Guy Burtonboy

Evidence for persistence of human parvovirus B19 DNA in bone marrow

A nested polymerase chain reaction assay (nPCR) was used to investigate the potential of human parvovirus B19 DNA to persist in blood or bone marrow samples obtained either from blood donors or cadaveric bone donors or from patients presenting with clinical signs of parvovirus B19 infection. The presence of parvovirus B19 specific antibody in blood was tested by enzyme immunoassay (EIA). B19 virus genome was not detected in any blood sample of 115 blood donors, of whom 92 (80%) had anti‐B19 IgG antibody only as an indication of past infection. In contrast, B19 virus DNA was detected in the bone marrow of 4 out of 45 bone donors. Each one of the serum samples available for 3 of these 4 individuals contained anti‐B19 IgG antibody. Among 84 patients with clinical manifestations of parvovirus B19 infection, 17 (20%) had B19 virus DNA in bone marrow. Eight of the latter patients had anti‐B19 IgG antibody in their blood but neither anti‐B19 IgM nor B19 virus DNA. These data document the ability of parvovirus B19 DNA to persist in the bone marrow of asymptomatic individuals and patients with parvovirus B19 infection suspected on clinical grounds. J. Med. Virol. 53:229–232, 1997.

Rat monoclonal antibodies. I. Rapid purification from in vitro culture supernatants.

A technique for purifying rat monoclonal antibodies quickly and efficiently from in vitro culture supernatants is described. It is based on the fact that more than 95% of rat immunoglobulins carry kappa light chains. A mouse monoclonal antibody with suitable binding affinity for rat kappa light chains is immobilized on solid support and used to purify rat immunoglobulins. Milligrams of rat monoclonal antibodies may be rapidly concentrated from culture supernatants with high recovery. Rat monoclonal antibodies expressing lambda light chains (about 5% of the total) may be purified in a similar way with an appropriate anti-rat lambda chain monoclonal antibody.

Acute parvovirus B19 infection associated with fulminant hepatitis of favourable prognosis in young children

BACKGROUNDnThe cause of fulminant hepatitis (FH) in children is unexplained in up to 50% of cases. We report parvovirus B19 as an agent associated with FH in children and compare clinical characteristics of these patients with those of age-matched patients with FH of other origin.nnnMETHODSn45 patients presented with FH. No cause was apparent in 21 patients. Parvovirus B19 genome was retrospectively sought by PCR in serum collected at admission in 41 patients.nnnFINDINGSnParvovirus B19 genome was detected in serum from four of 21 patients with unexplained FH (four of 11 younger than 5 years). No B19 DNA was detected in serum from patients with other types of FH or from 82 patients with biliary atresia. Parvovirus B19 IgM was detected in one of the four patients. Patients with parvovirus B19 infection had significantly lower bilirubin concentrations than age-matched patients with FH due to hepatitis A (nine) or other causes (nine) (poisoning with amanita excluded). All patients with parvovirus B19 survived without orthotopic liver transplantation, with restoration of normal liver function within 17 days.nnnINTERPRETATIONnIn patients younger than 5 years with FH of unexplained origin, evidence of acute parvovirus B19 was associated with a distinct clinical pattern. In particular, low bilirubin concentrations and rapid recovery of liver function without transplantation were distinctive features.

Canine hemorrhagic enteritis: Detection of viral particles by electron microscopy

SummaryAt necropsy, several dogs which died showing symptoms of hemorrhagic diarrhea, had significant lesions of the mucosa that were found especially in the duodenum and upper part of the small bowel.Study of ultrathin sections from the diseased mucosa revealed particles resembling parvoviruses in altered nuclei of cells of the intestinal crypts.Electron microscopic examination of intestinal contents by negative staining has shown the presence of many viral particles which have a diameter of 24 nm and whose profile is consistent with an icosahedral shape. These virions float at a density of 1.43 g/cm3 in cesium chloride and agglutinate rhesus monkey and swine red blood cells at 4° C.A possible etiological role in discussed.This virus is compared with the minute virus of canines and the Feline Panleukopenia virus.

The effect of repeated treatment with dexamethasone on the re-excretion pattern of infectious bovine rhinotracheitis virus and humoral immune response

Abstract Two cattle, free of antibody to infectious bovine rhinotracheitis virus (IBRV) were infected intranasally with IBRV, and developed specific antibody to the virus. Ten weeks later, both animals were given an intravenous course of dexamethasone (DM). Nasal excretion of physical particles of virus, as judged by electron microscopy, occurred in both animals, as early as 24 h after the first DM injection, and high levels of infectious particles appeared several days later. Neutralizing antibody titre to IBRV increased following excretion of virus. Further courses of DM given at 20 and 32 weeks following initial infection were not associated with excretion of physical, ‘non-infectious’ particles or significant changes in specific antibody titre, although on each occasion one of the two animals excreted low levels of infectious particles.

Comparison of DNA Sequencing and a Line Probe Assay for Detection of Human Immunodeficiency Virus Type 1 Drug Resistance Mutations in Patients Failing Highly Active Antiretroviral Therapy

ABSTRACT The resistance of human immunodeficiency virus type 1 (HIV-1) to drugs is a major cause of antiretroviral treatment failure. We have compared direct sequencing to a line probe assay (LiPA) for the detection of drug resistance-related mutations in 197 clinical samples, and we have investigated the sequential appearance of mutations under drug pressure. For 26 patients with virological failure despite the use of two nucleoside analogues and one protease inhibitor (indinavir [n = 6], ritonavir [n = 10], and saquinavir [n = 10]), genotypic resistance assays were carried out retrospectively every 3 months for up to 2 years by using direct sequencing (TruGene; Visible Genetics) and a LiPA for detection of mutations in the reverse transcriptase (INNO-LiPA HIV-1 RT; Innogenetics) and the protease (INNO-LiPA HIV Protease, prototype version; Innogenetics) genes. Comparison of the results from both assays found rare major discrepancies (<1% of codons analyzed). INNO-LiPA detected more wild-type–mutant mixtures than sequencing but suffered from a high rate of codon hybridization failures for the reverse transcriptase. LiPA detected earlier and more frequently than sequencing the transient mixed virus population that contained I84V, which appears before V82A in the protease sequence. Mutations M461, G48V, and L90M were often transient and drug pressure related. In conclusion, direct sequencing and LiPAs give concordant results for most clinical isolates. LiPAs are more sensitive for the detection of mixed virus populations. Mutation I84V appears in minor populations in the early steps of the pathways of resistance to indinavir and ritonavir. The fact that some mutations can be found only transiently and in minor virus populations highlights the importance of a low detection limit for resistance assays.

Comparison Between Strains of Infectious Bovine-rhinotracheitis Virus (bovid-herpesvirus 1), From Respiratory and Genital Origins, Using Polyacrylamide-gel Electrophoresis of Structural Proteins

Abstract The polypeptide composition of three strains of infectious bovine rhinotracheitis virus, isolated from typical respiratory disease (IBR), has been compared with that of three strains isolated from the genital tract of cattle suffering from infectious pustular vulvovaginitis (IPV). All the IBR strains are similar to each other, but different from the IPV strains, which in turn were similar to each other. IBR isolates and IPV isolates differed in three polypeptides.

Characterization of a Nonhemagglutinating Mutant of Canine Parvovirus

A nonhemagglutinating mutant of a 1978 isolate of canine parvovirus (CPV) was derived after repeated passages in the NLFK feline kidney cell line. The mutant CPV was antigenically indistinguishable from wild-type virus when tested with 82 monoclonal antibodies, and it replicated in cat and dog cell lines in culture. Sequences of the VP-1 and VP-2 genes revealed two nucleotide and two predicted amino acid sequence differences at 77 and 88 genome map units in the mutant compared to hemagglutinating viruses. One or both of those two mutations must determine the difference in the ability of the virus to agglutinate erythrocytes.

Variant human immunodeficiency virus type 1 proteases and response to combination therapy including a protease inhibitor

ABSTRACT The objective of this observational study was to assess the genetic variability in the human immunodeficiency virus (HIV) protease gene from HIV type 1 (HIV-1)-positive (clade B), protease inhibitor-naı̈ve patients and to evaluate its association with the subsequent effectiveness of a protease inhibitor-containing triple-drug regimen. The protease gene was sequenced from plasma-derived virus from 116 protease inhibitor-naı̈ve patients. The virological response to a triple-drug regimen containing indinavir, ritonavir, or saquinavir was evaluated every 3 months for as long as 2 years (n = 40). A total of 36 different amino acid substitutions compared to the reference sequence (HIV-1 HXB2) were detected. No substitutions at the active site similar to the primary resistance mutations were found. The most frequent substitutions (prevalence, >10%) at baseline were located at codons 15, 13, 12, 62, 36, 64, 41, 35, 3, 93, 77, 63, and 37 (in ascending order of frequency). The mean number of polymorphisms was 4.2. A relatively poorer response to therapy was associated with a high number of baseline polymorphisms and, to a lesser extent, with the presence of I93L at baseline in comparison with the wild-type virus. A71V/T was slightly associated with a poorer response to first-line ritonavir-based therapy. In summary, within clade B viruses, protease gene natural polymorphisms are common. There is evidence suggesting that treatment response is associated with this genetic background, but most of the specific contributors could not be firmly identified. I93L, occurring in about 30% of untreated patients, may play a role, as A71V/T possibly does in ritonavir-treated patients.

HIV-1 detection by nested PCR and viral culture in fresh or cryopreserved postmortem skin: potential implications for skin handling and allografting.

AIMS: To date, the risk relating to the handling or allografting of human immunodeficiency virus type 1 (HIV-1) infected postmortem skin remains hypothetical. While blood screening for HIV antibodies is still the key safety procedure to detect HIV infected cadavers, false negative results are a concern. Conversely, false positive results may hamper the collection of skin allografts. Accordingly, viral culture was used to clarify skin infectivity and the nested polymerase chain reaction (PCR) was used to assess the reliability of skin PCR testing. METHODS: Viral culture and nested PCR performed with gag and pol specific primers were investigated in cadaveric skin and blood from 12 HIV-1 infected patients. Samples were collected repeatedly between one and five days in seven patients. In most cases, analyses were performed on triplicate skin samples: fresh (n = 26); cryopreserved in 5% dimethylsulphoxide (n = 21), or cryopreserved in 30% glycerol (n = 26). RESULTS: HIV was isolated in two of 26 cultures of fresh skin specimens (8%), seven of 47 cryopreserved skin specimens (15%), and eight of 26 blood specimens (31%). The nested PCR detected HIV-1 in all skin samples (n = 73), regardless of the postmortem interval or cryopreservation. In blood, a positive signal was found in eight of 12 patients but two of them had discordant results on successive samples. CONCLUSIONS: These data suggest that nested PCR on postmortem skin samples can detect HIV more reliably than on blood. They also demonstrate the potential viral infectivity of fresh or stored skin postmortem samples in HIV infected patients. They underscore the need for caution during the handling of skin tissue from HIV infected cadavers and confirm the potential risk related to accidental allografting of HIV contaminated skin.