Completely assembled virus particles detected by transmission electron microscopy in proximal and mid-axons of neurons infected with herpes simplex virus type 1, herpes simplex virus type 2 and pseudorabies virus

The morphology of alphaherpesviruses during anterograde axonal transport from the neuron cell body towards the axon terminus is controversial. Reports suggest that transport of herpes simplex virus type 1 (HSV-1) nucleocapsids and envelope proteins occurs in separate compartments and that complete virions form at varicosities or axon termini (subassembly transport model), while transport of a related alphaherpesvirus, pseudorabies virus (PRV) occurs as enveloped capsids in vesicles (assembled transport model). Transmission electron microscopy of proximal and mid-axons of primary superior cervical ganglion (SCG) neurons was used to compare anterograde axonal transport of HSV-1, HSV-2 and PRV. SCG cell bodies were infected with HSV-1 NS and 17, HSV-2 2.12 and PRV Becker. Fully assembled virus particles were detected intracellularly within vesicles in proximal and mid-axons adjacent to microtubules after infection with each virus, indicating that assembled virions are transported anterograde within axons for all three alphaherpesviruses.     

A nanoparticle-assisted PCR assay to improve the sensitivity for rapid detection and differentiation of wild-type pseudorabies virus and gene-deleted vaccine strains

Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the rapid amplification of DNA and has been adopted for the detection of virus because of its simplicity, rapidity, and specificity. A nanoPCR assay was developed to detect and differentiate wild-type and gene-deleted pseudorabies virus (PRV). Three pairs of primers for nanoPCR developed in this study were selected from conserved regions of PRV, producing specific amplicons of 431 bp (gB), 316 bp (gE), and 202 bp (gG). The sensitivity of this assay using purified plasmid constructs containing the specific gene fragments was 100–1000 fold higher than conventional PCR. The PRV nanoPCR assay did not amplify porcine parvovirus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, porcine teschovirus, or African swine fever virus but produced three bands of expected size with PRV and two bands of expected size with the gene-deleted PRV-Bartha-K61. Of 110 clinical samples collected from seven provinces in China, 53% and 48% were positive for wild-type PRV according to the nanoPCR assay and virus isolation, respectively.     

Virus inactivation of plasma-derived proteins by pasteurization in the presence of guanidine hydrochloride

BACKGROUND: Viruses, among them parvovirus B19 and other small, nonenveloped viruses, may be present in human blood and may contaminate plasma-derived therapeutics. Efficient inactivation or removal of such viruses, especially parvoviruses, represents a current problem and corresponding technologies are under investigation. In this report, such a technology is described. STUDY DESIGN AND METHODS: A recently developed pasteurization of human apolipoprotein A-I (apoA-I), which is performed at 60 degrees C for 10 hours in the presence of guanidine hydrochloride (GdnHCl), was validated by using a series of model viruses, including members of the families parvoviridae and picornaviridae. The model viruses were spiked into the apoA-I- and GdnHCl-containing solutions, and virus inactivation was evaluated by infectivity assays in cell cultures. The mechanism of virus inactivation was studied by virus sedimentation analysis using the picornavirus model. RESULTS: All viruses tested were inactivated to levels below the limit of detection, although different inactivation kinetics were obtained for the different viruses. The mechanism of virus inactivation by this pasteurization was disassembly of the virus particles into single proteins or small noninfectious viral subunits. CONCLUSION: The pasteurization validated in this report has the potential to inactivate a wide range of transfusion-relevant viruses including parvoviruses and picornaviruses.     

鼻咽癌放射治疗视觉通路PRV的探讨

目的研究鼻咽癌在常规放疗、适形调强放疗和全程调强放疗中,视觉通路危险器官的PRV确定,特别是晶体的PRV确定和剂量控制。方法我院自2005.7.1直线加速器开展精确放疗以来,进行鼻咽癌治疗病例55例,用1-2个周期的DDP+5-Fu新辅助诱导化疗后,进行分组治疗。A组:常规放疗,B组:适形+调强放疗,C组:全程调强放疗。考虑到眼球在5-10分钟的放疗过程中眼的不自主的活动,根据30例肿瘤患者和30例正常人群的眼的活动度的测定,确定眼的活动范围,并对眼晶体、眼球、视神经等视觉通路的PRV进行勾画并TPS计算的剂量结果比较。结果由于眼的活动,进行合理的眼各部位PRV确定对可以较好的避免眼晶体和其他视路的放射损伤,确保精确放疗除了对靶区能达到临床的控制要求的同时,还对危险器官能更好的保护。     

Detection of piscine orthoreoviruses (PRV‐1 and PRV‐3) in Atlantic salmon and rainbow trout farmed in Germany

Piscine orthoreoviruses (PRVs) are emerging pathogens causing circulatory disorders in salmonids. PRV‐1 is the etiological cause of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar), characterized by epicarditis, inflammation and necrosis of the myocardium, myositis and necrosis of red skeletal muscle. In 2017, two German breeding farms for Atlantic salmon and rainbow trout (Oncorhynchus mykiss) experienced disease outbreaks with mortalities of 10% and 20% respectively. The main clinical signs were exhaustion and lethargic behaviour. During examinations, PRV‐1 in salmon and PRV‐3 in trout were detected for the first time in Germany. Further analyses also indicated the presence of Aeromonas salmonicida in internal tissues of both species. While PRV‐1 could be putatively linked with the disease in Atlantic salmon, most of the rainbow trout suffered from an infection with A. salmonicida and not with PRV‐3. Interestingly, the sequence analysis suggests that the German PRV‐3 isolate is more similar to a Chilean PRV‐3 isolate from Coho salmon (Oncorhynchus kisutch) than to PRV‐3 from rainbow trout from Norway. This indicates a wide geographic distribution of this virus or dispersal by global trade. These findings indicate that infections with PRVs should be considered when investigating disease outbreaks in salmonids.     

Inputs to serotonergic neurons revealed by conditional viral transneuronal tracing

Descending projections arising from brainstem serotonergic (5HT) neurons contribute to both facilitatory and inhibitory controls of spinal cord “pain” transmission neurons. Unclear, however, are the brainstem networks that influence the output of these 5HT neurons. To address this question, here we used a novel neuroanatomical tracing method in a transgenic line of mice in which Cre recombinase is selectively expressed in 5HT neurons (ePet‐Cre mice). Specifically, we injected the conditional pseudorabies virus recombinant (BA2001) that can replicate only in Cre‐expressing neurons. Because BA2001 transports exclusively in a retrograde manner, we were able to reveal a subset of the neurons and circuits that are located upstream of the Cre‐expressing 5HT neurons. We show that diverse brainstem regions differentially target the 5HT neurons of the dorsal raphe (DR) and the nucleus raphe magnus of the rostroventral medulla (RVM). Among these are several catecholaminergic and cholinergic cell groups, the periaqueductal gray, several brainstem reticular nuclei, and the nucleus of the solitary tract. We conclude that a brainstem 5HT network integrates somatic and visceral inputs arising from various areas of the body. We also identified a circuit that arises from projection neurons of deep spinal cord laminae V–VIII and targets the 5HT neurons of the NRM, but not of the DR. This spinoreticular pathway constitutes an anatomical substrate through which a noxious stimulus can activate 5HT neurons of the NRM and in turn could trigger descending serotonergic antinociceptive controls. J. Comp. Neurol. 514:145–160, 2009. © 2009 Wiley‐Liss, Inc.     

Translation of papaya ringspot virus RNA in vitro: detection of a possible polyprotein that is processed for capsid protein, cylindrical-inclusion protein, and amorphous-inclusion protein

The genomic RNA of papaya ringspot virus (PRV), a member of the potyvirus group, was translated in a rabbit reticulocyte cell-free system as an approach to determining the translation strategy of the virus. The RNA directed synthesis of more than 20 distinct polypeptides ranging from apparent molecular weight of 26,000 (26K) to 220K. Antiserum to PRV capsid protein (CP) reacted with a subset of these polypeptides, including a 36K protein that comigrated with PRV CP during electrophoresis. Immunoprecipitation with antiserum to PRV cylindrical-inclusion protein (CIP) defined another set of polypeptides including 70K, 108K, 205K, and 220K proteins as major precipitates. The 70K protein comigrated with authentic CIP, and the 205K and 220K proteins were related to both CP and CIP. Immunoprecipitation with antiserum to PRV amorphous-inclusion protein (AIP) defined a unique set of polypeptides which contained a 112K protein as the major precipitate and 51K, 65K, and 86K proteins as minor precipitates. The 51K protein comigrated with authentic AIR A major product of 330K was observed when translation was done without the reducing agent, dithiothreitol. Immunological analyses and kinetic studies indicated that the 330K protein zone was related to the presumed CP, CIP, and AIP zones and 330K possibly is the common precursor for these viral proteins. The presence of a polyprotein of Mr corresponding to the entire coding capacity of the genomic RNA and its likely precursor relationship to the other polypeptides suggest that proteolytic processing is involved in the translation of PRV RNA.     

Planning organ at risk volume margins for organ motion of the intestine

BACKGROUND AND PURPOSE: To account for internal organ motion and set-up uncertainties around organs at risk (OR) in radiotherapy (RT), the ICRU report no 62 introduced the planning organ at risk volume (PRV). In the present study, we have quantified PRV margins for the intestine, which is an important OR in pelvic RT. MATERIALS AND METHODS: The present study was based on intestine contours outlined in a total of 149 CT scans of 20 male bladder cancer patients (20 planning scans, 129 during treatment). From these data, we created location probability maps of the intestine for each patient. A commercial treatment planning system was used to add 3D isotropic intestine PRV margins (from 5 to 30 mm, in intervals of 5 mm) around the intestine planning outline. We then derived the fraction of patients for which a given PRV encompassed various degrees of intestine motion (85%, 90% and 95% of volumes with different probabilities of intestinal occupancy). As a measure of the specificity of the PRV, we also derived the fraction of the PRV containing volumes with zero probability of intestinal occupancy. RESULTS: Isotropic margins of up to 30 mm are required to account for all intestine motion in 90% of the patients, while isotropic margins of 5 - 10 mm will encompass 85 - 95% of the volumes having a probability of intestinal occupancy of > or = 75% in the same fraction of patients. Intestine PRVs are not very specific and will also include volumes where the intestine will rarely or never be located. CONCLUSIONS: Large intestinal motion was found, but isotropic PRV margins of 5-10 mm will include the major part of volumes with a large probability of intestinal occupancy in most patients.     

Construction and immunogenicity of a gE/gI/TK-deleted PRV based on porcine pseudorabies virus variant

Pseudorabies (PR) caused by re-emerging pseudorabies virus (PRV) variant has outbroken among PRV vaccine-immunized swine herds on many Chinese pig farms, with severe socioeconomic consequences since late 2011. Here, a gE/gI/TK-deleted recombinant virus (rPRV NY-gE⁻/gI⁻/TK⁻) was constructed based on PRV NY strain from 2012 through homologous DNA recombination and gene-editing technology termed clustered regularly interspaced palindromic repeats (CRISPR)/associated (Cas9) system. The rPRV NY-gE⁻/gI⁻/TK⁻ strain showed similar growth kinetics to the parental PRV NY strain in vitro, and was safe for mice. Sixty mice were injected subcutaneously (s.c.) twice with 10^6.0 TCID₅₀ of rPRV NY-gE⁻/gI⁻/TK⁻ and DMEM, respectively, with two-week interval. The levels of PRV gB antibodies and neutralizing antibodies against PRV NY in mice immunized with rPRV NY-gE⁻/gI⁻/TK⁻ were higher than those in the DMEM control group. The number of T lymphocyte subclasses CD3⁺, CD4⁺ and CD8⁺ in rPRV NY-gE⁻/gI⁻/TK⁻-immunized mice was higher than that in DMEM-injected mice. After challenge with 10^6.0 TCID₅₀ PRV NY at 42 dpi, all rPRV NY-gE⁻/gI⁻/TK⁻-immunized mice survived without exhibiting any pathological lesions in different tissues and intranuclear eosinophilic inclusions of the brain, and the viral genomic copy numbers in various organs of mice were obviously lower than DMEM group. These results showed the rPRV NY-gE⁻/gI⁻/TK⁻ could be a promising next-generation vaccine to control now epidemic PR in China.