Peter Kabat

Evolution and Distribution of Class II-Related Endogenous Retroviruses

ABSTRACT Endogenous retroviruses (ERVs) are widespread in vertebrate genomes and have been loosely grouped into “classes” on the basis of their phylogenetic relatedness to the established genera of exogenous retroviruses. Four of these genera—the lentiviruses, alpharetroviruses, betaretroviruses, and deltaretroviruses—form a well-supported clade in retroviral phylogenies, and ERVs that group with these genera have been termed class II ERVs. We used PCR amplification and sequencing of retroviral fragments from more than 130 vertebrate taxa to investigate the evolution of the class II retroviruses in detail. We confirm that class II retroviruses are largely confined to mammalian and avian hosts and provide evidence for a major novel group of avian retroviruses, and we identify additional members of both the alpha- and the betaretrovirus genera. Phylogenetic analyses demonstrated that the avian and mammalian viruses form distinct monophyletic groups, implying that interclass transmission has occurred only rarely during the evolution of the class II retroviruses. In contrast to previous reports, the lentiviruses clustered as sister taxa to several endogenous retroviruses derived from rodents and insectivores. This topology was further supported by the shared loss of both the class II PR-Pol frameshift site and the class II retrovirus G-patch domain.

Complete nucleotide sequence of an endogenous retrovirus from the amphibian, Xenopus laevis

We report the first full-length sequence of an endogenous amphibian retrovirus derived from the African clawed toad Xenopus laevis. The virus, termed Xen1, has one of the largest endogenous retroviral genomes described to date of over 10 kb in length and it also has a relatively complex genomic organisation consisting of LTR-orf1, orf2, gag, pol, env-LTR. Orfs 1 and 2 are novel, duplicated genes of unknown function. Phylogenetic analysis indicates that Xen1 is most closely related to the epsilon -retroviruses WDSV and WEHV types 1 and 2, which are large, complex exogenous retroviruses present within Walleye fish.

Using nested RT-PCR analyses to determine the prevalence of avian influenza viruses in passerines in western Slovakia, during summer 2007

The prevalence of avian influenza virus (AIV), together with the distribution of different AIV subtypes, was studied in migratory waterfowl and terrestrial birds caught in western Slovakia during summer 2007. Both oropharyngeal and cloacal swabs were collected. Screening of samples revealed that 18% of oropharyngeal and 18% of cloacal samples were positive for AIV. Samples from both the oropharynx and cloaca were positive in only 6.6% of cases. A total of 10 different subtypes of haemagglutinin (H2, H3, H4, H6, H7, H9, H10, H11, H12, and H13) and 4 different subtypes of neuraminidase (N1, N2, N3, and N5) were detected in 32 samples from this location. The most abundant subtypes of HA in the samples were H12 and H9 (25% each), followed by H11 and H10 (15% each), and H13 (9%). There were 3 cases where different AIV infections were detected in oropharyngeal and cloacal samples originating from the same bird (H13N1 and H12N5; H13N3 and H9N5; H10N2 and H9N5 in the oropharynx and cloaca, respectively).

EASEL, A GYPSY LTR-RETROTRANSPOSON IN THE SALMONIDAE

Despite the close similarities between retroviruses and the gypsy/Ty3 group of LTR-retrotransposons their host ranges are largely distinct: the retroviruses are found only in vertebrates, whereas the gypsy LTR-retrotransposons are almost exclusively restricted to invertebrates, plants and fungi. Here we report the amplification by PCR, and characterisation, of one of the first LTR-retrotransposons to be discovered in vertebrates - in several members of the piscine family Salmonidae. Phylogenetic analysis of this retroelement, termed easel, indicates that it is probably a phylogeneticaly basal member of the gypsy group of LTR-retrotransposons and occurs in some of the same species from which retroviruses have previously been isolated. Thus some members of the Salmonidae are the first organisms known to harbour both retroviral branch elements and the gypsy LTR-retrotransposon branch elements. This creates an overlap in the host ranges of the two retroelement families.

Prevalence of avian influenza viruses, Borrelia garinii, Mycobacterium avium, and Mycobacterium avium subsp. paratuberculosis in waterfowl and terrestrial birds in Slovakia, 2006

The prevalence of Borrelia, Mycobacteria and avian influenza virus (AIV) infections, together with the distribution of different AIV subtypes, was studied in migratory waterfowl and terrestrial birds trapped in three localities in Slovakia during 2006. Samples obtained from waterfowl captured in the Senianske Ponds area of Eastern Slovakia showed the highest diversity of AIV isolates. A total of 13 different subtypes were detected in 19 samples from this location (H1N2, H2N2, H3N2, H6N6, H7N6, H9N2, H9N5, H9N6, H10N5, H10N6, H12N6, H13N6, and H16N6). H3N5 virus was detected in 50% of passerines testing positive for AIV in the Parizske Wetlands, with H7N2, H9N2, H9N5, H12N1, and H13N2 infections also recorded at this locality. H9N5 virus predominated in passerines captured at Trnava Ponds, with isolates H1N6, H6N5, H7N2, H7N6, H10N3, and H10N6 also detected at this location. There were five cases where different AIV infections were detected in oropharyngeal and cloacal samples originating from the same bird (H13N6 and H1N2; H10N5 and H12N6; H9N5 and H6N5; H10N6 and H7N6; and H9N2 and H3N5 in the oropharynx and cloaca, respectively). Between 21% and 52% of captured birds tested positive for Borrelia burgdorferi sensu lato, with the proportion infected depending on bird species and locality. Samples were characterized by polymerase chain reaction-restriction fragment length polymorphism analysis and identified as Borrelia garinii species (either B/B′ or R/R′ pattern). Mycobacteria were detected in 42% and 26% of waders captured at Senianske Ponds and marsh-dwelling passerines captured in the Parizske Wetlands, respectively. Interestingly, forest-dwelling passerine species caught in the Trnava Ponds region were tested negative for Mycobacteria.

Influenza A virus replication is inhibited in IFN-λ2 and IFN-λ3 transfected or stimulated cells.

Interferons lambda (IFN-λ) are the most recently defined members of the class III cytokine family. To investigate whether IFN-λ2 and IFN-λ3 displayed antiviral activity against influenza A virus (IAV), a number of cell lines induced with IFNs - as well as two established cell lines (A549-IFN-λ2 and A549-IFN-λ3) - were infected with IAV. Our results indicate that IFN-λ2 has statistically significant antiviral activity in A549-IFN-λ2 (P=0.0028) although less so than IFN-λ3, which reduced viral titer to 10% (P<0.0001). The reverse was observed for cells treated with IFNs, with IFN-λ2-treated A549 cells inhibiting IAV infection more efficiently than IFN-λ3-treated A549 cells. The antiviral effect on IFN-stimulated cells was most apparent on Vero cells (compared with MDCK and HeLa). Both IFNs significantly inhibited IAV replication and inhibition was observed in a dose-dependent manner, with an optimal IFN concentration of 20 ng/ml. IFN-λ2 was more potent than IFN-λ3 on Vero cells while IFN-λ3 appeared more efficient than IFN-λ2 on MDCK and HeLa cells.

Characterization and Complete Nucleotide Sequence of an Unusual Reptilian Retrovirus Recovered from the Order Crocodylia

ABSTRACT A novel group of retroviruses found within the order Crocodylia are described. Phylogenetic analyses demonstrate that they are probably the most divergent members of the Retroviridae described to date; even the most conserved regions of Pol show an average of only 23% amino acid identity when compared to other retroviruses.

Cytotoxicity testing of scaffolds potentially suitable for the preparation of three-dimensional skin substitutes

The preparation and study of three-dimensional functional skin substitutes has been the focus of intense research for several decades. Dermal substitutes are now commonly used in medical practice for a variety of applications. Here, we assess the toxicity of seven selected acellular dermal matrix materials to establish their potential for use in future three-dimensional skin substitute studies. The cytotoxicity of acellular dermis (of Allo- and Xenograft origin) prepared in our lab and biomaterials based on collagen and hyaluronic acid (Coladerm H and Coladerm H–L) were compared to that seen in three commercially available products (Xe-Derma, AlloDerm and Xeno-Impl). Murine fibroblasts NIH-3T3 and human dermal fibroblasts were used in cytotoxicity tests, with any resultant cytotoxic effects caused by the seven tested dermal scaffolds visualised using an inverted microscope system and confirmed in parallel using colorimetric MTT cell proliferation assays. While most of the dermal substitutes did not demonstrate a cytotoxic effect on our two cell types, Xeno and Xeno-Impl scaffolds clearly did. The cytotoxic effect of acellular Xeno dermal matrix could essentially be removed through a regime of multiple washes, but we were unable to remove the cytotoxic effect of Xeno-Impl. Thus, Xeno-Impl alone has been excluded from our future work on preparation of 3D skin substitutes.

Skin explant cultures as a source of keratinocytes for cultivation.

Cultivated human keratinocytes can be used successfully in the treatment of burn patients, but efforts to heal burns and other wounds can be hampered by the very small skin biopsies available for cultivation of transplantable keratinocyte sheets. A small biopsy (and correspondingly small number of enzymatically isolated keratinocytes for use in classical cultivation techniques) can lead to a low yield of multilayer sheets for clinical application or unacceptably long cultivation times. One way of addressing this is to make use of skin remnants remaining after enzymatic digestion and culture cells migrating out of these skin explants. Sufficient numbers of explant-derived keratinocytes can be obtained to facilitate additional routine cultivation of these cells. Biopsy remnants can be used to initiate explant cultures repeatedly (we were able to re-use pieces of skin 10 times and still obtain useful numbers of keratinocytes) and this “passaging” yields substantially more cells for classical cultivation than would be available from conventional methodology alone, and in a comparable timeframe. Another advantage of this method is that it does not require additional biopsies to be procured from already-compromised patients and overcomes problems associated with contamination of skin samples with resistant hospital-acquired bacterial infections common during prolonged hospitalization.

Prevalence of avian influenza viruses, Mycobacterium avium, and Mycobacterium avium, subsp. paratuberculosis in marsh-dwelling passerines in Slovakia, 2008

Prevalence of the infectious respiratory agens, avian influenza virus (AIV), Mycobacterium avium (M. avium), and Mycobacterium avium subspecies paratuberculosis (MAP), was studied in migratory marsh-dwelling passerines captured in the Parížske močiare wetlands in Western Slovakia during 2008. Surveillance of 650 birds revealed a lower prevalence of AIV in spring (13.6%) than in summer (17.5%). A total of 14 different subtypes were detected in samples obtained from birds captured during the spring, with the most prevalent subtypes being H8N3, H6N4, H11N6 and H12N6. Subtypes H12N6, H6N6 and H2N5 were predominant in passerines captured during summer months. In eight cases, different AIV infections were detected in the oropharyngeal and cloacal samples originating from a single bird (H1N1 and H8N3; H1N3 and H9N3; H2N3 and H12N6; H2N1 and H8N1; H4N2 and H9N6; H5N5 and H11N6; H6N4 and H11N6; H7N1 and H10N3 in the oropharynx and cloaca, respectively). M. avium was detected in 9.2% and 0.8% of marsh-dwelling passerines captured during spring and summer, respectively. Only two birds were co-infected with AIV and M. avium. All birds were negative for MAP.