Traveling Waves and Spread Rates for a West Nile Virus Model

A reaction–diffusion model for the spatial spread of West Nile virus is developed and analysed. Infection dynamics are based on a modified version of a model for cross infection between birds and mosquitoes (Wonham et al., 2004, An epidemiological model for West-Nile virus: Invasion analysis and control application. Proc. R. Soc. Lond. B 271), and diffusion terms describe movement of birds and mosquitoes. Working with a simplified version of the model, the cooperative nature of cross-infection dynamics is utilized to prove the existence of traveling waves and to calculate the spatial spread rate of infection. Comparison theorem results are used to show that the spread rate of the simplified model may provide an upper bound for the spread rate of a more realistic and complex version of the model.     

Discovery of Bovine Digital Dermatitis-Associated Treponema spp. in the Dairy Herd Environment by a Targeted Deep-Sequencing Approach

The bacteria associated with the infectious claw disease bovine digital dermatitis (DD) are spirochetes of the genus Treponema; however, their environmental reservoir remains unknown. To our knowledge, the current study is the first report of the discovery and phylogenetic characterization of rRNA gene sequences from DD-associated treponemes in the dairy herd environment. Although the spread of DD appears to be facilitated by wet floors covered with slurry, no DD-associated treponemes have been isolated from this environment previously. Consequently, there is a lack of knowledge about the spread of this disease among cows within a herd as well as between herds. To address the issue of DD infection reservoirs, we searched for evidence of DD-associated treponemes in fresh feces, in slurry, and in hoof lesions by deep sequencing of the V3 and V4 hypervariable regions of the 16S rRNA gene coupled with identification at the operational-taxonomic-unit level. Using treponeme-specific primers in this high-throughput approach, we identified small amounts of DNA (on average 0.6% of the total amount of sequence reads) from DD-associated treponemes in 43 of 64 samples from slurry and cow feces collected from six geographically dispersed dairy herds. Species belonging to the Treponema denticola/Treponema pedis-like and Treponema phagedenis-like phylogenetic clusters were among the most prevalent treponemes in both the dairy herd environment and the DD lesions. By the high-throughput approach presented here, we have demonstrated that cow feces and environmental slurry are possible reservoirs of DD-associated treponemes. This method should enable further clarification of the etiopathogenesis of DD.     

Validation of a Demographic Model for Woodland Caribou

Abstract: Wildlife population models are potentially valuable for conservation planning. Validation is necessary to ensure that models are sufficiently robust for predicting management outcomes consistent with conservation objectives. Sorensen et al. (2008) produced a model of woodland caribou (Rangifer tarandus) population growth rate that was recently modified and used as a predictive tool at several scales. We computed confidence intervals and evaluated the performance of this model using novel data. Confidence intervals were wide, and results suggested that the model may have a positive bias, resulting in over-estimation of population growth rates, as well as low predictive power. Wide confidence intervals mean that current understanding of factors governing woodland caribou herd dynamics is not sufficient for wildlife managers to make reliable projections of responses to management.     

A Quantitative Assay for Measuring of Bovine Immunodeficiency Virus Using a Luciferase-based Indicator Cell Line

In order to quantitate the bovine immunodeficiency virus (BIV) infection in vitro, a BIV indicator cell line (BIVL) was established by transfecting baby hamster kidney cells with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the B...     

A Stochastic Regression Model for General Trend Analysis of Longitudinal Continuous Data

A predictive continuous time model is developed for continuous panel data to assess the effect of time‐varying covariates on the general direction of the movement of a continuous response that fluctuates over time. This is accomplished by reparameterizing the infinitesimal mean of an Ornstein–Uhlenbeck processes in terms of its equilibrium mean and a drift parameter, which assesses the rate that the process reverts to its equilibrium mean. The equilibrium mean is modeled as a linear predictor of covariates. This model can be viewed as a continuous time first‐order autoregressive regression model with time‐varying lag effects of covariates and the response, which is more appropriate for unequally spaced panel data than its discrete time analog. Both maximum likelihood and quasi‐likelihood approaches are considered for estimating the model parameters and their performances are compared through simulation studies. The simpler quasi‐likelihood approach is suggested because it yields an estimator that is of high efficiency relative to the maximum likelihood estimator and it yields a variance estimator that is robust to the diffusion assumption of the model. To illustrate the proposed model, an application to diastolic blood pressure data from a follow‐up study on cardiovascular diseases is presented. Missing observations are handled naturally with this model.     

乙型肝炎病毒急性感染小鼠模型的建立

采用高压水注射方法,通过尾静脉将具有复制能力的HBV质粒导入BABL/cJ小鼠体内,应用real-time PCR、ELISA、RIA、Southern Blot、Northern Blot,以及免疫组化等方法,检测小鼠病毒血症、血清和肝组织中HBV 抗原表达动态变化、肝组织中HBV转录和复制情况,以及小鼠免疫应答状况。结果HBV基因可以在小鼠体内表 达和复制,并诱导小鼠产生特异性免疫应答,其应答模式及HBV清除过程与人类的HBV急性感染类似。实验显 示高压注射具有复制能力的HBV质粒可以在小鼠体内建立HBV急性感染模型,这种模型可以用于HBV病毒 学、免疫学以及抗病毒药物筛选等方向的研究。     

新甲型H1N1流感病毒小鼠致死模型的建立

建立新甲型H1N1流感病毒小鼠致死模型,为研究致病性、宿主适应性以及疫苗保护性提供动物模型,并寻找病毒在适应宿主过程中影响毒力和适应性的关键位点。将新甲型H1N1流感病毒A/四川/SWL1/2009 H1N1在小鼠中连续传15代,各代次毒株均在MDCK细胞上增殖后进行测序,根据序列分析结果选择6个传代毒株感染小鼠,连续监测14 d体重和死亡情况;并对第14代和15代病毒在噬斑实验纯化后克隆和测序分析。原代病毒不致死BABL/C小鼠,经动物体内连续传代适应宿主动物后,其毒力增强,具体表现为所选的6个传代毒株中第7、11、15代毒株可以100%致死试验小鼠;分析这6个传代毒株的全基因组表明这些毒株的部分氨基酸位点发生突变。新甲型H1N1流感病毒经小鼠体内连续传代后,建立了小鼠致死模型,病毒毒力增强可能与某些氨基酸位点的改变有关。     

Validation of a mathematical model of the bovine estrous cycle for cows with different estrous cycle characteristics

A recently developed mechanistic mathematical model of the bovine estrous cycle was parameterized to fit empirical data sets collected during one estrous cycle of 31 individual cows, with the main objective to further validate the model. The a priori criteria for validation were (1) the resulting model can simulate the measured data correctly (i.e. goodness of fit), and (2) this is achieved without needing extreme, probably non-physiological parameter values. We used a least squares optimization procedure to identify parameter configurations for the mathematical model to fit the empirical in vivo measurements of follicle and corpus luteum sizes, and the plasma concentrations of progesterone, estradiol, FSH and LH for each cow. The model was capable of accommodating normal variation in estrous cycle characteristics of individual cows. With the parameter sets estimated for the individual cows, the model behavior changed for 21 cows, with improved fit of the simulated output curves for 18 of these 21 cows. Moreover, the number of follicular waves was predicted correctly for 18 of the 25 two-wave and three-wave cows, without extreme parameter value changes. Estimation of specific parameters confirmed results of previous model simulations indicating that parameters involved in luteolytic signaling are very important for regulation of general estrous cycle characteristics, and are likely responsible for differences in estrous cycle characteristics between cows.     

Transgenic Melon and Squash Expressing Coat Protein Genes of Aphid-borne Viruses do not Assist the Spread of an Aphid Non- transmissible Strain of Cucumber Mosaic Virus in the Field

Transgenic melon and squash containing the coat protein (CP) gene of the aphid transmissible strain WL of cucumber mosaic cucumovirus (CMV) were grown under field conditions to determine if they would assist the spread of the aphid non-transmissible strain C of CMV, possibly through heterologous encapsidation and recombination. Transgenic melon were susceptible to CMV strain C whereas transgenic squash were resistant although the latter occasionally developed chlorotic blotches on lower leaves. Transgenic squash line ZW-20, one of the parents of commercialized cultivar Freedom II, which expresses the CP genes of the aphid transmissible strains FL of zucchini yellow mosaic (ZYMV) and watermelon mosaic virus 2 (WMV 2) potyviruses was also tested. Line ZW-20 is resistant to ZYMV and WMV 2 but is susceptible to CMV. Field experiments conducted over two consecutive years showed that aphid-vectored spread of CMV strain C did not occur from any of the CMV strain C-challenge inoculated transgenic plants to any of the uninoculated CMV-susceptible non- transgenic plants. Although CMV was detected in 3% (22/764) of the uninoculated plants, several assays including ELISA, RT- PCR-RFLP, identification of CP amino acid at position 168, and aphid transmission tests demonstrated that these CMV isolates were distinct from strain C. Instead, they were non-targeted CMV isolates that came from outside the field plots. This is the first report on field experiments designed to determine the potential of transgenic plants expressing CP genes for triggering changes in virus-vector specificity. Our results indicate that transgenic plants expressing CP genes of aphid transmissible strains of CMV, ZYMV, and WMV 2 are unlikely to mediate the spread of aphid non-transmissible strains of CMV. This finding is of practical relevance because transgenic crops expressing the three CP genes are targeted for commercial release, and because CMV is economically important, has a wide host range, and is widespread worldwide.     

Development and Validation of a Gene-Based Model for Outcome Prediction in Germ Cell Tumors Using a Combined Genomic and Expression Profiling Approach

Germ Cell Tumors (GCT) have a high cure rate, but we currently lack the ability to accurately identify the small subset of patients who will die from their disease. We used a combined genomic and expression profiling approach to identify genomic regions and underlying genes that are predictive of outcome in GCT patients. We performed array-based comparative genomic hybridization (CGH) on 53 non-seminomatous GCTs (NSGCTs) treated with cisplatin based chemotherapy and defined altered genomic regions using Circular Binary Segmentation. We identified 14 regions associated with two year disease-free survival (2yDFS) and 16 regions associated with five year disease-specific survival (5yDSS). From corresponding expression data, we identified 101 probe sets that showed significant changes in expression. We built several models based on these differentially expressed genes, then tested them in an independent validation set of 54 NSGCTs. These predictive models correctly classified outcome in 64–79.6% of patients in the validation set, depending on the endpoint utilized. Survival analysis demonstrated a significant separation of patients with good versus poor predicted outcome when using a combined gene set model. Multivariate analysis using clinical risk classification with the combined gene model indicated that they were independent prognostic markers. This novel set of predictive genes from altered genomic regions is almost entirely independent of our previously identified set of predictive genes for patients with NSGCTs. These genes may aid in the identification of the small subset of patients who are at high risk of poor outcome.     

Phylogeography Reveals Association between Swine Trade and the Spread of Porcine Epidemic Diarrhea Virus in China and across the World

The ongoing SARS (severe acute respiratory syndrome)-CoV (coronavirus)-2 pandemic has exposed major gaps in our knowledge on the origin, ecology, evolution, and spread of animal coronaviruses. Porcine epidemic diarrhea virus (PEDV) is a member of the genus Alphacoronavirus in the family Coronaviridae that may have originated from bats and leads to significant hazards and widespread epidemics in the swine population. The role of local and global trade of live swine and swine-related products in disseminating PEDV remains unclear, especially in developing countries with complex swine production systems. Here, we undertake an in-depth phylogeographic analysis of PEDV sequence data (including 247 newly sequenced samples) and employ an extension of this inference framework that enables formally testing the contribution of a range of predictor variables to the geographic spread of PEDV. Within China, the provinces of Guangdong and Henan were identified as primary hubs for the spread of PEDV, for which we estimate live swine trade to play a very important role. On a global scale, the United States and China maintain the highest number of PEDV lineages. We estimate that, after an initial introduction out of China, the United States acted as an important source of PEDV introductions into Japan, Korea, China, and Mexico. Live swine trade also explains the dispersal of PEDV on a global scale. Given the increasingly global trade of live swine, our findings have important implications for designing prevention and containment measures to combat a wide range of livestock coronaviruses.     

Validation of a full body finite element model (THUMS) for running-type impacts to the lower extremity

The purpose of this study was to determine whether modifying an existing, highly biofidelic full body finite element model [total human model for safety (THUMS)] would produce valid amplitude and temporal shock wave characteristics as it travels proximally through the lower extremity. Modifying an existing model may be more feasible than developing a new model, in terms of cost, labour and expertise. The THUMS shoe was modified to more closely simulate the material properties of a heel pad. Relative errors in force and acceleration data from experimental human pendulum impacts and simulated THUMS impacts were 22% and 54%, respectively, across the time history studied. The THUMS peak acceleration was attenuated by 57.5% and took 19.7 ms to travel proximally along the lower extremity. Although refinements may be necessary to improve force and acceleration timing, the modified THUMS represented, to a certain extent, shock wave propagation and attenuation demonstrated by living humans under controlled impact conditions.     

对虾白斑综合征杆状病毒体内增殖模型的建立

应用对虾白斑综合征杆状病毒（WSSV）,对淡水克氏螯虾、罗氏沼虾、日本沼虾和两种淡水蟹（中华绒螯蟹、长江华溪蟹）进行人工感染实验。结果除淡水克氏螯虾之外,其它受试的虾蟹均不能感染WSSV。克氏螯虾3个不同剂量级感染至12d平均死亡率为94％。从发病或死亡个体采集血淋巴,经电镜负染色可观察到完整的病毒粒子,其形态大小、靶细胞组织病理均与从中国对虾中分离的WSSV相似或相同。同时,通过原位杂交技术进一步证明该实验的可靠性。克氏螯虾重复感染效果良好,有可能成为研究WSSV的一种理想的病毒体内增殖模型。     

丙型肝炎病毒NS3/4A丝氨酸蛋白酶瞬时表达小鼠模型的建立

目的:建立丙型肝炎病毒NS3/4A丝氨酸蛋白酶体内活性评价模型。方法:利用NS4A/B是NS3/4A丝氨酸蛋白酶作用底物的特性,构建融合基因NS3/NS4A/B-SEAP,底物片段NS4A/B插在NS3/4A和人分泌性碱性磷酸酶(SEAP)之间,融合基因表达后SEAP的分泌依赖于有活性的NS3/4A在NS4A/B位点的切割。将含融合基因的质粒NS3/4A(△4AB)SEAP通过水动力转染技术转染到小鼠体内,检测小鼠血清中SEAP的活性,高活性的SEAP是该评价体系成立的证据。结果与结论:在瞬时表达NS3/4A的小鼠血清中检测到了高活性的SEAP,建立了可用于评价抗NS3/4A的小鼠体内瞬时模型。     

Functional and Biochemical Characterization of Hepatitis C Virus (HCV) Particles Produced in a Humanized Liver Mouse Model

Lipoprotein components are crucial factors for hepatitis C virus (HCV) assembly and entry. As hepatoma cells producing cell culture-derived HCV (HCVcc) particles are impaired in some aspects of lipoprotein metabolism, it is of upmost interest to biochemically and functionally characterize the in vivo produced viral particles, particularly regarding how lipoprotein components modulate HCV entry by lipid transfer receptors such as scavenger receptor BI (SR-BI). Sera from HCVcc-infected liver humanized FRG mice were separated by density gradients. Viral subpopulations, termed HCVfrg particles, were characterized for their physical properties, apolipoprotein association, and infectivity. We demonstrate that, in contrast to the widely spread distribution of apolipoproteins across the different HCVcc subpopulations, the most infectious HCVfrg particles are highly enriched in apoE, suggesting that such apolipoprotein enrichment plays a role for entry of in vivo derived infectious particles likely via usage of apolipoprotein receptors. Consistent with this salient feature, we further reveal previously undefined functionalities of SR-BI in promoting entry of in vivo produced HCV. First, unlike HCVcc, SR-BI is a particularly limiting factor for entry of HCVfrg subpopulations of very low density. Second, HCVfrg entry involves SR-BI lipid transfer activity but not its capacity to bind to the viral glycoprotein E2. In conclusion, we demonstrate that composition and biophysical properties of the different subpopulations of in vivo produced HCVfrg particles modulate their levels of infectivity and receptor usage, hereby featuring divergences with in vitro produced HCVcc particles and highlighting the powerfulness of this in vivo model for the functional study of the interplay between HCV and liver components.     

Human Exposure to Malathion during a Possible Vector-Control Intervention against West Nile Virus. II: Evaluation of the Toxicological Risks Using a Probabilistic Approach

In order to prevent the propagation of West Nile Virus (WNV), insecticide sprayings have been carried out in several locations in North America since 1999 with the objective of controlling the mosquito populations that transmit this pathogen. An attempt to quantitatively compare the risk of developing a health response to WNV infection with the toxicological risk of insecticides is presented here. First, the acute and subchronic environmental concentrations resulting from repeated spraying events were modeled according to a reasonable worst-case spraying sequence established in an intervention program proposed by the Government of Quebec (Canada). Second, probability density functions (PDF) were established for some exposure parameters according to the data for the concerned population, when feasible. Monte Carlo analyses were performed by incorporating these PDF into the equations used to calculate the daily absorbed doses resulting from the exposure scenarios presented in the companion article (this issue). The results showed that for a significant proportion of the population, aerial and, to a lesser extent, ground sprayings of malathion can generate acute and subchronic exposure that may exceed some levels of toxicological concern based on the USEPA's reference values. Indeed, in the case of acute exposure following aerial spraying for infants, toddlers, and children, these proportions were respectively 37.1%, 59.5%, and 32.8% of the individuals, and 27.3%, 41.3%, and 24.9% following subchronic exposure. For ground spraying, these values were 12.5%, 24.2%, 8.8%; and 9.8%, 16.5%, and 7.4%. These results allowed the comparison of the probability of exceeding a level of toxicological concern for malathion exposure with the probability of developing WNV symptoms. This comparison shows that in some circumstances, the toxicological risk of malathion may exceed the infectious risk of WNV.     

免疫抑制剂地塞米松可明显延长乙型肝炎病毒抗原在水动力法转染HBV小鼠模型中的表达

建立基于高压水动力法的乙型肝炎病毒(HBV)转染小鼠模型,并进一步建立和优化乙肝动物模型研究方法。首先构建了含腺相关病毒倒转末端重复序列元件与包含1.3个拷贝HBV基因组(ayw亚型)的HBV表达质粒(pAAV-HBV1.3);并将pAAV-HBV1.3质粒经高压水动力法尾静脉注射C57BL/6小鼠,不同时间点采集血液和肝组织标本,ELISA检测血清HBsAg、HBeAg表达;Real-time PCR检测血清及肝组织病毒载量;HE染色、免疫组化染色检测肝组织病理学改变及病毒抗原在肝组织中的定位及表达;最后采用免疫抑制剂地塞米松注射液(DEX)腹腔注射小鼠,建立免疫功能抑制小鼠模型,在此基础上制备乙肝病毒转染小鼠模型,并进行血清HBsAg、HBeAg检测。结果是正常免疫状态下,小鼠转染pAAV-HBV1.3 10d时血清及肝组织HBV相关抗原阳性,30d后HBV相关抗原检测阴性,但30d和60d血清及肝组织病毒载量检测均为阳性,且与对照组差异显著(P0.01,P0.05);经地塞米松注射后处于免疫抑制状态下的高压水动力法建立的乙肝病毒转染小鼠,则在60d仍可检测到HBsAg、HBeAg的表达。以上结果表明通过高压水动力法建立了急性乙肝小鼠模型,通过抑制小鼠免疫状态,可延长病毒在小鼠体内存留时间。该模型建立为HBV疫苗评价、药物开发及乙肝相关致病机理研究奠定了基础。     

虎源H5N1亚型禽流感病毒感染小鼠模型的建立

为研究H5N1亚型禽流感病毒的病原特性、致病机理及对其疫苗与救治药物效果评价提供平台,利用本室分离鉴定的虎源A/Tiger/Harbin/01/2002株(简称HAB/01)H5N1亚型禽流感病毒进行连续10倍稀释后,对4～6周龄雄性BALB/c小鼠经乙醚麻醉后进行滴鼻攻毒,每个稀释度接种10只实验小鼠,测定其MLD50,检测小鼠感染、致病的多项指标,观察期为14d。结果感染小鼠呈现出规律的以肺炎为主的临床症状、病理变化及病死率;测得该病毒对小鼠的MLD50为10-7.1/0.05mL。成功建立了虎源H5N1亚型禽流感病毒感染BALB/c小鼠的实验模型。