The susceptibility of Atlantic salmon fry to freshwater infectious pancreatic necrosis is largely explained by a major QTL.

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.

Phylogenetic and antigenic characterization of new fish nodavirus isolates from Europe and Asia.

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.

Enterotoxin production by Salmonella typhimurium strains of different virulence.

Six strains of Salmonella typhimurium (TML, W118, LT7, SL1027, M206 and Thax-1) of known virulence and ability to induce fluid secretion when inoculated into the rabbit ileum were examined for enterotoxin production. Enterotoxic activity, assayed in the rabbit ileal-loop test, was detected in polymyxin-B extracts from all strains (with the possible exception of Thax-1) cultured for 6 h in casamino acid-yeast extract medium. The extracts were inactive in tissue-culture assays with CHO, Y-1 adrenal and Vero cells, and in the infant mouse assay for enterotoxin. There was no correlation between enterotoxigenicity in vitro and the ability of whole organisms to induce fluid secretion in vivo. The significance of these results in relation to salmonellosis is discussed.

The nature and role of mucosal damage in relation to Salmonella typhimurium-induced fluid secretion in the rabbit ileum.

The time course and nature of mucosal damage induced in rabbit ileal loops by two strains of Salmonella typhimurium (TML and W118) isolated from human infections was assessed by immunofluorescence microscopy and by scanning and transmission electronmicroscopy. Salmonella-induced fluid secretion occurred in the presence or absence of gross mucosal architectural damage. Neither strain caused mucosal ulceration. When damage did occur, the villi were shortened by loss of their tip regions with concomitant reforming of an intact mucosal surface. Immediately preceding the onset of fluid secretion, marked infiltration of the mucosa with polymorphonuclear leukocytes and occasional macrophages was seen. This revives an earlier suggestion that interaction between invading salmonellae and acute inflammatory cells may be an important factor in initiation of fluid secretion. Brush-border invasion by salmonellae cannot per se be the immediate cause of fluid secretion, because the latter occurred several hours after initial invasion.

Expression of an antigen in strains of Salmonella typhimurium which reacts with antibodies to cholera toxin.

Six strains of Salmonella typhimurium (W118, TML, SL1027, LT7, M206 and Thax 1) of different virulence were examined for the presence of antigens which react with antibodies to cholera toxin (anti-CT). A fluorescent-antibody-labelling technique employing anti-CT was used to analyse antigen expression. A rapid increase in the proportion of cells producing a CT-related antigen was demonstrated in cells in early log phase (1-4 h growth) followed by a rapid decline during mid-late log phase in each of the six strains. The nature of the CT-related antigen was analysed by immunoblotting using anti-CT. An antigen of mol. wt equivalent to a high-mol. wt species of CT B subunit was detected in polymyxin-B extracts of all strains but greater amounts were observed in the strains that we consider avirulent. Nothing equivalent to a CT A-related subunit was observed in any of the strains. The relatedness of the salmonella antigen to CT was limited. The high-mol. wt antigen was not disrupted in the denaturing conditions of SDS-PAGE; nothing was detected by enzyme-linked immunosorbent assays with either ganglioside or anti-CT as anchor.

Detection of human papillomavirus type-16 DNA utilising microtitre-plate based amplification reactions and a solid-phase enzyme-immunoassay detection system.

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.

Serological and molecular evidence of enterovirus infection in patients with end-stage dilated cardiomyopathy.

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.

Identification of B-cell epitopes on the betanodavirus capsid protein.

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.

Detection of human papillomavirus type 16 in microtitre plate based immuno-enzymatic assays: use to determine E5 gene expression in cervical carcinomas.

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.

Disaccharidase activities in small intestine of rotavirus-infected suckling mice: a histochemical study.

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.

Salmonellosis: in retrospect and prospect.

Despite years of study and the accumulation of much potentially relevant information, neither the microbial determinants nor the pathophysiological mechanisms of salmonella-induced enteritis are known with precision. Earlier work is reviewed on the experimental pathology of salmonellosis, the pathophysiology of the disease and the biotyping of salmonella strains in closed rabbit ileal loops. The same strains have been confirmed by us to be (i) invasive and diarrhoeagenic, (ii) invasive and non-diarrhoeagenic, and (iii) non-invasive and non-diarrhoeagenic. At least two mechanisms have been put forward to explain fluid exsorption. One implicates prostaglandins released from polymorphonuclear cells (PMNs) interacting with invading organisms, whereas the second involves salmonella enterotoxin(s). This subject is in a state of confusion and requires clarification. The toxin has been shown by some to bear partial likeness to either cholera toxin (although the evidence is in fact contradictory) or Shiga toxin. Since both 'cholera-like' and 'Shiga-like' toxins are produced by all three biotypes in vitro, production of toxin (of whatever class or subclass) cannot per se be the sole explanation for salmonella-induced fluid secretion. In our experiments the onset of fluid secretion in rabbit ileal loops was coincident with the appearance of large numbers of infiltrating PMNs. We have also shown that organisms from all three biotypes, grown for 6 h in iron-containing but not in iron-deficient media, yielded polymyxin B extracts which are enterotoxic in rabbit ileal loops; culture supernatants were negative. Structural damage occurred to villus tips but not crypts in infected loops, succeeded the onset of fluid secretion, and was not reproduced by polymyxin B enterotoxic extracts. Thus salmonella diarrhoea may be a complex phenomenon with multiple determinants which might include the release of endogenous secretagogues and bacterial enterotoxin(s), if such are shown to be synthesized and released in vivo at appropriate times and in appropriate sites. Structural damage to villus tips leading to shortened villi may also contribute to diarrhoea by altering absorption (tip function)/secretion (crypt function) ratios as well as to the expulsion of those organisms which have not migrated to deeper tissues.

Transport of water and electrolytes by rotavirus-infected mouse intestine: a time course study.

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.

An in vitro model to study aspects of the pathophysiology of murine rotavirus-induced diarrhoea.

An in vitro system is described and validated for studying transport of solutes and water in both uninfected and rotavirus-infected neonatal mouse intestine. Control intestine exhibited stable water absorption for periods of up to 40 min. Water absorption was temperature-dependent. Na-dependent, and enhanced by glucose-containing perfusion solutions. Theophylline induced net secretion of water by control intestinal tissue. Water transport by rotavirus-infected lower small intestine was significantly depressed as compared to control levels, and rotavirus-infected middle small intestine exhibited net secretion of water. Upper small intestine and colon from infected animals did not differ significantly from control tissues in their ability to transport water. Water secretion by infected middle small intestine was reversed to absorption by glucose-containing solutions.

Use of rubella virus E1 fusion proteins for detection of rubella virus antibodies.

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.

Human papillomavirus type 16 in infants: use of DNA sequence analyses to determine the source of infection.

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.

Nucleotide sequence analysis of a major antigenic domain of the E1 glycoprotein of 22 rubella virus isolates.

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.

Kinetics, tissue specificity and pathological changes in murine rotavirus infection of mice.

Mice that did not contain antibodies to rotavirus were orally infected with murine rotavirus (EDIM strain) and observed over 7 days. As judged by ELISA, only the small intestine was infected, not the colon. The infection was biphasic, viral antigen peaks being observed at 48 h and approximately 120 h post-infection. Clinically evident diarrhoea was maximal at 72 h. Virus in the upper, middle and lower regions of the small intestine was mainly tissue-associated; most virus was found in the middle small intestine. Two peaks (48 h and 120 h post-infection) of virus antigen were observed in the colon, but these corresponded to luminal, not tissue-associated viral antigen. Only enterocytes in the upper two-thirds of villus epithelia were infected as judged by fluorescent-antibody analysis and transmission electron microscopy. Scanning electron microscopy revealed morphological appearances not hitherto correlated with the progress of the infection: villus tips were convoluted, corresponding to the shedding of virus-infected cells but the lower regions of infected villi were shrunken and considerably narrowed compared to tips.

A study of the microcirculation in whole villi of neonatal mice using a peroxidase histochemical staining method.

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.

Rotavirus-induced changes in the microcirculation of intestinal villi of neonatal mice in relation to the induction and persistence of diarrhea.

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.