Evaluation of the RIDA®GENE RT-PCR assays for detection of sapovirus, astrovirus, adenovirus, and rotavirus in stool samples of adults in Switzerland.

Sapovirus (SaV) and astrovirus (AstV) increasingly are recognized as cause of acute viral gastroenteritis (AGE). We evaluated the real-time RT-PCR assays RIDA®GENE SaV and viral stool panel II (RGN RT-PCR) for detection of SaV, AstV, adenovirus (AdV) F40/41 and rotavirus (RoV) in clinical stool samples (n = 69). Results were compared with reference singleplex RT-PCRs. The sensitivity for SaV, AstV and RoV are 100%, the specificity ranges from 98.1% to 100%. In 10 out of 11 AdV (all types) samples, the RGN RT-PCR for AdV F40/41 displayed negative results. Retrospectively, 196 stool specimens from adult patients previously tested negative for norovirus (NoV) were analyzed. In about 10% of NoV-negative stool samples, AdV (n = 9), RoV (n = 6), AstV (n = 3) or SaV (n = 3) were found. The RGN RT-PCR assays are useful for detection of enteric viruses other than NoV. This study emphasizes the need for further testing of NoV-negative stool samples in patients with AGE.

Resolution of plasma sample mix-ups through comparison of patient antibody patterns to E. coli.

BACKGROUND: Accidental sample mix-ups and the need for their swift resolution is a challenge faced by every analytical laboratory. To this end, we developed a simple immunoblot-based method, making use of a patient's characteristic plasma antibody profile to Escherichia coli (E. coli) proteins. METHODS: Nitrocellulose strips of size-separated proteins from E. coli whole-cell lysates were incubated with patient plasma and visualised with an enzyme-coupled secondary antibody and substrate. Plasma samples of 20 random patients as well as five longitudinal samples of three patients were analysed for antibody band patterns, to evaluate uniqueness and consistency over time, respectively. For sample mix-ups, antibody band patterns of questionable samples were compared with samples of known identity. RESULTS: Comparison of anti-E. coli antibody patterns of 20 random patients showed a unique antibody profile for each patient. Antibody profiles remained consistent over time, as shown for three patients over several years. Three example cases demonstrate the use of this methodology in mis-labelling or -pipetting incidences. CONCLUSION: Our simple method for resolving plasma sample mix-ups between non-related individuals can be performed with basic laboratory equipment and thus can easily be adopted by analytical laboratories.

Investigations on the diagnosis and retroviral aetiology of renal neoplasia in budgerigars (Melopsittacus undulatus).

The high susceptibility of budgerigars (Melopsittacus undulatus) to neoplasia, and specifically renal neoplasia, has often been reported. Further investigations led to a suspicion of a retrovirus as the causative agent for renal neoplasia in budgerigars, but definitive proof has yet to be found. In the present study, 32 budgerigars suspected of having renal neoplasia (based on the clinical presentation) were examined. The objectives were to investigate the use of different diagnostic methods for the ante-mortem diagnosis of this condition and to find more supporting evidence of a retroviral aetiology. The predominant clinical signs observed in budgerigars with renal neoplasia were lameness and absence of deep pain sensation of one leg. Alterations in haematology, plasma chemistry, and urine analyses could not pinpoint the cases of renal neoplasia. Contrast radiography of the intestinal tract proved to be diagnostically more useful compared with plain radiographic studies. Histology confirmed the renal neoplasia as adenocarcinoma. Investigations for virus identification included product-enhanced reverse transcriptase assay and enzyme-linked immunosorbent assay for the detection of avian leucosis virus group-specific antigen. Cell cultures and electron microscopy were performed on a limited number of patients. These investigations could find no presence of an exogenous, replicating retrovirus, neither could viral particles be detected by electron microscopy. Based on the current findings, it can be concluded that there is no evidence of retroviral involvement in the occurrence of renal neoplasia in budgerigars.

Detection of reverse transcriptase activity in human melanoma cell lines and identification of a murine leukemia virus contaminant.

BACKGROUND: Stimulated by earlier reports on the presence of retroviruses in mouse and hamster melanoma cell lines, we addressed the question as to whether human melanoma cell lines might also harbour a retrovirus. METHODS AND RESULTS: The melanoma cell lines SK-MEL-25, SK-MEL-28, MEL-JUSO, MML-I, MeWo, A-375, Colo-38, BS-780 were confirmed to be human by human leucocyte antigen (HLA) typing, and supernatants were tested by the product-enhanced reverse transcriptase (PERT) assay for reverse transcriptase (RT) activity. Cell lines SK-MEL-25, SK-MEL-28, MEL-JUSO and MML-I were positive, whereas cell lines MeWo, A-375, Colo-38 and BS-780 were negative. The RT activity peaked at a buoyant density in sucrose typical for retroviruses. From this peak fraction an R-U5 sequence indistinguishable from murine leukemia virus (MLV) was identified by particle-associated retrovirus RNA amplification (PARRA). Semiquantitative MLV-specific RNA-PCR demonstrated colocalization of the MLV-like RNA and RT activity on the sucrose gradient of SK-Mel-25. MLV RNA and DNA were also detectable in culture supernatants of SK-MEL-28, MEL-JUSO and MML-I, but not of MeWo, A-375, Colo-38 and BS-780 by semiquantitative polymerase chain reaction (PCR). Sequence comparison revealed highest homology with the RET sequence previously identified in mouse myeloma SP2/0-AG14 cells. Taken together, our data strongly suggest that certain human melanoma cell lines are productively infected by a MLV which was probably introduced during tumour passage in mice or by laboratory contamination many years ago and subsequently spread to other lines. CONCLUSION: We recommend mandatory testing of melanoma and other human cell lines for contamination with infectious MLV or other animal retroviruses, similar to mycoplasma screening, in order to avoid artificial experimental data.

Identification and characterization of two closely related unclassifiable endogenous retroviruses in pythons (Python molurus and Python curtus).

Boid inclusion body disease (BIBD) is a fatal disorder of boid snakes that is suspected to be caused by a retrovirus. In order to identify this agent, leukocyte cultures (established from Python molurus specimens with symptoms of BIBD or kept together with such diseased animals) were assessed for reverse transcriptase (RT) activity. Virus from cultures exhibiting high RT activity was banded on sucrose density gradients, and the RT peak fraction was subjected to highly efficient procedures for the identification of unknown particle-associated retroviral RNA. A 7-kb full retroviral sequence was identified, cloned, and sequenced. This virus contained intact open reading frames (ORFs) for gag, pro, pol, and env, as well as another ORF of unknown function within pol. Phylogenetic analysis showed that the virus is distantly related to viruses from both the B and D types and the mammalian C type but cannot be classified. It is present as a highly expressed endogenous retrovirus in all P. molurus individuals; a closely related, but much less expressed virus was found in all tested Python curtus individuals. All other boid snakes tested, including Python regius, Python reticulatus, Boa constrictor, Eunectes notaeus, and Morelia spilota, were virus negative, independent of whether they had BIBD or not. Virus isolated from P. molurus could not be transmitted to the peripheral blood mononuclear cells of B. constrictor or P. regius. Thus, there is no indication that this novel virus, which we propose to name python endogenous retrovirus (PyERV), is causally linked with BIBD.

Feline leukaemia provirus load during the course of experimental infection and in naturally infected cats.

Feline leukaemia virus (FeLV) infection in domestic cats can vary in its outcome (persistent, transient, no infection) for reasons that are not entirely known. It was hypothesized that the initial virus and provirus load could significantly influence the course of retrovirus infection. To determine the role of provirus loads, two methods of PCR, a nested PCR and a fluorogenic probe-based (TaqMan) real-time quantitative PCR, which were specific to the U3 region of FeLV-A were established. FeLV provirus in naturally and experimentally infected cats was then measured. Only 3 weeks after experimental FeLV-A infection, persistently infected cats demonstrated higher provirus loads and lower humoral immune responses than cats that had overcome antigenaemia. Lower initial provirus loads were associated with successful humoral immune responses. Unexpectedly, provirus in the buffy-coat cells of two cats that tested negative for the p27 antigen (a marker for viraemia) was also detected. In 597 Swiss cats, comparison of p27 antigen levels with PCR results revealed broad agreement. However, similar to the experimental situation, a significant number of animals (10%) was negative for the p27 antigen and FeLV-positive by PCR. These cats had a mean provirus load 300-fold lower than that of animals testing positive for the p27 antigen. In conclusion, an association between the provirus load and the outcome of FeLV infection was found. Detection of provirus carriers should contribute to further the control of FeLV. In addition, quantification of provirus loads will lead to a better understanding of FeLV pathogenesis and anti-retrovirus protective mechanisms.