HF Strain Research Articles

Discovery of in vivo Virulence Genes of Obligatory Intracellular Bacteria by Random Mutagenesis.

Ehrlichia spp. are emerging tick-borne obligatory intracellular bacteria that cause febrile and sometimes fatal diseases with abnormal blood cell counts and signs of hepatitis. Ehrlichia HF strain provides an excellent mouse disease model of fatal human ehrlichiosis. We recently obtained and established stable culture of Ehrlichia HF strain in DH82 canine macrophage cell line, and obtained its whole genome sequence and annotation. To identify genes required for in vivo virulence of Ehrlichia, we constructed random insertional HF strain mutants by using Himar1 transposon-based mutagenesis procedure. Of total 158 insertional mutants isolated via antibiotic selection in DH82 cells, 74 insertions were in the coding regions of 55 distinct protein-coding genes, including TRP120 and multi-copy genes, such as p28/omp-1, virB2, and virB6. Among 84 insertions mapped within the non-coding regions, seven are located in the putative promoter region since they were within 50 bp upstream of the seven distinct genes. Using limited dilution methods, nine stable clonal mutants that had no apparent defect for multiplication in DH82 cells, were obtained. Mouse virulence of seven mutant clones was similar to that of wild-type HF strain, whereas two mutant clones showed significantly retarded growth in blood, livers, and spleens, and the mice inoculated with them lived longer than mice inoculated with wild-type. The two clones contained mutations in genes encoding a conserved hypothetical protein and a staphylococcal superantigen-like domain protein, respectively, and both genes are conserved among Ehrlichia spp., but lack homology to other bacterial genes. Inflammatory cytokine mRNA levels in the liver of mice infected with the two mutants were significantly diminished than those infected with HF strain wild-type, except IL-1β and IL-12 p40 in one clone. Thus, we identified two Ehrlichia virulence genes responsible for in vivo infection, but not for infection and growth in macrophages.

Molecular survey of neglected bacterial pathogens reveals an abundant diversity of species and genotypes in ticks collected from animal hosts across Romania

BackgroundTicks are transmitting a wide range of bacterial pathogens that cause substantial morbidity and mortality in domestic animals. The full pathogen burden transmitted by tick vectors is incompletely studied in many geographical areas, and extensive studies are required to fully understand the diversity and distribution of pathogens transmitted by ticks.ResultsWe sampled 824 ticks of 11 species collected in 19 counties in Romania. Ticks were collected mainly from dogs, but also from other domestic and wild animals, and were subjected to molecular screening for pathogens. Rickettsia spp. was the most commonly detected pathogen, occurring in 10.6% (87/824) of ticks. Several species were detected: Rickettsia helvetica, R. raoultii, R. massiliae, R. monacensis, R. slovaca and R. aeschlimannii. A single occurrence of the zoonotic bacterium Bartonella vinsonii berkhoffii was detected in a tick collected from a dog. Anaplasma phagocytophilum occurred in four samples, and sequences similar to Anaplasma marginale/ovis were abundant in ticks from ruminants. In addition, molecular screening showed that ticks from dogs were carrying an Ehrlichia species identical to the HF strain as well as the enigmatic zoonotic pathogen “Candidatus Neoehrlichia mikurensis”. An organism similar to E. chaffeensis or E. muris was detected in an Ixodes ricinus collected from a fox.ConclusionsWe describe an abundant diversity of bacterial tick-borne pathogens in ticks collected from animal hosts in Romania, both on the level of species and genotypes/strains within these species. Several findings were novel for Romania, including Bartonella vinsonii subsp. berkhoffii that causes bacteremia and endocarditis in dogs. “Candidatus Neoehrlichia mikurensis” was detected in a tick collected from a dog. Previously, a single case of infection in a dog was diagnosed in Germany. The results warrant further studies on the consequences of tick-borne pathogens in domestic animals in Romania.

Dominant Inheritance of Field-Evolved Resistance to Fipronil in Plutella xylostella (Lepidoptera: Plutellidae).

A field-collected strain (HF) of Plutella xylostella (L.) showed 420-fold resistance to fipronil compared with a susceptible laboratory strain (Roth). The HF-R strain, derived from the HF strain by 25 generations of successive selection with fipronil in the laboratory, developed 2,200-fold resistance to fipronil relative to the Roth strain. The F(1) progeny of the reciprocal crosses between HF-R and Roth showed 640-fold (R♀ × S♂) and 1,380-fold (R♂ × S♀) resistance to fipronil, indicating resistance is inherited as an incompletely dominant trait. Analysis of progeny from a backcross (F1♂ × S♀) suggests that resistance is controlled by one major locus. The LC(50) of the R♂ × S♀ cross F(1) progeny is slightly but significantly higher than that of the R♀ × S♂ cross F(1) progeny, suggesting a minor resistance gene on the Z chromosome. Sequence analysis of PxGABARα1 (an Rdl-homologous GABA receptor gene of P. xylostella) from the HF-R strain identified two mutations A282S and A282G (corresponding to the A302S mutation of the Drosophila melanogaster Rdl gene), which have been previously implicated in fipronil resistance in several insect species including P. xylostella. PxGABARα1 was previously mapped to the Z chromosome of P. xylostella. In conclusion, fipronil resistance in the HF-R strain of P. xylostella was incompletely dominant, and controlled by a major autosomal locus and a sex-linked minor gene (PxGABARα1) on the Z chromosome.

Inheritance of photoperiodic control of larval diapause in the Asian corn borer Ostrinia furnacalis (Guenée).

The Asian corn borer, Ostrinia furnacalis enters diapause as fully grown larvae. Owing to geographical variation in photoperiodic control of diapause, the subtropical strain from Hefei city (HF) enters diapause in response to short daylengths, whereas the tropical strain from Ledong county (LD) exhibits almost no diapause under the same conditions. The two strains were used in crosses to study the inheritance of diapause. The HF strain showed a typical long-day response with a critical daylength of approximately 14.97 h at 22 °C, 14.60 h at 25 °C and 13.68 h at 28 °C. The LD strain showed weak photoperiodic responses at 22 and 25 °C; and the F1 progeny also showed a long-day response with significantly shorter critical daylength compared with the HF strain. However, the LD × HF (F × M) crosses had significantly longer critical daylengths than HF × LD crosses, indicating a sexual bias in the inheritance of diapause induction, with the male parent having more influence on the F1 progeny. The critical daylength in a backcross to HF was significantly longer than a backcross to LD. Whether the inheritance of diapause fits an additive hypothesis or not depended on photoperiod, with results from different photoperiods showing additive inheritance or incomplete dominance of either diapause or non-diapause. Unlike diapause induction, the duration of diapause for reciprocal crosses was equally influenced by each parent, suggesting that diapause incidence and maintenance are controlled by separate systems in O. furnacalis.

High-frequency stimulation of the medial prefrontal cortex decreases cellular firing in the dorsal raphe.

High-frequency deep brain stimulation (HFS-DBS) of the subcallosal cingulate (SCC) region has been investigated as a treatment for refractory forms of depression with a ~50% remission rate in open label studies. However, the therapeutic mechanisms of DBS are still largely unknown. Using anaesthetized Sprague Dawley rats, we recorded neuronal spiking activity in 102 neurons of the dorsal raphe (DR) before, during and after the induction of a 5-min HFS train in the infralimbic region (IL) of the medial prefrontal cortex (mPFC), the rodent homologue of the human SCC. The majority of DR cells (82%) significantly decreased firing rate during HFS (P<0.01, 55.7±4.5% of baseline, 35 rats). To assess whether mPFC-HFS mediates inhibition of DR cellular firing by stimulating local GABAergic interneurons, the GABAA antagonist bicuculline (Bic, 100μm) was injected directly into the DR during HFS. Neurons inhibited by HFS recovered their firing rate during Bic+HFS (P<0.01, n=15, seven rats) to levels not different from baseline. Cells that were not affected by HFS did not change firing rate during Bic+HFS (P=0.968, n=7, three rats). These results indicate that blocking GABAA reverses HFS-mediated inhibition of DR neurons. As the cells that were not inhibited by HFS were also unaffected by HFS+Bic, they are probably not innervated by local GABA. Taken together, our results suggest that mPFC-HFS may exert a preferential effect on DR neurons with GABAA receptors.

Prevalence and genetic heterogeneity of porcine group C rotaviruses in nursing and weaned piglets in Ohio, USA and identification of a potential new VP4 genotype.

Swine fecal samples collected from seven farms were screened for group C rotaviruses (RVCs) using a reverse transcription-polymerase chain reaction assay. A total of 380 samples were tested and 19.5% were positive. Of the 128 samples collected in 2012, 23.5% from nursing piglets and 8.5% from weaned piglets were RVC positive, with a higher RVC frequency in diarrheic (28.4%) than in non-diarrheic (6.6%) piglets. Two strains (RVC/Pig-wt/USA/RV0104/2011/G3PX and RVC/Pig-wt/USA/RV0143/2012/G6Px) from two different farms were characterized genetically to gain information on virus diversity based on full length sequences of the inner capsid VP6, enterotoxin NSP4 and the outer capsid VP7 and VP4 (partial for RV0104) genes. The VP6 gene of the two strains showed high (99%) nucleotide identity to one another, 84-91% identity to other porcine RVCstrains and 81-82% identity to human and bovine RVC strains. The NSP4 gene analysis revealed that RVC/Pig-wt/USA/RV0104/2011/G3PX and RVC/Pig-wt/USA/RV0143/2012/G6Px strains were not closely related to each other (87% identity), but shared higher identity with prototype RVC/Pig-wt/USA/Cowden/1980/G1Px strain (93% and 89%, respectively) and were more distantly related to human strains (72-76% identity). The VP7 gene analysis indicated that the two strains were distantly related to one another (72% identity). RVC/Pig-wt/USA/RV0143/2012/G6Px was most closely related to porcine RVC G6 strains (82-86% identity), whereas RVC/Pig-wt/USA/RV0104/2011/G3PX was most closely related to porcine HF (G3) strain (94% identity). Analysis of the full length nucleotide sequence of the VP4 gene revealed that RVC/Pig-wt/USA/RV0143/2012/G6Px was distantly related to porcine (75%), bovine (74%) and human (70%) strains. The deduced amino acid identities (69.5-75.6%) of VP4 between RVC/Pig-wt/USA/RV0143/2012/G6Px and other RVCs were low; hence, we propose that this strain comprises a new VP4 genotype. Our results indicate high genetic heterogeneity in RVCs genes and the concurrent co-circulation of different genotypes at the same time. Our findings are useful for the development of more accurate diagnostic tools, for basic research to understand gene function and to provide information for RVC diversity germane to vaccine development.

Lack of DREAM Protein Enhances Learning and Memory and Slows Brain Aging

Memory deficits in aging affect millions of people and are often disturbing to those concerned. Dissection of the molecular control of learning and memory is paramount to understand and possibly enhance cognitive functions. Old-age memory loss also has been recently linked to altered Ca(2+) homeostasis. We have previously identified DREAM (downstream regulatory element antagonistic modulator), a member of the neuronal Ca(2+) sensor superfamily of EF-hand proteins, with specific roles in different cell compartments. In the nucleus, DREAM is a Ca(2+)-dependent transcriptional repressor, binding to specific DNA signatures, or interacting with nucleoproteins regulating their transcriptional properties. Also, we and others have shown that dream mutant (dream(-/-)) mice exhibit marked analgesia. Here we report that dream(-/-) mice exhibit markedly enhanced learning and synaptic plasticity related to improved cognition. Mechanistically, DREAM functions as a negative regulator of the key memory factor CREB in a Ca(2+)-dependent manner, and loss of DREAM facilitates CREB-dependent transcription during learning. Intriguingly, 18-month-old dream(-/-) mice display learning and memory capacities similar to young mice. Moreover, loss of DREAM protects from brain degeneration in aging. These data identify the Ca(2+)-regulated "pain gene" DREAM as a novel key regulator of memory and brain aging.

Distribution of Ehrlichiae in Tissues as Determined by In-situ Hybridization

Specific identification of ehrlichiae in the tissues and determination of their distribution is difficult. In this study, an in-situ hybridization method was developed to detect ehrlichial 16S rRNA in tissue specimens from mice experimentally infected with the HF strain. This strain is closely related to Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis. HF strain-specific 16S rRNA was detected in endothelial cells and monocyte–macrophages in the liver, lungs, bone marrow, spleen, lymph nodes, and large and small intestinal tissues. The results suggest that the in-situ hybridization method with a digoxigenin-labelled RNA probe specific to ehrlichial 16S rRNA will be useful for post-mortem diagnosis and for the histopathological investigation of ehrlichial infection.

Effect of immunity on gene delivery into anterior horn motor neurons by live attenuated herpes simplex virus vector.

Efficient and prolonged foreign gene expression has been demonstrated in the bilateral anterior horn motor neurons of the spinal cord by intramuscular inoculation with attenuated herpes simplex virus (HSV) expressing latency associated transcript promoter-driven beta-galactosidase (betaH1). To examine the effect of immunity on the gene delivery, betaH1 was applied in rats immunized subcutaneously or intramuscularly with the parent HF strain. Rats were immunized subcutaneously with HF strain and 28 days later when the high antibody titer was maintained, betaH1 was inoculated into the right gastrocnemius muscle. Second, 35 days after inoculation with HF strain into the right gastrocnemius muscle, betaH1 was inoculated at the same site. In both ways of immunization, immunity did not abolish or prevent the transgene expression in the anterior horn motor neurons, but attenuated the range and the number of the beta-galactosidase-positive neurons from about 85% to 50-65% on 28 days after inoculation with betaH1. However, beta-galactosidase activity was observed in a wide range of the bilateral anterior horn motor neurons without significant pathological changes. These findings support the feasibility of the attenuated HSV vector in gene delivery into the central nervous system, even in the presence of immunity.

Ehrlichial Proliferation and Acute Hepatocellular Necrosis in Immunocompetent Mice Experimentally Infected with the HF Strain of Ehrlichia, Closely Related to Ehrlichia chaffeensis

Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis, is closely related to the HF strain of Ehrlichia isolated from ticks in Japan. In this study, BALB/c mice inoculated intraperitoneally with the HF strain developed severe illness and died at about day 9 post-inoculation. At necropsy, diffuse liver necrosis was evident. Ehrlichial microcolonies were observed in endothelial cells, monocytes and macrophages of the liver, bone marrow, spleen, thymus, and large and small intestine. Immunocompetent mice infected with the HF strain would provide a useful model for studying pathogenesis and immunity in acute and severe ehrlichiosis caused by E. chaffeensis and related Ehrlichia spp.

Differentiation of herpes simplex virus types 1 and 2 by plaque appearances on semicontinuous rabbit lens epithelial cells in the clinical laboratory.

Herpes simplex virus type 1 (HSV1) and type 2 (HSV2) were differentiated on the basis of different plaque appearance on semicontinuous rabbit lens epithelial (RLE) cells. Plaques produced by HSV1 strains were small; the mean diameter was 1.29 +/- 0.37 mm 3 days after inoculation. HSV2 strains produced large and small plaques, with the ratio of large to small about 20:1. The mean diameters of the large and the small plaques of HSV2 were 3.34 +/- 0.56 mm and 0.97 +/- 0.31 mm respectively 3 days after inoculation. The clones from the large plaques consistently produced large and small plaques and the small-plaque clones produced only small plaques. Round cells plus heterokaryotes were characteristic of the CPE of HSV1. Large plaques of HSV2 were produced by a large membranous syncytium that was liable to lyse. Small round cells were characteristic of the CPE of the small-plaque clones of HSV2. Glycoprotein C-negative (gC-) strains produced intermediate-sized plaques and a few pin point ones that consisted of membranous syncytia and round cells, respectively. Except for the HF strain (a reference strain of HSV1 producing a membranous syncytium on RLE cells), the result of the differentiation of HSV1 (179 strains) and HSV2 (40 strains) with the RLE plaque assay system was consistent with that of Syva's monoclonal antibody assay system and the restriction endonuclease digestion method.

Temporal development of the behavioral effects of herpes encephalitis in mice

The temporal development of behavioral effects due to central nervous system infection with the herpes simplex type 1 virus was examined in two mouse models. Following infection of adult female NYA/Nylar mice with the HF strain of herpes, or adult female BALB/c mice with the F strain, the majority of animals survived. An increase in motor activity observed 7 days following infection of NYA/Nylar mice coincided in time with a decline in brain virus titers. Likewise, an increase in errors occurring during serial-reversal performance in a water Y-maze was observed 8 days following infection of Balb/c mice and coincided with the declining phase of the viral growth curve. Taken together, these results suggest that processes involved in the elimination of the virus from the brain, such as the cellular immune response, may be important in the development of behavioral effects produced by nonfatal herpes encephalitis.

Eclipse of Coxsackievirus Infectivity: the Restrictive Event for a Non-fusing Myogenic Cell Line

Coxsackieviruses A2, A5 and B3 did not replicate in L8CL3-U cells (a non-fusing variant of the rat L8 myogenic cell line) although these cells possessed a common receptor for coxsackieviruses A2 and A5, and a different receptor for coxsackievirus B3. The restriction in replication was identified as a block in viral eclipse, since 6 M-LiCl treatment permitted recovery of the coxsackievirus A2 inoculum from L8CL3-U cells after 2 h at 37 degrees C, and the cells could be transfected by viral RNA. Cellular fusion which was induced in L8CL3-U cultures by herpes simplex virus type 1 (HF strain) facilitated coxsackievirus A2 and A5 replication. Differentiating myogenic L8 cells acquired full susceptibility to infection concurrently with the appearance of acetylcholine receptors, the muscle-specific isoenzyme of creatine phosphokinase, prominent myotube formation and the acquired capacity of the cells to eclipse virus.

Experimental Herpesvirus Keratitis in the Rabbit: Topical versus Intrastromal Infection Routes

Intrastromal and topical routes of infection of rabbit corneas with the HF strain of herpes simplex virus type 1 were compared clinically to determine which route of infection would present the best model of disciform edema and of deep stromal keratitis for our further studies on how the immune response of the infected animals may influence the expression of clinical disease. In addition, virus clearing and persistence of viral antigens as immunologic stimuli were monitored by virologic and electron microscopic immunocytochemical studies. We found topical infection to be preferable to the intrastromal infection route for presenting stromal disease in that it resulted in a higher incidence of epithelial disease with less anterior chamber involvement. The topical infection route model should be valuable in studies of cell-mediated immune response against HSV-infected cells.

Interaction of Ultraviolet-Irradiated Herpes Simplex Virus Type 1 with BSC-1 cells

Ultraviolet irradiation of herpes simplex virus (HSV) did not affect the transfer of uncoated virus DNA to the nuclei of infected cells but the synthesis of virus DNA was suppressed. The virus-specific DNA polymerase was synthesized in cells infected with the u.v.-irradiated HF strain of HSV. In cells infected with the u.v.-irradiated KOS strain, the virus DNA polymerase activity was hardly detectable. The two strains of HSV differ in the sensitivity of the virus DNA polymerase gene to u.v.-irradiation.

Inheritance and recombination of strain-specific characters in tobacco streak virus

The genetics of symptom determinants and serological type of tobacco streak virus (TSV) were investigated by picking single-lesion hybrid clones of virus originating from heterologous combinations of nucleoprotein components of strains with contrasting characters. A dwarfing of chronically diseased Xanthi-nc tobacco leaves required two factors from different components of the HF strain. In hybrid clones between HF and other strains, each factor was usually, but not always, perpetuated in the component that had provided it to the cross, and was masked. Its presence was demonstrated by backcrossing the hybrid to the HF parent or by crossing with other hybrid clones carrying a single dwarfing determinant in the complementary component. Factors conditioning toothed (t) or entire (e) margins on chronically diseased leaves were carried in each of the particle types. These factors were mutually exclusive when both were present in heterologous combinations of particles, the functional type apparently being determined by chance. The presence of two d (dwarfing) determinants masked t or e determinants, but did not prevent their replication. A determinant conditioning failure of Xanthi tobacco to recover from necrotic symptoms was carried only in the top component, and disappeared from functional particles on aging. Determinants for two serological types were also mutually exclusive and were carried by all three particle types. There appeared to be a linkage between the dwarfing determinants and the serological determinants. Recombinations of other characteristics (lesion type on Vigna cylindrica, chronic symptoms on Xanthi tobacco, and serological type) appeared about as often as would be expected on the basis of random assortment. Redundance in genetic information among particle types of TSV is common; recombination greatly increases variation.

Herpes simplex virus infection in rabbit eye.

Interferon or interferon-like virus inhibitor appears in rabbit cornea soon after infection with the HF strain of herpesvirus and prior to detection of local antibody. The early appearance of the virus inhibitor at the site of infection suggests it probably plays an important role in recovery from herpetic keratitis. Local antibody produced after initial herpesvirus infection may not be required for recovery. On the other hand, local antibody produced<i>after</i>primary virus infection plays a major role in resistance to<i>reinfection</i>with the same or related strain of herpesvirus. Serum antibody is of minor importance in resistance to reinfection and perhaps may be involved in controlling the severity of herpetic keratitis. Corneal resistance to reinfection herpetic keratitis wanes with time, and by day 90, corneas recovered from infection with herpesvirus are fully susceptible to reinfection.

Serological studies on herpes simplex viruses by neutralization kinetic test.

Neutralization kinetic analysis for appropriate seven strains of herpes simplex virus (HSV) was performed using guinea pig antisera. Six strains among those were different in the diagnosis or plaque sizes in African grivet monkey kidney (GMK) cells. In the first place, the reliability of neutralization rate constant (K values) was examined. K values of each antiserum against every virus strain were determined under the suitable condition and the normalized neutralization constants (NK values) were calculated for antigenic comparison. The average NK value against the standard HF strain of six heterologous antisera was 41, which was the lowest of those against the virus strains tested. On the other hand, Z strain was neutralized most rapidly by heterologous antisera. The average NK value was 94 which was the highest value. For the serological differentiation by NK value, anti-HF serum was unsuitable. On the contrary, NK values of anti-Z serum were the lowest and the most separate values against the heterologous strains, so that it will be the most excellent for serological differentiation. The results were not so successively feasible as might have been expected. The some reasons were discussed.

Thermoinactivation of HF and M strains of herpes simplex virus in various conditions.

A comparison was made for HF and M strains of herpes simplex virus for their thermosensitivity in various conditions. The infectivity of herpes viruses were preserved well by 10% bovine serum and 1% skim milk in PBS at 45°C for 10 minutes. The titers of HF and M strains showed pretty well preservations by PBS, MLE and 10% glycerin in PBS. The thermostabilizing effects of 10% DMSO, 1 M MgCl2, 1 M Na2SO4 in PBS and distilled water were fairly inferior. The thermostability of M strain under the various conditions was greater than HF strain. The range of optimal pH for high thermostability was more comprehensive in M strain as compared to HF strain.

Plaque assay for herpes simplex virus in L-929 (Earle) mouse fibroblasts.

SummaryA plaque assay method for the HF strain of herpes simplex virus using L-cells under methyl cellulose has been described. Its ease in preparation, the reproducibility of results, the distinctness of the plaques, and the short period of time required for plaques to appear make it a suitable technic for use in classroom experiments and in research.