A single amino acid is critical for the expression of B-cell epitopes on the helicase domain of the pestivirus NS3 protein.

Truncated NS3 proteins, expressed by recombinant baculoviruses, were used to investigate the location of conserved B-cell epitopes on this non-structural bovine viral diarrhoea virus (BVDV) protein. A goat anti-pestivirus antiserum, and a panel of anti-NS3 monoclonal antibodies, including the BVDV-1 specific antibody P1D8, were used to verify the presence or absence of the epitopes. Interestingly, the monoclonal antibodies reacted only with the truncated protein encompassing the helicase domain of NS3. Expression of the B-cell epitopes was dependent on, but not within, a 57 amino acid sequence at the carboxy-terminal end of this protein, supporting observations that these conserved epitopes are conformational in nature. A comparison of deduced amino acid sequences of the helicase domain from BVDV-1, BVDV-2, BDV and CSFV isolates highlighted a single amino acid that appeared to be unique to P1D8-reactive BVDV-1 isolates. Site-directed mutagenesis studies confirmed that this amino acid is critical for the expression of the BVDV-1 specific NS3 epitope recognised by the P1D8 monoclonal antibody. Surprisingly, the amino acid was also important for an epitope recognised by two group-specific monoclonal antibodies, P1H11 and P4A11. Protein modelling studies, based on the structure of the hepatitis C NS3 helicase domain, indicated that this amino acid occupies a prominent position on the surface of the protein.

Phylogenetic analysis of pestiviruses from domestic and wild ruminants.

Infections with pestiviruses occur in cattle, sheep, pigs and also in numerous other ungulate species. In the present study, pestiviruses from goat, buffalo, deer and giraffe were analysed at the molecular level; unusual strains from cattle and pigs were also included. A phylogenetic analysis of the respective pestiviruses was undertaken on the basis of a fragment from the 5' noncoding region as well as the gene encoding autoprotease Npro. Statistical analyses of the respective phylogenetic trees-based on the 5' NCR revealed low confidence levels for most of the branches, while the structure of the tree based on the Npro gene was supported by high bootstrap values. Accordingly, the isolates from goat, buffalo and deer can be grouped together with bovine viral diarrhoea virus (pestivirus type 1); within this genotype three subgroups and one disparate virus have been identified. One isolate from pig and one from cattle belong to the group of 'true' border disease virus (pestivirus type 3), which can be further subdivided into two major subgroups. Interestingly, the giraffe isolate does not belong to one of the four established pestivirus genotypes. The phylogenetic analysis strongly suggests that genotype 1 pestiviruses occur world-wide in many ruminant species. Furthermore, phylogenetic trees based on the Npro gene nucleotide sequences show that the respective sequences do not segregate into discrete lineages based on host-species origin.

Characterization of RNA synthesis during a one-step growth curve and of the replication mechanism of bovine viral diarrhoea virus.

The noncytopathic Australian bovine viral diarrhoea virus (BVDV) Trangie isolate was used to establish a one-step growth curve and to investigate previously uncharacterized aspects of pestivirus replication. The eclipse phase was found to be approximately 8 to 10 h and the first appearance of viral antigen assayed by immunofluorescence occurred around 6 h post-infection (p.i.). Both positive- and negative-sense virus RNAs were first detected at 4 h p.i. by Northern blot hybridization using strand-specific RNA probes generated by in vitro transcription from cDNA cloned from the NS3 region. The ratio of positive- to negative-sense virus RNA changed from 2:1 at 4 h p.i. to 10:1 at 12 h p.i. and thereafter. The kinetics of synthesis showed that the rate of synthesis of positive-strand viral RNA increased rapidly from 6 h p.i., whereas the rate of synthesis of the negative-strand remained constant. The copy number of genomic RNA determined by Northern blot hybridization analysis was estimated to be 1.5 x 10(4) copies per cell, 16 to 24 h p.i. Viral RNA species that were thought to represent replicative intermediate (RI) and replicative forms (RF) were detected after electrophoretic separation by urea-PAGE. Confirmation of the identity of the RI and RF was obtained using LiCl precipitation and RNase A digestion of [3H]uridine-labelled RNA. Pulse-chase labelling of BVDV-infected cells was consistent with synthesis of nascent BVDV RNA through an RI derived by strand displacement from an RF template and thus the synthesis of BVDV RNA is likely to be similar to the model proposed for flavivirus replication.

Cytopathogenicity of border disease virus is correlated with integration of cellular sequences into the viral genome.

Two border disease virus (BDV) pairs each consisting of cytopathogenic (cp) and non-cp viruses have been analyzed at the molecular level. Within the NS2-3 (p125) encoding region of both cp viruses, insertions of cellular sequences were identified which were absent in the corresponding non-cp isolates. A comparative sequence analysis revealed that within each pair the cp and non-cp viruses are almost identical. This strongly suggests that the cp BDV isolates developed from the non-cp viruses by RNA recombination between the viral genome and cellular sequences. Nonstructural protein NS3 (p80) was demonstrated after infection with both cp BDV strains. In addition, fusion proteins composed of cellular and viral sequences were identified. In contrast, expression of NS3 and the fusion proteins was not found after infection with the respective non-cp counterparts.

Molecular characterization of border disease virus, a pestivirus from sheep.

Three serologically different pestivirus strains isolated from sheep were selected for molecular analysis. cDNA and deduced amino acid sequences of the genomic regions encoding glycoproteins E1 and E2 were obtained from the three strains. A comparison with amino acid sequences of bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) revealed that one of the three ovine pestivirus strains can be grouped together with BVDV. The other two strains, however, were clearly different from both BVDV and CSFV. Surprisingly, the amino acid sequences from these two viruses were more similar to CSFV than to BVDV. The identity between so-called "true" BDV strains at the amino acid level is about 95% for E1 and 86% for E2 and thus similar to homologies found between CSFV strains. For one "true" BDV strain the genomic region encompassing the nonstructural protein p125 was also cloned and sequenced. The respective comparative analysis led to results which are similar to the ones obtained for the two structural glycoproteins. Taken together the data demonstrate that "true" border disease virus strains represent a separate group within the genus pestivirus.

Detection of hog cholera virus antigens in experimentally-infected pigs using an antigen-capture ELISA.

An antigen-capture ELISA was used to detect hog cholera virus (HCV) antigens in blood and tissues taken from pigs infected with 2 different strains of virus. Specific antigens were demonstrated in peripheral blood leucocytes (PBLs) and a wide range of tissue samples 4-6 days after infection of pigs with a moderate-high virulent HCV strain (Weybridge virus). Strong signal to noise (S/N) ratios were obtained in the ELISA for PBLs and lymphoid tissues such as spleen, tonsil and mesenteric lymph nodes at 5-7 days after infection with the Weybridge virus, S/N ratios varying between 8.1-19.7 for blood samples and 4.3-19.1 for spleen samples. High positive ELISA results were also obtained for duodenum and ileum samples (S/N ratios 10.3-18.6) taken from these pigs, reflecting severe pathological changes observed in the gut at post mortem. In contrast, the antigen-capture ELISA gave strong positive results for PBLs and spleen samples only at 7-9 days after infection of pigs with a low virulent strain of HCV (New South Wales virus). The ELISA S/N ratios averaged 9.5 for PBLs and 8.9 for spleen samples in these animals. Although virus isolation detected infection earlier in the infected pigs, the ELISA returned positive results on PBLs and spleen samples around the time all of the animals first showed typical signs of classical swine fever. The technique does not require tissue culture and takes less than 36 h to return a definitive result.

An antigen-capture ELISA detects pestivirus antigens in blood and tissues of immunotolerant carrier cattle.

An antigen-capture ELISA was developed for the detection of pestivirus-specific antigens in peripheral blood leucocytes (PBLs), blood clots and tissue samples of immunotolerant cattle persistently infected with virus. The ELISA demonstrated complete agreement with conventional virus isolation procedures undertaken on specimens from a total of 58 carrier animals and 360 uninfected animals. The technique is based on capturing antigen with a high-titred goat polyclonal antiserum and detecting the bound antigen with a combination of 3 broadly-reactive monoclonal antibodies. Increased sensitivity was obtained with the use of an avidin-biotin complex (ABC) amplification method. On average, ELISA optical densities (ODs) for PBL and blood clot samples derived from carrier animals were 1.53 and 0.95, respectively, while uninfected animals had corresponding values of less than 0.15 for all blood samples. Tissue samples from carrier cattle had OD values ranging from an average of 0.95 for liver to 1.77 for spleen, with negative values for all tissues again averaging less than 0.20. Signal-to-noise (S/N) ratios calculated from the ELISA OD readings for carrier cattle showed an average of 15.6 for blood samples and 16.4 for tissues. In contrast, all samples from negative cattle had S/N ratios less than 2.0. The antigen-capture ELISA has been validated on field samples and is suitable for routine diagnostic and certification testing.

Lysosomal enzyme activities in sheep plasma and lymph.

The activities of acid phosphatase, beta-glucuronidase and N-acetyl-beta-glucosaminidase were measured in blood plasma, afferent and efferent popliteal lymph, intestinal lymph and in four different organs of the sheep. Acid phosphatase had an optimum activity at pH 4-5 while beta-glucuronidase and N-acetyl-beta-glucosaminidase had optimal activities at pH 5-0 and pH 4-5-5-0, respectively. Comparative studies showed that the sheep had very low activities of acid phosphatase and relatively low activities of N-acetyl-beta-glucosaminidase in the blood plasma compartment in relation to the activities of these enzymes in the plasma of the rabbit and the rat. The tissue activities of all three enzymes were relatively high when compared with those in the two non-ruminant species. It is considered that the low plasma activities of acid phosphatase in the sheep reflect a rapid turnover of this enzyme in the plasma compartment. The activities of the three enzymes in regional lymph indicated that acid phosphatase was being added to the capillary filtrate at a regional tissue level, whereas beta-glucuronidase and N-acetyl-beta-glucosaminidase in lymph were derived from filtration from the blood plasma compartment on a molecular weight basis. The high lymph: plasma ratios observed for acid phosphatase activity in intestinal lymph may indicate a function of this enzyme in lipid absorption in the sheep.

The influence of the lymph node on the protein concentration of efferent lymph leaving the node.

1. Experiments have been performed in sheep to determine the contribution of lymph formed within a lymph node to the total protein output in lymph leaving the node. 2. The lymphatic duct leaving the popliteal lymph node was cannulated and the protein and lymphocyte output in efferent lymph determined. The afferent lymph flow to the popliteal node was then diverted and lymph formed only within the lymph node collected from the efferent cannula. It appeared from the results that the popliteal lymph node forms lymph at the rate of approximately 1 ml. per hour and may contribute 30-50% of the protein output observed in efferent lymph. 3. The importance of lymph formation within the lymph node varied between nodes found in different regions of the body. This was due in part to the different protein concentrations in the afferent lymph to the different nodes. 4. A positive correlation was found between the protein and lymphocyte concentrations in efferent lymph from the popliteal lymph node in seven out of eleven sheep and in lymph formed within the popliteal lymph node in two out of three sheep. It is suggested that this relationship may be due to an increased transfer of plasma proteins through the post-capillary venules in the lymph node accompanying the continual traffic of lymphocytes across the wall of these vessels. The results indicated that the protein transfer across the post-capillary venules was not an indiscriminate transfer of plasma per se but a selective transport from the blood plasma compartment based on molecular size.

Red blood cell catabolism in lymph nodes of the sheep and rat: quantitation.

Estimates have been made of the number of red blood cells catabolised in the lymph nodes of the sheep and the rat. These estimates were based on observations on the output of bilirubin in intestinal lymph of the sheep and on bilirubin output in thoracic duct lymph of the rat. It was calculated that some 2 to 15 X 10(9) red blood cells were catabolised in a 24 hour period by the lymph nodes of the intestinal region of the sheep, representing approximately 3% of all the red cells destroyed in this period. Similarly, 0.9 to 4.6 X 10(8) red blood cells were estimated to be catabolised every 24 hours in the lymph nodes of those areas drained by the thoracic duct of the rat, representing approximately 6% of all the red cells destroyed in a 24 hour period. Calculations extending these results to include all areas of the body in both species indicated that the lymph nodes may account for some 6-7% of the total number of red blood cells catabolised in the animal.

Response of the regional lymphatic system of the sheep to acute stress and adrenaline.

An acute pain stimulus resulted in elevated lymph flow and output of cells from the popliteal lymph node of the sheep in the first 15 min after the stress. Efferent lymph flow increased by an average of 93% above the mean resting flow and cell output rose by an average of 170% during this period, but by 30 min after the stress, values for both lymph flow and cell output had returned to normal. The cell content of the efferent lymph was significantly higher in the first 15 min after the acute stress and it is suggested that there is a sizeable pool of lymphocytes within the resting popliteal node which can be mobilized into the lymph by an acute stress. A single intravenous injection of 1 mg adrenaline the efferent lymph flow in all the sheep examined but gave rise to an increased cell output in only 50% of the sheep. This indicated that there may be other factors, possibly hormonal, involved in the movement of the pool of lymphocytes out of the regional lymph node following acute stress. Both acute pain stress and adrenaline resulted in an increased afferent popliteal lymph flow and output of cells from the regional tissues in the first 15 min after administration. The results are suggestive of a small pool of lymphocytes in the regional tissues which may be readily mobilized by either acute stress or adrenaline. Part of the increases in efferent and afferent lymph flow observed following acute stress and adrenaline appeared to be due to an increased lymph formation, presumably as a result of an increased capillary pressure. Nevertheless, it is considered that the greater part of the increased flow of lymph from both regions resulted from an accelerated movement of performed lymph.

High concentration of bilirubin in post-nodal lymph associated with red blood cell catabolism in lymph nodes of the sheep.

Qualitative and quantitative analysis of post-nodal lymph of the sheep has shown that the distinct yellow colour of this fluid pool is due to the presence of relatively large amounts of bilirubin. In efferent lymph from thepopliteal, prefemoral, prescapular, renal and intestinal lymph nodes total bilirubin concentrations were 3-8 times higher than the corresponding concentrations in blood plasma. In contrast the total bilirubin concentrations in afferent lymph from the lower leg and kidney were less than the corresponding concentrations in blood plasma. Histological examination of several popliteal and mesenteric lymph nodes revealed the presence of free iron and bilirubin in the cytoplasm of cells located near the lymphatic sinuses of the node. In addition, the concentration of bilirubin in efferent lymph from the popliteal node was observed to increase following an induced rise in the number of red blood cells reaching the node by way of the afferent lymphatic duct. These latter observations suggest that the bilirubin in post-nodal lymph is associated with the catabolism of extravascular red cells by reticulo-endothelial cells within the lymph nodes.

The effect of anaesthesia and surgery on lymph flow, protein and leucocyte concentration in lymph of the sheep.

Anaesthesia and the trauma of surgery, associated with the cannulation of lymphatic ducts in various regions of the body of the sheep, had a profound effect on lymph flow, protein concentration and leucocyte concentration of lymph. In general lymph flow was depressed and the protein concentration elevated in lymph collected at the time of cannulation, or within the first 24 hours of recovery from surgery. The changes in protein concentration in lymph draining the peripheral regions of the body appeared to be due to surgical interference in the region of drainage. The greatest changes in lymph flow were observed in lymph draining peripheral regions (skin, tendon, muscular areas) while lymph draining soft tissues in central regions (kidney, liver) was less affected by the anaesthesia and surgical stress. A neutrophilia was observed in venous blood collected under anaesthesia while the overall numbers of lymphocytes in three sources of efferent lymph were depressed. It is suggested that corticosteroid hormones may play a role in the changes in leucocyte migration observed during anaesthesia and surgical stress. Changes observed in the cellular content of afferent lymph appeared to be due to a low grade inflammation associated with surgical interference in the region of lymphatic drainage.

A postcoupling method for the demonstration of N-acetyl-beta-D-glucosaminidase in unfixed frozen tissue sections.

The localization and activity of the lysosomal enzyme, N-acetyl-beta-glucosaminidase, has been studied in unfixed frozen sections of tissues from normal and hemorrhaged rats with the use of a modified postcoupling technique. Discrete localization of the end product of the reaction was achieved in this method by the incorporation of 20% (w/v) polyvinyl alcohol (molecular weight 14,000) in the incubation medium. Advantages of the present method include the ability to overcome the inhibitory effects on enzyme activity of both tissue fixation and the presence of diazonium salts in the incubation medium. The staining reaction obtained with this technique demonstrates the enzyme activity at specific cytoplasmic sites within cells and has a wider applicability to the comparative study of N-acetyl-beta-glucosaminidase activity in normal and injured tissues.

The lysosomal enzyme N-acetyl-beta-glucosaminidase in rabbit muscle following a period of ischaemia.

N-acetyl-beta-glucosaminidase activity increased in injured plantar muscle during the interval of 1 to 7 days after a period of 6 hours ischaemia of the rabbit hindpaw. Histochemical visualization demonstrated that this increased activity was not only associated with infiltrating inflammatory cells but also with the appearance of discrete lysosomal-like granules within damaged muscle fibres. Seven days after injury, the enzyme activity had reached a maximum with levels in the injured muscle 11 times higher than in normal muscle. The activity at this time was visualized in granules longitudinally arranged along regenerating muscle fibres, seemingly implicating the longitudinal sarcotubular system in their origin. Residual histochemical activity was evident at 14 and 28 days after injury. It is suggested that, in addition to its hydrolytic activity, N-acetyl-beta-glucosaminidase may have a role in the formation of mucopolysaccharides in the regenerative processes of muscle.