Infection, Dissemination, and Transmission of a West Nile Virus Green Fluorescent Protein Infectious Clone byCulex pipiens quinquefasciatusMosquitoes

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b’, is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.

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Preparation of North American Type II PRRSV Infectious Clone Expressing Green Fluorescent Protein

BioMed Research International ◽

10.1155/2014/368581 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Liyue Wang ◽

Kao Zhang ◽

Hongyu Lin ◽

Wenyan Li ◽

Jiexia Wen ◽

...

Keyword(s):

Green Fluorescent Protein ◽

North American ◽

Fluorescent Protein ◽

Infectious Clone ◽

Type Ii ◽

Nsp2 Gene ◽

Gfp Gene ◽

Overlap Pcr ◽

Green Fluorescent ◽

North American Type

Porcine reproductive and respiratory syndrome virus (PRRSV) is still one of the most important infectious diseases threatening the swine industry. To construct North American type II PRRSV infectious clone containing green fluorescent protein (GFP) gene, we amplify gfp gene, flanked by PRRSV Nsp2 gene fragments upstream and downstream, using overlap PCR method from pcDNA-EF1-GFP plasmid and FL12 plasmid containing PRRSV infectious genome as the templates. The Nsp2 fragment-flanked gfp gene was inserted into Nsp2 gene of the FL12 plasmid bySpeI andXhoI sites to generate PRRSV infectious recombinant plasmid (FL12-GFP) containing gfp gene. The recombinant PRRSV expressing GFP (PRRSV-GFP) was rescued in baby hamster kidney-21 (BHK-21) cells by transfecting PRRSV mRNA synthesizedin vitroand amplified in Marc-145 cells. The PRRSV-GFP infectivity and replication capacity were identified. Results showed that, by adopting overlap PCR strategy, the gfp gene was successfully inserted into and fused with PRRSV Nsp2 gene in the PRRSV infectious clone plasmid FL-12 to generate FL12-GFP plasmid. The recombinant PRRSV-GFP was generated through transfecting PRRSV mRNA in BHK-2 cells. Like its parental virus, the recombinant PRRSV-GFP maintains its infectivity to Marc-145 cells and porcine alveolar macrophages (PAMs). This study provides essential conditions for further investigation on PRRSV.

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A Full-Length cDNA Infectious Clone of North American Type 1 Porcine Reproductive and Respiratory Syndrome Virus: Expression of Green Fluorescent Protein in the Nsp2 Region

Journal of Virology ◽

10.1128/jvi.01032-06 ◽

2006 ◽

Vol 80 (23) ◽

pp. 11447-11455 ◽

Cited By ~ 96

Author(s):

Ying Fang ◽

Raymond R. R. Rowland ◽

Michael Roof ◽

Joan K. Lunney ◽

Jane Christopher-Hennings ◽

...

Keyword(s):

United States ◽

Green Fluorescent Protein ◽

North American ◽

Fluorescent Protein ◽

Infectious Clone ◽

The United States ◽

Respiratory Syndrome Virus ◽

Green Fluorescent ◽

North American Type

ABSTRACT The recent emergence of a unique group of North American type 1 porcine reproductive and respiratory syndrome virus (PRRSV) in the United States presents new disease control problems for a swine industry that has already been impacted seriously by North American type 2 PRRSV. In this study, a full-length cDNA infectious clone was generated from a low-virulence North American type 1 PRRSV isolate, SD01-08. In vitro studies demonstrated that the cloned virus maintained growth properties similar to those of the parental virus. Virological, pathological, and immunological observations from animals challenged with cloned viruses were similar to those from animals challenged with the parental virus and a modified live virus vaccine. To further explore the potential use as a viral backbone for expressing foreign genes, the green fluorescent protein (GFP) was inserted into a unique deletion site located at amino acid positions 348 and 349 of the predicted Nsp2 region in the virus, and expression of the Nsp2-GFP fusion protein was visualized by fluorescent microscopy. The availability of this North American type 1 infectious clone provides an important research tool for further study of the basic viral biology and pathogenic mechanisms of this group of type 1 PRRSV in the United States.

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Ly6c+ “inflammatory monocytes” are microglial precursors recruited in a pathogenic manner in West Nile virus encephalitis

Journal of Experimental Medicine ◽

10.1084/jem.20080421 ◽

2008 ◽

Vol 205 (10) ◽

pp. 2319-2337 ◽

Cited By ~ 208

Author(s):

Daniel R. Getts ◽

Rachael L. Terry ◽

Meghann Teague Getts ◽

Marcus Müller ◽

Sabita Rana ◽

...

Keyword(s):

West Nile Virus ◽

Fluorescent Protein ◽

Central Nervous System Infection ◽

West Nile ◽

Inflammatory Monocyte ◽

Inflammatory Monocytes ◽

Monocyte Migration ◽

Microglial Phenotype ◽

West Nile Virus Encephalitis ◽

Green Fluorescent

In a lethal West Nile virus (WNV) model, central nervous system infection triggered a threefold increase in CD45int/CD11b+/CD11c− microglia at days 6–7 postinfection (p.i.). Few microglia were proliferating, suggesting that the increased numbers were derived from a migratory precursor cell. Depletion of “circulating” (Gr1−(Ly6Clo)CX3CR1+) and “inflammatory” (Gr1hi/Ly6Chi/CCR2+) classical monocytes during infection abrogated the increase in microglia. C57BL/6 chimeras reconstituted with cFMS–enhanced green fluorescent protein (EGFP) bone marrow (BM) showed large numbers of peripherally derived (GFP+) microglia expressing GR1+(Ly6C+) at day 7 p.i., suggesting that the inflammatory monocyte is a microglial precursor. This was confirmed by adoptive transfer of labeled BM (Ly6Chi/CD115+) or circulating inflammatory monocytes that trafficked to the WNV-infected brain and expressed a microglial phenotype. CCL2 is a chemokine that is highly expressed during WNV infection and important in inflammatory monocyte trafficking. Neutralization of CCL2 not only reduced the number of GFP+ microglia in the brain during WNV infection but prolonged the life of infected animals. Therefore, CCL2-dependent inflammatory monocyte migration is critical for increases in microglia during WNV infection and may also play a pathogenic role during WNV encephalitis.

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