A comparative, BAC end sequence enabled map of the genome of the American mink (Neovison vison)

In this report we present the results of the analysis of approximately 2.7 Mb of genomic information for the American mink (Neovison vison) derived through BAC end sequencing. Our study, which encompasses approximately 1/1000^th of the mink genome, suggests that simple sequence repeats (SSRs) are less common in the mink than in the human genome, whereas the average GC content of the mink genome is slightly higher than that of its human counterpart. The 2.7 Mb mink genomic dataset also contained 2,416 repeat elements (retroids and DNA transposons) occupying almost 31% of the sequence space. Among repeat elements, LINEs were over-represented and endogenous viruses (aka LTRs) under-represented in comparison to the human genome. Finally, we present a virtual map of the mink genome constructed with reference to the human and canine genome assemblies using a comparative genomics approach and incorporating over 200 mink BESs with unique hits to the human genome.     

Characterization of an enteropathogenic bovine calicivirus representing a potentially new calicivirus genus

Bovine enteric caliciviruses (BEC) are associated with diarrhea in young calves. The BEC strains detected in Europe form a third genogroup within the genus "Norwalk-like viruses" (NLV) of the family Caliciviridae. In this report, we present sequence, clinical, and histological data characterizing a novel enteropathogenic BEC strain, NB, detected in fecal specimens from calves in the United States. The complete RNA genome of the NB virus is 7,453 bases long and is organized into two open reading frames (ORFs). ORF-1 is 2,210 amino acids long and encodes a large nonstructural polyprotein contiguous with the major capsid protein (VP1), similar to the lagoviruses and "Sapporo-like viruses" (SLV). The conserved calicivirus motifs were identified in the nonstructural proteins. ORF-2 is located at the 3' end of the genome and encodes a small basic protein (VP2) of 225 amino acids. The 5' and 3' untranslated regions are 74 and 67 bases long, respectively. Among caliciviruses, NB virus shows amino acid identities of 14.1 to 22.6% over the entire ORF-1 nonstructural-protein sequence with NLV, SLV, vesivirus, and lagovirus strains, while the overall sequence identity of the complete NB VP-1 with other caliciviruses is low, varying between 14.6 and 26.7%. Phylogenetic analysis of the complete VP1 protein, including strains from all four calicivirus genera, showed the closest grouping of NB virus to be with viruses in the genus Lagovirus, which cause liver infections and systemic hemorrhage in rabbits. In gnotobiotic calves, however, NB virus elicited only diarrhea and intestinal lesions that were most severe in the upper small intestine (duodenum and jejunum), similar to the NLV BEC strains. The tissues of major organs, including the lung, liver, kidney, and spleen, had no visible microscopic lesions.     

Complete Genome of a Porcine Calicivirus Strain in Anhui Province,China, Is Significantly Shorter than That of the Other Chinese Strain

Caliciviruses that cause diarrhea have been reported in both industrial and developing countries, including China, in recent years. Here, we report the complete genome of a porcine calicivirus strain, Ah-1, which is prevalent in swine groups in Anhui Province. This viral genome is 7,342 nucleotides (nt) long, excluding the poly(A) of the 3′ end, which is 202 nt shorter in the 3′ untranslated region (UTR) than that of the other Chinese porcine calicivirus strain (Ch-sw-sav1; GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"FJ387164","term_id":"227976393","term_text":"FJ387164"}}FJ387164), previously isolated in the Shanghai area, China, though they shared 98.8% sequence identity over the whole genome excluding the 202-nt-shorter region.     

B2-like repetitive sequence from the X Chromosome of the American mink (Mustela vison)

In analysis of the repeats from the mink X Chromosome (Chr), we have identified a B2-like repetitive sequence of 195 base pairs (bp) flanked by short direct repeats of 14 bp. It contains regions homologous to the split intragenic RNA polymerase III promoter and a 3 A-rich region followed by an oligo(dA) sequence. A feature of the repeat is the presence of a perfect polypyrimidine tract 22 bp in length absent from the known Alu- and Alu-like sequences. Alignment of the mink B2-like sequence and mouse B2-consensus sequence allowed us to estimate their similarity as 55%. The repeat is present in 1–2×10⁵ copies per mink genome and 2–4×10³ copies per X Chr. In situ hybridization analysis demonstrated a similar distribution pattern of the B2-like repeat along the length of all the mink chromosomes including the X. We also observed the presence of mink B2-like hybridizable sequence in the genomes of other Carnivora species.The nucleotide sequence data reported in this paper have been submitted to EMBL Data Library and have been assigned the accession number X52381 (MVB2RPT).     

Complete Genome Sequence of a Newly Identified Porcine Astrovirus Genotype 3 Strain US-MO123

Astrovirus (AstV) infections are among the most common causes of gastroenteritis and are also associated with extraintestinal manifestations in humans and many animals. Herein, for the first time, the complete genome sequence of newly identified porcine astrovirus genotype 3 (PAstV3) strain US-MO123 was determined. Sequence comparison and phylogenetic analysis showed that PAstV3 has the closest relationship with mink AstV and the human AstV strains VA1, VA2, and SG, indicating the same ancestral origin and zoonotic potential of the virus.     

Molecular Characterization of a Bovine Enteric Calicivirus: Relationship to the Norwalk-Like Viruses

Jena virus (JV) is a noncultivatable bovine enteric calicivirus associated with diarrhea in calves and was first described in Jena, Germany. The virus was serially passaged 11 times in colostrum-deprived newborn calves and caused diarrheal disease symptoms at each passage. The complete JV genome sequence was determined by using cDNA made from partially purified virus obtained from a single stool sample. JV has a positive-sense single-stranded RNA genome which is 7,338 nucleotides in length, excluding the poly(A) tail. JV genome organization is similar to that of the human Norwalk-like viruses (NLVs), with three separate open reading frames (ORFs) and a 24-nucleotide sequence motif located at the 5′ terminus of the genome and at the start of ORF 2. The polyprotein (ORF 1) consists of 1,680 amino acids and has the characteristic 2C helicase, 3C protease, and 3D RNA polymerase motifs also found in the NLVs. However, comparison of the N-terminal 100 amino acids of the JV polyprotein with those of the group 1 and group 2 NLVs showed a considerable divergence in sequence. The capsid protein (ORF 2) at 519 amino acids is smaller than that of all other caliciviruses. JV ORF 2 was translated in vitro to produce a 55-kDa protein that reacted with postinfection serum but not preinfection serum. Phylogenetic studies based on partial RNA polymerase sequences indicate that within the Caliciviridae JV is most closely related to the group 1 NLVs.     

Dog chromosome-specific paints reveal evolutionary inter- and intrachromosomal rearrangements in the American mink and human

Forty chromosome-specific paint probes of the domestic dog (Canis familiaris, 2n = 78) were used to delineate conserved segments on metaphase chromosomes of the American mink (Mustela vison, 2n = 30) by fluorescence in situ hybridisation. Half of the 38 canine autosomal probes each painted one pair of homologous segments in a diploid mink metaphase, whereas the other 19 dog probes each painted from two to five pairs of discrete segments. In total, 38 canine autosomal paints highlighted 71 pairs of conserved segments in the mink. These painting results allow us to establish a complete comparative chromosome map between the American mink and domestic dog. This map demonstrates that extensive chromosome rearrangements differentiate the karyotypes of the dog and American mink. The 38 dog autosomes could be reconstructed from the 14 autosomes of the American mink through at least 47 fissions, 25 chromosome fusions, and six inversions. Furthermore, comparison of the current dog/mink map with the published human/dog map discloses 23 cryptic intrachromosomal rearrangements in 10 regions of conserved synteny in the human and American mink genomes and thus further refined the human/mink comparative genome map.     

Conversion of Friend mink cell focus-forming virus to Friend spleen focus-forming virus by modification of the 3'' half of the env gene.

The 3' half of the env gene of the dualtropic Friend mink cell focus-forming virus was modified by replacing the restriction enzyme fragment of the genome DNA with the corresponding fragment of the acutely leukemogenic, polycythemia-inducing strain of Friend spleen focus-forming virus (F-SFFVP) genome DNA. Replacement with the fragment of F-SFFVP env containing the 585-bp deletion, the 6-bp duplication, and the single-base insertion converted the resulting chimeric genome so that the mutant had a pathogenic activity like that of F-SFFVP. Replacement with the fragment containing only the 585-bp deletion did not result in a pathogenic virus. However, when this virus pseudotyped by Friend murine leukemia virus was passaged in newborn DBA/2 mice, we could recover weakly pathogenic viruses with a high frequency. Molecular analysis of the genome of the recovered virus revealed the presence of a single-base insertion in the same T5 stretch where the wild-type F-SFFV env has the single-base insertion. These results provided evidence that the unique genomic structures present in the 3' half of F-SFFV env are the sole determinants that distinguish the pathogenicity of F-SFFV from that of Friend mink cell focus-forming virus. The importance of the dualtropic env-specific sequence present in the 5' half of F-SFFV env for the pathogenic activity was evaluated by constructing a mutant F-SFFV genome in which this sequence was replaced by the ecotropic env sequence of Friend murine leukemia virus and by examining its pathogenicity. The results indicated that the dualtropic env-specific sequence was essential to pathogenic activity.     

Frequent site-specific deletion of coliphage lambda murine sarcoma virus recombinants and its use in the identification of a retrovirus integration site.

Stocks of hybrid lambda phages carrying the complete integrated provirus of either m1 or HT1 Moloney murine sarcoma virus, as well as flanking host sequences, frequently contain significant numbers of phages carrying a specific deletion. This deletion arises from a recombination event between the terminally repeated sequences in the provirus that deletes the unique Moloney murine sarcoma virus sequences bracketed by the terminally repeated sequences. Physical mapping has shown that the deletion phage retains one complete copy of the terminally repeated sequence and the flanking mink host sequences. One such deletion, lambdaHT1r+, was used to characterize a mink genomic DNA sequence that contains an HT1 Moloney murine sarcoma virus integration site. This integration site sequence from normal mink cells was also cloned into phage lambda. An analysis of the heteroduplexes between the integration site and the lambdaHT1r+ deletion indicated that no major rearrangement of host sequences occurred upon integration of the Moloney murine sarcoma provirus.     

Molecular properties of a gag- pol- env+ murine leukemia virus from cultured AKR lymphoma cells.

We have described the isolation of a replication-defective murine leukemia virus from a culture of AKR lymphoma cells [Rein et al., Nature (London) 282:753-754, 1979]. To facilitate the characterization of this murine leukemia virus, we transmitted it to mink cells and analyzed its genome by restriction mapping of the mink cellular DNA. This genome resembled the Akv genome quite closely, but it had an additional KpnI cleavage site at 1.3 kilobase pairs from the 5' end of the provirus and a small (approximately 50-base-pair) deletion between 1.8 and 3.0 kilobase pairs from the 5' end. When we tested these mink cells by immune precipitation or by competition radioimmunoassay, we found that they synthesized gPr82env, but contained no detectable gag or pol proteins. It seems likely that the KpnI cleavage site at 1.3 kilobase pairs reflects an abnormal sequence at or near the beginning of the gag gene, which prevents gag or pol translation by introducing a frameshift or termination codon into this region.     

An insect picornavirus may have genome organization similar to that of caliciviruses.

Computer-assisted analysis of the amino acid sequence of the product encoded by the sequenced 3' portion of the cricket paralysis virus (CrPV), an insect picornavirus, genome showed that this protein is homologous not to the RNA-directed RNA polymerases, as originally suggested, but to the capsid proteins of mammalian picornaviruses. Alignment of the CrPV protein sequence with those of picornavirus and calicivirus capsid proteins demonstrated that the sequenced portion of the insect picornavirus genome encodes the C-terminal part of VP3 and the entire VP1. Thus CrPV seems to have a genome organization distinct from that of other picornaviruses but closely resembling that of caliciviruses, with the capsid proteins encoded in the 3' part of the genome. On the other hand, the tentative phylogenetic trees generated from the VP3 alignment revealed grouping of CrPV with hepatitis A virus, a true picornavirus, not with caliciviruses. Thus CrPV may be a picornavirus with a calicivirus-like genome organization. Different options for CrPV genome expression are discussed.     

Characteristics and contributions of defective, ecotropic, and mink cell focus-inducing viruses involved in a retrovirus-induced immunodeficiency syndrome of mice.

LP-BM5 murine leukemia virus, a derivative of Duplan-Laterjet virus, contains a mixture of replication-competent B-tropic ecotropic and mink cell focus-inducing (MCF) viruses and a defective genome that is the proximal cause of a syndrome, murine AIDS (MAIDS), characterized by lymphoproliferation and immunodeficiency. The defective (BM5d) and ecotropic components of this mixture were molecularly cloned, and complete (BM5d) or partial (ecotropic) nucleotide sequences were determined. BM5d closely resembled the Du5H genome cloned from the Duplan virus, featuring a highly divergent p12 sequence in the gag open reading frame. In MAIDS-sensitive C57BL/6 mice, BM5d was detected in tissues within 2 weeks of infection but was absent from tissues of the MAIDS-resistant strain, A/J, 12 weeks after infection. B-cell-lineage tumors from mice with MAIDS contained and expressed BM5d, and clonal integrations of this genome were variably associated with clonal expansions of B cells in infected mice. Finally, mRNA crosshybridizing with a probe for BM5d was present in spleen but not kidney cells of uninfected B6 mice.     

A re-assigned American mink (Neovison vison) map optimal for genome-wide studies

Our previously published second generation genetic map for the American mink (Neovison vison) has been used and redesigned in its best for genome-wide studies with maximum of efficiency. A number of 114 selected markers, including 33 newly developed microsatellite markers from the CHORI-231 mink Bacterial Artificial Chromosome (BAC) library, have been genotyped in a two generation population composed of 1200 individuals. The outcome reassigns the position of some markers on the chromosomes and it produces a more reliable map with a convenient distance between markers. A total of 104 markers mapped to 14 linkage groups corresponding to the mink autosomes. Six markers are unlinked and four markers are allocated to the X chromosome by homology but no linkage was detected. The sex-average linkage map spans 1192 centiMorgans (cM) with an average intermarker distance of 11.4 cM and 1648 cM when the ends of the linkage groups and the autosomal unlinked markers are added. Sex-specific genetic linkage maps were also generated. The male sex-specific map had a total length of 1014.6 cM between the linked markers and an average inter-marker interval of 9.7 cM. The female map has a corresponding length of 1378.6 cM and an average inter-marker interval of 13.3 cM. The study is complemented with additional anchorage for most of the chromosomes of the map by BAC in situ hybridization with clones containing microsatellites strategically selected from the various parts of the genome. This map provides an improved tool for genetic mapping and comparative genomics in mink, also useful for the future assembly of the mink genome sequence when this will be taken forward.     

Escherichia Coli and Virus isolated from “Sticky Kits”

A total of 121 Escherichia coli strains isolated from 3-week-old mink kits were serotyped and examined for virulence factors. 56 strains were isolated from healthy kits while 65 were from “sticky kits”. Among these, 34 different serotypes were detected. No difference in serotypes or the presence of virulence factors could be detected between healthy and diseased kits. By electron microscopy of faecal samples corona-, rota-, and calicivirus were demon-strated among healthy as well as diseased kits.