RESUMEN

Sapovirus (SaV) is a member of the Caliciviridae family, which causes acute gastroenteritis in humans and animals. Human sapoviruses (HuSaVs) are genetically and antigenically diverse, but the lack of a viral replication system and structural information has hampered the development of vaccines and therapeutics. Here, we successfully produced a self-assembled virus-like particle (VLP) from the HuSaV GI.6 VP1 protein, and the first atomic structure was determined using single-particle cryo-electron microscopy (cryo-EM) at a 2.9-Å resolution. The atomic model of the VP1 protein revealed a unique capsid protein conformation in caliciviruses. All N-terminal arms in the A, B, and C subunits interacted with adjacent shell domains after extending through their subunits. The roof of the arched VP1 dimer was formed between the P2 subdomains by the interconnected ß strands and loops, and its buried surface was minimized compared to those of other caliciviruses. Four hypervariable regions that are potentially involved in the antigenic diversity of SaV formed extensive clusters on top of the P domain. Potential receptor binding regions implied by tissue culture mutants of porcine SaV were also located near these hypervariable clusters. Conserved sequence motifs of the VP1 protein, "PPG" and "GWS," may stabilize the inner capsid shell and the outer protruding domain, respectively. These findings will provide the structural basis for the medical treatment of HuSaV infections and facilitate the development of vaccines, antivirals, and diagnostic systems. IMPORTANCE SaV and norovirus, belonging to the Caliciviridae family, are common causes of acute gastroenteritis in humans and animals. SaV and norovirus infections are public health problems in all age groups, which occur explosively and sporadically worldwide. HuSaV is genetically and antigenically diverse and is currently classified into 4 genogroups consisting of 18 genotypes based on the sequence similarity of the VP1 proteins. Despite these detailed genetic analyses, the lack of structural information on viral capsids has become a problem for the development of vaccines or antiviral drugs. The 2.9-Å atomic model of the HuSaV GI.6 VLP presented here not only revealed the location of the amino acid residues involved in immune responses and potential receptor binding sites but also provided essential information for the design of stable constructs needed for the development of vaccines and antivirals.

Asunto(s)

Proteínas de la Cápside , Cápside , Sapovirus , Animales , Cápside/ultraestructura , Proteínas de la Cápside/ultraestructura , Microscopía por Crioelectrón , Conformación Proteica , Sapovirus/ultraestructura , Porcinos

RESUMEN

Vesivirus 2117 is an adventitious agent that, in 2009, was identified as a contaminant of Chinese hamster ovary cells propagated in bioreactors at a pharmaceutical manufacturing plant belonging to Genzyme. The consequent interruption in supply of Fabrazyme and Cerezyme (drugs used to treat Fabry and Gaucher diseases, respectively) caused significant economic losses. Vesivirus 2117 is a member of the Caliciviridae, a family of small icosahedral viruses encoding a positive-sense RNA genome. We have used cryo-electron microscopy and three-dimensional image reconstruction to calculate a structure of vesivirus 2117 virus-like particles as well as feline calicivirus and a chimeric sapovirus. We present a structural comparison of several members of the Caliciviridae, showing that the distal P domain of vesivirus 2117 is morphologically distinct from that seen in other known vesivirus structures. Furthermore, at intermediate resolutions, we found a high level of structural similarity between vesivirus 2117 and Caliciviridae from other genera: sapovirus and rabbit hemorrhagic disease virus. Phylogenetic analysis confirms vesivirus 2117 as a vesivirus closely related to canine vesiviruses. We postulate that morphological differences in virion structure seen between vesivirus clades may reflect differences in receptor usage.

Asunto(s)

Cápside/ultraestructura , Lagovirus/ultraestructura , Sapovirus/ultraestructura , Vesivirus/ultraestructura , Animales , Cricetulus , Microscopía por Crioelectrón , Imagenología Tridimensional , Filogenia , ARN Viral/genética , Análisis de Secuencia de ADN , Vesivirus/genética

RESUMEN

UNLABELLED: The capsid protein (VP1) of all caliciviruses forms an icosahedral particle with two principal domains, shell (S) and protruding (P) domains, which are connected via a flexible hinge region. The S domain forms a scaffold surrounding the nucleic acid, while the P domains form a homodimer that interacts with receptors. The P domain is further subdivided into two subdomains, termed P1 and P2. The P2 subdomain is likely an insertion in the P1 subdomain; consequently, the P domain is divided into the P1-1, P2, and P1-2 subdomains. In order to investigate capsid antigenicity, N-terminal (N-term)/S/P1-1 and P2/P1-2 were switched between two sapovirus genotypes GI.1 and GI.5. The chimeric VP1 constructs were expressed in insect cells and were shown to self-assemble into virus-like particles (VLPs) morphologically similar to the parental VLPs. Interestingly, the chimeric VLPs had higher levels of cross-reactivities to heterogeneous antisera than the parental VLPs. In order to better understand the antigenicity from a structural perspective, we determined an intermediate-resolution (8.5-Å) cryo-electron microscopy (cryo-EM) structure of a chimeric VLP and developed a VP1 homology model. The cryo-EM structure revealed that the P domain dimers were raised slightly (∼5 Å) above the S domain. The VP1 homology model allowed us predict the S domain (67-229) and P1-1 (229-280), P2 (281-447), and P1-2 (448-567) subdomains. Our results suggested that the raised P dimers might expose immunoreactive S/P1-1 subdomain epitopes. Consequently, the higher levels of cross-reactivities with the chimeric VLPs resulted from a combination of GI.1 and GI.5 epitopes. IMPORTANCE: We developed sapovirus chimeric VP1 constructs and produced the chimeric VLPs in insect cells. We found that both chimeric VLPs had a higher level of cross-reactivity against heterogeneous VLP antisera than the parental VLPs. The cryo-EM structure of one chimeric VLP (Yokote/Mc114) was solved to 8.5-Å resolution. A homology model of the VP1 indicated for the first time the putative S and P (P1-1, P2, and P1-2) domains. The overall structure of Yokote/Mc114 contained features common among other caliciviruses. We showed that the P2 subdomain was mainly involved in the homodimeric interface, whereas a large gap between the P1 subdomains had fewer interactions.

Asunto(s)

Microscopía por Crioelectrón , Sapovirus/química , Sapovirus/ultraestructura , Virosomas/química , Virosomas/ultraestructura , Secuencia de Aminoácidos , Anticuerpos Antivirales/inmunología , Antígenos Virales/genética , Antígenos Virales/inmunología , Antígenos Virales/metabolismo , Antígenos Virales/ultraestructura , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/ultraestructura , Reacciones Cruzadas , Datos de Secuencia Molecular , Multimerización de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Recombinación Genética , Sapovirus/genética , Sapovirus/inmunología , Virosomas/genética , Virosomas/inmunología

RESUMEN

Acute gastroenteritis accounts for a significant burden of medically attended illness in children under the age of five. For this study, four multiplex reverse transcription PCR assays were used to determine the incidence of adenovirus, astrovirus, coronavirus, norovirus GI and GII, rotavirus, and sapovirus in stool samples submitted for viral electron microscopy (EM) to the Children's Hospital Colorado. Of 1105 stool samples available, viral RNA/DNA was detected in 247 (26.2%) of 941 pediatric samples (median age = 2.97 years, 54% male) with 28 (3.0%) positive for more than one virus. Adenovirus, astrovirus, norovirus GI, norovirus GII, rotavirus, and sapovirus were detected in 95 (10.0%), 33 (3.5%), 8 (0.9%), 90 (9.6%), 49 (5.2%), and 2 (0.2%) of the pediatric samples, respectively. No coronaviruses were identified. Sequencing of norovirus positive samples indicated an outbreak of norovirus strain GII.4 in 2006 with evidence of numerous circulating strains. Multiple samples from the same immunocompromised patients demonstrated symptomatic shedding of norovirus for up to 32 weeks and astrovirus for 12 weeks. RT-PCR detected 99 of 111 (89%) adenovirus-positive samples versus 12 (11%) by EM, and 186 of 192 (97%) sapovirus/astrovirus/norovirus-positive samples versus 21 (11%) by EM. Noroviruses and adenoviruses are common causes of gastroenteritis in children. Immunocompromised patients can be infected with multiple viruses and shed viruses in their stools for prolonged periods. This data support the superiority of RT-PCR compared to EM for diagnosis of viral gastroenteritis.

Asunto(s)

Infecciones por Adenoviridae/epidemiología , Adenoviridae/aislamiento & purificación , Infecciones por Adenovirus Humanos/epidemiología , Infecciones por Caliciviridae/epidemiología , Infecciones por Enterovirus/epidemiología , Gastroenteritis/epidemiología , Gastroenteritis/virología , Infecciones por Rotavirus/epidemiología , Adenoviridae/genética , Adenoviridae/ultraestructura , Niño , Preescolar , Colorado/epidemiología , Coronavirus/aislamiento & purificación , Coronavirus/ultraestructura , Brotes de Enfermedades , Heces/virología , Femenino , Gastroenteritis/etiología , Humanos , Lactante , Masculino , Microscopía Electrónica , Reacción en Cadena de la Polimerasa Multiplex , Norovirus/aislamiento & purificación , Norovirus/ultraestructura , Virus ARN/genética , Virus ARN/aislamiento & purificación , Virus ARN/ultraestructura , ARN Viral/genética , ARN Viral/aislamiento & purificación , Rotavirus/genética , Rotavirus/aislamiento & purificación , Sapovirus/aislamiento & purificación , Sapovirus/ultraestructura , Factores de Tiempo , Esparcimiento de Virus

RESUMEN

Human sapovirus (SaV) is uncultivable, but expression of the recombinant capsid protein (rVP1) in insect cells results in the formation of virus-like particles (VLPs) that are morphologically similar to the native viruses. However, the SaV rVP1 expression levels are considerably low. We have found that inclusions of short foreign nucleotide sequences inserted directly upstream from the predicted rVP1 AUG start codon lead to increased yield of VLPs. This method allowed us to express a SaV rVP1, which could not have been expressed to measurable or practical levels otherwise.

Asunto(s)

Proteínas de la Cápside/biosíntesis , Proteínas Recombinantes/biosíntesis , Sapovirus , Animales , Cápside/ultraestructura , Proteínas de la Cápside/genética , Línea Celular , Codón Iniciador , Expresión Génica , Humanos , Recién Nacido , Masculino , Mutagénesis Insercional , Proteínas Recombinantes/genética , Sapovirus/genética , Sapovirus/ultraestructura , Spodoptera/citología

RESUMEN

The family Caliciviridae is divided into four genera and consists of single-stranded RNA viruses with hosts ranging from humans to a wide variety of animals. Human caliciviruses are the major cause of outbreaks of acute nonbacterial gastroenteritis, whereas animal caliciviruses cause various host-dependent illnesses with a documented potential for zoonoses. To investigate inter- and intragenus structural variations and to provide a better understanding of the structural basis of host specificity and strain diversity, we performed structural studies of the recombinant capsid of Grimsby virus, the recombinant capsid of Parkville virus, and San Miguel sea lion virus serotype 4 (SMSV4), which are representative of the genera Norovirus (genogroup 2), Sapovirus, and Vesivirus, respectively. A comparative analysis of these structures was performed with that of the recombinant capsid of Norwalk virus, a prototype member of Norovirus genogroup 1. Although these capsids share a common architectural framework of 90 dimers of the capsid protein arranged on a T=3 icosahedral lattice with a modular domain organization of the subunit consisting of a shell (S) domain and a protrusion (P) domain, they exhibit distinct differences. The distally located P2 subdomain of P shows the most prominent differences both in shape and in size, in accordance with the observed sequence variability. Another major difference is in the relative orientation between the S and P domains, particularly between those of noroviruses and other caliciviruses. Despite being a human pathogen, the Parkville virus capsid shows more structural similarity to SMSV4, an animal calicivirus, suggesting a closer relationship between sapoviruses and animal caliciviruses. These comparative structural studies of caliciviruses provide a functional rationale for the unique modular domain organization of the capsid protein with an embedded flexibility reminiscent of an antibody structure. The highly conserved S domain functions to provide an icosahedral scaffold; the hypervariable P2 subdomain may function as a replaceable module to confer host specificity and strain diversity; and the P1 subdomain, located between S and P2, provides additional fine-tuning to position the P2 subdomain.

Asunto(s)

Caliciviridae/clasificación , Caliciviridae/ultraestructura , Cápside/química , Variación Genética , Secuencia de Aminoácidos , Animales , Caliciviridae/química , Caliciviridae/genética , Cápside/metabolismo , Cápside/ultraestructura , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Microscopía por Crioelectrón , Cristalización , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Norovirus/química , Norovirus/genética , Norovirus/ultraestructura , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sapovirus/química , Sapovirus/genética , Sapovirus/ultraestructura , Especificidad de la Especie , Vesivirus/química , Vesivirus/genética , Vesivirus/ultraestructura , Virión/química , Virión/metabolismo , Virión/ultraestructura

RESUMEN

OBJECTIVES: To review and assess the role of electron microscopy in the investigation of new infectious diseases. DESIGN: To design a screening strategy to maximize the likelihood of detecting new or emerging pathogens in clinical samples. RESULTS: Electron microscopy remains a useful method of investigating some viral infections (infantile gastroenteritis, virus-induced outbreaks of gastroenteritis and skin lesions) using the negative staining technique. In addition, it remains an essential technique for the investigation of new and emerging parasitic protozoan infections in the immunocompromised patients from resin-embedded tissue biopsies. Electron microscopy can also have a useful role in the investigation of certain bacterial infections. CONCLUSIONS: Electron microscopy still has much to contribute to the investigation of new and emerging pathogens, and should be perceived as capable of producing different, but equally relevant, information compared to other investigative techniques. It is the application of a combined investigative approach using several different techniques that will further our understanding of new infectious diseases.

Asunto(s)

Enfermedades Transmisibles Emergentes/microbiología , Enfermedades Transmisibles Emergentes/virología , Microscopía Electrónica/métodos , Animales , Gastroenteritis/virología , Humanos , Microsporidios/ultraestructura , Norovirus/ultraestructura , Orthopoxvirus/ultraestructura , Sapovirus/ultraestructura , Adhesión del Tejido

RESUMEN

To evaluate the performance of an automated specimen search system in the detection of caliciviruses such as Norwalk-like viruses and Sapporo-like viruses, a suitable negative staining method was developed and the viruses were examined using the system installed in a transmission electron microscope (TEM). Clear images of the viruses were obtained by staining with 2% uranyl acetate at pH 4.0 as compared with 2% phosphotungstic acid staining at any pH. When the image parameter of 30+/-6 nm for the diameter of a single virus-like particle of 2% uranyl-acetate-stained Norwalk-like virus was set on the automated specimen search system, 95% of the virus-like particles that were counted by the conventional TEM technique were detected. The system was used to detect Norwalk-like viruses in five semipurified stool samples in which Norwalk-like viruses had already been detected by reverse transcription-polymerase chain reaction assay and conventional electron microscopy. The positive detection rate for Norwalk-like viruses, which had been counted by the conventional technique, ranged from 56.2 to 77.9% using this system. Our findings indicate that the automated specimen search system installed in a TEM is suitable for the detection of caliciviruses in semipurified stool samples. The system is useful for clinical diagnosis without the need for operator intervention.

Asunto(s)

Infecciones por Caliciviridae/virología , Gastroenteritis/virología , Norovirus/aislamiento & purificación , Sapovirus/aislamiento & purificación , Automatización , Infecciones por Caliciviridae/patología , Gastroenteritis/patología , Humanos , Microscopía Electrónica/métodos , Norovirus/genética , Norovirus/ultraestructura , Compuestos Organometálicos , Ácido Fosfotúngstico , Sapovirus/genética , Sapovirus/ultraestructura , Coloración y Etiquetado/métodos