Continuous porcine cell lines developed from alveolar macrophages

Weingartl, Hana M.; Sabara, Marta; Pasick, John; Moorlehem, E van; Babiuk, L. A.

doi:10.1016/s0166-0934(02)00085-x

Cited by 128 publications

(104 citation statements)

References 23 publications

(23 reference statements)

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“…PAMs were isolated from lung lavage fluid samples from 4-to 5-week-old simian immunodeficiency virus (SIV)-seronegative piglets according to a standard protocol described previously (27). The isolated PAMs were characterized by flow cytometric analysis staining with fluorescein isothiocyanate-conjugated mouse anti-pig macrophage monoclonal antibody (MAb) clone BA4D5 (AbD Serotec) and cultivated in RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS), 1% HEPES, 50 g/ml gentamicin, and 1ϫ Antibiotic-Antimycotic (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Influenza A Virus Panhandle Structure Is Directly Involved in RIG-I Activation and Interferon Induction

Liu¹,

Park²,

Pyo

et al. 2015

J Virol

110

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Retinoic acid-inducible gene I (RIG-I) is an important innate immune sensor that recognizes viral RNA in the cytoplasm. Its nonself recognition largely depends on the unique RNA structures imposed by viral RNA. The panhandle structure residing in the influenza A virus (IAV) genome, whose primary function is to serve as the viral promoter for transcription and replication, has been proposed to be a RIG-I agonist. However, this has never been proved experimentally. Here, we employed multiple approaches to determine if the IAV panhandle structure is directly involved in RIG-I activation and type I interferon (IFN) induction. First, in porcine alveolar macrophages, we demonstrated that the viral genomic coding region is dispensable for RIG-I-dependent IFN induction. Second, using in vitro-synthesized hairpin RNA, we showed that the IAV panhandle structure could directly bind to RIG-I and stimulate IFN production. Furthermore, we investigated the contributions of the wobble base pairs, mismatch, and unpaired nucleotides within the wild-type panhandle structure to RIG-I activation. Elimination of these destabilizing elements within the panhandle structure promoted RIG-I activation and IFN induction. Given the function of the panhandle structure as the viral promoter, we further monitored the promoter activity of these panhandle variants and found that viral replication was moderately affected, whereas viral transcription was impaired dramatically. In all, our results indicate that the IAV panhandle promoter region adopts a nucleotide composition that is optimal for balanced viral RNA synthesis and suboptimal for RIG-I activation. IMPORTANCEThe IAV genomic panhandle structure has been proposed to be an RIG-I agonist due to its partial complementarity; however, this has not been experimentally confirmed. Here, we provide direct evidence that the IAV panhandle structure is competent in, and sufficient for, RIG-I activation and IFN induction. By constructing panhandle variants with increased complementarity, we demonstrated that the wild-type panhandle structure could be modified to enhance RIG-I activation and IFN induction. These panhandle variants posed moderate influence on viral replication but dramatic impairment of viral transcription. These results indicate that the IAV panhandle promoter region adopts a nucleotide composition to achieve optimal balance of viral RNA synthesis and suboptimal RIG-I activation. Our results highlight the multifunctional role of the IAV panhandle promoter region in the virus life cycle and offer novel insights into the development of antiviral agents aiming to boost RIG-I signaling or virus attenuation by manipulating this conserved region.

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Section: Methodsmentioning

confidence: 99%

Influenza A Virus Panhandle Structure Is Directly Involved in RIG-I Activation and Interferon Induction

Liu¹,

Park²,

Pyo

et al. 2015

J Virol

110

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“…The continuous porcine monocytic cell line 3D4/31, developed from porcine alveolar m (63), was grown in a 1:1 mixture of RPMI 1640 (GIBCO-BRL) and minimal essential medium containing Earle's salts (GIBCO-BRL) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 0.3 mg/ml glutamine (BDH Chemicals Ltd., Poole, England), 1% nonessential amino acids (100ϫ; GIBCO-BRL), and antibiotics (monocyte/macrophage medium). Cells were seeded at 2 ϫ 10 5 cells/ml of monocyte/macrophage medium in 96-well cell culture plates (Nunc, Roskilde, Denmark).…”

Section: Methodsmentioning

confidence: 99%

Porcine Circovirus 2 Uses Heparan Sulfate and Chondroitin Sulfate B Glycosaminoglycans as Receptors for Its Attachment to Host Cells

Misinzo

Delputte

Meerts

et al. 2006

J Virol

139

105

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“…A published example of an immortalized PAM cell line, however, is not permissive for PRRSV infection (33). In addition, monocytes from peripheral blood are largely refractory to PRRSV infection.…”

mentioning

confidence: 99%

CD163 Expression Confers Susceptibility to Porcine Reproductive and Respiratory Syndrome Viruses

Calvert

Slade

Shields

et al. 2007

J Virol

294

239

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Direct functional screening of a cDNA expression library derived from primary porcine alveolar macrophages (PAM) revealed that CD163 is capable of conferring a porcine reproductive and respiratory syndrome virus (PRRSV)-permissive phenotype when introduced into nonpermissive cells. Transient-transfection experiments showed that full-length CD163 cDNAs from PAM, human U937 cells (histiocytic lymphoma), African green monkey kidney cells (MARC-145 and Vero), primary mouse peritoneal macrophages, and canine DH82 (histocytosis) cells encode functional virus receptors. In contrast, CD163 splice variants without the C-terminal transmembrane anchor domain do not provide PRRSV receptor function. We established several stable cell lines expressing CD163 cDNAs from pig, human, and monkey, using porcine kidney (PK 032495), feline kidney (NLFK), or baby hamster kidney (BHK-21) as the parental cell lines. These stable cell lines were susceptible to PRRSV infection and yielded high titers of progeny virus. Cell lines were phenotypically stable over 80 cell passages, and PRRSV could be serially passed at least 60 times, yielding in excess of 10 5 50% tissue culture infective doses/ml.

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Continuous porcine cell lines developed from alveolar macrophages

Cited by 128 publications

References 23 publications

Influenza A Virus Panhandle Structure Is Directly Involved in RIG-I Activation and Interferon Induction

Influenza A Virus Panhandle Structure Is Directly Involved in RIG-I Activation and Interferon Induction

Porcine Circovirus 2 Uses Heparan Sulfate and Chondroitin Sulfate B Glycosaminoglycans as Receptors for Its Attachment to Host Cells

CD163 Expression Confers Susceptibility to Porcine Reproductive and Respiratory Syndrome Viruses

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