RESUMO
Infectious pancreatic necrosis (IPN) is a viral disease with a significant negative impact on the global aquaculture of Atlantic salmon. IPN outbreaks can occur during specific windows of both the freshwater and seawater stages of the salmon life cycle. Previous research has shown that a proportion of the variation seen in resistance to IPN is because of host genetics, and we have shown that major quantitative trait loci (QTL) affect IPN resistance at the seawater stage of production. In the current study, we completed a large freshwater IPN challenge experiment to allow us to undertake a thorough investigation of the genetic basis of resistance to IPN in salmon fry, with a focus on previously identified QTL regions. The heritability of freshwater IPN resistance was estimated to be 0.26 on the observed scale and 0.55 on the underlying scale. Our results suggest that a single QTL on linkage group 21 explains almost all the genetic variation in IPN mortality under our experimental conditions. A striking contrast in mortality is seen between fry classified as homozygous susceptible versus homozygous resistant, with QTL-resistant fish showing virtually complete resistance to IPN mortality. The findings highlight the importance of the major QTL in the genetic regulation of IPN resistance across distinct physiological lifecycle stages, environmental conditions and viral isolates. These results have clear scientific and practical implications for the control of IPN.
Assuntos
Suscetibilidade a Doenças/veterinária , Doenças dos Peixes/genética , Pancreatopatias/veterinária , Locos de Características Quantitativas , Salmo salar/genética , Animais , Mapeamento Cromossômico , Doenças dos Peixes/transmissão , Água Doce , Genótipo , Repetições de Microssatélites , Necrose , Pancreatopatias/genéticaRESUMO
The pepscan procedure was used to identify betanodavirus B-cell epitopes recognized by neutralizing mouse monoclonal antibodies (MAbs) and serum samples obtained from sea bass, Dicentrarchus labrax, naturally infected with betanodavirus. Pepscan was performed with a panel of thirty-four 12-mer synthetic peptides that mimicked the entire betanodavirus capsid protein. Sea bass serum samples reacted strongly with three regions of the capsid protein comprising amino acid residues 1-32, 91-162 and 181-212. The latter region was also recognized by neutralizing MAbs and coincided with a region of high antigenic propensity identified by an antigen prediction algorithm. These data suggest that a region of the betanodavirus capsid protein spanning amino acid residues 181-212 may represent a neutralization domain that could potentially be used to inform the development of nodavirus vaccines and immunodiagnostic reagents.
Assuntos
Proteínas do Capsídeo/imunologia , Epitopos de Linfócito B/imunologia , Nodaviridae/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Bass/sangue , Bass/virologia , Proteínas do Capsídeo/química , Mapeamento de Epitopos , Epitopos de Linfócito B/química , Camundongos , Camundongos Endogâmicos BALB C , Nodaviridae/químicaAssuntos
Aquicultura , Doenças dos Peixes/virologia , Linguados/virologia , Nodaviridae/patogenicidade , Infecções por Vírus de RNA/veterinária , Animais , Sequência de Bases , DNA Viral , Dados de Sequência Molecular , Nodaviridae/genética , Infecções por Vírus de RNA/genética , Infecções por Vírus de RNA/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Reino UnidoRESUMO
Nodaviruses are widespread causative agents of viral nervous necrosis in fish. Based on the coat protein sequence, fish nodaviruses are categorized into four different genotypes. In this study, we present data on the phylogenetic and antigenic characterization of 12 new isolates, eight European and four of Asian origin, from farmed and wild species of fish. Phylogenetic analysis based on the nucleotide sequence (688 bases) of the coat protein classified the majority of these new isolates to the RGNNV genotype. Geographic or host-species specificities were not revealed by this study. Neutralizing assay experiments, further confirmed the genotypic classification, supporting the possibility that the different nodavirus genotypes can also be serologically distinguishable.
Assuntos
Capsídeo/genética , Peixes/virologia , Vírus de RNA/classificação , Sequência de Aminoácidos , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Ásia , Capsídeo/imunologia , Clonagem Molecular , Europa (Continente) , Genoma Viral , Genótipo , Soros Imunes , Dados de Sequência Molecular , Testes de Neutralização , Filogenia , Vírus de RNA/genética , Vírus de RNA/imunologia , Coelhos , Alinhamento de SequênciaRESUMO
Human papillomavirus type 16 (HPV-16) is a major cause of cervical neoplasia, but only a minority of HPV-16 infections result in cancer. Whether particular HPV-16 variants are associated with cervical disease has not yet been clearly established. An investigation of whether cervical neoplasia is associated with infection with HPV-16 intratypic variants was undertaken by using RFLP analyses in a study of 100 HPV-16 DNA-positive women with or without neoplasia. RFLP variant 2 was positively associated [odds ratio (OR)=2.57] and variant 5 was negatively associated with disease (OR=0.2). Variant 1, which resembles the reference isolate of HPV-16, was found at a similar prevalence among those with and without neoplasia. Variants 1 and 2 were also more likely to be associated with detectable viral mRNA than variant 5 (respectively P=0.03 and P=0.00). When HPV-16 E5 ORFs in 50 clones from 36 clinical samples were sequenced, 19 variant HPV-16 E5 DNA sequences were identified. Twelve of these DNA sequences encoded variant E5 amino acid sequences, 10 of which were novel. Whilst the associations between HPV-16 E5 RFLP variants and neoplasia could not be attributed to differences in amino acid sequences, correlation was observed in codon usage. DNA sequences of RFLP variant 2 (associated with greatest OR for neoplasia) had a significantly greater usage of common mammalian codons compared with RFLP pattern 1 variants.
Assuntos
Colo do Útero/virologia , Variação Genética , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Transcrição Gênica , Infecções Tumorais por Vírus/virologia , Displasia do Colo do Útero/virologia , Neoplasias do Colo do Útero/virologia , Sequência de Aminoácidos , Sequência de Bases , Colo do Útero/patologia , Códon , DNA Viral , Feminino , Humanos , Dados de Sequência Molecular , Infecções por Papillomavirus/patologia , Infecções Tumorais por Vírus/patologia , Neoplasias do Colo do Útero/patologia , Displasia do Colo do Útero/patologiaRESUMO
E1 gene nucleotide sequences of 63 rubella virus isolates from North America, Europe, and Asia isolated between 1961 and 1997 were compared phylogenetically. Two genotypes were evident: Genotype I contained 60 viruses from North America, Europe, and Japan, and genotype II contained 3 viruses from China and India. The genotype I isolates prior to 1970 grouped into a single diffuse clade, indicating intercontinental circulation, while most post-1975 viruses segregated into geographic clades from each continent, indicating evolution in response to vaccination programs. The E1 amino acid sequences differed by no more than 3%; thus, no major antigenic variation was apparent. Among 4 viruses from congenital rubella syndrome that occurred following reinfection, only one amino acid substitution occurred in several important epitopes, indicating that antigenic drift is not important in this phenomenon. However, 2 viruses isolated from chronic arthritis exhibited changes in these epitopes. Isolates of the RA 27/3 vaccine strain were readily identifiable by nucleotide sequence.
Assuntos
Vírus da Rubéola/genética , Rubéola (Sarampo Alemão)/epidemiologia , Rubéola (Sarampo Alemão)/virologia , Sequência de Aminoácidos , Ásia/epidemiologia , Europa (Continente)/epidemiologia , Humanos , Dados de Sequência Molecular , América do Norte/epidemiologia , Filogenia , Vírus da Rubéola/classificação , Vírus da Rubéola/isolamento & purificação , Proteínas do Envelope Viral/genéticaRESUMO
We have determined the nucleotide sequence of the region of the rubella virus genome which encodes amino acids 195-296 of the E1 glycoprotein (E1-195-296) from a panel of 22 rubella viruses obtained from Europe, USA and Asia between 1963-1995. E1-195-296 contains neutralizing and haemagglutinating determinants, and may represent a major antigenic domain. The nucleotide sequence divergence of the 22 rubella viruses compared to the Therien strain sequence ranged from 0.65-7.14%. The greatest sequence divergence occurred in two rubella viruses of Indian origin, and was more than twofold greater than that previously reported for rubella virus. The majority of nucleotide changes occurring in the 22 viruses did not effect the deduced amino acid sequence of E1-195-296. Two rubella viruses isolated from cases of reinfection in pregnancy did not exhibit nucleotide sequence variation resulting in changes in the deduced amino acid sequence of E1-195-296, suggesting that antigenic change within this region of E1 is not associated with rubella reinfection. A rubella virus isolated from a synovial fluid sample exhibited a nucleotide substitution in a putative neutralization domain contained within E1-195-296. Phylogenetic analysis was performed to study the relationship between E1-195-296 coding sequences of the 22 viruses in this report and corresponding sequences of other rubella viruses in the databank.
Assuntos
Antígenos Virais/genética , DNA Viral , Vírus da Rubéola/genética , Análise de Sequência de DNA , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Sequência de Bases , Humanos , Dados de Sequência Molecular , Filogenia , Vírus da Rubéola/classificação , Vírus da Rubéola/imunologia , Vírus da Rubéola/isolamento & purificação , Homologia de Sequência do Ácido NucleicoRESUMO
OBJECTIVE: To study the relative diagnostic value of enterovirus-specific molecular biological and serological assays in patients with end-stage dilated cardiomyopathy, and to investigate the possible role of other cardiotropic viruses in dilated cardiomyopathy. DESIGN: Analysis of recipient myocardial tissue and serum from patients with dilated cardiomyopathy and controls undergoing cardiac transplantation for end-stage cardiac disease. SETTING: University virology department and transplantation unit. METHODS: Reverse transcriptase-polymerase chain reaction and nucleotide sequence analysis of myocardial RNA and DNA; enterovirus-specific in situ hybridization; enterovirus-specific immunoglobulin M detection. RESULTS: Enterovirus RNA was detected in myocardial tissue from only a small proportion of (five of 75) hearts. However, although enterovirus-specific immunoglobulin M responses were detected in 22 (28%) of 39 controls patients, a significantly higher prevalence was observed among patients with dilated cardiomyopathy (22 (56%) of 39 patients; P < 0.005). All enteroviruses detected in myocardium showed greatest nucleotide sequence homology with coxsackievirus type B3. Detection of enterovirus RNA in myocardium by the polymerase chain reaction and by in situ hybridisation gave comparable results. Other potentially cardiotropic virus genomes, including human cytomegalovirus, influenzaviruses, and coronaviruses were not detected in myocardium. CONCLUSION: This study found that enterovirus-specific immunoglobulin M responses provided the strongest evidence of enterovirus involvement in patients with end-stage dilated cardiomyopathy. However, the high background prevalence of these responses limits their diagnostic value. The finding that enteroviruses detected in myocardium were coxsackievirus type B3 accords with recent findings in patients with acute myocarditis, and indicates that this serotype is the major cardiotropic human enterovirus.
Assuntos
Cardiomiopatia Dilatada/virologia , Infecções por Enterovirus/diagnóstico , Enterovirus/genética , Enterovirus/imunologia , Coração/virologia , Anticorpos Antivirais/sangue , Sequência de Bases , Primers do DNA/genética , Enterovirus Humano B/genética , Humanos , Imunoglobulina M/sangue , Hibridização In Situ , Dados de Sequência Molecular , RNA Viral/análise , Alinhamento de SequênciaRESUMO
Human papillomavirus type 16 (HPV-16) DNA is detected commonly in cervical carcinomas; in this study, we have determined the analytical sensitivities of Hybrid Capture, HPV-consensus PCR, and three HPV-16-specific polymerase chain reactions (PCRs) for the detection of HPV-16 DNA. Samples investigated included a cervical cancer cell line, cervical scrapes from 20 patients attending colposcopy clinics, and buccal swabs from eight immunosuppressed children. HPV-16 E7 and E5-nested PCRs [Cavuslu et al. (1996): Journal of Virological Methods, in press] produced positive signals from samples containing fewer than ten HPV-16 genomes per reaction. HPV-consensus PCR [Manos et al. (1989): Cancer Cells 7:209-214] and HPV-16 PCR using primers of van den Brule et al. [(1990): Journal of Clinical Microbiology 25:2739-2743] were of intermediate sensitivity (i.e., produced positive signals from samples containing 250 and 2,500 HPV-16 genoms/reaction, respectively) and Hybrid Capture could detect just 50,000 HPV-16 genomes/reaction. Highest rates of positivity for cervical samples were detected with HPV-16 E7 or E5-nested PCRs [50% (10 of 20 samples) and 60% (12 of 20 samples) positive, respectively], intermediate rates with HPV-consensus PCR and PCRs using the primers of van den Brule et al. [both 35% (7 of 20 samples)], and lowest rates of positivity [25% (5 of 20 samples)] with Hybrid Capture. None of eight buccal swab samples from immunosuppressed children were positive by Hybrid Capture, yet three (37.5%) were positive by HPV-16 E5-nested PCR. These data indicate that HPV-16 type-specific PCRs should be used for the investigation of specimens that may contain low amounts of HPV-16 DNA.
Assuntos
DNA Viral/análise , Proteínas Oncogênicas Virais/genética , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase/métodos , Infecções Tumorais por Vírus/virologia , Adolescente , Adulto , Colo do Útero/patologia , Colo do Útero/virologia , Criança , Feminino , Humanos , Pessoa de Meia-Idade , Mucosa Bucal/patologia , Mucosa Bucal/virologia , Papillomaviridae/genética , Proteínas E7 de Papillomavirus , Infecções por Papillomavirus/patologia , Sensibilidade e Especificidade , Infecções Tumorais por Vírus/patologiaRESUMO
We have searched, using a sensitive nested-PCR, for enterovirus RNA in cerebrospinal fluid and post mortem central nervous system (CNS) tissue from patients with previous poliomyelitis with or without late functional deterioration, patients with motor neuron disease (MND), and control patients with other neurological disease or without neurological disease. Enterovirus RNA was detected in patients with previous poliomyelitis and MND, but also in control patients with and without neurological disease. Our results do not provide any evidence that such enterovirus infection is related to late functional deterioration in patients with previous poliomyelitis, which could be attributed to other medical conditions in most instances, and do not support the hypothesis that MND is associated with enterovirus infection of the CNS. Nucleotide sequence analysis of enterovirus RNA sequences detected indicated that enteroviruses detected were of the non-polio type.
Assuntos
Infecções por Enterovirus/virologia , Doença dos Neurônios Motores/virologia , Síndrome Pós-Poliomielite/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Doença de Alzheimer/virologia , Sequência de Bases , Criança , Doença Crônica , DNA Viral/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Córtex Motor/patologia , Córtex Motor/virologia , Doença dos Neurônios Motores/patologia , Mudanças Depois da Morte , Homologia de Sequência do Ácido Nucleico , Medula Espinal/patologia , Medula Espinal/virologiaRESUMO
Perinatal transmission of human papillomavirus type 16 (HPV-16) and persistence of virus DNA in infants until 6 months of age has been described. To confirm the origin of infant infections as maternal, we determined the nucleotide sequence of the upstream regulatory region (URR; bp 7540 to 157) of HPV-16 in samples from 13 HPV-16 DNA-positive mothers and their infants at 6 weeks and 2 years of age. Identical HPV-16 variant URR sequences were found in two mother/infant samples and similar variants were found in three sets. Four mothers with samples which contained prototypic HPV-16 sequences delivered infants who also had the prototypic sequence. Four mothers with variant URRs delivered infants who harboured either prototypic or different URR variants. Thus, concordant variants or prototypic sequences were detected in nine of 13 mother/infant samples, indicating that up to 69.2% of HPV-16-positive infants acquire virus from their mothers.
Assuntos
DNA Viral/isolamento & purificação , Transmissão Vertical de Doenças Infecciosas , Papillomaviridae/genética , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Sequências Reguladoras de Ácido Nucleico , Infecções Tumorais por Vírus/diagnóstico , Sequência de Bases , Primers do DNA , Feminino , Seguimentos , Variação Genética , Humanos , Lactente , Recém-Nascido , Masculino , Dados de Sequência Molecular , Mães , Papillomaviridae/classificação , Infecções por Papillomavirus/transmissão , Reação em Cadeia da Polimerase/métodos , Gravidez , Complicações Infecciosas na Gravidez/virologia , Infecções Tumorais por Vírus/transmissãoRESUMO
BACKGROUND: E5-based nested polymerase chain reaction (PCR) assays and a PCR-enzyme immunoassay (EIA) to detect human papillomavirus type 16 (HPV-16) DNA have been developed. These assays were designed to detect small amounts of HPV-16 DNA for epidemiological studies of subclinical infection. OBJECTIVES: The E5 gene of HPV-16 may be lost in some cell lines derived from cervical carcinomas. The aim of this study was to determine if, and how frequently, E5 gene loss occurs in biopsy samples from patients with cervical lesions. STUDY DESIGN: Sixteen HPV-16 (E7) DNA positive and five HPV-16 DNA negative cervical lesions (nineteen cervical carcinomas, two cervical intraepithelial neoplasias) were investigated by E5 nested PCR and EIA. RESULTS: Overall, 15 of the 16 (93.75%) HPV-16 E7 positive samples were positive for HPV-16 E5 DNA: 14 of 16 (87.5%) were positive by E5 PCR and 15 of 16 (93.75%) were positive by E5 PCR, nested PCR and by PCR-EIA. One of 14 HPV-16 (E7) DNA positive cervical carcinomas was negative for E5 DNA in all three assays. CONCLUSION: Loss of the HPV-16 E5 open reading frame (ORF) is a rare event in HPV-16 positive cervical carcinomas and was detected in just one of 14 (7.1%) cases.
RESUMO
The development of a nested polymerase chain reaction (PCR) assay to detect low concentrations of human papillomavirus type-16 (HPV-16) DNA for epidemiological studies is described. The PCR utilises primers located in the E5 open reading frame, has an analytical sensitivity of 4 HPV-16 genomes and does not produce amplicons from other common genital HPVs (types-6, -11, -18, -31 and 33). This assay was carried out in 96-well plates utilising internal primers labelled with dinitrophenol (DNP) and biotin so that amplicons can be captured onto streptavidincoated plates and detected using an alkaline phosphatase-labelled monoclonal antibody to DNP. The assay was effective for detecting HPV-16 DNA in plasmids, cell-lines and, both freshly collected or archival (formalin-fixed/paraffin embedded) clinical specimens. This system is therefore suitable for epidemiological studies to identify individuals infected with HPV-16 DNA in episomal form who may be at increased risk of developing anogenital carcinomas.
Assuntos
DNA Viral/análise , Técnicas Imunoenzimáticas , Proteínas Oncogênicas Virais/genética , Papillomaviridae/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Proteínas Repressoras , Displasia do Colo do Útero/virologia , Neoplasias do Colo do Útero/virologia , Primers do DNA , Estudos de Avaliação como Assunto , Feminino , Células HeLa , Humanos , Papillomaviridae/genética , Proteínas E7 de Papillomavirus , Sensibilidade e Especificidade , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/patologia , Displasia do Colo do Útero/patologiaRESUMO
OBJECTIVE: To determine whether enterovirus RNA can be demonstrated in archival necropsy material in acute myocarditis. DESIGN: Analysis of paraffin embedded myocardial tissue from cases of acute myocarditis. SETTING: University virology department. METHODS: Extraction of RNA from tissue followed by polymerase chain reaction (PCR) and DNA sequence analysis. PATIENTS: Six patients with histologically proven myocarditis and eight controls. RESULTS: Enterovirus RNA was identified in 5 of 6 patients with myocarditis and in none of the controls. The nucleotide sequences of the PCR products showed greatest similarity to group B coxsackieviruses, particularly coxsackievirus B3. CONCLUSION: This study indicates that archival tissue samples, even histologically stained tissue sections, can be used to study the role of enteroviruses in myocardial disease using molecular detection techniques. If a predominant role for coxsackievirus B3 in myocarditis is confirmed by further study, this may have implications for the development of a specific vaccine.
Assuntos
Infecções por Enterovirus , Enterovirus/isolamento & purificação , Miocardite/virologia , Reação em Cadeia da Polimerase , RNA Viral/análise , Doença Aguda , Adolescente , Adulto , Idoso , Anticorpos Antivirais/sangue , Sequência de Bases , Estudos de Casos e Controles , Criança , Enterovirus/classificação , Feminino , Coração/virologia , Humanos , Hibridização In Situ , Lactente , Recém-Nascido , Masculino , Dados de Sequência MolecularRESUMO
Two glutathione S-transferase fusion proteins containing 44 (p1503) and 75 (p1509) amino acid residues of the rubella virus E1 glycoprotein were expressed in Escherichia coli with the aim of producing a recombinant rubella virus antigen for use in serological assays. p1503 contained three neutralizing and hemagglutinating epitopes (G. M. Terry, L. M. Ho-Terry, P. Londesborough, and K. R. Rees, Arch. Virol. 98:189-197, 1988); p1509 contained the putative neutralization domain described by Mitchell et al. (L. A. Mitchell, T. Zhang, M. Ho, D. Decarie, A. Tingle, M. Zrein, and M. Lacroix, J. Clin. Microbiol. 30:1841-1847, 1992) in addition to the three epitopes present in p1503. Both fusion proteins were soluble and affinity purified on glutathione-Sepharose 4B. In Western blots (immunoblots), p1503 and p1509 reacted with human sera containing rubella virus-specific immunoglobulin G. When used as antigens in indirect enzyme immunoassays to detect rubella virus-specific immunoglobulin G, p1503 correctly identified the rubella virus antibody status of 43 (76.8%) and p1509 correctly identified that of 48 (85.7%) of 56 serum samples received for routine rubella virus antibody screening. The results obtained with p1509 compare well with those obtained with a latex agglutination assay.
Assuntos
Anticorpos Antivirais/sangue , Vírus da Rubéola/imunologia , Rubéola (Sarampo Alemão)/imunologia , Proteínas do Envelope Viral/imunologia , Antígenos Virais/genética , Antígenos Virais/isolamento & purificação , Sequência de Bases , Clonagem Molecular , DNA Viral/genética , Escherichia coli/genética , Reações Falso-Negativas , Glutationa Transferase/genética , Glutationa Transferase/isolamento & purificação , Humanos , Técnicas Imunoenzimáticas/estatística & dados numéricos , Imunoglobulina G/sangue , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Reprodutibilidade dos Testes , Rubéola (Sarampo Alemão)/prevenção & controle , Vírus da Rubéola/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/isolamento & purificaçãoRESUMO
The anatomy of the microcirculation of intestinal villi from the upper, middle, and lower small intestine of neonatal mice from 8 to 14 days old was studied using a histochemical peroxidase technique that specifically stained erythrocytes. Over 8-14 days, there was little chronological variation between the same regions of gut; the exception was the lower intestine, which, in younger mice, was noticeably less well perfused with erythrocytes. Vascular beds in the middle and lower intestine comprised a hairpin loop with cross-connections. In the upper intestine, the capillary beds were generally more complex, particularly in apical regions of the villi. Most villi were well perfused with erythrocytes, but a minority (less than 10%) contained considerably fewer red cells, some to the point of being totally ischemic. Other villi (less than 5%) were hyperemic, and the vascular beds packed and engorged with red cells. Usually, the packing density (hematocrit) of red cells within blood vessels increased progressively from villus base to apex. Red-cell deformation was more prevalent at villus apices, with marked crenation in some villi, yet in the basal regions of these same villi, the red cells were of normal discoid shape. The peroxidase staining technique produces a reliable histological picture of red cells circulating through villi. It also reveals differential perfusion of erythrocytes between and within villi, and that blood vessels pass through hypertonic zones in the apical regions of villi.
Assuntos
Animais Recém-Nascidos/fisiologia , Mucosa Intestinal/irrigação sanguínea , Animais , Hematócrito , Técnicas Imunoenzimáticas , Mucosa Intestinal/ultraestrutura , Intestino Delgado/irrigação sanguínea , Camundongos , Camundongos Endogâmicos BALB C , Microcirculação , Microscopia Eletrônica , MicrovilosidadesRESUMO
Using a histochemical peroxidase technique, under conditions that preferentially stain erythrocytes, we have shown changes in the microcirculation of villi of neonatal mice infected with murine rotavirus. Between 18 and 48 h postinfection (PI), throughout all areas of the small intestine there occurred, sequentially, a marked ischemia and atrophy of villi. By 72 h PI, villi had recovered their normal height and showed incipient hyperemic microcirculation. At 96 h PI, hyperemic microcirculation was most marked. Between 120 and 144 h PI, a second phase of villus atrophy occurred, which was more attenuated and confined to the upper and middle regions of the intestine. This phase was not accompanied by a wide-spread ischemia of villi: a minority of villi were short and ischemic but many appeared hyperemic. Recovery of villus microcirculation occurred at 168 h PI, which coincided with recovery from diarrhea. These changes in villus microcirculation are discussed in relation to the pathology and pathophysiology of rotavirus infection. We make two novel suggestions. First, the reduction in red cells flowing through villi in the early stages of the infection instigates hypoxia and hence atrophy of villi. The ensuing but ephemeral increase in rate of cell division, necessary for the reconstitution of villi, induces hypersecretion. Second, the increase in numbers of erythrocytes found in villi during their regrowth phase and throughout the remaining time course of the infection perturbs the countercurrent system, lowering the osmolality of the hyperosmotic zone located at villus tips, thereby impairing water absorption and prolonging diarrhea.
Assuntos
Diarreia/etiologia , Mucosa Intestinal/irrigação sanguínea , Infecções por Rotavirus/fisiopatologia , Animais , Animais Recém-Nascidos , Atrofia/etiologia , Estudos de Casos e Controles , Diarreia/veterinária , Técnicas Imunoenzimáticas , Mucosa Intestinal/patologia , Isquemia/etiologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Microvilosidades , Fatores de TempoRESUMO
A histochemical study of the time course of the appearance and location of lactase and alpha-glucosidase (used to detect sucrase and maltase) activities was carried out on control and rotavirus-infected mice from 7 to 14 days old. The overall pattern of enzyme activity was in agreement with previous quantitative studies on the activities of these enzymes. No evidence was obtained to support the idea that lactase deficiency was the result of repopulation of villi (denuded of lactase-producing villus cells) with immature lactase-negative cells. Low lactase activity was more likely to reflect profound changes in metabolically crippled cells, and recovery of lactase activity with recovery of normal metabolic functions. The location of enzyme activity to brush border regions rather than the cytoplasm of villus enterocytes enhances the significance of previous quantitative studies on these enzymes. The timing and duration of diminished lactase activities were such that they were unlikely to cause the induction or perpetuation of diarrhea in murine rotavirus diarrhea. The appearance in infected animals of alpha-glucosidase 3 days earlier than normal indicates that, in addition to reversible changes seen with lactase, developmental changes were accelerated that affected both crypt and villus cells.
Assuntos
Dissacaridases/biossíntese , Intestino Delgado/enzimologia , Infecções por Rotavirus/metabolismo , Animais , Animais Lactentes , Citoplasma/metabolismo , Diarreia/etiologia , Intestino Delgado/ultraestrutura , Lactase , Camundongos , Camundongos Endogâmicos BALB C , Microvilosidades/metabolismo , Microvilosidades/ultraestrutura , Infecções por Rotavirus/complicações , alfa-Glucosidases/biossíntese , beta-Galactosidase/biossínteseRESUMO
The movement of water and transport of Na+ and Cl- by mid-small intestine (M-SI) of rotavirus-infected neonatal mice was investigated by an in vitro perfusion technique. The concentrations of Na+, K+, and Cl- in the luminal contents of upper, middle, and lower small intestine and colon of infected mice were determined by flame photometry (Na+, K+) and an ion selective microelectrode (Cl-). In M-SI, maximal disturbance of water transport occurred at 72 h postinfection (PI): Infected tissue exhibited net water secretion. Water transport was also impaired at 144 h PI. Net secretion of Cl- occurred at 72 h PI, with some evidence of a second phase of reduced magnitude at 120-144 h PI. The magnitude and statistical significance of changes in Na+ transport were both less than those for Cl-, but the pattern of change was similar to that for Cl-. Luminal concentrations of Na+ were elevated between 48 and 144 h PI in the small intestine; this was particularly so in distal regions. Luminal Cl- concentration was maximally elevated to a considerable degree at 72 h PI and remained high at 96 h PI throughout the small intestine; thereafter, Cl- concentration returned to near normal. K+ concentration was unchanged in the small intestinal lumen; in the colon, however, K+ concentrations were depressed 72-168 h PI. In the light of previous data from this laboratory, the present data are interpreted as evidence for a secretory component in rotavirus-induced diarrhea.
Assuntos
Diarreia/fisiopatologia , Intestino Delgado/fisiopatologia , Infecções por Rotavirus/fisiopatologia , Equilíbrio Hidroeletrolítico , Animais , Transporte Biológico Ativo , Diarreia/metabolismo , Eletrólitos/farmacocinética , Intestino Delgado/metabolismo , Camundongos , Infecções por Rotavirus/metabolismo , Fatores de Tempo , ÁguaRESUMO
Neonatal mice were infected at 7 days of age with rotavirus [epizootic diarrhea of infant mice (EDIM) virus] and killed at 24-h intervals postinfection (PI). Cytoplasmic concentrations of Na, Mg, P, S, Cl, K, and Ca intestinal epithelial cells from infected and age-matched control animals were measured by x-ray microanalysis. In villus tip cells, Ca concentration increased at 24-96 h PI; Na concentration increased at 24-72 h PI; Ca and Na concentrations were near normal by 168 h PI. K concentration decreased 24-72 h PI, and Cl concentration decreased 48-96 h PI. In crypt cells, changes were observed without a discernible pattern: at 96 h PI, Na, Mg, S, and Cl concentrations increased and K concentration decreased; at 120 h PI, the concentrations of all elements except Na and Ca increased. In villus base cells, the mean concentrations of all elements except Ca peaked at 48-72 h PI and at 120 h PI. Na and Cl concentrations increased dramatically in some cells from 48 h PI onward. All the above concentration values were obtained from freeze-dried specimens and expressed in millimoles per kilogram of dry weight. Conversion of a limited number of data, pertaining to villus base cells, from dry weight to wet weight was possible. This conversion revealed that villus base cells in infected animals were more hydrated than corresponding cells from control animals. Also, the Na and Cl concentrations in mmol/kg H2O were significantly higher in villus base cells from infected animals than in those from corresponding controls: 137 +/- 7 versus 38 +/- 4 (Na) and 121 +/- 5 versus 89 +/- 6 (Cl). Wet weight concentrations of other elements were either the same (Mg) or lower (P, S, and K) after infection with virus. From these studies, a new concept of the pathophysiology of rotavirus-induced diarrhoeal secretion is proposed: stimulation of villus base cells to rapid cell division is accompanied by transient accumulation of Na and Cl; excess NaCl is secreted into the lumen, which is the driving force for fluid loss.