scholarly journals Laboratory Strains of Murine Cytomegalovirus Are Genetically Similar to but Phenotypically Distinct from Wild Strains of Virus

2008 ◽  
Vol 82 (13) ◽  
pp. 6689-6696 ◽  
Author(s):  
L. M. Smith ◽  
A. R. McWhorter ◽  
L. L. Masters ◽  
G. R. Shellam ◽  
A. J. Redwood
Keyword(s):  
Inbred Mice ◽  
Laboratory Strain ◽  
Murine Cytomegalovirus ◽  
Phenotypic Characteristics ◽  
Phenotypic Differences ◽  
Resistance Patterns ◽  
Wild Strains ◽  
Clinical Models ◽  
Low Passage

ABSTRACT Murine cytomegalovirus (MCMV) is widely used to model human cytomegalovirus (HCMV) infection. However, it is known that serially passaged laboratory strains of HCMV differ significantly from recently isolated clinical strains of HCMV. It is therefore axiomatic that clinical models of HCMV using serially passaged strains of MCMV may not be able to fully represent the complexities of the system they are attempting to model and may not fully represent the complex biology of MCMV. To determine whether genotypic and phenotypic differences also exist between laboratory strains of MCMV and wild derived strains of MCMV, we sequenced the genomes of three low-passage strains of MCMV, plus the laboratory strain, K181. We coupled this genetic characterization to their phenotypic characteristics. In contrast to what is seen with HCMV (and rhesus CMV), there were no major genomic rearrangements in the MCMV genomes. In addition, the genome size was remarkably conserved between MCMV strains with no major insertions or deletions. There was, however, significant sequence variation between strains of MCMV, particularly at the genomic termini. These more subtle genetic differences led to considerable differences in in vivo replication with some strains of MCMV, such as WP15B, replicating preferentially in otherwise-MCMV-resistant C57BL/6 mice. CBA mice were no more resistant to MCMV than C57BL/6 mice and for some MCMV strains appeared to control infection less well than C57BL/6 mice. It is apparent that the previously described host resistance patterns of inbred mice and MCMV are not consistently applicable for all MCMV strains.

scholarly journals Production Strategies for Pentamer-Positive Subviral Dense Bodies as a Safe Human Cytomegalovirus Vaccine

Vaccines ◽  
2019 ◽  
Vol 7 (3) ◽  
pp. 104 ◽  
Author(s):  
Gogesch ◽  
Penner ◽  
Krauter ◽  
Büscher ◽  
Grode ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Clinical Manifestations ◽  
Laboratory Strain ◽  
Viral Reactivation ◽  
Reading Frame ◽  
Dense Bodies ◽  
Large Numbers ◽  
Genetic Modifications ◽  
Clinical Models

Infections with the human cytomegalovirus (HCMV) are associated with severe clinical manifestations in children following prenatal transmission and after viral reactivation in immunosuppressed individuals. The development of an HCMV vaccine has long been requested but there is still no licensed product available. Subviral dense bodies (DB) are immunogenic in pre-clinical models and are thus a promising HCMV vaccine candidate. Recently, we established a virus based on the laboratory strain Towne that synthesizes large numbers of DB containing the pentameric protein complex gH/gL/UL128-131 (Towne-UL130repΔGFP). The work presented here focuses on providing strategies for the production of a safe vaccine based on that strain. A GMP-compliant protocol for DB production was established. Furthermore, the DB producer strain Towne-UL130rep was attenuated by deleting the UL25 open reading frame. Additional genetic modifications aim to abrogate its capacity to replicate in vivo by conditionally expressing pUL51 using the Shield-1/FKBP destabilization system. We further show that the terminase inhibitor letermovir can be used to reduce infectious virus contamination of a DB vaccine by more than two orders of magnitude. Taken together, strategies are provided here that allow for the production of a safe and immunogenic DB vaccine for clinical testing.

scholarly journals Repair of an Attenuated Low-Passage Murine Cytomegalovirus Bacterial Artificial Chromosome Identifies a Novel Spliced Gene Essential for Salivary Gland Tropism

2020 ◽  
Vol 94 (22) ◽  
Author(s):  
Alec James Redwood ◽  
Laura Lee Masters ◽  
Baca Chan ◽  
Shay Leary ◽  
Cathy Forbes ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Salivary Gland ◽  
Hcmv Infection ◽  
Nk Cell ◽  
Transcriptional Analysis ◽  
Murine Cytomegalovirus ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes ◽  
Low Passage

ABSTRACT The cloning of herpesviruses as bacterial artificial chromosomes (BACs) has revolutionized the study of herpesvirus biology, allowing rapid and precise manipulation of viral genomes. Several clinical strains of human cytomegalovirus (HCMV) have been cloned as BACs; however, no low-passage strains of murine CMV (MCMV), which provide a model mimicking these isolates, have been cloned. Here, the low-passage G4 strain of was BAC cloned. G4 carries an m157 gene that does not ligate the natural killer (NK) cell-activating receptor, Ly49H, meaning that unlike laboratory strains of MCMV, this virus replicates well in C57BL/6 mice. This BAC clone exhibited normal replication during acute infection in the spleen and liver but was attenuated for salivary gland tropism. Next-generation sequencing revealed a C-to-A mutation at nucleotide position 188422, located in the 3′ untranslated region of sgg1, a spliced gene critical for salivary gland tropism. Repair of this mutation restored tropism for the salivary glands. Transcriptional analysis revealed a novel spliced gene within the sgg1 locus. This small open reading frame (ORF), sgg1.1, starts at the 3′ end of the first exon of sgg1 and extends exon 2 of sgg1. This shorter spliced gene is prematurely terminated by the nonsense mutation at nt 188422. Sequence analysis of tissue culture-passaged virus demonstrated that sgg1.1 was stable, although other mutational hot spots were identified. The G4 BAC will allow in vivo studies in a broader range of mice, avoiding the strong NK cell responses seen in B6 mice with other MCMV BAC-derived MCMVs. IMPORTANCE Murine cytomegalovirus (MCMV) is widely used as a model of human CMV (HCMV) infection. However, this model relies on strains of MCMV that have been serially passaged in the laboratory for over four decades. These laboratory strains have been cloned as bacterial artificial chromosomes (BACs), which permits rapid and precise manipulation. Low-passage strains of MCMV add to the utility of the mouse model of HCMV infection but do not exist as cloned BACs. This study describes the first such low-passage MCMV BAC. This BAC-derived G4 was initially attenuated in vivo, with subsequent full genomic sequencing revealing a novel spliced transcript required for salivary gland tropism. These data suggest that MCMV, like HCMV, undergoes tissue culture adaptation that can limit in vivo growth and supports the use of BAC clones as a way of standardizing viral strains and minimizing interlaboratory strain variation.

Comparison of Pseudomonas aeruginosa strains reveals that Exolysin A toxin plays an additive role in virulence

2020 ◽  
Vol 78 (1) ◽  
Author(s):  
Maria Medina-Rojas ◽  
William Stribling ◽  
Erik Snesrud ◽  
Brittany I Garry ◽  
Yuanzhang Li ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Galleria Mellonella ◽  
Multidrug Resistant ◽  
Secretion System ◽  
Phenotypic Characteristics ◽  
Phenotypic Differences ◽  
Transporter Protein ◽  
Successful Infection ◽  
Infection Models

ABSTRACT Background Pseudomonas aeruginosa possesses an array of virulence genes ensuring successful infection development. A two-partner secretion system Exolysin BA (ExlBA) is expressed in the PA7-like genetic outliers consisting of ExlA, a pore-forming toxin and ExlB transporter protein. Presence of exlBA in multidrug-resistant (MDR) strains has not been investigated, particularly in the strains isolated from wounded soldiers. Methods We screened whole genome sequences of 2439 MDR- P. aeruginosa strains for the presence of exlBA. We compiled all exlBA positive strains and compared them with a diversity set for demographics, antimicrobial profiles and phenotypic characteristics: surface motility, biofilm formation, pyocyanin production and hemolysis. We compared the virulence of strains with comparable phenotypic characteristics in Galleria mellonella. Results We identified 33 exlBA-positive strains (1.5%). These strains have increased antibiotic resistance, they are more motile, produce more robust biofilms and have comparable pyocianin production with the diversity set despite the phenotypic differences within the group. In in vivo infection models, these strains were less virulent than Type III Secretion System (T3SS) positive counterparts. Conclusions exlBA-positive strains are wide spread among the PA7-like outliers. While not as virulent as strains possessing T3SS, these strains exhibit phenotypic features associated with virulence and are still lethal in vivo.

Negative cross resistance of Spodoptera litura Fabricius population of tomato to newer molecule spinetoram

ENTOMON ◽  
2019 ◽  
Vol 44 (2) ◽  
pp. 127-132
Author(s):  
M. Visnupriya ◽  
N. Muthukrishnan
Keyword(s):  
Spodoptera Litura ◽  
Laboratory Strain ◽  
Field Population ◽  
Resistance Ratio ◽  
Cross Resistance ◽  
In Vivo Toxicity

Field population of Spodoptera litura from tomato ( resistant to the majority of the conventional insecticide molecules) were subjected to the in vivo toxicity of spinetoram 12 SC to assess whether cross resistance exists or not. Untreated larvae of both field and laboratory strains showed no mortality during 48 hours of feeding. After 48 hours of feeding on spinetoram 12 SC treated leaves, LC50s of field larvae were 0.28, 0.93, 3.71 and 7.11 ppm for the 2nd, 3rd, 4th and 5th instars of S. litura respectively. However, in the laboratory strain these values were 1.12, 5.86, 36.72 and 91.55 ppm for 2nd, 3rd, 4th and 5th instars of S. litura respectively. Resistance ratio was 0.25, 0.16, 0.10 and 0.08 for the 2nd instar up to the 5th instar of S. litura.

scholarly journals Delivery of melittin-loaded niosomes for breast cancer treatment: an in vitro and in vivo evaluation of anti-cancer effect

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Farnaz Dabbagh Moghaddam ◽  
Iman Akbarzadeh ◽  
Ehsan Marzbankia ◽  
Mahsa Farid ◽  
Leila khaledi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Treatment ◽  
Cell Lines ◽  
Encapsulation Efficiency ◽  
Inbred Mice ◽  
Breast Cancer Treatment ◽  
Anticancer Effect ◽  
Anti Cancer

Abstract Background Melittin, a peptide component of honey bee venom, is an appealing candidate for cancer therapy. In the current study, melittin, melittin-loaded niosome, and empty niosome had been optimized and the anticancer effect assessed in vitro on 4T1 and SKBR3 breast cell lines and in vivo on BALB/C inbred mice. "Thin-layer hydration method" was used for preparing the niosomes; different niosomal formulations of melittin were prepared and characterized in terms of morphology, size, polydispersity index, encapsulation efficiency, release kinetics, and stability. A niosome was formulated and loaded with melittin as a promising drug carrier system for chemotherapy of the breast cancer cells. Hemolysis, apoptosis, cell cytotoxicity, invasion and migration of selected concentrations of melittin, and melittin-loaded niosome were evaluated on 4T1 and SKBR3 cells using hemolytic activity assay, flow cytometry, MTT assay, soft agar colony assay, and wound healing assay. Real-time PCR was used to determine the gene expression. 40 BALB/c inbred mice were used; then, the histopathology, P53 immunohistochemical assay and estimate of renal and liver enzyme activity for all groups had been done. Results This study showed melittin-loaded niosome is an excellent substitute in breast cancer treatment due to enhanced targeting, encapsulation efficiency, PDI, and release rate and shows a high anticancer effect on cell lines. The melittin-loaded niosome affects the genes expression by studied cells were higher than other samples; down-regulates the expression of Bcl2, MMP2, and MMP9 genes while they up-regulate the expression of Bax, Caspase3 and Caspase9 genes. They have also enhanced the apoptosis rate and inhibited cell migration, invasion in both cell lines compared to the melittin samples. Results of histopathology showed reduce mitosis index, invasion and pleomorphism in melittin-loaded niosome. Renal and hepatic biomarker activity did not significantly differ in melittin-loaded niosome and melittin compared to healthy control. In immunohistochemistry, P53 expression did not show a significant change in all groups. Conclusions Our study successfully declares that melittin-loaded niosome had more anti-cancer effects than free melittin. This project has demonstrated that niosomes are suitable vesicle carriers for melittin, compare to the free form.

Varying influence of the autolysin, N-acetyl muramidase, and the cell envelope proteinase on the rate of autolysis of six commercial Lactococcus lactis cheese starter bacteria grown in milk

2000 ◽  
Vol 67 (4) ◽  
pp. 585-596 ◽  
Author(s):  
SELVARANI GOVINDASAMY-LUCEY ◽  
PRAMOD K. GOPAL ◽  
PATRICK A. SULLIVAN ◽  
CHRISTOPHER J. PILLIDGE
Keyword(s):  
Lactococcus Lactis ◽  
Cell Walls ◽  
Cell Envelope ◽  
Laboratory Strain ◽  
Proteolytic Cleavage ◽  
Zymogram Analysis ◽  
Sds Page ◽  
Derivatives Of ◽  
Free Strains

The autolysin, N-acetyl muramidase (AcmA), of six commercial Lactococcus lactis subsp. cremoris starter strains and eight Lc. lactis subsp. cremoris derivatives or plasmid-free strains was shown by renaturing SDS-PAGE (zymogram analysis) to be degraded by the cell envelope proteinase (lactocepin; EC 3.4.21.96) after growth of strains in milk at 30 °C for 72 h. Degradation of AcmA was less in starter strains and derivatives producing lactocepin I/III (intermediate specificity) than in strains producing lactocepin I. This supports previous observations on AcmA degradation in derivatives of the laboratory strain Lc. lactis subsp. cremoris MG1363 (Buist et al. Journal of Bacteriology180 5947–5953 1998). In contrast to the MG1363 derivatives, however, the extent of autolysis in milk of the commercial Lc. lactis subsp. cremoris starter strains in this study did not always correlate with lactocepin specificity and AcmA degradation. The distribution of autolysins within the cell envelope of Lc. lactis subsp. cremoris starter strains and derivatives harvested during growth in milk was compared by zymogram analysis. AcmA was found associated with cell membranes as well as cell walls and some cleavage of AcmA occurred independently of lactocepin activity. An AcmA product intermediate in size between precursor (46 kDa) and mature (41 kDa) forms of AcmA was clearly visible on zymograms, even in the absence of lactocepin I activity. These results show that autolysis of commercial Lc. lactis subsp. cremoris starter strains is not primarily determined by AcmA activity in relation to lactocepin specificity and that proteolytic cleavage of AcmA in vivo is not fully defined.

Pre-clinical models of cellular therapy combined with cytokines demonstrate in vitro and in vivo ovarian cancer cell killing

2012 ◽  
Vol 125 ◽  
pp. S45
Author(s):  
S. Ingersoll ◽  
S. Ahmad ◽  
G. Stoltzfus ◽  
M. Merchant ◽  
A. Ahmed ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cell ◽  
Cellular Therapy ◽  
Ovarian Cancer Cell ◽  
Cell Killing ◽  
Clinical Models

In vivo proof-of-concept for two experimental antiviral drugs, both directed to cellular targets, using a murine cytomegalovirus model

2019 ◽  
Vol 161 ◽  
pp. 63-69 ◽  
Author(s):  
Eric Sonntag ◽  
Friedrich Hahn ◽  
Luca D. Bertzbach ◽  
Lisa Seyler ◽  
Christina Wangen ◽  
...  
Keyword(s):  
Antiviral Drugs ◽  
Murine Cytomegalovirus ◽  
Proof Of Concept ◽  
Cellular Targets

scholarly journals Essential nucleotide- and protein-dependent functions of Actb/β-actin

2018 ◽  
Vol 115 (31) ◽  
pp. 7973-7978 ◽  
Author(s):  
Xiaobai Patrinostro ◽  
Pallabi Roy ◽  
Angus Lindsay ◽  
Christopher M. Chamberlain ◽  
Lauren J. Sundby ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Null Mouse ◽  
Cellular Functions ◽  
Embryonic Fibroblasts ◽  
High Frequency Hearing Loss ◽  
Phenotypic Differences ◽  
Functional Differences ◽  
Actin Protein

The highly similar cytoplasmic β- and γ-actins differ by only four functionally similar amino acids, yet previous in vitro and in vivo data suggest that they support unique functions due to striking phenotypic differences between Actb and Actg1 null mouse and cell models. To determine whether the four amino acid variances were responsible for the functional differences between cytoplasmic actins, we gene edited the endogenous mouse Actb locus to translate γ-actin protein. The resulting mice and primary embryonic fibroblasts completely lacked β-actin protein, but were viable and did not present with the most overt and severe cell and organismal phenotypes observed with gene knockout. Nonetheless, the edited mice exhibited progressive high-frequency hearing loss and degeneration of actin-based stereocilia as previously reported for hair cell-specific Actb knockout mice. Thus, β-actin protein is not required for general cellular functions, but is necessary to maintain auditory stereocilia.

scholarly journals Comparison of the Susceptibilities of C57BL/6 and A/J Mouse Strains to Streptococcus suis Serotype 2 Infection

2008 ◽  
Vol 76 (9) ◽  
pp. 3901-3910 ◽  
Author(s):  
María de la Cruz Domínguez-Punaro ◽  
Mariela Segura ◽  
Danuta Radzioch ◽  
Serge Rivest ◽  
Marcelo Gottschalk
Keyword(s):  
Septic Shock ◽  
Inbred Mice ◽  
Late Phase ◽  
Streptococcus Suis ◽  
Mouse Strains ◽  
Necrosis Factor Alpha ◽  
Central Nervous System Damage ◽  
Proinflammatory Mediators ◽  
Outbred Mice

ABSTRACT Streptococcus suis is an important swine and human pathogen. Assessment of susceptibility to S. suis using animal models has been limited to monitoring mortality rates. We recently developed a hematogenous model of S. suis infection in adult CD1 outbred mice to study the in vivo development of an early septic shock-like syndrome that leads to death and a late phase that clearly induces central nervous system damage, including meningitis. In the present study, we compared the severities of septic shock-like syndrome caused by S. suis between adult C57BL/6J (B6) and A/J inbred mice. Clinical parameters, proinflammatory mediators, and bacterial clearance were measured to dissect potential immune factors associated with genetic susceptibility to S. suis infection. Results showed that A/J mice were significantly more susceptible than B6 mice to S. suis infection, especially during the acute septic phase of infection (100% of A/J and 16% of B6 mice died before 24 h postinfection). The greater susceptibility of A/J mice was associated with an exaggerated inflammatory response, as indicated by their higher production of tumor necrosis factor alpha, interleukin-12p40/p70 (IL-12p40/p70), gamma interferon, and IL-1β, but not with different bacterial loads in the blood. In addition, IL-10 was shown to be responsible, at least in part, for the higher survival in B6 mice. Our findings demonstrate that A/J mice are very susceptible to S. suis infection and provide evidence that the balance between pro- and anti-inflammatory mediators is crucial for host survival during the septic phase.

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