Reverse Genetics System for Porcine Enteric Calicivirus, a Prototype Sapovirus in the Caliciviridae

ABSTRACT A porcine enteric calicivirus (PEC), strain Cowden in the genus Sapovirus of the Caliciviridae family, can be propagated in a porcine kidney continuous cell line (LLC-PK) in the presence of bile acids in the cell culture medium. A full-length cDNA copy of the Cowden PEC genome was cloned into a plasmid vector directly downstream from the T7 RNA polymerase promoter, and capped RNA transcripts derived from this clone were infectious when transfected into LLC-PK cells. The recovery of PEC after transfection of RNA transcripts was dependent on the presence of bile acids, consistent with our recent identification of a bile acid-mediated signaling pathway required for PEC replication (Chang et al., Proc. Natl. Acad. Sci. USA 101:8733-8788, 2004). Recovery of virus was verified by detection of PEC antigen in transfected cells by immunofluorescence and enzyme-linked immunosorbent assays, direct observation of recovered viral particles by electron microscopy, and partial sequence analysis of their genomes (first 1,070 nucleotides) to differentiate them from tissue culture-adapted parental virus. The recovered virus retained its ability to infect piglets when administered by the oral route and showed an attenuated phenotype similar to that of the tissue culture-adapted parental virus. This reverse genetics system for PEC provides a new tool to study the molecular basis of replication and pathogenesis for caliciviruses associated with diarrheal disease.

Download Full-text

Generation of Infectious Pancreatic Necrosis Virus from Cloned cDNA

Journal of Virology ◽

10.1128/jvi.72.11.8913-8920.1998 ◽

1998 ◽

Vol 72 (11) ◽

pp. 8913-8920 ◽

Cited By ~ 44

Author(s):

Kun Yao ◽

Vikram N. Vakharia

Keyword(s):

Rna Polymerase ◽

Reverse Genetics ◽

In Vitro Transcription ◽

Infectious Pancreatic Necrosis Virus ◽

Rna Dependent Rna Polymerase ◽

Rna Transcripts ◽

Recovered Virus ◽

Cloned Cdna ◽

Pancreatic Necrosis Virus

ABSTRACT We developed a reverse genetics system for infectious pancreatic necrosis virus (IPNV), a prototype virus of theBirnaviridae family, with the use of plus-stranded RNA transcripts derived from cloned cDNA. Full-length cDNA clones of the IPNV genome that contained the entire coding and noncoding regions of RNA segments A and B were constructed. Segment A encodes a 106-kDa precursor protein which is cleaved to yield mature VP2, nonstructural protease, and VP3 proteins, whereas segment B encodes the RNA-dependent RNA polymerase VP1. Plus-sense RNA transcripts of both segments were prepared by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of chinook salmon embryo (CHSE) cells with combined transcripts of segments A and B generated infectious IPNV particles 10 days posttransfection. Furthermore, a transfectant virus containing a genetically tagged sequence was generated to confirm the feasibility of this system. The presence and specificity of the recovered virus were ascertained by immunofluorescence staining of infected CHSE cells with rabbit anti-IPNV serum and by nucleotide sequence analysis. In addition, 3′-terminal sequence analysis of RNA from the recovered virus showed that extraneous nucleotides synthesized at the 3′ end during in vitro transcription were precisely trimmed or excluded during replication, and hence these were not incorporated into the genome. An attempt was made to determine if RNA-dependent RNA polymerase of IPNV and infectious bursal disease virus (IBDV), another birnavirus, can support virus rescue in heterologous combinations. Thus, CHSE cells were transfected with transcripts derived from IPNV segment A and IBDV segment B and Vero cells were transfected with transcripts derived from IBDV segment A and IPNV segment B. In either case, no infectious IPNV or IBDV particles were generated even after a third passage in cell culture, suggesting that viral RNA-dependent RNA polymerase is species specific. However, the reverse genetics system for IPNV that we developed will greatly facilitate studies of viral replication and pathogenesis and the design of a new generation of live attenuated vaccines.

Download Full-text

Identification and Characterization of a Poliovirus Capsid Mutant with Enhanced Thermal Stability

Journal of Virology ◽

10.1128/jvi.01510-18 ◽

2018 ◽

Vol 93 (6) ◽

Cited By ~ 3

Author(s):

Y Nguyen ◽

Palmy R. Jesudhasan ◽

Elizabeth R. Aguilera ◽

Julie K. Pfeiffer

Keyword(s):

Thermal Stability ◽

High Temperatures ◽

Oral Route ◽

Mutant Virus ◽

Single Amino Acid ◽

Bacterial Surface ◽

Virus Particles ◽

Heat Resistant ◽

Viral Particles ◽

Very High

ABSTRACTEnteric viruses, including poliovirus, are spread by the fecal-oral route. In order to persist and transmit to a new host, enteric virus particles must remain stable once they are in the environment. Environmental stressors such as heat and disinfectants can inactivate virus particles and prevent viral transmission. It has been previously demonstrated that bacteria or bacterial surface glycans can enhance poliovirus virion stability and limit inactivation from heat or bleach. While investigating the mechanisms underlying bacterially enhanced virion thermal stability, we identified and characterized a poliovirus (PV) mutant with increased resistance to heat inactivation. The M132V mutant harbors a single amino acid change in the VP1 capsid coding that is sufficient to confer heat resistance but not bleach resistance. Although the M132V virus was stable in the absence of bacteria or feces at most temperatures, M132V virus was stabilized by feces at very high temperatures. M132V PV had reduced specific infectivity and RNA uncoating compared with those of wild-type (WT) PV, but viral yields in HeLa cells were similar. In orally inoculated mice, M132V had a slight fitness cost since fecal titers were lower and 12.5% of fecal viruses reverted to the WT. Overall, this work sheds light on factors that influence virion stability and fitness.IMPORTANCEViruses spread by the fecal-oral route need to maintain viability in the environment to ensure transmission. Previous work indicated that bacteria and bacterial surface polysaccharides can stabilize viral particles and enhance transmission. To explore factors that influence viral particle stability, we isolated a mutant poliovirus that is heat resistant. This mutant virus does not require feces for stability at most temperatures but can be stabilized by feces at very high temperatures. Even though the mutant virus is heat resistant, it is susceptible to inactivation by treatment with bleach. This work provides insight into how viral particles maintain infectivity in the environment.

Download Full-text

Sindbis Virus with a Tricomponent Genome

Journal of Virology ◽

10.1128/jvi.79.1.637-643.2005 ◽

2005 ◽

Vol 79 (1) ◽

pp. 637-643 ◽

Cited By ~ 13

Author(s):

Rafik Fayzulin ◽

Rodion Gorchakov ◽

Olga Petrakova ◽

Evgenia Volkova ◽

Ilya Frolov

Keyword(s):

Tissue Culture ◽

Virus Replication ◽

Genetic Information ◽

Sindbis Virus ◽

Viral Particles ◽

Rna Fragments

ABSTRACT We established a system for propagation of Sindbis virus (SIN)-based replicons in tissue culture in the form of a tricomponent genome virus. Three RNA fragments containing complementing genetic information required for virus replication are packaged into separate viral particles, and each cell produces at least 1,000 packaged replicons and the number of packaged helpers sufficient to perform the next passage. This system can be used to generate large stocks of packaged replicons. The formation of infectious recombinant SIN virus was not detected in any experiments. These features make multicomponent genome SIN an attractive system for a variety of research and biotechnology applications.

Download Full-text

Cholesterol and bile acids regulate cholesterol 7 alpha-hydroxylase expression at the transcriptional level in culture and in transgenic mice.

Molecular and Cellular Biology ◽

10.1128/mcb.14.4.2809 ◽

1994 ◽

Vol 14 (4) ◽

pp. 2809-2821 ◽

Cited By ~ 47

Author(s):

M I Ramirez ◽

D Karaoglu ◽

D Haro ◽

C Barillas ◽

R Bashirzadeh ◽

...

Keyword(s):

Tissue Culture ◽

Transgenic Mice ◽

Bile Acids ◽

Transcriptional Activation ◽

Fusion Gene ◽

Proximal Promoter ◽

Transcriptional Level ◽

Endogenous Gene ◽

Hydroxylase Gene

Cholesterol 7 alpha-hydroxylase (7 alpha-hydroxylase) is the rate-limiting enzyme in bile acid biosynthesis. It is subject to a feedback control, whereby high levels of bile acids suppress its activity, and cholesterol exerts a positive control. It has been suggested that posttranscriptional control plays a major part in that regulation. We have studied the mechanisms by which cholesterol and bile acids regulate expression of the 7 alpha-hydroxylase gene and found it to be solely at the transcriptional level by using two different approaches. First, using a tissue culture system, we localized a liver-specific enhancer located 7 kb upstream of the transcriptional initiation site. We also showed that low-density lipoprotein mediates transcriptional activation of chimeric genes, containing either the 7 alpha-hydroxylase or the albumin enhancer in front of the 7 alpha-hydroxylase proximal promoter, to the same extent as the in vivo cholesterol-mediated regulation of 7 alpha-hydroxylase mRNA. In a second approach, using transgenic mice, we have found that expression of an albumin enhancer-7 alpha-hydroxylase-lacZ fusion gene is restricted to the liver and is regulated by cholesterol and bile acids in a manner quantitatively similar to that of the endogenous gene. We also found, that a liver-specific enhancer is necessary for expression of the rat 7 alpha-hydroxylase gene, in agreement with the tissue culture experiments. Together, these results demonstrate that cholesterol and bile acids regulate the expression of the 7 alpha-hydroxylase gene solely at the transcriptional level.

Download Full-text

Cholesterol and bile acids regulate cholesterol 7 alpha-hydroxylase expression at the transcriptional level in culture and in transgenic mice

Molecular and Cellular Biology ◽

10.1128/mcb.14.4.2809-2821.1994 ◽

1994 ◽

Vol 14 (4) ◽

pp. 2809-2821

Author(s):

M I Ramirez ◽

D Karaoglu ◽

D Haro ◽

C Barillas ◽

R Bashirzadeh ◽

...

Keyword(s):

Tissue Culture ◽

Transgenic Mice ◽

Bile Acids ◽

Transcriptional Activation ◽

Fusion Gene ◽

Proximal Promoter ◽

Transcriptional Level ◽

Endogenous Gene ◽

Hydroxylase Gene

Download Full-text

Development and characterization of an infectious cDNA clone of the virulent Bucyrus strain of Equine arteritis virus

Journal of General Virology ◽

10.1099/vir.0.82415-0 ◽

2007 ◽

Vol 88 (3) ◽

pp. 918-924 ◽

Cited By ~ 38

Author(s):

Udeni B. R. Balasuriya ◽

Eric J. Snijder ◽

Hans W. Heidner ◽

Jianqiang Zhang ◽

Jessika C. Zevenhoven-Dobbe ◽

...

Keyword(s):

Reverse Genetics ◽

Severe Disease ◽

Plasmid Vector ◽

High Titre ◽

Equine Arteritis Virus ◽

Progeny Virus ◽

Cdna Clones ◽

Virulence Determinants ◽

Severity Of The Disease

Strains of Equine arteritis virus (EAV) differ in the severity of the disease that they induce in horses. Infectious cDNA clones are potentially useful for identification of genetic determinants of EAV virulence; to date, two clones have been derived from a cell culture-adapted variant of the original (Bucyrus) isolate of EAV, and it has previously been shown that recombinant virus derived from one of these (rEAV030) is attenuated in horses. A complete cDNA copy of the genome of the virulent Bucyrus strain of EAV has now been assembled into a plasmid vector. In contrast to rEAV030, recombinant progeny virus derived from this clone caused severe disease in horses, characterized by pyrexia, oedema, leukopenia, high-titre viraemia and substantial nasal shedding of virus. The availability of infectious cDNA clones that produce recombinant viruses of different virulence to horses will facilitate characterization of the virulence determinants of EAV through reverse genetics.

Download Full-text

Pancreatitis Associated with an Adenovirus in a Rhesus Monkey

Veterinary Pathology ◽

10.1177/030098587401100208 ◽

1974 ◽

Vol 11 (2) ◽

pp. 165-171 ◽

Cited By ~ 15

Author(s):

F. W. Chandler ◽

C. S. Callaway ◽

S. R. Adams

Keyword(s):

Electron Microscopy ◽

Tissue Culture ◽

Rhesus Monkey ◽

Nonhuman Primate ◽

Nonhuman Primates ◽

Necrotizing Pancreatitis ◽

Acinar Cells ◽

Pancreatic Acinar Cells ◽

Culture Studies ◽

Viral Particles

A juvenile Rhesus monkey died suddenly while being conditioned for tissue culture studies. A diagnosis of necrotizing pancreatitis associated with adenovirus was made on the basis of the demonstration by electron microscopy of paracrystalline arrays of viral particles and granular and fibrillar inclusions in necrotic pancreatic acinar cells. This is the first description of necrotizing pancreatitis in a nonhuman primate. Adenovirus should be considered in the etiology of pancreatitis in nonhuman primates.

Download Full-text

Development of Reverse Genetics Systems for Bluetongue Virus: Recovery of Infectious Virus from Synthetic RNA Transcripts

Journal of Virology ◽

10.1128/jvi.00808-08 ◽

2008 ◽

Vol 82 (17) ◽

pp. 8339-8348 ◽

Cited By ~ 140

Author(s):

Mark Boyce ◽

Cristina C. P. Celma ◽

Polly Roy

Keyword(s):

Bluetongue Virus ◽

Reverse Genetics ◽

Replication Cycle ◽

Cdna Clones ◽

Viral Mrnas ◽

Rna Transcripts ◽

Complete Set ◽

Virus Recovery ◽

Outer Capsid

ABSTRACT Bluetongue virus (BTV), an insect-vectored emerging pathogen of both wild ruminants and livestock, has had a severe economic impact in agriculture in many parts of the world. The investigation of BTV replication and pathogenesis has been hampered by the lack of a reverse genetics system. Recovery of infectious BTV is possible by the transfection of permissive cells with the complete set of 10 purified viral mRNAs derived in vitro from transcribing cores (M. Boyce and P. Roy, J. Virol. 81:2179-2186, 2007). Here, we report that in vitro synthesized T7 transcripts, derived from cDNA clones, can be introduced into the genome of BTV using a mixture of T7 transcripts and core-derived mRNAs. The replacement of genome segment 10 and the simultaneous replacement of segments 2 and 5 encoding the two immunologically important outer capsid proteins, VP2 and VP5, are described. Further, we demonstrate the recovery of infectious BTV entirely from T7 transcripts, proving that synthetic transcripts synthesized in the presence of cap analogue can functionally substitute for viral transcripts at all stages of the BTV replication cycle. The generation of BTV with a fully defined genome permits the recovery of mutations in a defined genetic background. The ability to generate specific mutants provides a new tool to investigate the BTV replication cycle as well as permitting the generation of designer vaccine strains, which are greatly needed in many countries.

Download Full-text

Bile Acids and Bicarbonate Inversely Regulate Intracellular Cyclic di-GMP in Vibrio cholerae

Infection and Immunity ◽

10.1128/iai.01664-14 ◽

2014 ◽

Vol 82 (7) ◽

pp. 3002-3014 ◽

Cited By ~ 43

Author(s):

Benjamin J. Koestler ◽

Christopher M. Waters

Keyword(s):

Small Intestine ◽

Bile Acids ◽

Vibrio Cholerae ◽

Diarrheal Disease ◽

Intracellular Concentration ◽

Intestinal Epithelial ◽

Content Type ◽

Proximal Small Intestine ◽

Ph Buffer ◽

Messenger Molecule

ABSTRACTVibrio choleraeis a Gram-negative bacterium that persists in aquatic reservoirs and causes the diarrheal disease cholera upon entry into a human host.V. choleraeemploys the second messenger molecule 3′,5′-cyclic diguanylic acid (c-di-GMP) to transition between these two distinct lifestyles. c-di-GMP is synthesized by diguanylate cyclase (DGC) enzymes and hydrolyzed by phosphodiesterase (PDE) enzymes. Bacteria typically encode many different DGCs and PDEs within their genomes. Presumably, each enzyme senses and responds to cognate environmental cues by alteration of enzymatic activity. c-di-GMP represses the expression of virulence factors inV. cholerae, and it is predicted that the intracellular concentration of c-di-GMP is low during infection. Contrary to this model, we found that bile acids, a prevalent constituent of the human proximal small intestine, increase intracellular c-di-GMP inV. cholerae. We identified four c-di-GMP turnover enzymes that contribute to increased intracellular c-di-GMP in the presence of bile acids, and deletion of these enzymes eliminates the bile induction of c-di-GMP and biofilm formation. Furthermore, this bile-mediated increase in c-di-GMP is quenched by bicarbonate, the intestinal pH buffer secreted by intestinal epithelial cells. Our results lead us to propose thatV. choleraesenses distinct microenvironments within the small intestine using bile and bicarbonate as chemical cues and responds by modulating the intracellular concentration of c-di-GMP.

Download Full-text