Acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein

In feline coronavirus (FCoV) pathogenesis, the ability to infect macrophages is an essential virulence factor. Whereas the low-virulence feline enteric coronavirus (FECV) isolates primarily replicate in the epithelial cells of the enteric tract, highly virulent feline infectious peritonitis virus (FIPV) isolates have acquired the ability to replicate efficiently in macrophages, which allows rapid dissemination of the virulent virus throughout the body. FIPV 79-1146 and FECV 79-1683 are two genetically closely related representatives of the two pathotypes. Whereas FECV 79-1683 causes at the most a mild enteritis in young kittens, FIPV 79-1146 almost invariably induces a lethal peritonitis. The virulence phenotypes correlate with the abilities of these viruses to infect and replicate in macrophages, a feature of FIPV 79-1146 but not of FECV 79-1683. To identify the genetic determinants of the FIPV 79-1146 macrophage tropism, we exchanged regions of its genome with the corresponding parts of FECV 79-1683, after which the ability of the FIPV/FECV hybrid viruses to infect macrophages was tested. Thus, we established that the FIPV spike protein is the determinant for efficient macrophage infection. Interestingly, this property mapped to the C-terminal domain of the protein, implying that the difference in infection efficiency between the two viruses is not determined at the level of receptor usage, which we confirmed by showing that infection by both viruses was equally blocked by antibodies directed against the feline aminopeptidase N receptor. The implications of these findings are discussed.     

ARROGANT: an application to manipulate large gene collections

ARROGANT (ARRay OrGANizing Tool) is a software tool developed to facilitate the identification, annotation and comparison of large collections of genes or clones. The objective is to enable users to compile gene/clone collections from different databases, allowing them to design experiments and analyze the collections as well as associated experimental data efficiently. ARROGANT can relate different sequence identifiers to their common reference sequence using the UniGene database, allowing for the comparison of data from two different microarray experiments. ARROGANT has been successfully used to analyze microarray expression data for colon cancer, to compile genes potentially related to cardiac diseases for subsequent resequencing (to identify single nucleotide polymorphisms, SNPs), to design a new comprehensive human cDNA microarray for cancer, to combine and compare expression data generated by different microarrays and to provide annotation for genes on custom and Affymetrix chips.     

Factors that increase the effective concentration of CXCR4 dictate feline immunodeficiency virus tropism and kinetics of replication

The surface glycoprotein (gp95) of the feline immunodeficiency virus (FIV) binds in a strain-specific manner to several cell surface molecules, including CXCR4, heparan sulfate proteoglycans (HSPGs), DC-SIGN, and a 43-kDa cell surface receptor on T cells recently identified as CD134 by M. Shimojima et al. (Science 303:1192-1195, 2004). CXCR4 is the entry receptor in all known cases, and the other molecules act as binding receptors to help facilitate infection. In this report, we confirm and extend the findings regarding CD134 as a primary receptor for FIV. In addition, we show that temperature critically influences the binding properties of FIV gp95 to CXCR4 and HSPGs. The data show that gp95 of the field strain FIV-PPR bound to CXCR4 at 22 degrees C, whereas binding was not detected at 4 degrees C. In contrast, binding of the laboratory adapted FIV-34TF10 gp95 was observed at either 4 degrees C or 22 degrees C, albeit at increased levels at the higher temperature. The level of CXCR4 increased after the temperature was switched from 4 to 22 degrees C, whereas the level of HSPGs decreased, resulting in higher binding of gp95 from both strains to CXCR4 and lower binding of gp95 of FIV-34TF10 to HSPGs (FIV-PPR gp95 does not bind to these molecules). The findings also show that HSPGs facilitate the CXCR4-mediated infectivity of CrFK and G355-5 cells by FIV-34TF10. These two nonlymphoid cell lines express very low levels of CXCR4 and are permissive to FIV-34TF10 but not to productive infection by FIV-PPR. However, overexpression of human CXCR4 in CrFK or G-355-5 cells resulted in extensive cell fusion and infection by FIV-PPR. Taken together, these findings indicate that factors that increase the effective concentration of CXCR4 enhance FIV infectivity and may involve (i) temperature or ligand-induced conformational changes in CXCR4 that enhance SU binding, (ii) coreceptor interactions with gp95 that either alter gp95 conformation to enhance CXCR4 binding and/or raise the localized concentration of receptor or ligand, or (iii) direct increase in CXCR4 concentration via overexpression.     

The Dictyostelium genome project: an invitation to species hopping.

Dictyostelium allows some of the general problems of eukaryotic biology to be addressed by using molecular genetic tools that are more normally associated with yeast. The genome project, now underway, marks an important increase in the attractiveness of Dictyostelium as an experimental organism and will invite increased 'species hopping' by experimenters. We provide a brief guide to the problems that are being addressed in Dictyostelium, to the genome project itself and to the molecular genetic tools available for its exploitation.     

地下水脱卤过程中的微生物种间代谢互作：提高原位氯代烯烃厌氧脱氯效能的有效途径

有机卤呼吸细菌(organohalide-respiring bacteria, OHRB)在氯代烯烃污染地下水的原位生物修复中扮演着关键性的角色,提高其丰度及活性对氯代烯烃的完全去除具有重要意义。在实际环境中,有机卤呼吸细菌往往与多种微生物共存,微生物种间代谢互作现象十分普遍,有机污染物的完全无害化往往需要通过微生物菌群的协同代谢作用来实现。因此,本文围绕微生物种间代谢互作进行综述,对目前获得的脱氯微生物菌种资源及脱氯机理进行了回顾,重点阐述了专性OHRB、非专性OHRB和非OHRB的种间代谢互作行为及机制,并提出以种间代谢互作为指导进行合成微生物群落的构建来有效提高氯代烯烃厌氧生物降解效率,为实现环境氯代烯烃类有机污染物的快速、彻底无害化提供理论指导。     

An effective way to determine the melting curve

A new ‘two-phase’ simulation method with which to accurately predict the melting curve is proposed. The method requires, as a starting configuration, generating a two-phase coexistence state by employing a suitable ensemble. Examining a change in volume ratio of the two phases upon varying temperature (pressure) under a fixed pressure (temperature) allows us to determine the phase transition point. The Clausius–Clapeyron relationship can then be implemented as a guide to predict the nearby phase transition point. The method was applied to determine the solid–liquid phase boundary of the modified Lennard–Jones system. A better accuracy, as that achieved by the non-equilibrium relaxation method (Asano Y, Fuchizaki K. J Phys Soc Jpn. 2017;86:025001), was obtained but with much less computational cost.     

PEPCRE: an interactive program to create,manipulate and display oligopeptides

PEPCRE is an interactive computer graphics program for the rapid construction, manipulation and display of oligopeptides. Essentially any desired conformation of an oligopeptide can be constructed in a simple and straightforward manner. The program provides various display and output possibilities. It is user-friendly and is written in FORTRAN 77 for use on inexpensive, monochrome graphics terminals.     

引用宗教是保护好野生动植物的有效途径

引用宗教是保护好野生动植物的有效途径野生动植物保护是一项社会公益事业,其兴衰成败直接影响到人类的生存和发展。我国野生动植物资源十分丰富,面临的保护任务艰巨而繁重。要保护好野生动植物,从措施上讲,一靠充裕有效的资金投入,二靠广泛自觉的公众参与。由于我...     

Growth dynamics of Dracaena cinnabari under controlled conditions as the most effective way to protect endangered species

Dracaena cinnabari Balf. fil. is an endangered endemic species growing on the Yemeni island of Soqotra. Dracaena woodlands are considered as one of the oldest forest communities on Earth. Uncontrolled grazing unfortunately caused a lack of naturally occurring regeneration. Our two-year research was focused on the growth dynamics of Dracaena seedlings from two separate populations. One hundred of germinated seeds from two different altitudes from the island were sown and planted under the same conditions. Average increment and difference between the growth dynamics of plants from the two localities were investigated. The observed data on this plant species revealed very interesting, hitherto unknown results. (1) The seedlings germinated within a time period from four to ten weeks. Germination rate was 90% on the Firmihin highland plateau and 78% on the Scand Mountain. (2) Average plant length from both localities was almost the same (24.9 cm) at the end of measurement. Differences in values between the two populations proved as non-significant. (3) A significant difference was found in the number of leaves and in the sum of lengths of all leaves on one plant. While the seedlings from Firmihin featured a wide spreading above-ground part with a large number of leaves, the plants from Scand invested more energy into faster leaves elongation rate. (4) Growth dynamics reflected seasonal changes. Increments were slower or ceased during the period of vegetative rest from autumn to spring. (5) Average mortality rate was 13%. Most of the plants died during the period of vegetative rest. Further study on germination and regeneration under artificial conditions seems like the only way to prevent species extinction.     

Binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity

The binding of viruses to host cells is the first step in determining tropism and pathogenicity. While avian infectious bronchitis coronavirus (IBV) infection and avian influenza A virus (IAV) infection both depend on α2,3-linked sialic acids, the host tropism of IBV is restricted compared to that of IAV. Here we investigated whether the interaction between the viral attachment proteins and the host could explain these differences by using recombinant spike domains (S1) of IBV strains with different pathogenicities, as well as the hemagglutinin (HA) protein of IAV H5N1. Protein histochemistry showed that S1 of IBV strain M41 and HA of IAV subtype H5N1 displayed sialic acid-dependent binding to chicken respiratory tract tissue. However, while HA bound with high avidity to a broad range of α2,3-linked sialylated glycans, M41 S1 recognized only one particular α2,3-linked disialoside in a glycan array. When comparing the binding of recombinant IBV S1 proteins derived from IBV strains with known differences in tissue tropism and pathogenicity, we observed that while M41 S1 displayed binding to cilia and goblet cells of the chicken respiratory tract, S1 derived from the vaccine strain H120 or the nonvirulent Beaudette strain had reduced or no binding to chicken tissues, respectively, in agreement with the reduced abilities of these viruses to replicate in vivo. While the S1 protein derived from the nephropathogenic IBV strain B1648 also hardly displayed binding to respiratory tract cells, distinct binding to kidney cells was observed, but only after the removal of sialic acid from S1. In conclusion, our data demonstrate that the attachment patterns of the IBV S proteins correlate with the tropisms and pathogenicities of the corresponding viruses.     

2019新型冠状病毒基因组的生物信息学分析

2019年12月,中国武汉报道了冠状病毒引起的肺炎,其临床症状与2003年爆发的严重急性呼吸综合征(Severe Acute Respiratory Syndrome, SARS)不同,因此推断该病毒可能是冠状病毒的一个新变种。不同于简单使用全基因组序列的其它研究,我们于2018年在国际上首次提出分子功能与进化分析相结合的研究思想,并应用于Beta冠状病毒B亚群(BB冠状病毒)基因组的研究。在这一思想指导下,本研究使用BB冠状病毒基因组中的一个互补回文序列(命名为Nankai complemented palindrome)与其所在的编码区(命名为Nankai CDS)对新发布的2019新型冠状病毒基因组(GenBank:MN908947)进行分析以期准确溯源,并对BB冠状病毒的跨物种传播和宿主适应性进行初步研究。溯源分析的结果支持2019新型冠状病毒源自蝙蝠,但与SARS冠状病毒差异巨大,这一结果与两者临床症状差异一致。本研究的最重要发现是BB冠状病毒存在大量的可变翻译,从分子水平揭示了BB冠状病毒变异快、多样性高的特点。从BB冠状病毒可变翻译中获取的信息可应用于(但不限于)其快速检测、基因分型、疫苗开发以及药物设计。另外,我们推断BB冠状病毒可能通过可变翻译以适应不同宿主。基于大量基因组数据的实证分析,本研究在国际上首次从分子水平尝试解释了BB冠状病毒变异快、宿主多且具有较强的宿主适应性的原因。     

Targeted genotyping by sequencing: a new way to genome profile the cat

Targeted GBS is a recent approach for obtaining an effective characterization for hundreds to thousands of markers. The high throughput of next‐generation sequencing technologies, moreover, allows sample multiplexing. The aims of this study were to (i) define a panel of single nucleotide polymorphisms (SNPs) in the cat, (ii) use GBS for profiling 16 cats, and (iii) evaluate the performance with respect to the inference using standard approaches at different coverage thresholds, thereby providing useful information for designing similar experiments. Probes for sequencing 230 variants were designed based on the Felis_catus_8.0. 8.0 genome. The regions comprised anonymous and non‐anonymous SNPs. Sixteen cat samples were analysed, some of which had already been genotyped in a large group of loci and one having been whole‐genome sequenced in the 99_Lives Cat Genome Sequencing Project. The accuracy of the method was assessed by comparing the GBS results with the genotypes already available. Overall, GBS achieved good performance, with 92–96% correct assignments, depending on the coverage threshold used to define the set of trustable genotypes. Analyses confirmed that (i) the reliability of the inference of each genotype depends on the coverage at that locus and (ii) the fraction of target loci whose genotype can be inferred correctly is a function of the total coverage. GBS proves to be a valid alternative to other methods. Data suggested a depth of less than 11× is required for greater than 95% accuracy. However, sequencing depth must be adapted to the total size of the targets to ensure proper genotype inference.     

The first karyogram of a Bromeliaceae species: an allopolyploid genome

The Bromeliaceae family has been traditionally distributed in the subfamilies Bromelioideae, Tillandsioideae and Pitcairnioideae. However, phylogenetic studies have provided other classifications, highlighting the need for analyses in order to characterize the genome of different species from this family. In this sense, the present work aimed to determine nuclear 2C-value and base composition, characterize the chromosomes and establish the karyogram of Pitcairnia flammea. Flow cytometry yielded 2C = 1.44 pg, AT = 64.28 % and GC = 35.72 % for this species, indicating its relatively small genome size. Despite reduced length and morphological similarity of the chromosomes, P. flammea metaphases presented well-spread chromosomes, with well-defined primary constriction, without chromatin damage and cytoplasmic background. These aspects allowed morphometric chromosomal characterization and assembly of the first karyogram of a Bromeliaceae species. The karyogram displayed 2n = 50 chromosomes, of which all were submetacentric. Karyomorphological analysis revealed grouped pairs of cytogenetically identical chromosomes (2–3, 4–5, 6–9, 10–17, 18–19, 20–23 and 24–25), plus one isolated chromosome (1), not identical to any other. This result suggests an allopolyploid origin for the P. flammea genome. Thus, the present investigation contributed with karyotype data for taxonomic and evolutionary aspects of this group.     

Phyto-activity of biocides used to manipulate herbivory: tests of three pesticides on fourteen plant species

1. We tested three pesticides used for field manipulations of herbivory for direct phytoactive effects on the germination and growth of 14 herbaceous plant species selected to provide a range of life-history strategies and functional groups.2. We report three companion experiments: (A) Two insecticides, chlorpyrifos (granular soil insecticide) and dimethoate (foliar spray), were applied in fully-factorial combination to pot-germinated individuals of 12 species. (B) The same fully-factorial design was used to test for direct effects on the germination of four herbaceous legumes. (C) The molluscicide, metaldehyde, was tested for direct effects on the germination and growth of six plant species.3. The insecticides had few significant effects on growth and germination. Dimethoate acted only on growth stimulating Anisantha sterilis, Sonchus asper and Stellaria graminea. In contrast, chlorpyrifos acted on germination increasing the germination of Trifolium dubium and Trifolium pratense. There was also a significant interactive effect of chlorpyrifos and dimethoate on the germination of T. pratense. However, all effects were relatively small in magnitude and explanatory power. The molluscicide had no significant effect on plant germination or growth.4. The small number and size of direct effects of the pesticides on plant performance is encouraging for the use of these pesticides in manipulative experiments on herbivory, especially for the molluscicide. However, a small number of direct (positive) effects of the insecticides on some plant species need to be taken into account when interpreting field manipulations of herbivory with these compounds, and emphasises the importance of conducting tests for direct phyto-active effects.

Zusammenfassung

1. Wir untersuchten den phytoaktiven Einfluss von drei, in Feldexperimenten eingesetzten Pestiziden, auf die Keimfähigkeit und auf das Wachstum von 14 Krautpflanzen Arten mit verschiedenen Life-history Strategien und aus unterschiedlichen funktionalen Gruppen.2. Wir führten drei Experimente durch: (A) Zwei Insektizide, chlorpyrifos (ein Bodenkörniges Insektizid) und dimethoate (ein Blatt-Insektizid), wurden in allen möglichen Kombinationen (fully-factorial) bei Topfpflanzen von 12 Arten angewendet. (B) Dasselbe Design wurde verwendet, um direkte Einflüsse auf die Keimfähigkeit von vier krautigen Leguminosen zu untersuchen. (C) Die Wirkung des Molluskizids metaldehyde auf Keimfähigkeit und Wachstum wurde auf sechs Planzenarten getestet.3. Bei den Insektiziden fanden wir wenige signifikante Einflüsse auf die Keimfähigkeit und das Wachstum. Dimethoate hatte einen positiven Einfluss auf das Wachstum von Anisantha sterilis, Sonchus asper und Stellaria graminea. Während Chlorpyrifos eine erhöhte Keimfähigkeit bei Trifolium dubium und Trifolium pratense bewirkte. Chlorpyrifos und dimethoate zeigten ausserdem eine signifikante interaktion, auf die Keimfähigkeit von T. pratense. Alle gemessenen Einflüsse waren relativ klein und mit nur geringen Erklärungswort. Das Molluskizid hatte keinen signifikanten Effekt auf die Keimfähigkeit und das Wachstum der Pflanzen.4. Die geringen direkten Auswirkungen der Pestizide auf die Pflanzenfitness den Einsatz dieser Mittel bei Herbivore Experimentieren. Besonders das Molluskizid eignet sich zur Anwendung. Trotzdem müssen die wenigen (positiven) Einflüsse der Insektizide auf einige Pflanzenarten bei der Interpretation von Pflanzenfrassexperimenten, die diese Arten untersuchen, berücksichtigt werden. Unser Ergebnis zeigt wie wichtig es ist direkte phytoaktive Auswirkungen von Pestiziden testen.     

Functional differences in the peplomer glycoproteins of feline coronavirus isolates.

The feline coronaviruses can be divided into two distinct antigenic groups on the basis of antigenic differences found on the peplomer (E2) glycoprotein of the virus. Because the E2 glycoprotein is responsible for many of the biological functions of coronaviruses, experiments were done to determine whether there were any E2 functional differences between these two antigenic groups. The avirulent feline infectious peritonitis virus (FIPV) isolate FIPV-UCD-2, which has one antigenic type of E2, was less rapidly internalized and could not spread from cell to cell in the presence of neutralizing antibody. Two virulent isolates, FIPV-DF2 and FIPV-79-1146, as well as the non-FIP-causing feline enteric coronavirus (FECV) isolate FECV-79-1683, all of which have the second antigenic type of E2, were very rapidly internalized and were able to spread from cell to cell despite the presence of neutralizing antibody. The avirulent FIPV-UCD-2 and FECV-79-1683 isolates were more labile at 37 degrees C at pHs of 6.5 and above than were the virulent FIPV-DF2 and FIPV-79-1146 isolates.