RESUMO
In this study we report the complete sequence and genome organization of the serotype I feline coronavirus (FCoV) strain Black. Furthermore, a reverse genetic system was established for this FCoV strain by cloning a full-length cDNA copy into vaccinia virus. This clone served as basis for the generation of recombinant FCoV (recFCoV) that was shown to bear the same features in vitro as the parental FCoV. Using this system, accessory 3abc genes in the FCoV genome were replaced by green fluorescent protein (recFCoV-GFP) and Renilla luciferase genes (recFCoV-RL). In addition, we showed that feline CD14(+) blood monocytes and dendritic cells can be easily detected after infection with recFCoV-GFP. Thus, our established reverse genetic system provides a suitable tool to study the molecular biology of serotype I FCoV.
Assuntos
Coronavirus Felino/genética , Genoma Viral , Análise de Sequência de DNA/métodos , Animais , Sequência de Bases , Gatos , Clonagem Molecular , DNA Complementar/genética , Células Dendríticas/imunologia , Ordem dos Genes , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Receptores de Lipopolissacarídeos/análise , Luciferases de Renilla/análise , Luciferases de Renilla/genética , Dados de Sequência Molecular , Monócitos/imunologia , SorotipagemRESUMO
The genes encoding accessory proteins 3a, 3b, 3c, 7a and 7b, the S2 domain of the spike (S) protein gene and the membrane (M) protein gene of feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV) samples were amplified, cloned and sequenced. For this faeces and/or ascites samples from 19 cats suffering from feline infectious peritonitis (FIP) as well as from 20 FECV-infected healthy cats were used. Sequence comparisons revealed that 3c genes of animals with FIP were heavily affected by nucleotide deletions and point mutations compared to animals infected with FECV; these alterations resulted either in early termination or destruction of the translation initiation codon. Two ascites-derived samples of cats with FIP which displayed no alterations of ORF3c harboured mutations in the S2 domain of the S protein gene which resulted in amino acid exchanges or deletions. Moreover, changes in 3c were often accompanied by mutations in S2. In contrast, in samples obtained from faeces of healthy cats, the ORF3c was never affected by such mutations. Similarly ORF3c from faecal samples of the cats with FIP was mostly intact and showed only in a few cases the same mutations found in the respective ascites samples. The genes encoding 3a, 3b, 7a and 7b displayed no mutations linked to the feline coronavirus (FCoV) biotype. The M protein gene was found to be conserved between FECV and FIPV samples. Our findings suggest that mutations of 3c and spike protein genes correlate with the occurrence of FIP.
Assuntos
Coronavirus Felino/genética , Cisteína Endopeptidases/genética , Peritonite Infecciosa Felina/virologia , Glicoproteína da Espícula de Coronavírus/genética , Proteínas Virais de Fusão/genética , Proteínas Virais/genética , Proteases Virais 3C , Animais , Sequência de Bases , Gatos , Clonagem Molecular , Primers do DNA/genética , Fezes/virologia , Dados de Sequência Molecular , Mutação/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Análise de Sequência de DNA/veterináriaRESUMO
Expression of the kdpFABC operon coding for the high affinity K+ -translocating KdpFABC complex of Escherichia coli is induced by K+ limitation or high osmolality. This process is controlled by the sensor kinase/response regulator system KdpD/KdpE. To study the importance of the transmembrane domains of KdpD for stimulus perception, each amino acid residue of the transmembrane domain 1 and Asp-424 of the adjacent periplasmic loop were replaced with Cys in a KdpD derivative devoid of native Cys residues. In vivo analysis of KdpD proteins with a single Cys residue revealed that 14 out of 18 amino acid replacements caused an altered response towards an osmotic upshift imposed by NaCl, whereby only four replacements also altered the response towards changes in the K+ concentration. The in vitro activities of most of the KdpD derivatives were in the range of KdpD devoid of native Cys residues. The results reveal that the osmosensing and K+ -sensing properties of KdpD can be dissected. Furthermore, the data support the hypothesis that osmosensing involves amino acid residues of the transmembrane domains.