scholarly journals 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

2006 ◽  
Vol 80 (23) ◽  
pp. 11447-11455 ◽  
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.

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

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
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.

scholarly journals Phylogeny-Based Evolutionary, Demographical, and Geographical Dissection of North American Type 2 Porcine Reproductive and Respiratory Syndrome Viruses

2010 ◽  
Vol 84 (17) ◽  
pp. 8700-8711 ◽  
Author(s):  
Mang Shi ◽  
Tommy Tsan-Yuk Lam ◽  
Chung-Chau Hon ◽  
Michael P. Murtaugh ◽  
Peter R. Davies ◽  
...  
Keyword(s):  
United States ◽  
North American ◽  
Phylogenetic Analyses ◽  
The United States ◽  
Respiratory Syndrome Virus ◽  
Viral Spread ◽  
Comprehensive Picture ◽  
North American Type ◽  
Orf5 Sequences

ABSTRACT Type 2 (or North American-like) porcine reproductive and respiratory syndrome virus (PRRSV) was first recorded in 1987 in the United States and now occurs in most commercial swine industries throughout the world. In this study, we investigated the epidemiological and evolutionary behaviors of type 2 PRRSV. Based on phylogenetic analyses of 8,624 ORF5 sequences, we described a comprehensive picture of the diversity of type 2 PRRSVs and systematically classified all available sequences into lineages and sublineages, including a number of previously undescribed lineages. With the rapid growth of sequence deposition into the databases, it would be technically difficult for veterinary researchers to genotype their sequences by reanalyzing all sequences in the databases. To this end, a set of reference sequences was established based on our classification system, which represents the principal diversity of all available sequences and can readily be used for further genotyping studies. In addition, we further investigated the demographic histories of these lineages and sublineages by using Bayesian coalescence analyses, providing evolutionary insights into several important epidemiological events of type 2 PRRSV. Moreover, by using a phylogeographic approach, we were able to estimate the transmission frequencies between the pig-producing states in the United States and identified several states as the major sources of viral spread, i.e., “transmission centers.” In summary, this study represents the most extensive phylogenetic analyses of type 2 PRRSV to date, providing a basis for future genotyping studies and dissecting the epidemiology of type 2 PRRSV from phylogenetic perspectives.

scholarly journals Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b’ Is Not Required for In Vitro Virus Replication

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 632
Author(s):  
Yingyun Cai ◽  
Shuiqing Yu ◽  
Ying Fang ◽  
Laura Bollinger ◽  
Yanhua Li ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Lines ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Infectious Clone ◽  
Hemorrhagic Fever ◽  
Simian Hemorrhagic Fever Virus ◽  
Hemorrhagic Fever Virus ◽  
Green Fluorescent

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.

scholarly journals Replication-Competent Rhabdoviruses with Human Immunodeficiency Virus Type 1 Coats and Green Fluorescent Protein: Entry by a pH-Independent Pathway

1999 ◽  
Vol 73 (8) ◽  
pp. 6937-6945 ◽  
Author(s):  
Eli Boritz ◽  
Jennifer Gerlach ◽  
J. Erik Johnson ◽  
John K. Rose
Keyword(s):  
Human Immunodeficiency Virus ◽  
Green Fluorescent Protein ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Neutralizing Antibodies ◽  
Fluorescent Protein ◽  
Gene Encoding ◽  
Immunodeficiency Virus ◽  
Green Fluorescent

ABSTRACT We describe a replication-competent, recombinant vesicular stomatitis virus (VSV) in which the gene encoding the single transmembrane glycoprotein (G) was deleted and replaced by anenv-G hybrid gene encoding the extracellular and transmembrane domains of a human immunodeficiency virus type 1 (HIV-1) envelope protein fused to the cytoplasmic domain of VSV G. An additional gene encoding a green fluorescent protein was added to permit rapid detection of infection. This novel surrogate virus infected and propagated on cells expressing the HIV receptor CD4 and coreceptor CXCR4. Infection was blocked by SDF-1, the ligand for CXCR4, by antibody to CD4 and by HIV-neutralizing antibody. This virus, unlike VSV, entered cells by a pH-independent pathway and thus supports a pH-independent pathway of HIV entry. Additional recombinants carrying hybrid env-G genes derived from R5 or X4R5 HIV strains also showed the coreceptor specificities of the HIV strains from which they were derived. These surrogate viruses provide a simple and rapid assay for HIV-neutralizing antibodies as well as a rapid screen for molecules that would interfere with any stage of HIV binding or entry. The viruses might also be useful as HIV vaccines. Our results suggest wide applications of other surrogate viruses based on VSV.

scholarly journals Development of genetic markers in the non-structural protein 2 region of a US type 1 porcine reproductive and respiratory syndrome virus: implications for future recombinant marker vaccine development

2008 ◽  
Vol 89 (12) ◽  
pp. 3086-3096 ◽  
Author(s):  
Ying Fang ◽  
Jane Christopher-Hennings ◽  
Elizabeth Brown ◽  
Haixia Liu ◽  
Zhenhai Chen ◽  
...  
Keyword(s):  
Recombinant Virus ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Infectious Clone ◽  
Wild Type Virus ◽  
Respiratory Syndrome Virus ◽  
Type Virus ◽  
Wild Type ◽  
Marker Virus

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a major problem in the pork industry worldwide. The limitations of current PRRSV vaccines require the development of a new generation of vaccines. One of the key steps in future vaccine development is to include markers for diagnostic differentiation of vaccinated animals from those naturally infected with wild-type virus. Using a cDNA infectious clone of type 1 PRRSV, this study constructed a recombinant green fluorescent protein (GFP)-tagged PRRSV containing a deletion of an immunogenic epitope, ES4, in the nsp2 region. In a nursery pig disease model, the recombinant virus was attenuated with a lower level of viraemia in comparison with that of the parental virus. To complement the marker identification, GFP and ES4 epitope-based ELISAs were developed. Pigs immunized with the recombinant virus lacked antibodies directed against the corresponding deleted epitope, but generated a high-level antibody response to GFP by 14 days post-infection. These results demonstrated that this recombinant marker virus, in conjunction with the diagnostic tests, enables serological differentiation between marker virus-infected animals and those infected with the wild-type virus. This rationally designed marker virus will provide a basis for further development of PRRSV marker vaccines to assist with the control of PRRS.

scholarly journals Characterization of Emerging European-Like Porcine Reproductive and Respiratory Syndrome Virus Isolates in the United States

2004 ◽  
Vol 78 (7) ◽  
pp. 3684-3703 ◽  
Author(s):  
Susan L. Ropp ◽  
Carrie E. Mahlum Wees ◽  
Ying Fang ◽  
Eric A. Nelson ◽  
Kurt D. Rossow ◽  
...  
Keyword(s):  
United States ◽  
North American ◽  
Reference Strain ◽  
Genomic Sequence ◽  
Nonstructural Protein ◽  
The United States ◽  
Amino Acid Identity ◽  
Nucleotide Identity ◽  
Respiratory Syndrome Virus ◽  
The North

ABSTRACT European-like field isolates of porcine reproductive and respiratory syndrome virus (PRRSV) have recently emerged in North America. The full-length genomic sequence of an index isolate characterized in 1999, strain EuroPRRSV, served as the reference strain for further studies of the evolution and epidemiology of European-like isolates (type 1) in the United States. Strain EuroPRRSV shared 90.1 to 100% amino acid identity with the prototype European strain, Lelystad, within the structural and nonstructural open reading frames (ORFs) and 95.3% overall nucleotide identity. The 5′ untranslated region and two nonstructural regions within ORF 1 were closely examined due to significant divergence from strain Lelystad. A 51-bp deletion in a region within ORF 1a, coding for nonstructural protein 2 (NSP2), was observed. Sequence analysis of the structural ORFs 2 to 7 of additional European-like isolates indicated that these isolates share 93% nucleotide identity with one another and 95 to 96% identity with the Lelystad strain but only 70% identity with the North American reference strain VR-2332. Phylogenetic analysis with published PRRSV ORF 3, 5, and 7 nucleotide sequences indicated that these newly emerging isolates form a clade with the Lelystad and United Kingdom PRRSV isolates. Detailed analysis of four of these isolates with a panel of 60 monoclonal antibodies directed against the structural proteins confirmed a recognition pattern that was more consistent with strain Lelystad than with other North American isolates.

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

2010 ◽  
Vol 10 (3) ◽  
pp. 267-274 ◽  
Author(s):  
Charles E. McGee ◽  
Alexandr V. Shustov ◽  
Konstantin Tsetsarkin ◽  
Ilya V. Frolov ◽  
Peter W. Mason ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
West Nile Virus ◽  
Fluorescent Protein ◽  
Infectious Clone ◽  
West Nile ◽  
Green Fluorescent

scholarly journals Intracellular green fluorescent protein–polyalanine aggregates are associated with cell death

2000 ◽  
Vol 348 (1) ◽  
pp. 15-19 ◽  
Author(s):  
Julia RANKIN ◽  
Andreas WYTTENBACH ◽  
David C. RUBINSZTEIN
Keyword(s):  
Cell Death ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Cag Repeat ◽  
Cag Repeats ◽  
Spinocerebellar Ataxia Type ◽  
Aggregate Formation ◽  
Green Fluorescent ◽  
Intracellular Aggregates

Eight diseases, exemplified by Huntington's disease and spinocerebellar ataxia type 1, are caused by CAG-repeat expansion mutations. The CAG repeats are translated into expanded polyglutamine tracts, which are associated with deleterious novel functions. While these diseases are characterized by intraneuronal aggregate formation, it is unclear whether the aggregates cause disease. We have addressed this debate by generating intracellular aggregates with green fluorescent protein (GFP) fused to 19-37 alanines. No aggregates were seen in cells expressing native GFP or GFP fused to seven alanines. Aggregate-containing cells expressing GFP fused to 19-37 polyalanines show high rates of nuclear fragmentation compared with cells expressing the same constructs without aggregates, or cells expressing GFP fused to seven alanines. This suggests an association between aggregate formation and cell death.

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