Solid-phase Immune Electron Microscopy Research Articles

Antibody-based affinity cryo-EM grid

The Affinity Grid technique combines sample purification and cryo-Electron Microscopy (cryo-EM) grid preparation into a single step. Several types of affinity surfaces, including functionalized lipids monolayers, streptavidin 2D crystals, and covalently functionalized carbon surfaces have been reported. More recently, we presented a new affinity cryo-EM approach, cryo-SPIEM, which applies the traditional Solid Phase Immune Electron Microscopy (SPIEM) technique to cryo-EM. This approach significantly simplifies the preparation of affinity grids and directly works with native macromolecular complexes without need of target modifications. With wide availability of high affinity and high specificity antibodies, the antibody-based affinity grid would enable cryo-EM studies of the native samples directly from cell cultures, targets of low abundance, and unstable or short-lived intermediate states.

Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies.

Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (non-purified His-tagged bacteriophage T7, His-tagged Escherichiacoli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures.

Characterisation of the bovine enteric calici-like virus, Newbury agent 1.

The bovine enteric calici‐like virus, Newbury agent 1 (NA1) was characterised to determine if it is a member of the Caliciviridae and to establish its antigenic relationship to the established bovine enteric calicivirus Newbury agent 2 (NA2). Solid phase immune electron microscopy (SPIEM) allowed quantification of NA1 virions and identification of faecal samples with optimal virus levels. NA1 particles were 36.6 nm in diameter, had an indefinite surface structure resembling that of human small round structured viruses (SRSVs), and a buoyant density of 1.34 g ml−1. A single capsid protein of 49.4 kDa was detected by Western blotting in purified NA1 preparations prepared from post‐infection but not pre‐infection faecal samples and with post‐ but not pre‐infection sera. NA1 was antigenically unrelated to the bovine enteric calicivirus NA2 by SPIEM. These properties were consistent with classification of NA1 within the Caliciviridae but demonstrated heterogeneity in the capsid composition of bovine enteric caliciviruses.

Characterisation of the bovine enteric calici-like virus, Newbury agent 1

The bovine enteric calici-like virus, Newbury agent 1 (NA1) was characterised to determine if it is a member of the Caliciviridae and to establish its antigenic relationship to the established bovine enteric calicivirus Newbury agent 2 (NA2). Solid phase immune electron microscopy (SPIEM) allowed quantification of NA1 virions and identification of faecal samples with optimal virus levels. NA1 particles were 36.6 nm in diameter, had an indefinite surface structure resembling that of human small round structured viruses (SRSVs), and a buoyant density of 1.34 g ml −1. A single capsid protein of 49.4 kDa was detected by Western blotting in purified NA1 preparations prepared from post-infection but not pre-infection faecal samples and with post- but not pre-infection sera. NA1 was antigenically unrelated to the bovine enteric calicivirus NA2 by SPIEM. These properties were consistent with classification of NA1 within the Caliciviridae but demonstrated heterogeneity in the capsid composition of bovine enteric caliciviruses.

Investigation of an outbreak of gastroenteritis caused by Norwalk-like virus, using solid phase immune electron microscopy

In February 1993, 95 persons (47 patients and 48 staff members) were affected by an hospital outbreak of viral gastroenteritis. Using direct electron microscopy (EM) the causative agent was identified as a small round structured virus. This was confirmed as a Norwalk-like virus using solid phase immune electron microscopy (SPIEM). Of 94 stool samples examined, 12 (13%) samples containing small round structured viruses (SRSV) were SPIEM positive for Norwalk-like virus. A further 25 (27%) samples contained small round featureless virus (SRFV) identified by direct EM and were negative on SPIEM. The illness was characterized by preceding influenza-like symptoms in 76% of cases followed by vomiting (76%), diarrhoea (79%) and abdominal pain (79%). One fatality was recorded. The outbreak lasted for 15 days, with a peak incidence of new cases amongst patients and staff occurring on day 5. It was controlled through a combination of ward closures, patient cohorting, suspension of duties for affected staff and disinfection procedures. Difficulties were encountered in the education of staff and in the implementation of environmental control measures. Screening of hospital catering services and a case control study, carried out among affected staff members, failed to identify a foodborne source. Consumption of tap water in the hospital was commoner among affected staff members than among controls, but this did not reach significance (P = 0·1).

The Bovine Newbury Agent-2 Is Genetically More Closely Related to Human SRSVs Than to Animal Caliciviruses

The hypothesis that the enteric bovine calici-like virus Newbury agent (NA-2) belongs to the familyCaliciviridaewas examined by genome sequence analysis. Use of solid-phase immune electron microscopy allowed samples with good levels of virus to be identified and amplification of the genome was achieved by reverse transcription–polymerase chain reaction. Examination of a 216-amino-acid sequence in the RNA-dependent RNA polymerase gene and a 116-amino-acid sequence in the capsid gene showed that NA-2 had the closest deduced amino acid identity (77 to 80% for the polymerase region and 67 to 73% for the capsid region) to the morphologically indistinguishable human SRSVs (small round structured viruses) of genogroup 1, which are classified as members of theCaliciviridae.It had a weak relationship (<34.5% deduced amino acid identity) in both the polymerase and the capsid regions to animal caliciviruses, all of which have classical morphology. This is the first genomic data from a nonhuman virus with SRSV morphology. It confirms the hypothesis that the bovine enteric calici-like virus NA-2 is a member of the familyCaliciviridaeand endorses the observation to date that viruses with SRSV morphology are genomically distinct.

A Part of Amino Acid Sequence of the Gastro-colonic Cancer Inducing Virus Protein in WF-Osaka (WF/Osp) Rats.

Wistar-Furth-Osaka (WF/Osp) rat strain has been considered to induce spontaneous gastro-colonic adenocarcinoma. ACI rats, fosterbred by WF/Osp had developed the same type of colon adenocarcinoma. The serum from colon cancer-carrying WF/Osp rats induced the same tumors in Wistar/Shionogi (Wistar/Shi) rats. The serum from colon cancer-carrying WF/Osp rats also induced the analogous tumors in ACI rats by intra-peritoneal injection at newborn period respectively. Negative staining on solid-phase immune electron microscopy study revealed numerous particles in the tissue culture medium of transplantable gastric carcinoma. To bring to light, the amino acid sequence of virus antigen protein, using affinity chromatography column, we succeeded to obtain pure antigen on PVDF membrane. Protein structure of the antigen was analyzed, and amino acid sequence of Valine-Proline-Asparatic acid-Lysine-Threonine-Valine-Lysine-Tryptophan-Asparagine (VPDKTVKWN) turned out to be the novel structure among protein sequence by FASTA E-mail Server on GenomeNet.

Role of long-persisting human hepatitis E virus antibodies in protection

The role of long-persisting HEV antibodies in humans was investigated. A well-characterized human volunteer anti-HEV (IgG) after 4 years of the recovery of the disease was passively immunized (200 mg kg −1 body wt) intramuscularly into three rhesus monkeys (nos 7, 8 and 9), while two monkeys (nos 5 and 6) were injected with normal immunoglobulin preparation (negative for anti-HEV IgG). At 16 h later all the animals were challenged intravenously with live HEV, as 10% stool extract prepared from the volunteer (positive for HEV by solid-phase immune electron microscopy). It was seen that transaminases (ALT/AST) were elevated in immunized monkeys on day 49 (no. 8), 60 (no. 7) and 86 (no. 9) after the virus challenge. On serological examination, all immunized (nos 7, 8 and 9) and one unimmunized (no. 6) monkeys were found positive for HEV IgM on day 10 post-challenge, while monkey no. 5 was negative. Further, all the animals from the immunized and unimmunized group seroconverted to HEV-IgG when tested on days 25 and 55 post-challenge. This has clearly shown that there is no protection. Therefore, these long-persisting HEV antibodies alone in humans, may not be protective in passive immunization.

Outbreak of gastroenteritis in military recruits associated with serotype 3 astrovirus infection.

A serotype 3 astrovirus was identified in stool samples from an outbreak of acute gastroenteritis that occurred among military recruits in France. Sixteen stools samples and eight pairs of acute- and convalescent-phase serum were collected from affected individuals. Astrovirus was detected in two stool samples by electron microscopy and in four stool samples by reverse transcriptase-polymerase chain reaction (RT-PCR). Seroconversion to the astrovirus present in one stool was detected in seven patients by using solid-phase immune electron microscopy (SPIEM) and dot blot. For three patients, the serological results were consistent with the PCR results, indicating that astrovirus was a cause of gastroenteritis in these young adults. This study describes the characterization of the serotype 3 astrovirus associated with this outbreak. The virus has a buoyant density in cesium chloride of 1.365 gm/ml and contains two proteins immuno-precipitated with rabbit serum. Only the larger protein was recognized by immunoblotting using a convalescent-phase human serum. The protein composition of this virus differs from that reported for serotype 1 astrovirus, indicating heterogeneity in the capsid composition among astrovirus serotypes.

Establishment of a Persistently Infected Cell Line with the Colon Cancer Inducing Virus of WF-Osaka (WF/Osp) Rats.

Wistar-Furth-Osaka (WF/Osp) rat strain has been considered to induce spontaneous gastrocolonic adenocar-cinoma. ACI rats, fosterbred from WF/Osp also developed the same type of colon adenocarcinoma. The serum from colon cancer-carrying rats induced the same tumors in Wistar/Shionogi (Wistar/Shi) rat strain, and the serum of which also induced analogous tumors in ACI rat strain by intraperitoneal injection at newborn period. Negative staining on solid-phase immune electron microscopy study revealed numerous particles in the tissue culture medium of transplantable gastric carcinoma. To bring to light the infectious cell line, susceptible to colon cancer inducing virus was the consequential element to analyze transmissible agents. Using monoclonal antibody against colon cancer inducing virus, IF studies were carried out on all organs, mainly the chest, abdominal, and urogenital organs of cancer carrying WF/Osp rat. We found that the NBT-II cell line originating from the urinary bladder cancer cell is susceptible to gastrocolonic cancer inducing virus.

Evaluation of a degenerate primer for the PCR detection of human caliciviruses

Numerous outbreaks of gastroenteritis have been associated with Norwalk virus and Small Round Structured Viruses (SRSVs). These single-stranded RNA viruses, recently classified in the Caliciviridae, have been divided into three genogroups. Antigenic relationships also have been established among the different strains. As both an in vitro culture system and an animal model are lacking for these viruses, virus detection depends primarily on electron microscopy, immunological assays or molecular detection. In this study we first analyzed the genetic homology of the RNA polymerase region for 40 SRSV strains. From a consensus sequence for these strains, we designed a degenerate oligonucleotide to prime cDNA synthesis from viral RNA. We evaluated the degenerate primer in combination with three previously described primers in PCR reactions. A panel of 15 stools containing SRSVs, typed when possible by solid phase immune electron microscopy (SPIEM), were selected to represent all three genogroups and four different SPIEM antigenic types. Serial dilutions of the purified viral nucleic acids were amplified using the three different primer sets. Virus-specific probes were used to characterize the amplicons obtained. Virus-specific amplicons were obtained with at least one primer pair for each strain, but apparent viral RNA titers differed as much as 1000-fold between primer sets. Amplicons from all but one of the 15 strains were confirmed as virus-specific using a panel of 10 different probes. Correlations between the most sensitive primer pair and SPIEM type were seen. This study showed that a single degenerate primer could be used in cDNA synthesis for a variety of SRSVs but that the sensitivity of the RT-PCR assay depended upon the second primer and virus-specific probes used.

Enzyme immunoassay using baculovirus-expressed human calicivirus (Mexico) for the measurement of IgG responses and determining its seroprevalence in London, UK.

The use of an enzyme immunoassay (EIA) employing a baculovirus-expressed recombinant human calicivirus (Mexico virus, MxV) for the detection of IgG-specific antibodies is described. MxV appeared to be related antigenically to a strain of small round structured virus, SRSV/UK4/Leeds/91, which had previously been shown by solid phase immune electron microscopy (SPIEM) to be related to Snow Mountain agent (SMA). One other outbreak which occurred in San Anita, USA in 1980 and was due to consumption of contaminated water was caused by a virus antigenically related to MxV. Volunteers and patients who developed significant IgG responses to rMxV showed anamnestic IgG responses (2 to 4-fold) in the recombinant Norwalk virus (rNV) IgG assay. Patients and volunteers who were known to have been infected with several other strains of calicivirus/small round spherical viruses (SRSV) including NV and SRSV UK3 showed no significant antibody response to rMxV in the EIA. A seroepidemiological survey of sera from 338 children in London showed that infection with MxV occurred earlier in life than NV. Primary infections with MxV were common after the age of 6 months. Over 70% of children had evidence of infection by the ages of 2 years, whereas only 12% of these children had been infected with NV. High concentrations of maternal antibody were present during the first month of life which was detected in 96% of the neonates. The results suggest that the high sensitivity of the EIA may be detecting maternal antibody throughout the first 8 months of life.

Genomic diversity of small round structured viruses in the United Kingdom.

Fifty-two faecal specimens collected in the United Kingdom between 1986 and 1992, which contained small round structured virus (SRSV) particles, were tested by reverse transcriptase polymerase chain reaction assays using two primer pairs derived from sequences of Snow Mountain Agent and Norwalk virus. There was poor correlation between results obtained with each primer pair. Twenty specimens (38%) gave positive bands with SM51/31 primers and 18 (34%) were positive with SM52/32 primers, with a total of 30 specimens (57.7%) giving amplification products of the expected size with one or both primer pairs. Genomic variation was investigated by sequencing a 266 bp region of the RNA polymerase gene from nine strains which had been antigenically typed by solid phase immune electron microscopy (SPIEM). RNA sequence identities ranged from 53 to 99%. Three genomic groups were suggested by phylogenic analysis, the first of which contained Norwalk virus, Southampton virus, and strains typed by SPIEM as SRSV UK2. The second contained Snow Mountain agent and strains typed as either SRSV UK3 or UK4. The third contained strains typed as SRSV UK1 and strains untypeable by SPIEM. Some correlation was demonstrated when antigen typing by SPIEM and phylogenic grouping based on sequence data were compared.

Atypical strain of hepatitis E virus (HEV) from North India

Hepatitis E virus (HEV) infection was detected during an epidemic in North India. Virus particles present in the stool of an acutely ill patient (YAM-67) was transmitted intravenously into rhesus monkeys (M. mulata) and orally to a human volunteer. Virus-like particles (VLPs) of 32-34 nm were detected in the bile of monkeys and in the stools of the human volunteer by means of solid phase immune electron microscopy (SPIEM) with acute homologous and heterologous sera. The VLPs were confirmed to be HEV by a reverse transcription polymerase chain reaction (RT-PCR). Virus-like particles from human volunteer stools were passaged further into rhesus monkeys. A bimodal rise in aminotransferase levels were observed in the animals, and liver histopathology indicated mild to severe form of hepatitis. Further, SPIEM and RT-PCR analysis in monkey bile revealed presence of virus from 15 to 45 days post-inoculation. Rechallenge of the animals 6 months after recovery with the same viral inoculum failed to produce abnormal liver function tests indicating the presence of protective immunity during this period. The VLPs in the stool from the patient (YAM-67) with epidemic hepatitis were found to retain infectivity even after several cycles of freeze-thawing and exposure at 37 degrees C for 2 days. Moreover, these VLPs from the patient, human volunteer, and monkeys did not react with an anti-HEV chimpanzee serum from NIH, Bethesda, MD. These findings indicate that this North India isolate of HEV is an atypical strain of HEV. The present study further validates that the rhesus monkey is a suitable experimental model for HEV.

A Large Outbreak of Acute Gastroenteritis Associated with Astrovirus among Students and Teachers in Osaka, Japan

In June 1991, a large outbreak of acute nonbacterial gastroenteritis occurred among students and teachers at 10 primary and 4 junior high schools in Katano City, Osaka, Japan. The outbreak affected > 4700 persons, lasted 5 days, and was believed to have been linked to contaminated food from a common supplier. Astrovirus, identified as the etiologic agent, was detected by direct electron microscopy in 10 of 38 fecal samples obtained from patients with diarrhea. Detection was confirmed by solid-phase immune electron microscopy (IEM), EIA, reverse transcription-polymerase chain reaction, and virus isolation in CaCo-2 cells. Several patients who had astrovirus in their stool also demonstrated a significant antibody response to a reference strain of astrovirus by IEM and EIA and to their own isolate by IEM. Astrovirus can be an important agent of epidemic acute nonbacterial gastroenteritis in school-aged children and adults in Japan.

Characterization of a variant strain of Norwalk virus from a food-borne outbreak of gastroenteritis on a cruise ship in Hawaii

A gastroenteritis outbreak affecting at least 217 (41%) of 527 passengers on a cruise ship was caused by a variant strain of Norwalk virus (NV) that is related to but distinct from the prototype NV strain. Consumption of fresh-cut fruit served at two buffets was significantly associated with illness (P < or = 0.01), and a significant dose-response relationship was evident between illness and the number of various fresh-cut fruit items eaten. Seven (58%) of 12 paired serum specimens from ill persons demonstrated at least fourfold rises in antibody response to recombinant NV capsid antigen. A 32-nm small round-structured virus was visualized by electron microscopy in 4 (29%) of 14 fecal specimens, but none of the 8 specimens that were examined by an enzyme immunoassay for NV antigen demonstrated antigen. Four (40%) of 10 fecal specimens were positive by reverse transcriptase-PCR by using primer pairs selected from the polymerase region of NV. In a 145-bp region, the PCR product shared only 72% nucleotide sequence identity with the reference NV strain and 77% nucleotide sequence identity with Southampton virus but shared 95% nucleotide sequence identity with UK2 virus, a United Kingdom reference virus strain. In addition, the outbreak virus was serotyped as UK2 virus by solid-phase immune electron microscopy. The genetic and antigenic divergence of the outbreak strain from the reference NV strain highlights the need for more broadly reactive diagnostic assays and for improved understanding of the relatedness of the NV group of agents.

Transmissible colon carcinoma of WF-Osaka strain rats.

A litter of ACI rat strain, foster bred by a female colon cancer-prone WF-Osaka rat, developed colon carcinoma in the ascending colon at the age of 4 months. We assumed that some microbial infection during their suckling might cause colon carcinoma of WF-Osaka rats, and that the high incidence of spontaneous regression of the colon carcinoma might be an immunologic reaction against microbial agent in the cancer cells. In this report, we showed that new born rats of ACI, LE and W/Shi strain developed colon carcinoma by the intraperitoneal injection of the serum from cancer carrying WF-Osaka rat within 2 months after birth, and that this microbial agent was transmissible among rats regardless the strain. The etiology of colon carcinoma of WF-Osaka rat was considered to be virus-like infection as we found FITC positive granular materials in the cytoplasm of tissue culture cells of colon carcinoma (C2) and tissue specimen of transplantable gastric carcinoma (S3). Supernatant of tissue culture medium of colon carcinoma (C2) also induced colon carcinoma. Solid-phase immune electronmicroscopy study revealed round to oval particles, measuring approximately 40-50 nm in diameter, in the sediment of tissue culture medium of the transplantable gastric carcinoma (S3), which explain that oncogenic viral agent is substantially related to the induction of colon carcinoma in WF-Osaka rat strain.

Norwalk-like viruses: demonstration of genomic diversity by polymerase chain reaction.

A reverse transcription-polymerase chain reaction (RT-PCR) amplification procedure was developed for the detection of Norwalk-like viruses in fecal specimens. Ninety-nine fecal specimens collected in the United Kingdom and containing small round-structured virus particles as determined by electron microscopy were tested. They came from 50 outbreaks and 16 sporadic cases of viral gastroenteritis. RT-PCR products of the appropriate size for Norwalk virus RNA were detected in 15 specimens from three outbreaks, suggesting that viruses closely related to Norwalk virus have not been circulating widely in the United Kingdom in recent years. From four isolates, the RT-PCR amplification products of two genomic regions were sequenced and the degree of genomic variation was compared. DNA sequencing of the PCR products revealed strong similarities among strains from the United Kingdom (approximately 97% for both regions amplified) but significant differences from Norwalk virus (67 to 78%). All of the viruses detected by RT-PCR were classified as serotype UK2 by solid-phase immune electron microscopy or enzyme-linked immunosorbent assay. These findings provide evidence of a genomic relationship between Norwalk virus and serotype UK2 small round-structured viruses.

Detection of Norwalk virus in the UK by the polymerase chain reaction

We have developed a polymerase chain reaction for the detection of Norwalk virus using the published sequence of the virus RNA dependent RNA polymerase gene and have used this to clone and sequence this region of a virus from a UK outbreak. We have applied this method to a panel of UK Norwalk-like viruses using both Tet-z and Taq DNA polymerases and found that amplification produces a multiplicity of bands from stool samples. However, combination with Southern blotting. Taq polymerase amplification detected virus in 13 of a panel of 30 clinical samples known to contain these viruses and also detected astroviruses in a mixed infection. Amplification using Tet-z DNA polymerase was less efficient (6/30) and detected predominantly viruses typed as UK type 2 by solid phase immune electron microscopy.

Detection of Norwalk virus in the UK by the polymerase chain reaction.

We have developed a polymerase chain reaction for the detection of Norwalk virus using the published sequence of the virus RNA dependent RNA polymerase gene and have used this to clone and sequence this region of a virus from a UK outbreak. We have applied this method to a panel of UK Norwalk-like viruses using both Tet-z and Taq DNA polymerases and found that amplification produces a multiplicity of bands from stool samples. However, in combination with Southern blotting, Taq polymerase amplification detected virus in 13 of a panel of 30 clinical samples known to contain these viruses and also detected astroviruses in a mixed infection. Amplification using Tet-z DNA polymerase was less efficient (6/30) and detected predominantly viruses typed as UK type 2 by solid phase immune electron microscopy.