Tibor Papp

Comparison of the Sensitivities of Noroviruses and Feline Calicivirus to Chemical Disinfection under Field-Like Conditions

ABSTRACT Noroviruses (NV), in the family Caliciviridae, are an important cause of gastroenteritis in humans worldwide. Measures for prevention and control of NV dissemination are therefore necessary to ensure public safety. The abilities of an organic acid (Venno Vet 1 Super), an aldehyde (Venno FF Super), a halogen compound (sodium hypochlorite solution), and a peroxide (Oxystrong FG) to inactivate feline calicivirus (FCV), a cultivable virus surrogate for NV, were studied. Molecular protocols were then used for the comparative evaluation of disinfectant efficacies against NV and FCV, which were tested by reproducing NV field conditions, using human fecal material as a protein load. Generally, disinfectant efficacy was strongly reduced by the organic impurities (feces) used during tests. All disinfectants, except the aldehyde, were effective on FCV, as measured by cell culture and reverse transcription-PCR (RT-PCR), with inactivation levels of ≥99.9%. The glutaraldehyde-based compound failed to adequately inactivate FCV according to RT-PCR results, although the infectivity in cell culture was completely abolished. Similar inactivation levels were achieved with NV, but generally NV appeared more resistant than FCV, and consequently, the suitability of FCV as a model for NV should be considered with caution. In conclusion, according to RT-PCR results, 5% Venno Vet 1 Super, 1% Oxystrong FG, and not less than 2% Venno FF Super, with a contact time of 1 h, and 1% sodium hypochlorite, with 6,000 ppm of free chlorine and a contact time of 15 min, are required for safe disinfection when a calicivirus-related outbreak is suspected.

PCR-sequence characterization of new adenoviruses found in reptiles and the first successful isolation of a lizard adenovirus

A consensus nested PCR was used to screen diagnostic samples from approximately 70 reptiles for the presence of adenoviruses (AdV) in the years 2006-2007. Classical virus isolation methods were also used with all samples. After adenoviruses were detected in a group of helodermatid lizards in a Danish zoo, a follow-up study was also carried out on lizards from this group (10 Mexican beaded lizards and 24 Gila monsters) over the period of a year. Adenoviruses were detected in a total of 26 lizards and snakes by PCR. The PCR amplicons from all positive animals were sequenced and the resulting polymerase gene sequences were used for phylogenetic analysis. Altogether six Agamid AdVs were amplified, with a minimal sequence variation between one another and between these and GenBank Agamid AdVs. The sequence obtained from one of the Gila monsters is identical with the GenBank Helodermatid AdV, while the sequences from the Mexican beaded lizards differ from this. In a snake collection we have detected a new AdV from an Asp viper. All of the above mentioned adenoviruses cluster in the Atadenovirus genus. However, the sequence from a new Varanid AdV detected in this study clusters outside this genus. On cell culture, viruses were isolated from three of the AdV positive helodermatid lizards (one Mexican beaded lizard and two Gila monsters) and identified as AdVs based on electron microscopy and PCR and sequencing using cell culture supernatant. This is the first report of the successful isolation of a lizard AdV.

Si(Li) Detector Lineshapes: Contributions from Atomic Physics and Detector Properties

The detailed response of an Si(Li) detector to the manganese K x-rays from a 55 Fe source was studied by least-squares fitting various spectrum models using a generalized variant of the GUPIX code. The peak-to-background ratio (>10 6 ) observed for the manganese Kα x-rays is superior in the detector used here to that observed in a crystal spectrometer, and very large spectrum intensities are recorded. In addition to well recognized effects such as the intrinsic Lorentzian distribution of an x-ray line and the approximately Gaussian detector response function, the Kα and Kβ peak shapes are influenced by multiplet structure. The Kβ shape is affected by 3p3d exchange coupling, and both Kα and Kβ are influenced by 3d spectator vacancy satellites. The flat shelf-like features below the peaks appear to be associated with escape of Auger and photoelectrons, and show a step-like structure that has not hitherto been discussed. Using an accurate energy calibration provided by simultaneously recorded rubidium K x-rays, the escape peak energy shift was shown to have the theoretical value of 1.74 keV. The escape peak width was significantly augmented, relative to the value expected for a diagram line at 4.2 keV energy, by shake-off satellites and by an additional component on its low-energy side; this observation explains the widely reported escape energy values in excess of 1.75 keV. Approximate determinations were made of the shape and intensity of the KLL radiative Auger feature and of the intensity of the KMM feature.

Comparison of paramyxovirus isolates from snakes, lizards and a tortoise.

Previously uncharacterized paramyxovirus (PMV) isolates from four snakes, three lizards and a tortoise were compared based on partial sequences of the L, HN, and U genes. Analysis of the sequences supported the classification of all reptilian PMVs in a separate genus (Ferlavirus) in the subfamily Paramyxovirinae. Within each of the gene segments, the squamatid isolates could be divided into two groups with a sequence divergence of 0.3-15.6% nt (0-6.8% aa) within the groups and 19.5-22.3% nt (5-7.4% aa) between the groups for the L gene, and 0.9-15.4% nt (0-6.9% aa) within the groups and 18.2-22.5% nt (4.4-9.5% aa) between the groups for the HN gene while higher values of 0.4-17.1% nt (0-13.3% aa) within the groups and 28.9-31.3% nt (25.5-27.8% aa) between the groups were found for the U gene. Isolates from lizards were found in both groups. There was no host species specificity in the grouping of the isolates from snakes and lizards. However, the L gene sequence obtained from the tortoise isolate differed significantly from the sequences obtained from the snake and lizard isolates. This isolate showed divergence values of 24.2-27% nt (18.5-20.9% aa) compared to the squamatid sequences. The tortoise isolate clustered together with the other reptilian PMVs, but not into any of the squamatid groups on the phylogenetic tree. It is hypothesized that this chelonian PMV has a more unique genome sequence as neither HN nor U gene parts could be amplified using newly designed consensus nested PCRs.

Development of Novel Adenoviral Vectors to Overcome Challenges Observed With HAdV-5-based Constructs.

Recombinant vectors based on human adenovirus serotype 5 (HAdV-5) have been extensively studied in preclinical models and clinical trials over the past two decades. However, the thorough understanding of the HAdV-5 interaction with human subjects has uncovered major concerns about its product applicability. High vector-associated toxicity and widespread preexisting immunity have been shown to significantly impede the effectiveness of HAdV-5–mediated gene transfer. It is therefore that the in-depth knowledge attained working on HAdV-5 is currently being used to develop alternative vectors. Here, we provide a comprehensive overview of data obtained in recent years disqualifying the HAdV-5 vector for systemic gene delivery as well as novel strategies being pursued to overcome the limitations observed with particular emphasis on the ongoing vectorization efforts to obtain vectors based on alternative serotypes.

New viruses from Lacerta monticola (Serra da Estrela, Portugal): further evidence for a new group of nucleo-cytoplasmic large deoxyriboviruses.

Lizard erythrocytic viruses (LEVs) have previously been described in Lacerta monticola from Serra da Estrela, Portugal. Like other known erythrocytic viruses of heterothermic vertebrates, these viruses have never been adapted to cell cultures and remain uncharacterized at the molecular level. In this study, we made attempts to adapt the virus to cell cultures that resulted instead in the isolation of a previously undetected Ranavirus closely related to FV3. The Ranavirus was subsequently detected by polymerase chain reaction (PCR) in the blood of infected lizards using primers for a conserved portion of the Ranavirus major capsid protein gene. Electron microscopic study of the new Ranavirus disclosed, among other features, the presence of intranuclear viruses that may be related to an unrecognized intranuclear morphogenetic process. Attempts to detect by PCR a portion of the DNA polymerase gene of the LEV in infected lizard blood were successful. The recovered sequence had 65.2/69.4% nt/aa% homology with a previously detected sequence from a snake erythrocytic virus from Florida, which is ultrastructurally different from the studied LEV. These results further support the hypothesis that erythrocytic viruses are related to one another and may represent a new group of nucleo-cytoplasmic large deoxyriboviruses.

Experimental Infection of Crickets (Gryllus Bimaculatus) with an Invertebrate Iridovirus Isolated from a High-Casqued Chameleon (Chamaeleo Hoehnelii)

Invertebrate iridoviruses (IIV) have been a regular problem for insect breeders. They have also recently been isolated from various lizard species. An iridovirus isolated from several tissues of a high-casqued chameleon (Chamaeleo hoehnelii) was identified as an IIV on the basis of electron microscopy (EM), sequencing of a portion of the major capsid protein (MCP) gene, and restriction endonuclease analysis of viral DNA. The pathogenicity of this isolate for crickets of the species Gryllus bimaculatus was tested by using 3 experimental infection studies. The mortality rates in the infected crickets ranged between 20% and 35%. The fat bodies of the crickets were examined on cell culture, with a nested PCR targeting the MCP gene, histologically, with in situ hybridization and by EM. Nested PCR was the most sensitive method for detecting IIV in the fat-body samples. Virus was re-isolated from several fat-body samples. In some fat bodies of infected crickets, massive arrays of viruses could be detected by EM. These findings support the hypothesis that IIV from insects are able to infect reptiles.

Whole-genome sequencing of a green bush viper reovirus reveals a shared evolutionary history between reptilian and unusual mammalian orthoreoviruses

In this study, we sequenced the whole genome of a reovirus isolated from a green bush viper (Atheris squamigera). The bush viper reovirus shared several features with other orthoreoviruses, including its genome organization. In phylogenetic analysis, this strain was monophyletic with Broome virus and baboon orthoreovirus, indicating that these viruses might have originated from a common ancestor. These new molecular data supplement previous information based mainly on biological properties of reptilian reoviruses, confirming their taxonomic position and broadening our knowledge of the evolution of members of the genus Orthoreovirus.

RT-PCR diagnosis followed by sequence characterization of paramyxoviruses in clinical samples from snakes reveals concurrent infections within populations and/or individuals

Paramyxoviruses (PMV) are important causes of disease in snakes in both private and zoologic collections and have been described in many different parts of the world. We have used the described L-gene nested RT-PCR (Ahne et al., 1999) as a diagnostic tool for the detection and characterization of PMV in clinical field samples from 102 snakes from Germany and Hungary. Fifty of the tested 203 samples were positive in the RT-PCR. 38 organs or swab samples from 25 snakes were selected for sequencing. They resulted in 13 different partial L-gene sequences, eight of which were identical to earlier characterized isolates, while five were new unique sequences, but also closely related to the earlier described PMV sequences. The comparison of sequences obtained from tissues from the same animal or from different animals from the same collection indicates that snakes can be infected with several different PMV during one outbreak. This is the first report of concurrent infection in a single snake or group of snakes with several distinct PMV.

Accumulation of a Low Pathogenic Avian Influenza Virus in Zebra Mussels (Dreissena polymorpha)

Abstract In order to investigate the potential role of mussels as a vector of influenza A viruses, we exposed zebra mussels (Dreissena polymorpha) to natural lake water containing a low pathogenic H5N1 avian influenza virus. Mussels were kept in water containing virus for 48 hr, then transferred into fresh water for another 14 days. Virus detection in mussels and water samples was performed by quantitative real-time reverse transcriptase-PCR (qRRT-PCR) and egg culture methods. Virus uptake was detected in all of the mussel groups that were exposed to virus. Even after 14 days in fresh water, virus could still be detected in shellfish material by both qRRT-PCR and egg culture methods. The present study demonstrates that zebra mussels are capable of accumulating influenza A viruses from the surrounding water and that these viruses remain in the mussels over an extended period of time.