Genomic structure and evolution of a novel gene (PLA2L) with duplicated phospholipase A2-like domains.

In a previous study, we isolated a novel human cDNA with two domains of homology to secreted phospholipase A2 (sPLA2) embedded within a much larger open reading frame. The corresponding gene, termed PLA2L, is also unusual in that it is transcribed from an endogenous retroviral long terminal repeat promoter in teratocarcinoma cell lines. The associated retroviral element, a member of the HERV-H family of sequences, is found within an intron of the human PLA2L gene and has apparently assumed transcriptional regulatory functions at this locus. In this study we have isolated genomic clones spanning the human PLA2L locus and have determined the intron/exon structure of the PLA2-like domains. This intron/exon structure is very similar to that of known sPLA2s despite the fact that the PLA2L gene is highly diverged and has a novel duplicated structure. We also mapped PLA2L to chromosome 8q24, a location that differs from the known locations of human sPLA2s. Genomic PCR across primate species was performed to determine the approximate time of integration of the HERV-H element. Results indicate that the element integrated 15-20 million years ago since it is present in chimpanzee and gorilla but absent in orangutan and lower primates. Although the function of the PLA2L gene is not known, genomic Southern analyses suggest evolutionary conservation in mammals. These results contribute to our understanding of the unique and complex evolutionary history of the PLA2L gene.

Gain of Sp1 sites and loss of repressor sequences associated with a young, transcriptionally active subset of HERV-H endogenous long terminal repeats.

HERV-H sequences comprise a large family of human endogenous retrovirus-like elements. Previous DNA sequence comparisons of HERV-H long terminal repeats (LTRs) have led to their classification into three subtypes, Types I, Ia, and II. Type Ia appears to have been generated by recombination between Type I and Type II LTRs. These subtypes differ in evolutionary age and transcriptional activity with Type Ia LTRs being younger in evolutionary terms and possessing stronger promoter function than the other two subtypes. In this study, possible mechanisms responsible for the functional difference between LTRs have been explored. Types I and II LTRs each contain different sets of repeated segments in their U3 regions which are disrupted in Type Ia LTRs. Using reporter gene assays, we have shown that both types of repeated segments can suppress activity of the human beta-globin gene promoter when cloned at a distant site. Both sets of repeats also repress promoter activity of a Type Ia LTR when directly inserted within its U3 region. In addition, using deletion constructs, we have localized two positive regulatory segments within the Type Ia LTR, both of which contain a potential binding site for the transcription factor Sp1. Gel mobility shift assays demonstrated that fragments containing these sites do bind Sp1. Although Type I LTRs are generally similar to Type Ia LTRs in the regions surrounding the Sp1 sites, there are sequence differences within the sites. Gel-shift analysis revealed no or much reduced Sp1 binding of Type I LTR fragments containing these sites. Thus, it appears that the loss of repeated suppresser elements and the acquisition of Sp1-binding sites have both contributed to the relatively strong transcriptional activity of the Type Ia LTRs.

Experimental infection of conventional dogs with canine parvovirus.

Four 6-week-old conventional pups were inoculated with a parvovirus (PV) isolated from the feces of a dog with naturally occurring enteritis. Blood for hematologic studies, virus isolation (VI), and antibody titration and feces for VI and negative-contrast electron microscopy were collected on day 0 and daily until necropsy. Beginning at postinoculation day 2, necropsies were done and specimens were collected for immunofluorescence, VI, and light microscopic examination. The PV infection was confirmed by VI, immunofluorescence, electron microscopy, and seroconversion. Clinical illness was not observed in inoculated pups, although mild intestinal lesions similar to those of naturally occurring PV enteritis were found. The failure to elicit severe disease in conventional pups indicates that one or more factors, such as intercurrent enteric or systemic infections, immune status, age, nutrition, virulence of virus, dose of infectious virus, and route of inoculation influence the clinical and pathologic manifestations of PV infection.

Lesions associated with coccidiosis in nursing piglets.

Of 45 piglets with diarrhea, 28 had coccidiosis, with no evidence of concurrent viral infection. Villous atrophy and necrotic enteritis were the characteristic lesions, and were more severe in piglets with combined viral and coccidial infections than with coccidiosis alone. Necrotic enteritis presumably was caused by bacterial invasion of the villous lamina propria at foci denuded of epithelium by coccidia, viruses or both. Consistent lesions associated with coccidia in piglets not infected by other primary enteric pathogens suggest that coccidia are the cause of significant clinical disease in nursing piglets 6 to 15 days old.

Lesions of spontaneous canine viral enteritis.

Spontaneous enteric disease characterized by hemorrhagic diarrhea and high mortality occurred in puppies from commercial kennels in three midwestern states. Microscopic lesions resembling those of panleukopenia in cats were seen in the intestines. The predominant features were necrosis of crypt epithelium, collapse or dilation of crypt lumina and villous atrophy. Viral particles morphologically resembling parvovirus were found in the feces by direct electron microscopy. The canine virus reacted with antibody to feline panleukopenia virus by immunoelectron microscopy and fluorescent antibody technique. Fluorescent antibody was used to detect virus in the crypt epithelium of affected dogs. Feline kidney cells inoculated with fecal preparations had cytopathic effect and positive fluorescence by fluorescent antibody technique.