Non-enveloped Virus-like Particles Research Articles

Size-selective downstream processing of virus particles and non-enveloped virus-like particles.

Non-enveloped virus-like particles (VLPs) are versatile protein nanoparticles with great potential for biopharmaceutical applications. However, conventional protein downstream processing (DSP) and platform processes are often not easily applicable due to the large size of VLPs and virus particles (VPs) in general. The application of size-selective separation techniques offers to exploit the size difference between VPs and common host-cell impurities. Moreover, size-selective separation techniques offer the potential for wide applicability across different VPs. In this work, basic principles and applications of size-selective separation techniques are reviewed to highlight their potential in DSP of VPs. Finally, specific DSP steps for non-enveloped VLPs and their subunits are reviewed as well as the potential applications and benefits of size-selective separation techniques are shown.

Nonenveloped Virus-Like Particles: A Promising Antigen-Delivery Strategy for the Induction of Antitumor Immune Responses

The use of antigen delivery systems is essential for inducing antitumor immune responses. Among these, virus-like particles (VLPs) increase the immunogenicity of coupled antigens to stimulate cellular and humoral immune responses. In particular, non-enveloped or capsid VLPs (cVLPs) are a promising antigen delivery system option due to their additional advantages of high production yields and low cost. This review summarizes the latest works on cVLPs in cancer vaccines, supporting cVLPs as a tumor antigen delivery system for immunotherapy and addressing some critical aspects of cVLP use such as production, assembly, decoration and immunization strategies, which can improve the effectiveness of cancer vaccines based on cVLPs.

Virus-like particle size and molecular weight/mass determination applying gas-phase electrophoresis (native nES GEMMA)

(Bio-)nanoparticle analysis employing a nano-electrospray gas-phase electrophoretic mobility molecular analyzer (native nES GEMMA) also known as nES differential mobility analyzer (nES DMA) is based on surface-dry analyte separation at ambient pressure. Based on electrophoretic principles, single-charged nanoparticles are separated according to their electrophoretic mobility diameter (EMD) corresponding to the particle size for spherical analytes. Subsequently, it is possible to correlate the (bio-)nanoparticle EMDs to their molecular weight (MW) yielding a corresponding fitted curve for an investigated analyte class. Based on such a correlation, (bio-)nanoparticle MW determination via its EMD within one analyte class is possible. Turning our attention to icosahedral, non-enveloped virus-like particles (VLPs), proteinaceous shells, we set up an EMD/MW correlation. We employed native electrospray ionization mass spectrometry (native ESI MS) to obtain MW values of investigated analytes, where possible, after extensive purification. We experienced difficulties in native ESI MS with time-of-flight (ToF) detection to determine MW due to sample inherent characteristics, which was not the case for charge detection (CDMS). nES GEMMA exceeds CDMS in speed of analysis and is likewise less dependent on sample purity and homogeneity. Hence, gas-phase electrophoresis yields calculated MW values in good approximation even when charge resolution was not obtained in native ESI ToF MS. Therefore, both methods-native nES GEMMA-based MW determination via an analyte class inherent EMD/MW correlation and native ESI MS-in the end relate (bio-)nanoparticle MW values. However, they differ significantly in, e.g., ease of instrument operation, sample and analyte handling, or costs of instrumentation.Graphical abstract

Enveloped and non-enveloped viral-like particles in Trypanosoma cruzi epimastigotes.

ABSTRACTElectron microscopy is routinely used to identify viral infections in protozoan parasites. These viruses have been described as non-enveloped and icosahedral structures with a diameter of 30-60 nm. Most of them are classified within the non-segmented dsRNA Totiviridae family. We observed virus-like particles (VLPs) through transmission electron microscopy in the cytoplasm of Trypanosoma cruzi epimastigotes grown in cultures. Clusters of electrodense enveloped VLPs having a diameter of 48 nm were also observed. These clusters appear to have been released from distended Golgi cisternae. Furthermore, a paracrystalline array of electrodense, non-enveloped VLPs (with a diameter of 32 nm) were found in distended Golgi cisternae or as smaller clusters at a distance from the RE or Golgi. We cannot rule out that the 48 nm enveloped VLPs belong to the ssRNA Flaviviridae family because they are within its size range. The localization of enveloped VLPs is consistent with the replication strategy of these viruses that transit through the Golgi to be released at the cell surface. Due to the size and shape of the 32 nm non-enveloped VLPs, we propose that they belong to the dsRNA Totiviridae family. This is the first description of cytoplasmic enveloped and non-enveloped VLPs in T. cruzi epimastigotes.

Plant-made vaccines against West Nile virus are potent, safe, and economically feasible.

The threat of West Nile virus (WNV) epidemics with increasingly severe neuroinvasive infections demands the development and licensing of effective vaccines. To date, vaccine candidates based on inactivated, live-attenuated, or chimeric virus, and viral DNA and WNV protein subunits have been developed. Some have been approved for veterinary use or are under clinical investigation, yet no vaccine has been licensed for human use. Reaching the milestone of a commercialized human vaccine, however, may largely depend on the economics of vaccine production. Analysis suggests that currently only novel low-cost production technologies would allow vaccination to outcompete the cost of surveillance and clinical treatment. Here, we review progress using plants to address the economic challenges of WNV vaccine production. The advantages of plants as hosts for vaccine production in cost, speed and scalability, especially those of viral vector-based transient expression systems, are discussed. The progress in developing WNV subunit vaccines in plants is reviewed within the context of their expression, characterization, downstream processing, and immunogenicity in animal models. The development of vaccines based on enveloped and non-enveloped virus-like particles is also discussed. These advancements suggest that plants may provide a production platform that offers potent, safe and affordable human vaccines against WNV.

Isolation of the Thosea asigna virus (TaV) from the epizootic Setothosea asigna larvae collected in South Sumatra and a study on its pathogenicity to Limacodidae larvae in Japan.

In 2005, natural epizootics were observed during an outbreak of Setothosea asigna (Lepidoptera: Limacodidae) larvae in an oil palm plantation in South Sumatra, Indonesia. The causative agent was filterable, which im plied it was a virus. Since preliminary testing using reverse PCR gave a positive result for the Thosea asigna virus (TaV), we experimented with the purification of the viral agent from the diseased larvae. Electron microsco py revealed nonenveloped virus-like particles that were spherical in shape and about 40 nm in diameter. cDNA cloning followed by sequencing demonstrated that RNA purified from the particles contained two large openreading-frames (ORFs) with a partly shared sequence and extensive homology (> 98% identity at the nucleotide level) with ORFs of TaV encoding an RNA-dependent RNA polymerase and the capsid protein, respectively. 3’ RACE suggested that there is, like in TaV genomic RNA, no poly(A) tract at the 3’-terminus of the RNA. The pathogenicity of the purified particles against Limacodidae larvae in Japan was demonstrated to be very strong for Monema flavescens and Austrapoda dentata. These results indicated that the agent causing the epizootic disease among S. asigna larvae in the oil palm plantations was TaV which also has potential as a biological control agent for Limacodidae pests in Japan. Keyword: Oil palm plantation, Limacodidae, Viruses, Tetraviridae, TaV

Identification of Adenovirus- and Dependovirus-like Agents in an Outbreak of Fatal Gastroenteritis in Captive Born California Mountain Kingsnakes, Lampropeltis zonata multicincta

ABSTRACT A group of seven juvenile California mountain kingsnakes, Lampropeltis zonata multicincta, developed severe acute gastroenteritis characterized by emesis, dehydration and sudden death. Histopathology demonstrated segmental mucosal necrosis and hyperplasia, villus blunting and fusion. Abundant basophilic Cowdry type-A intranuclear inclusions were demonstrated in enterocytes peripheral to the zone of acute necrosis throughout the small intestine. Electron microscopy demonstrated intranuclear crystalline arrays of non-enveloped virus-like particles that measured 65 to 70 nm in diameter. Clusters of smaller non-enveloped virus-like particles that measured 15 to 18 nm in diameter were found in association with the larger particles. The size, morphology and location of the two virus-like particles were consistent with an adenovirus and a parvovirus. The presence of the parvovirus-like particles in the same nuclei and in association with adenovirus-like particles suggested the parvovirus-like agent to b...

Enterically-transmitted non-A, non-B hepatitis

More than 50% of acute viral hepatitis occurring in some developing countries appears to be unrelated to infection by HAV or HBV and accumulating evidence suggests that a high proportion of this non-A, non-B hepatitis (NANB) is enterically transmitted. Epidemics or outbreaks of enterically-transmitted NANB (ET-NANB) have been documented in the Soviet Union, Nepal, Burma, Pakistan, India, Borneo, Somalia, Sudan, Ivory Coast, Algeria, and Mexico. These outbreaks primarily affect young to middle-age adults and are often associated with a high mortality rate in infected pregnant women, approaching 20% in most reported epidemics. Several investigators have reported finding 27 to 34 nm virus-like particles (VLPs) in stools of acutely infected cases. Stools containing these small, non-enveloped VLPs have been shown to cause NANB in experimentally infected cynomolgus macaques, African green monkeys, chimpanzees, rhesus monkeys, and Saguinus mystax monkeys (tamarins). Infected primates have been shown to seroconvert to 27-34 nm VLPs recovered from stools of cases occurring in the Soviet Union, India, Nepal, Burma, Pakistan and/or Mexico, suggesting that ET-NANB is caused by one virus or class of serologically related viruses. The morphological features and physicochemical properties of one candidate virus are very similar to those of some human caliciviruses, a group of viruses that is normally associated with outbreaks of severe diarrhoea.