Cold-passaged isolates and bat-swine influenza a chimeric viruses as modified live-attenuated vaccines against influenza a viruses in pigs

Swine influenza A virus (swIAV) infections in pig populations cause considerable morbidity and economic losses. Frequent reverse zoonotic incursions of human IAV boost reassortment opportunities with authentic porcine and avian-like IAV in swine herds potentially enhancing zoonotic and even pre-pandemic potential. Vaccination using adjuvanted inactivated full virus vaccines is frequently used in attempting control of swIAV infections. Accelerated antigenic drift of swIAV in large swine holdings and interference of maternal antibodies with vaccine in piglets can compromise these efforts. Potentially more efficacious modified live-attenuated vaccines (MLVs) bear the risk of reversion of MLV to virulence. Here we evaluated new MLV candidates based on cold-passaged swIAV or on reassortment-incompetent bat-IAV-swIAV chimeric viruses. Serial cold-passaging of various swIAV subtypes did not yield unambiguously temperature-sensitive mutants although safety studies in mice and pigs suggested some degree of attenuation. Chimeric bat-swIAV expressing the hemagglutinin and neuraminidase of an avian-like H1N1, in contrast, proved to be safe in mice and pigs, and a single nasal inoculation induced protective immunity against homologous challenge in pigs. Reassortant-incompetent chimeric bat-swIAV vaccines could aid in reducing the amount of swIAV circulating in pig populations, thereby increasing animal welfare, limiting economic losses and lowering the risk of zoonotic swIAV transmission.     

miRNA93和miRNA192在H3N2亚型SIV病毒复制及宿主抗病毒免疫中的作用

目的 探讨分离于广西猪流感病毒SW/Guangxi/NS2783/2010和SW/Guangxi/NS650/2012跨种属感染人肺腺癌A549细胞的miRNA93和miRNA192对病毒复制及宿主抗病毒免疫的影响。方法 通过测定不同稀释浓度的病毒感染细胞HA滴度值,确定病毒最佳稀释浓度。荧光定量PCR检测病毒感染细胞后miRNA93和miRNA192表达,Western blot检测病毒NP和HA蛋白表达水平;转染miRNA93和miRNA192抑制剂后,重新检测miRNA93、miRNA192、IFN-β及病毒NP、HA蛋白表达水平。结果 不同稀释度病毒感染人A549细胞后HA滴度的结果显示病毒SW2783的最佳稀释度为10^-3,而SW650的HA滴度无明显变化趋势,提示病毒SW2783对人A549细胞具有较好的适应性和感染能力。荧光定量PCR和Western blot结果提示两株病毒感染细胞后病毒NP和HA蛋白表达均先升高后降低,加入miRNA93抑制剂,两株病毒NP和HA蛋白的表达均上调;加入miRNA192抑制剂,病毒SW2783的HA蛋白表达下调（P=2.10×10^-4）,而SW650的HA蛋白表达上调（P=5.45×10^-5）,NP蛋白反而下调（P=0.034）;ELISA结果提示病毒SW2783和SW650感染细胞后炎症因子IFN-β表达水平随感染时间延长而升高。结论 研究表明miRNA93和miRNA192的表达与SIV病毒增殖及宿主抗病毒免疫有关,可作为干预猪流感病毒跨种属感染的新靶点。     

Electroporation enhances protective immune response of a DNA vaccine against Japanese encephalitis in mice and pigs

Japanese encephalitis virus (JEV) is a pathogenic cause of Japanese Encephalitis (JE), which is a zoonotic disease transmitted by mosquitoes and amplified by pigs. Infection of JEV may lead to severe neurological sequelae, even death in humans and reproductive disorders in pigs. Vaccination is the only way to control JEV infection in humans. For pigs play important role in the JEV transmission cycle, developing a new veterinary vaccine is considered as a useful strategy for cutting off the transmission route of JEV. We have previously reported that DNA vaccine pCAG-JME, expressing prM-E proteins of JEV, is effective in mice through intramuscular injection (IM). However, the poor immunogenicity, due to low expression of immunogen, is the major obstacle for the development of DNA vaccine in large animals. In the present study, therefore, we immunized mice and pigs with pCAG-JME intramuscularly accompanied with electroporation (EP) stimulation, the attractive gene delivery approach. As compared with IM, EP-mediated vaccination markedly increased the expression of immunogen in the injection site and induced a dose- and time-dependent immune response. 100% survival rate was observed in groups vaccinated with doses ranged from 10 to 100μg, indicating that 10μg of DNA with EP for individual was enough for inducing effective protection in mice. Surprisingly, survival rate and end-point titers of anti-JEV antibodies were higher in mice even at lower dose of DNA (5μg) than that in mice inoculated 100μg through IM. Notably, the prM-E antigens also induced high antibody response in pig, while the neutralizing antibody titer achieved 1:320. Our results suggested that EP-mediated DNA immunization might act as an effective strategy against JEV, at least in pig, and that EP has a potential application prospect in DNA vaccination.     

Efficacy of the NS1-truncated live attenuated influenza virus vaccine for swine against infection with viruses of major North American and European H3N2 lineages

Control of swine influenza A virus (swIAV) in North America and Europe is complicated because multiple antigenically distinct swIAV strains co-circulate in the field, and no vaccine is available that can provide broad cross-protection against all these swIAVs. In 2017, the first live attenuated influenza vaccine (LAIV) for swine was licensed in the US. The non-structural protein 1 (NS1)-truncated cluster I H3N2 strain A/swine/Texas/4199-2/98 NS1del126 (TX98 LAIV) in this vaccine provides partial cross-protection against heterologous North American cluster II and IV H3N2 swIAV strains. Its efficacy against European or more recent North American H3N2 lineages remains to be investigated. In this study, we evaluated the level of cross-protection against heterologous IAVs representative of the major H3N2 swIAV lineages in Europe and North America. TX98 LAIV prevented both nasal shedding and replication in the lungs of a North American cluster IV H3N2 swIAV for 2/4 pigs, prevented considerable nasal shedding of a North American novel human-like H3N2 swIAV for 2/4 pigs, and reduced replication of a European H3N2 swIAV in the lower respiratory tract to minimal titers for 1/3 pigs. Although TX98 LAIV elicited neutralizing antibodies against the homologous virus in serum and to a lesser extent in nose and lungs, no significant cross-reactive antibody titers against the heterologous swIAVs were detected. Partial cross-protection therefore likely relies on cellular and mucosal immune responses against conserved parts of the swIAV proteins. Since TX98 LAIV can offer partial protection against a broad range of H3N2 swIAVs, it might be a suitable priming vaccine for use in a heterologous prime-boost vaccination strategy.     

实验动物麻醉的应用研究（附1000例报告）

目的：通过分析1 000例实验动物猪的麻醉情况,总结较实用的麻醉方法。方法：对4年共1 000例猪选用咪达唑仑0.1～0.2 mg/kg及速眠新0.25～0.3 ml/kg复合肌注诱导镇静,琥珀酰胆碱、地西泮注射液、利多卡因加入至平衡液内,配制成麻醉混合液,采用缓慢静滴维持的方法实现实验猪的各种手术麻醉。结果：6例猪在麻醉过程中由于操作不当,于气管插管前后死亡,1例死于氧气供给缺乏,9例死于术中大出血,余均顺利维持至手术结束。结论：速眠新与地西泮复合用药,用于猪的麻醉是经济、实用的,具有很高的应用价值。     

Effects of a combination hemoglobin based oxygen carrier-hypertonic saline solution on oxygen transport in the treatment of traumatic shock

Background

Logistics complicate fluid resuscitation of traumatic shock on the battlefield. Traumatic shock can result in oxygen debt (O₂D) accumulation that is fatal. However, the ability of fluid strategies to repay O₂D are not commonly reported. This pilot study examined various resuscitation fluids, including a combination of PEGylated bovine hemoglobin and hypertonic saline (AfterShock™) on their ability to repay O₂D in traumatic shock.

Methods

41 anesthetized swine underwent hemorrhage to an O₂D of 80 mL/kg. Animals received one of the following: 500 mL whole blood, 500 mL AfterShock™, 500 mL hypertonic (7.2%) saline, 250 mL hypertonic (7.2%) saline, 500 mL Hetastarch (6%), or 500 mL lactated Ringer's. Oxygen transport variables (O₂D, oxygen consumption, oxygen delivery, central venous hemoglobin oxygen saturation, oxygen extraction ratios), lactate clearance, and survival were monitored for 3 h after treatment. Data were analyzed using mixed-model ANOVA and comparisons were made to the performance of whole blood.

Results

Only animals receiving AfterShock™, 500 mL hypertonic saline, and 500 mL Hetastarch survived to 180 min. While not statistically significant AfterShock™ demonstrated trends in improving the repayment of O₂D and in improving oxygen transport variables despite having lower levels of global oxygen delivery compared to whole blood, Hetastarch and 500 mL hypertonic saline groups.

Conclusion

Use of 500 mL AfterShock™, 500 mL of 7.2% saline or 500 mL of Hetastarch resulted in improved short-term survival. While not statistically significant, AfterShock™ demonstrated trends in improving O₂D. These findings may have implications for designing resuscitation fluids for combat casualty care.     

Ischaemia-modified albumin predicts the outcome of cardiopulmonary resuscitation: An experimental study

Introduction

Ischaemia-modified albumin (IMA) has recently been shown to be an early and sensitive marker of ischaemia. It is generally accepted that cardiac arrest causes the most severe form of global ischaemia. The aim of the present study was to identify whether IMA is an independent predictor of return of spontaneous circulation (ROSC) in a swine model of cardiac arrest.

Methods

Ventricular fibrillation (VF) was induced in 30 piglets, which were left untreated for 8 min before attempting resuscitation with precordial compression, mechanical ventilation and electrical defibrillation. Electrical defibrillation was attempted after 10 min of VF. Blood samples for IMA determination were drawn at baseline, after 8 min of VF and before delivery of each shock. A binary logistic regression model was implemented for the prediction of animals achieving ROSC from data available before the first defibrillation attempt. Backward stepwise selection was used to extract the final model. Inclusion and exclusion significance levels were 0.1 and 0.05, respectively. Receiver operating characteristic curves were used to determine the diagnostic accuracy, sensitivity and specificity of the parameters and to obtain the appropriate cut-off points.

Results

IMA exhibited 100% sensitivity and 93.8% specificity in defining the subgroup of animals that will achieve ROSC. This high-accuracy prediction had a very early onset (from eighth VF minute) and remained at the same level until the end of the experiment. When combining IMA and coronary perfusion pressure (CPP) measurements from the first CPR cycle in the form of the simple ratio IMA/CPP, a cut-off point of 7 could provide 100% sensitivity and specificity in distinguishing the animals that will achieve ROSC in the upcoming defibrillation attempts.

Conclusions

Until today, CPP has been found to be the only key determinant of successful resuscitation. Our study suggests that IMA can be a predictive index of ROSC even before the initiation of CPR.     

超声电子膀胱软镜在泌尿系疾病诊疗中的动物模型研究

目的 探讨超声电子膀胱软镜应用于泌尿系疾病诊疗的可行性. 方法 采用1 ～2岁雌性巴马香猪4只为实验模型,以超声支气管镜为雏形,通过B超模式观察不同膀胱充盈量下膀胱各层结构声像图,通过彩色多普勒超声模式观察膀胱周围重要血管的声像图,初步总结超声电子膀胱软镜的操作技巧和难点.采用专用一次性抽吸式活检针进行内镜超声引导下分层定位穿刺活检,活检组织送病理以验证超声分层定位的准确性,探讨超声电子膀胱软镜下穿刺的有效性和安全性. 结果 超声膀胱镜可同步显示内镜图像和超声图像.香猪膀胱充盈90 ml时,超声内镜下膀胱壁黏膜及黏膜下层、肌层、浆膜层层次清晰可辨并有助于穿刺针的分层定位,多普勒超声模式下,膀胱周围血管血流信号明显,超声引导穿刺可避免发生血管及周嗣脏器误伤.16处穿刺点送检组织病理结果与超声分层定位吻合. 结论 超声电子膀胱软镜诊断治疗泌尿系疾病安全可行,配套器械有待进一步改进.     

Swine Influenza A(H3N2) Virus Infection in Immunocompromised Man,Italy, 2014

Because swine influenza virus infection is seldom diagnosed in humans, its frequency might be underestimated. We report a immunocompromised hematologic patient with swine influenza A(H3N2) virus in 2014 in Italy. Local pigs were the source of this human infection.     

The first lack of evidence of H7N9 avian influenza virus infections among pigs in Eastern China

In this study, we sought to examine whether evidence existed suggesting that pigs were being infected with the novel H7N9 avian influenza virus. From November 2012 to November 2013, blood was drawn from 1560 pigs from 100 large farms in 4 provinces of eastern China. Many of these pigs were in close proximity to wild birds or poultry. Swine sera were studied using hemagglutinin inhibition (HI) assays and enzyme-linked immunosorbent assays (ELISAs) against the H7 antigen derived from the emergent H7N9 avian influenza virus (AIV). Only 29 of the 1560 samples had HI titers of 1:20 when using the H7N9 AIV antigens, and none of the 29 (H7N9 AIV) HI-positive samples were positive when using ELISA, indicating that no samples were positive for H7N9. The negative results were also verified using a novel competitive HA-ELISA. As pigs have been shown to be infected with other avian influenza viruses and as the prevalence of novel influenza A viruses (e.g., H7N9 AIV) may be increasing among poultry in China, similar seroepidemiological studies of pigs should be periodically conducted in the future.