Porcine reproductive and respiratory syndrome virus (PRRSV) infects fully differentiated cells of the monocyte/macrophage lineage. Recently, CD163 was shown to be a cellular receptor capable of ...mediating infection of otherwise PRRSV non-permissive cell lines. CD163 is a macrophage differentiation antigen belonging to the scavenger receptor cysteine-rich (SRCR) family of membrane proteins. We provide a brief review of current knowledge regarding CD163 in relation to PRRSV infection, and propose a structure-based prediction of amino acid sequences involved in PRRSV interaction.
Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus replicating in the cytoplasm, but the nucleocapsid (N) protein is specifically localized to the nucleus and nucleolus in ...virus-infected cells. A ‘pat7’ motif of 41-PGKK(N/S)KK has previously been identified in the N protein as the functional nuclear localization signal (NLS); however, the biological consequences of N protein nuclear localization are unknown. In the present study, the role of N protein nuclear localization during infection was investigated in pigs using an NLS-null mutant virus. When two lysines at 43 and 44 at the NLS locus were substituted to glycines, the modified NLS with 41-PGGGNKK restricted the N protein to the cytoplasm. This NLS-null mutation was introduced into a full-length infectious cDNA clone of PRRSV. Upon transfection of cells, the NLS-null full-length clone induced cytopathic effects and produced infectious progeny. The NLS-null virus grew to a titer 100-fold lower than that of wild-type virus. To examine the response to NLS-null PRRSV in the natural host, three groups of pigs, consisting of seven animals per group, were intranasally inoculated with wild-type, placebo, or NLS-null virus, and the animals were maintained for 4 weeks. The NLS-null-infected pigs had a significantly shorter mean duration of viremia than wild-type-infected pigs but developed significantly higher titers of neutralizing antibodies. Mutations occurred at the NLS locus in one pig during viremia, and four types of mutations were identified: 41-PGRGNKK, 41-PGGRNKK, and 41-PGRRNKK, and 41-PGKKSKK. Both wild-type and NLS-null viruses persisted in the tonsils for at least 4 weeks, and the NLS-null virus persisting in the tonsils was found to be mutated to either 41-PGRGNKK or 41-PGGRNKK in all pigs. No other mutation was found in the N gene. All types of reversions which occurred during viremia and persistence were able to translocate the mutated N proteins to the nucleus, indicating a strong selection pressure for reversion at the NLS locus of the N protein in vivo. Reversions from NLS-null to functional NLS in the tonsils suggest a possible correlation of viral persistence with N protein nuclear localization. These results show that N protein nuclear localization is non-essential for PRRSV multiplication but may play an important role in viral attenuation and in pathogenesis in vivo.
PRRSV (porcine reproductive and respiratory syndrome virus) nucleocapsid (N) protein is the most abundant structural protein of the virus. During infection, the N protein is specifically localized to ...the nucleus and nucleolus in addition to its normal cytoplasmic distribution. Previously, a nuclear localization signal (NLS, 41-PGKK(N/S)KKKN)-null mutant virus (41-PGGGNKKKN) showed reduced viremia and increased production of neutralizing antibodies in infected pigs. However, the mutagenized NLS underwent strong selection pressure in the pig that resulted in partial or complete reversion and reacquisition of NLS function, and thus the biological effect of the NLS-null mutation needed further investigation. In the present study, a total of 9 “reversion resistant” mutants were generated by amino acid deletions and substitutions using an infectious cDNA clone. Two mutant clones (PG-
-SKKKS and PG-
-S-KKS) that produced progeny viruses were genetically stable for at least 20 passages in cell culture. Infection of pigs with those mutants induced neutralizing antibodies to higher titers than with wild-type virus. Both mutant viruses induced viremia of lower titer and of shorter duration than wild-type virus. RT-PCR from tonsils showed that both mutants persisted at a reduced level. Virus transmission to contact pigs was also lower in the mutant virus infected groups. No reversion to functional NLS was detected in either mutant from any pig. These data demonstrate that N protein nuclear localization is indeed associated with viral pathogenesis and host response to PRRS.
Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which is characterized by late-term abortions in sows and respiratory disease in young pigs. Using ...an infectious cDNA clone of North American PRRSV strain P129, the viral genome was engineered to transcribe an additional subgenomic RNA initiating between non-structural and structural genes. Two unique restriction sites and a copy of the transcription regulatory sequence for ORF6 (TRS6) were inserted between ORFs 1b and 2a, yielding a general purpose expression vector. The enhanced green fluorescent protein (GFP) gene was cloned between the unique sites such that the inserted gene was transcribed from TRS2 which was located upstream within ORF1b, while the copy of TRS6 drives ORF2a/b transcription. Upon transfection of cells with this plasmid, PRRSV infection was initiated and progeny virus “P129-GFP” was obtained. Cells infected with P129-GFP showed fluorescence and the inserted gene was phenotypically stable for at least 37 serial in vitro passages. Subsequently, a capsid (C) protein gene was cloned from porcine circovirus type 2 (PCV2) recovered from an outbreak of porcine multisystemic wasting syndrome (PMWS) and inserted into the PRRSV infectious clone vector, generating virus “P129-PCV”. To determine the immunogenicity of the recombinant viruses, pigs were immunized intramuscularly with P129-WT (wild-type), P129-GFP, or P129-PCV2. By 5 weeks post-infection, specific antibody responses to GFP and PCV2 capsid were elicited. This is the first report of foreign gene expression using PRRSV from dedicated subgenomic RNAs and demonstrates the potential use of PRRSV as a vaccine vector for swine pathogens.
Reverse genetic systems were developed for a highly virulent ‘atypical’ porcine reproductive and respiratory syndrome virus (PRRSV). The full-length genome of 15
395 nucleotides was assembled as a ...single cDNA clone and placed under either the prokaryotic T7 or eukaryotic CMV promoter. Transfection of cells with the RNA transcripts or the DNA clone induced cytopathic effects and produced infectious progeny. The reconstituted virus was stable and grew to the titer of the parental virus in cells. Upon infection, pigs produced clinical signs and lung pathology typical for PRRSV and induced viremia and specific antibodies. Previously, we showed that the PRRSV nucleocapsid (N) protein forms homodimers via both noncovalent and covalent interactions and that cysteine at position 23 is responsible for the covalent interaction. The functional significance of cysteines of N for PRRSV infectivity was assessed using the infectious cDNA clone. Each cysteine of N at positions 23, 75, and 90 was replaced with serine and the individual mutation was incorporated into the cDNA clone such that three independent cysteine mutants were constructed. When transfected, the wild type and C75S clones induced cytopathic effects and produced infectious virus with indistinguishable plaque morphology. In contrast, the C23S mutation completely abolished infectivity of the clone, indicating that C23-mediated N protein homodimerization plays a critical role in PRRSV infectivity. Unexpectedly, the C90S mutation also appeared to be lethal for virus infectivity. Genome replication and mRNA transcription were both positive for the replication-defective C23S and C90S mutants. The data suggest that, in addition to homodimerization, the PRRSV N protein may also undergo heterodimerization with another structural protein using cysteine 90 and that the N protein heterodimerization is essential for PRRSV infectivity.
Bovine viral diarrhea virus (BVDV) infections are enzootic in the cattle population and continue to cause significant economic losses to the beef and dairy industries worldwide. Extent of the damages ...has stimulated increasing interest in control programs directed at eradicating BVDV infections. Use of a BVDV marker vaccine would facilitate eradication efforts as a negatively marked vaccine would enable differentiation of infected from vaccinated animals (DIVA). We describe here the construction of three chimeric BVDVs containing glycoprotein E(rns) of heterologous pestiviruses and the evaluation of the chimera viruses as potential marker vaccines against BVDV infections. Chimeric NADL/G-E(rns), NADL/R-E(rns), and NADL/P-E(rns) were constructed by replacing the E(rns) gene of the full-length BVDV (NADL strain) genome with the E(rns) genes of giraffe (G-E(rns)), reindeer (R-E(rns)), or pronghorn antelope (P-E(rns)) pestiviruses, respectively. Each chimeric NADL virus was viable and infectious in RD 420 (bovine testicular) and BK-6 (bovine kidney) cells. By immunohistochemistry assays, NADL/G-E(rns) and NADL/R-E(rns) chimeric viruses reacted to BVDV E(rns) specific monoclonal antibody (mAb) 15C5, whereas the NADL/P-E(rns) chimeric virus did not. In an animal vaccination study, inactivated vaccines made from two chimeric viruses and the wild type NADL BVDV induced similar neutralizing antibody responses. NADL/P-E(rns)-vaccinated animals were distinguished from animals vaccinated with the wild type virus by means of a companion serological DIVA assay. These results show that chimeric NADL/P-E(rns) virus containing the E(rns) gene of pronghorn antelope pestivirus could be a potential marker vaccine candidate for use in a BVDV control and eradication program.
Full-length infectious cDNA clones have recently become available for both European and North American genotypes of porcine reproductive and respiratory syndrome virus (PRRSV), and it is now possible ...to alter the PRRSV genome and create genetically defined mutant viruses. Among many possible applications of the PRRSV infectious cDNA clones, development of genetically modified vaccines is of particular interest. Using infectious clones, the PRRSV genome has been manipulated by changing individual amino acids, deleting coding regions, inserting foreign sequences, and generating arterivirus chimeras. The limited available data suggest that all structural proteins of PRRSV are essential for replication of the virus, and that PRRSV infectivity is relatively intolerant of subtle changes within the structural proteins. The major tasks in PRRSV research are to identify virulence factors and pathogenic mechanisms, and to understand the structure–function relationships of individual viral proteins. Utilizing these infectious clones as tools, a new generation of safe and efficacious PRRS vaccines may be constructed.
Highlights ► Three chimeric bovine viral diarrhea viruses were constructed using overlapping PCR methods. ► Chimeras contained glycoprotein Erns of heterologous pestiviruses. ► Each BVDV chimera was ...infectious in bovine cells and induced neutralizing antibody responses. ► BVDV/P-Erns (pronghorn antelope)-vaccinated cattle were uniquely detectable serologically. ► BVDV/P-Erns could be a potential BVDV marker vaccine candidate for BVDV eradication programs.
Highlights * Three chimeric bovine viral diarrhea viruses were constructed using overlapping PCR methods.*Chimeras contained glycoprotein Ernsof heterologous pestiviruses.*Each BVDV chimera was ...infectious in bovine cells and induced neutralizing antibody responses.*BVDV/P-Erns(pronghorn antelope)-vaccinated cattle were uniquely detectable serologically. * BVDV/P-Ernscould be a potential BVDV marker vaccine candidate for BVDV eradication programs.
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