Molecular epidemiology of rotaviral infection in South Indian children with acute diarrhea from 1995-1996 to 1998-1999

The distribution of VP7 (G-) and VP4 (P-) genotypes among 126 rotavirus strains from South Indian children, < 5 years of age and with acute diarrhoea, presenting to a single hospital during the months to November and December, from 1995 to 1998, was studied. Multiplex hemi-nested G- and P-typing polymerase chain reactions determined 101 (80%) G types and 78 (61%) P types, respectively. In order of frequency, the commonest G types were G1, G4, G2, G9, G3, and G8, and P types were P1B[4], P1A[8], and P2A[6] and the most common G:P combinations were G1:P1A[8], G1:P1B[4], G2P1B[4] and G4:P1A[8]. G1, G2, and G4 types were seen in all years. The single G3 isolate was seen in 1998. The single G8 isolate and the 5 G9 isolates were seen in 1997, after a period of heavy rain. Sequence analysis showed that the G8 isolate was related most closely to the bovine strain A5, and the G9 strains were distinct from the nonpathogenic Indian isolate 116E and similar to G9s isolated in Mysore and the United Kingdom described previously.     

Accumulation of p53 in response to adenovirus early region 1A sensitizes human cells to tumor necrosis factor alpha-induced apoptosis

Many tumor cells are resistant to tumor necrosis factor alpha (TNFalpha)-induced apoptosis. Adenovirus early region 1A (AdE1A) sensitizes the otherwise resistant cells to TNFalpha. AdE1A also stabilizes the p53 protein. The present study demonstrates a correlation between AdE1A-induced sensitization and stabilization of p53 in TNFalpha-induced apoptosis since the N-terminal and CR2 regions, the binding sites for CBP/p300, Rb and 26S proteasome regulatory components, are required for both these actions of AdE1A. TNFalpha does not induce apoptosis and AdE1A fails to sensitize TNFalpha cytotoxicity in p53-negative cells. However, introduction of exogenous p53 overcomes the cellular resistance to TNFalpha toxicity and enhances AdE1A sensitization, demonstrating that AdE1A sensitizes TNFalpha-induced apoptosis by its stabilization of p53. A proteasome inhibitor, lactacystin, enhances TNFalpha cytotoxicity in p53-positive and -negative cells, suggesting that accumulation of cellular proteins other than p53 might also regulate the cellular response to TNFalpha signaling.     

Diversity of enteric viruses detected in patients with gastroenteritis in a tertiary referral paediatric hospital

The genetic diversity of enteric viruses co-circulating in a cohort of patients with viral gastroenteritis in a large tertiary paediatric hospital in London, UK, was determined. Multiple strains of noroviruses (NV), sapoviruses (SV) and astroviruses (HAsV) were detected in these patients, indicating the likelihood of multiple introductions from different sources, possible sub-clinical infections and simultaneous infection with different viruses in immunocompromised and other patients. Routine screening of immunocompromised patients and infection control procedures are important to prevent nosocomial infection.     

The heteroduplex mobility assay (HMA) as a pre-sequencing screen for Norwalk-like viruses

Molecular epidemiological studies of Norwalk-like viruses (NLVs), previously known as small round structured viruses (SRSVs), are dependant currently on DNA sequencing of PCR amplicons, which is expensive and time consuming. The Heteroduplex Mobility assay (HMA) was evaluated as a method for identification of PCR amplicons from the commonly circulating NLV strains without DNA sequencing. The procedure was developed for use with two reference strains, a Mexico virus-like strain (MXV-like; Hu?NLV?RBH?1993?UK) and the Grimsby virus strain (Hu?NLV?Gimsby?1995?UK), and was optimised with regards to the annealing and electrophoresis conditions and the electrophoresis gel matrix. Using the optimised conditions, amplicons of less than 90% sequence identity formed visible heteroduplexes, allowing the strains to be placed into three categories; Mexico-like, Grimsby-like and non-Mexico virus/non-Grimsby virus strains. Outbreak strains 'genotyped' previously by DNA sequencing as Mexico virus or Grimsby virus were identified correctly by the heteroduplex mobility assay. The procedure was applied prospectively to strains from 130 outbreaks occurring in the UK between 1997 and 1998. Heteroduplex mobility assay was successful on 120 (92%) strains of which 68 (57%) were GRV-like strains, three (2.5%) were Mexico virus-like strains and 49 (41%) were categorised as non- Mexico/non-Grimsby virus strains. Amplicons from 50 of the 120 strains were sequenced and there was perfect correlation between the heteroduplex mobility assay categorisation and phylogenetic analysis. HMA offers a rapid, robust and far cheaper alternative to sequencing for the identification of prevalent Norwalk-like virus genotypes for molecular epidemiological studies.     

Genomic characterisation of the large segment of a rabbit picobirnavirus and comparison with the atypical picobirnavirus of Cryptosporidium parvum

Summary.  The 2362 base pair sequence of the larger of the two double stranded RNA genome segments of a rabbit strain of picobirnavirus (PBV) has a major open reading frame (ORF) of 591 amino acids and two smaller ORFs of 55 and 155 amino acids. A clone of the segment did not hybridise with other viral bisegmented ds RNAs from faecal samples. There is no relationship in sequence or organisation between this PBV sequence and the bisegmented dsRNAs found associated with Cryptosporidium parvum. This suggests that there are at least two distinct classes of bisegmented dsRNA viruses or viral-like agents in faeces. Received January 27, 1999/Accepted June 30, 1999     

Changes in antiplasmin and plasmin activities during extreme hemodilution and open heart surgery in dogs.

The effects of extreme hemodilution and open heart surgery on antiplasmin and plasmin activities were investigated in dogs. Assays for plasmin and antiplasmin activities were performed with a chromogenic peptide substrate for plasmin. After hemodilution and during cardiopulmonary bypass (CPB), plasmin activity on the substrate was observed and levels of both "fast-reacting" and "total" antiplasmins fell. The plasmin activity was shown to be due to the formation of alpha2-macroglobulin-plasmin complex which exhibited amidolytic activity on the chromogenic substrate. The falls in antiplasmin activity together with the appearance of the alpha2-macroglobulin-plasmin complex revealed that activation of the fibrinolytic system had occurred. After the operation and following reinfusion of autologous blood, decreased plasmin and increased antiplasmin activities were observed. On the first and sixth days following the operation plasmin activities were still slightly higher than pre-operation values, whilst antiplasmin activities were normal.     

Histochemical demonstration of desialation and desulphation of normal and inflammatory bowel disease rectal mucus by faecal extracts.

Experiments were carried out to assess the susceptibility of normal and inflammatory bowel disease rectal mucus to desulphation and desialation by faecal extracts and by bacterial sialidase. The effects were assessed histochemically using a combined high iron diamine (HID) and alcian blue (AB) stain for sulphomucins and sialomucins. Rectal mucus in biopsies from controls (irritable bowel syndrome) and patients with ulcerative colitis or Crohn's disease was resistant to desialation by Clostridium perfringens sialidase, but susceptible to desialation and desulphation by bacteria-free extracts of normal faeces. Periodic acid-Schiff (PAS) staining of adjacent sections similarly treated showed retention of neutral mucus. One faecal extract selectively desulphated all 42 biopsies, causing the goblet cells to change from HID positive to AB positive, suggesting that most, or all HID positive cells also contain sialomucins. This alters the interpretation of previous histochemical studies. Faecal extracts from patients with active ulcerative colitis (n = 6) had desialating and desulphating effects similar to faecal extracts from normal subjects (n = 6). Ulcerative colitis (n = 21), Crohn's disease (n = 18), and control (irritable bowel syndrome) (n = 17) rectal biopsies all showed similar susceptibility to desulphation by a pooled normal faecal extract, but rectal biopsies from patients with Crohn's disease proved more resistant to desialation than control or ulcerative colitis biopsies (p less than 0.02). These studies imply that colonic mucus undergoes continual desulphation and desialation in vivo as a result of faecal enzyme activity that is probably mainly of bacterial origin. Altered susceptibility of colonic mucus to this may be important in the pathogenesis of colonic disease.     

Reduced factor XII levels in patients with the antiphospholipid syndrome are associated with antibodies to factor XII

Antibodies to factor XII (FXII) have previously been identified in some patients who were lupus anti-coagulant-positive. The relationship between these antibodies and FXII levels appeared to be variable. The aim of the present study was to confirm the presence of antibodies to FXII in patients with well characterized antiphospholipid syndrome (APS) and to establish their potential effect on levels of FXII. Forty-two patients with APS were studied; 21 patients were found to have either immunoglobulin (Ig)G or IgM antibodies to FXII by enzyme-linked immunosorbent assay (ELISA) using a highly purified preparation of FXII (> 99% pure). Levels of FXII were statistically significantly lower (P = 0.02) in patients with antibodies to FXII when compared with patients without antibodies to FXII (median = 91 micro/dl, s.d. = 39.1, median = 122 micro/dl, s.d. = 41.1 respectively). Four of the 21 patients with antibodies to FXII were found to have FXII levels below the laboratory normal range. Antibodies to FXII are present in significant numbers of patients with APS and may lead to acquired FXII deficiency.