Adaptation of Vero cells to suspension growth for rabies virus production in different serum free media

Vero cells are nowadays widely used in the production of human vaccines. They are considered as one of the most productive and flexible continuous cell lines available for vaccine manufacturing. However, these cells are anchorage dependent, which greatly complicates upstream processing and process scale-up. Moreover, there is a recognized need to reduce the costs of vaccine manufacturing to develop vaccines that are affordable worldwide. The use of cell lines adapted to suspension growth contributes to reach this objective.The current work describes the adaptation of Vero cells to suspension culture in different serum free media according to multiple protocols based on subsequent passages. The best one that relies on cell adaption to IPT-AFM an in-house developed animal component free medium was then chosen for further studies. Besides, as aggregates have been observed, the improvement of IPT-AFM composition and mechanical dissociation were also investigated.In addition to IPT-AFM, three chemically defined media (CD293, Hycell CHO and CD-U5) and two serum free media (293SFMII and SFM4CHO) were tested to set up a serum free culture of the suspension-adapted Vero cells (VeroS) in shake flasks. Cell density levels higher than 2 × 10⁶ cells/mL were obtained in the assessed conditions. The results were comparable to those obtained in spinner culture of adherent Vero cells grown on Cytodex 1 microcarriers.Cell infection with LP-2061 rabies virus strain at an MOI (Multiplicity of Infection) of 0.1 and a cell density of 8 ± 0.5 × 10⁵ cells/mL resulted in a virus titer higher than 10⁷ FFU/mL in all media tested. Nevertheless, the highest titer equal to 5.2 ± 0.5 × 10⁷ FFU/mL, was achieved in IPT-AFM containing a reduced amount of Ca⁺⁺ and Mg⁺⁺. Our results demonstrate the suitability of the obtained VeroS cells to produce rabies virus at a high titer, and pave the way to develop VeroS cells bioreactor process for rabies vaccine production.     

Rabies virus glycoprotein serology ELISA for measurement of neutralizing antibodies in sera of vaccinated human subjects

BackgroundVaccination provides protection against infection by inducing VNAs mainly against RABV surface GP. The measurement of VNAs to RABV is commonly used to assess the level of immunity in humans and animals after vaccination. A VNA titer of ≥ 0.5 IU/mL of sera indicates adequate response to vaccination. Here, we report the development and validation of a RABV GP serology ELISA kit for semi-quantitative measurement of VNA titers in sera of vaccinated human subjects.MethodsUsing a recombinant RABV GP expressed in mammalian cells as the capture antigen, the ELISA method was established using HuMAb NM57 reference initially and HRIG reference subsequently. The limit of detection (LOD), linear range, reproducibility, and precision of the method were examined. Specificity and sensitivity were established to assess the diagnostic accuracy.ResultsRABV GP for ELISA plate coating and optimal dilution of human serum sample was 1 µg/mL and 1:20, respectively. Multiple assays were carried out by different technicians at different laboratories for assay standardization. Using the HRIG reference, the LOD was found to be 0.02–0.06 IU/mL and the linear range was 0.2–10.0 IU/ mL. The inter-assay CVs were in the range of 6.60–10.79%, indicating the reproducibility. None of the 12 known negative human sera, tested positive by ELISA, highlighting the specificity. A total of 415 unknown positive human sera were double-blind tested by the RFFIT and ELISA. The VNA titer cut-off value of ELISA was set at 1.5 IU/mL to ensure no false-positive. The diagnostic specificity and sensitivity were 100% and 91.1%, respectively.ConclusionsThe validation data characterize this ELISA as a suitable method for semi-quantitative measurement of VNA titers in human serum samples to assess vaccination status. The ELISA kit can offer simplicity, speed, low cost and high throughput, making it a practical tool for monitoring the immune response following vaccination.     

ONRAB® oral rabies vaccine is shed from,but does not persist in,captive mammals

ONRAB® is a human adenovirus rabies glycoprotein recombinant vaccine developed to control rabies in wildlife. To support licensing and widespread use of the vaccine, safety studies are needed to assess its potential residual impact on wildlife populations. We examined the persistence of the ONRAB® vaccine virus in captive rabies vector and non-target mammals. This research complements work on important rabies vector species (raccoon, striped skunk, and red fox) but also adds to previous findings with the addition of some non-target species (Virginia opossum, Norway rats, and cotton rats) and a prolonged period of post vaccination monitoring (41 days). Animals were directly inoculated orally with the vaccine and vaccine shedding was monitored using quantitative real-time PCR applied to oral and rectal swabs. ONRAB® DNA was detected in both oral and rectal swabs from 6 h to 3 days post-inoculation in most animals, followed by a resurgence of shedding between days 17 and 34 in some species. Overall, the duration over which ONRAB® DNA was detectable was shorter for non-target mammals, and by day 41, no animal had detectable DNA in either oral or rectal swabs. All target species, as well as cotton rats and laboratory-bred Norway rats, developed robust humoral immune responses as measured by competitive ELISA, with all individuals being seropositive at day 31. Similarly, opossums showed good response (89% seropositive; 8/9), whereas only one of nine wild caught Norway rats was seropositive at day 31. These results support findings of other safety studies suggesting that ONRAB® does not persist in vector and non-target mammals exposed to the vaccine. As such, we interpret these data to reflect a low risk of adverse effects to wild populations following distribution of ONRAB® to control sylvatic rabies.     

VIROLOGICAL AND SEROLOGICAL DIAGNOSIS OF RABIES IN BATS FROM AN URBAN AREA IN THE BRAZILIAN AMAZON

The outbreaks of rabies in humans transmitted by Desmodus rotundus in 2004 and 2005, in the northeast of the Brazilian State of Para, eastern Amazon basin, made this a priority area for studies on this zoonosis. Given this, the present study provides data on this phenomenon in an urban context, in order to assess the possible circulation of the classic rabies virus (RABV) among bat species in Capanema, a town in the Amazon basin. Bats were collected, in 2011, with mist nets during the wet and dry seasons. Samples of brain tissue and blood were collected for virological and serological survey, respectively. None of the 153 brain tissue samples analyzed tested positive for RABV infection, but 50.34% (95% CI: 45.67-55.01%) of the serum samples analyzed were seropositive. Artibeus planirostris was the most common species, with a high percentage of seropositive individuals (52.46%, 95% CI: 52.31 52.60%). Statistically, equal proportions of seropositive results were obtained in the rainy and dry seasons (c² = 0.057, d.f. = 1, p = 0.88). Significantly higher proportions of males (55.96%, 95% CI: 48.96-62.96%) and adults (52.37%, 95% CI: 47.35-57.39%) were seropositive. While none of the brain tissue samples tested positive for infection, the high proportion of seropositive specimens indicates that RABV may be widespread in this urban area.     

狂犬病病毒致病机制研究概况

狂犬病病毒（RV）是一种高度嗜神经性病毒，由RV引发的狂犬病通常是一种急性致死性神经系统损伤性疾病。RV通过周围神经末梢和中枢神经系统（CNS）的神经元进行病毒的复制和传播。绝大多数狂犬病例CNS病理学表现为急性脑脊髓炎，常常没有显著的显微镜下改变，脑部可以轻度肿胀，脑膜和脑实质血管轻度充血并伴有少量炎症细胞浸润，这也是其他急性病毒性脑炎常见的共同表现。     

Recombinant fowlpox virus inducing protective immunity in non-avian species

The natural host of fowlpox virus is limited to avian species. When inoculated into non-avian tissue culture cells, however, fowlpox virus can initiate an abortive infection. A fowlpox virus was engineered to express rabies virus glycoprotein. On inoculation of the recombinant virus into either avian (permissive) or non-avian (non-permissive) cells, the rabies glycoprotein was expressed as a membrane-associated antigen. Inoculation of the fowlpox virus recombinant into six different species of mammal resulted in specific immune responses to both fowlpox antigens and to rabies glycoprotein. In mice, cats and dogs the immune response was sufficient to protect against a live rabies virus challenge. The results demonstrate the utility of a fowlpox virus vector in immunizing non-avian species against rabies in the absence of productive viral replication of the fowlpox vector.     

110例嗜酸细胞增多性非变应性鼻炎的液氮冷冻治疗

１１０例嗜酸细胞增多性非变应性鼻炎的液氮冷冻治疗山西医学院第二附属医院（０３０００１）牛玉梅，刘学仁按Ｍｇｇｉｎｄ将常年性鼻炎分为三类：一类常年性变态反应性鼻炎；二类非变态反应性鼻炎伴鼻分泌物嗜酸细胞增多综合征也叫嗜酸细胞增多性非变态反应性鼻炎（Ｅｏｓｉｎｏｐｈｉｌｉｃｎｏｎａｌｌｅｒａｉｃｒｈｉｎｉｔｓ，ＥＮＲ）；三类自主神经性常年性鼻炎［１］。我们将ＥＮＲ中找不到致敏物，而与冷热空气有关与情绪无关的１１０例患者进行了液氮冷冻治疗。临床资料８０２例常年性鼻炎患者进行皮肤激发试验。４３７例找出了致敏物，进行特异性过敏诱因脱敏治疗或抗过敏药物治疗。３６５例试验阳性，其中有１３５例与冷热空气有关。ｌｌ０例行冷冻治疗。１ｌ０例中男５０例，女６０例；年龄最大６５岁，最小１６岁。１１０例均有间歇性的连续喷嚏发作，浆液性或粘液性鼻分泌物增多和鼻粘膜非充血性肿胀引起的堵塞，无因吸入致敏原诱发症状的病史；血清ＩｇＥ值不升高，特异性皮肤试验结果为阳性；鼻分泌物中嗜酸细胞阳性。方法：先将鼻腔内喷入１％地卡因溶液３次粘膜表面麻醉，用卷好的４厘米长、０．４～０．６厘米粗的棉棒沾上液氮在鼻镜直视下迅速插入鼻腔内。时间约１分钟左右（冷     

五例动物狂犬病脑部病理分析

1987年5月云南省部分地区发生狂犬病流行,首先在狗、马、牛之间,随后由动物传播给人。本文通过光镜及电镜观察狂犬病动物脑组织,探讨动物狂犬病的病理形态特点及讨论狂犬病诊断中的有关问题。     

慢性肾炎并腹泻引发急性心肌梗塞一例报告

慢性肾炎并腹泻引发急性心肌梗塞一例报告临汾地区卫校附属医院（０４１０００）马希贤患者男性，２３岁。９岁时曾因少尿，浮肿，血尿被诊断为“急性肾炎”，经中西药治疗痊愈。１９９１年１月因反复浮肿，少尿３个月，加重半月入院。入院后后经临床检查诊断为：慢性肾炎（肾病型），给予强的松（６０ｍｇ，顿服）、利尿剂及中药治疗，尿量增加，浮肿减轻，治疗一月后尿蛋白明显减轻，超声波检查腹水消失，将强的松量每周减少１０ｍｇ，至１０ｍｇ／日时作为维持量长期服用，入院第１４２天突然出现阵发性腹痛伴腹泻，呈稀水样便，５～６次／日，脐周压痛（＋），肠鸣音亢进。便常规：白细胞５～６个／ＨＰ，未见巨噬细胞及红细胞。考虑并急性肠炎，２天后病人自诉头晕。口干，血压９０／６０ｍｍＨｇ，当日中午睡眠时突发胸骨后闷痛，伴大汗、恶心及心悸，持续不缓解，急查心电图示频发室早，Ｖ１─３ＳＴ段呈弓背向上形抬高０．３～０．５ｍＶ，Ｒｖ１─３振幅降低，考虑“急性前间壁心肌梗塞”，经吸氧，静脉补液及应用低分子右旋糖酐、吗啡、硝酸甘油、尿激酶及利多卡因治疗疼痛消失。后查心电图Ｖ１─３出现ＱＳ波，ＳＴ─Ｔ符合急性心肌梗塞演变过程，心电向量示前间壁梗塞向量。讨论：本例突发     

我国狂犬病病原学和病理形态学观察

报告四例狂犬病病例及对其尸脑组织的病原学和病理形态学观察。４例病人均有被犬咬伤史；发病后临床病状典型，表现为发热、恐水、怕风等；潜伏期和病程分别在２０～１３２天和７～１４天；临床诊断明确。死亡后尸检脑组织病理改变为病毒性脑炎病变，在感染神经细胞胞浆内均检到内基氏体。从尸脑组织中分离出狂犬病毒４株。又从４例病人血清中和２例病人脑脊液中分别检测出特异性狂犬病毒抗体各１例。