. A. fumigatus- and A. terreus-specific real-time PCR assays may be useful in rapidly identifying the most common cause of IPA and a species that is intrinsically resistant to amphotericin B, respectively.
Assuntos
Aspergillus fumigatus/isolamento & purificação , Líquido da Lavagem Broncoalveolar/microbiologia , Técnicas de Laboratório Clínico/métodos , Aspergilose Pulmonar Invasiva/diagnóstico , Micologia/métodos , Reação em Cadeia da Polimerase/métodos , Adulto , Idoso , Aspergillus fumigatus/química , Aspergillus fumigatus/genética , Líquido da Lavagem Broncoalveolar/química , DNA Fúngico/genética , Feminino , Galactose/análogos & derivados , Humanos , Técnicas Imunoenzimáticas/métodos , Transplante de Pulmão , Masculino , Mananas/análise , Pessoa de Meia-Idade , Sensibilidade e Especificidade , TransplanteRESUMO
BACKGROUND: The relation between calf bovine leukosis virus (BLV) infection status and colostrum ingestion is unclear. Two conclusions have been drawn from previous studies. One suggests that colostrum ingestion transmits BLV to neonatal calves. The second suggests that colostral antibodies are protective. HYPOTHESIS: Colostrum from BLV-positive cattle is protective in naturally exposed calves. ANIMALS: Twelve colostrum-deprived Holstein calves and 20 colostrum-fed Holstein calves born to BLV-infected cows. METHODS: Prospective study. Colostrum-deprived calves were tested weekly by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) tests for BLV antibody and provirus for 12 weeks or until the animal became positive for BLV infection. Colostrum-fed calves were fed colostrum derived from BLV-positive cows. Thereafter, ELISA and PCR tests for BLV antibody and provirus were performed every other week until 2 consecutive negative ELISA tests or 1 positive PCR test was achieved. The proportion of calves that converted to BLV-positive status was calculated for each group and compared between groups by using the Fisher exact test. RESULTS: Four of 12 colostrum-deprived calves (33%) became BLV positive, whereas 0 of 20 colostrum-fed calves (0%) became BLV positive. The proportion of calves that became infected was significantly higher in the colostrum-deprived group (P = .014). CONCLUSIONS AND CLINICAL RELEVANCE: Calves born to BLV-positive cows are exposed during parturition, and a proportion of these calves will become infected with BLV. Administration of colostrum from BLV-positive cows greatly decreases the risk of infection.
Assuntos
Colostro/imunologia , Leucose Enzoótica Bovina/imunologia , Leucose Enzoótica Bovina/transmissão , Transmissão Vertical de Doenças Infecciosas/veterinária , Vírus da Leucemia Bovina/imunologia , Animais , Animais Recém-Nascidos , Anticorpos Antivirais/sangue , Bovinos , Colostro/virologia , DNA Viral/química , DNA Viral/genética , Leucose Enzoótica Bovina/virologia , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Incidência , Vírus da Leucemia Bovina/genética , Masculino , Reação em Cadeia da Polimerase/veterinária , Gravidez , Estudos Prospectivos , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
West Nile virus (WNV) has emerged as an important cause of encephalitis in humans and horses in North America. Although there is significant knowledge about the pathogenesis of disease caused by this flavivirus and about the immunity against it, no reports exist describing the sequence of pathological changes and their correlation to the immune response in the brain following infection with WNV. In this report the authors describe the major histopathological changes, as well as changes in cytokine and chemokine expression, in brains from WNV-infected C57Bl/6 mice. During the course of infection skin, spleen and kidney were all sites of WNV replication before virus reached the brain. In brain, increased expression of the chemokines monocyte chemoattractant protein (MCP)-5 (CCL12), interferon gamma inducible protein 10 (IP-10; CXCL10), and monokine induced by gamma interferon (MIG; CXCL9) preceded the expression of interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha), which have previously been considered to be key early cytokines in the pathogenesis and immune response of WNV encephalitis. These results suggest that the chemokines MCP-5, IP-10, and MIG are important triggers of inflammation in brain due to their early up-regulation following WNV infection.
Assuntos
Encéfalo/imunologia , Febre do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental , Animais , Encéfalo/patologia , Quimiocina CXCL10 , Quimiocina CXCL9 , Quimiocinas CC/biossíntese , Quimiocinas CXC/biossíntese , Feminino , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quimioatraentes de Monócitos/biossíntese , Fatores de Tempo , Regulação para Cima , Febre do Nilo Ocidental/patologiaRESUMO
As with a number of other viruses, Porcine reproductive and respiratory syndrome virus (PRRSV) has been shown to induce apoptosis, although the mechanism(s) involved remain unknown. In this study we have characterized the apoptotic pathways activated by PRRSV infection. PRRSV-infected cells showed evidence of apoptosis including phosphatidylserine exposure, chromatin condensation, DNA fragmentation, caspase activation (including caspase-8, 9, 3), and PARP cleavage. DNA fragmentation was dependent on caspase activation but blocking apoptosis by a caspase inhibitor did not affect PRRSV replication. Upregulation of Bax expression by PRRSV infection was followed by disruption of the mitochondria transmembrane potential, resulting in cytochrome c redistridution to the cytoplasm and subsequent caspase-9 activation. A crosstalk between the extrinsic and intrinsic pathways was demonstrated by dependency of caspase-9 activation on active caspase-8 and by Bid cleavage. Furthermore, in this study we provide evidence of the possible involvement of reactive oxygen species (ROS)-mediated oxidative stress in apoptosis induced by PRRSV. Our data indicated that cell death caused by PRRSV infection involves necrosis as well as apoptosis. In summary, these findings demonstrate mechanisms by which PRRSV induces apoptosis and will contribute to an enhanced understanding of PRRSV pathogenesis.
Assuntos
Apoptose , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Animais , Caspases/análise , Linhagem Celular , Chlorocebus aethiops , Citocromos c/análise , Citoplasma/química , Fragmentação do DNA , Humanos , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Necrose , Fosfatidilserinas/análise , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína X Associada a bcl-2/biossínteseRESUMO
OBJECTIVE: To determine the interval to provirus and serum antibody detection (via PCR assay and ELISA, respectively) in calves after experimental inoculation with bovine leukemia virus (BLV). ANIMALS: 8 colostrum-deprived, BLV-negative Holstein bull calves (> or = 6 weeks old). PROCEDURES: Via IM injection, each calf received a fresh whole-blood inoculum (day 0) calculated to contain 2 x 10(6) lymphocytes. Blood samples for the ELISA and PCR assay were collected from calves immediately prior to inoculation and weekly thereafter for 7 weeks. Mean and median number of weeks to PCR-detected conversion of BLV status and seroconversion were calculated. Point sensitivity and cumulative sensitivity of the 2 assays were calculated at each sample collection. At each sampling time, the proportion of calves identified as infected by the cumulative weekly ELISA and PCR assay results was compared by use of a Fisher exact test. RESULTS: In 5 calves, conversion of BLV status was detected via PCR assay before seroconversion was identified. However, seroconversion preceded PCR-detected conversion in 2 calves. In 1 calf, both assays yielded positive results at the same test date. These differences were not significant. CONCLUSIONS AND CLINICAL RELEVANCE: In experimentally inoculated BLV-negative calves, conversion of BLV status was detected via PCR assay more quickly than via ELISA; this difference was not significant and probably not clinically important. The PCR assay may be useful as a confirmatory test in animals of exceptional value; tests based on viral identification may become critically important if vaccines against BLV infection are developed and marketed.
Assuntos
Anticorpos Antivirais/sangue , Leucose Enzoótica Bovina/virologia , Vírus da Leucemia Bovina/crescimento & desenvolvimento , Provírus/crescimento & desenvolvimento , Animais , Bovinos , DNA Viral/química , DNA Viral/genética , Leucose Enzoótica Bovina/sangue , Leucose Enzoótica Bovina/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Vírus da Leucemia Bovina/genética , Vírus da Leucemia Bovina/imunologia , Masculino , Reação em Cadeia da Polimerase/veterinária , Provírus/genética , Provírus/imunologiaAssuntos
Genoma Viral , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Animais , Linhagem Celular , Cricetinae , DNA Complementar/metabolismo , Escherichia coli/metabolismo , Evolução Molecular , Variação Genética , Mutação , Fases de Leitura Aberta , Síndrome Respiratória e Reprodutiva Suína/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da EspécieRESUMO
OBJECTIVE: To determine the effect of dietary supplemental lipoic acid (LA) on serum concentrations of metabolic hormones and acute-phase proteins of steers challenged with infectious bovine rhinotracheitis virus (IBRV). ANIMALS: 32 steers. PROCEDURES: Steers were randomly assigned to 4 treatments: negative control (NC; no LA, no IBRV challenge), control (CON; no LA, IBRV challenge), 16 mg of LA/kg of body weight (BW)/d plus IBRV challenge (LA16), and 32 mg of LA/kg of BW/d plus IBRV challenge (LA32). Following a 21-day adaptation period, CON, LA16, and LA32 steers received IBRV (2 mL/nostril [day 0]); NC steers received saline (0.9% NaCl) solution. Blood samples, nasal swab specimens, BW, and rectal temperatures were obtained 0, 1, 3, 5, 7, 14, and 21 days after challenge. Serum was analyzed for concentrations of haptoglobin, amyloid-A, leptin, and anti-IBRV antibodies. RESULTS: Steers fed LA32 began gaining BW by day 7, whereas BW of CON and LA16 steers declined. Serum haptoglobin concentration of LA32 steers was lower than that of CON and LA16 steers on day 7. Serum neutralization titers for 30 of 32 steers were negative for anti-IBRV antibodies before challenge; however, all steers (including NCs) had antibodies on day 21. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that LA supplementation augmented certain aspects of the immune response; LA32 steers had a more rapid recovery from IBRV viral challenge than did others.
Assuntos
Proteínas de Fase Aguda/metabolismo , Rinotraqueíte Infecciosa Bovina/metabolismo , Rinotraqueíte Infecciosa Bovina/virologia , Ácido Tióctico/administração & dosagem , Ácido Tióctico/farmacologia , Aumento de Peso/efeitos dos fármacos , Ração Animal , Animais , Bovinos , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/farmacologia , MasculinoRESUMO
A cell model of primary monocytes and other mononuclear cells isolated from equine blood was used to study the kinetics of West Nile virus (WNV) replication in a natural host. West Nile virus has emerged on the North American continent as a significant cause of morbidity and mortality in a wide range of avian and mammalian species. While other flaviviruses are known to infect monocytes and lymphocytes, the ability of WNV to productively replicate in specific immune cells of peripheral blood has not been assessed. In this study, enriched populations of monocytes and lymphocytes as well as purified monocytes, CD4+, CD8+ and B lymphocytes were obtained from equine blood. Productive WNV replication was demonstrated by viral growth curves, quantitative RT-PCR for WNV RNA, and indirect immunofluorescence detection of a non-structural WNV protein. Enriched and purified monocytes consistently supported productive viral replication in blood from nine of nine horses tested while a minor subset of CD4+ lymphocytes supported productive replication in cells from three of the nine horses tested. Peak viral titers of 3.2-6.6 log10 PFU/ml were reached at 6 days post-inoculation (p.i.) and titers were maintained through 10-15 days p.i. Activation of monocytes with bacterial lipopolysaccharide, which resulted in activation of nuclear transcription factor kappaB (NF-kappaB) plus elevation of nitric oxide and type I interferon levels, reduced or eliminated WNV replication. These results suggest that immune cells of the peripheral blood may serve as target cells for initial replication of WNV and may play a role in subsequent viral dissemination. Furthermore, primary equine immune cell cultures represent a potentially useful model of a natural WNV host when testing compounds such as antivirals for use in WNV treatment.