Antony Hale

Capsid Protein Diversity among Norwalk-like Viruses

We have determined the complete capsid gene sequence of 20 Norwalk-like viruses (NLVs) collected predominantly from outbreaks in the UK between 1989 and 1996. These comprised nine genogroup I and eleven genogroup II strains. Phylogenetic analysis of these and 15 published sequences suggest seven genomic sub-groups within genogroup I, including three previously described. In genogroup II, eight sub-groups were apparent, of which four were novel. Amino acid identities between strains of distinct genogroups ranged from 37 to 44% while varying between 61 and 100% for strains within a genogroup. Separate phylogenetic analyses of the N-terminus and central variable region of the capsid showed good correlation. Sequence divergence between strains was greatest within the central variable region, with amino acid sequence identities as low as 28% within a genogroup. These 15 genomic sub-groups provide a framework for further investigations of genetic and antigenic relationships within this calicivirus clade.

Distinct epidemiological patterns of Norwalk-like virus infection.

Norwalk‐like viruses (NLV) are important economically as a cause of both sporadic gastroenteritis in the community and large outbreaks in hospitals and other institutional settings. Despite the description of several antigenic types relatively little is known about the epidemiology of these individual types. NLVs were detected by electron microscopy in faecal specimens from 706 outbreaks of gastroenteritis that represented 68% of all outbreaks of non‐bacterial gastroenteritis. These outbreaks took place in the counties of West and North Yorkshire and Humberside during six winter seasons between July 1992 and June 1998. NLV strains from 671 outbreaks were typed by antigen capture enzyme linked immunosorbent assays (ELISA) based on antisera made to recombinant virus‐like particles of three antigenically distinct NLVs; Norwalk (NV), Mexico (MXV) and Grimsby (GRV) viruses. GRV was the predominant strain for five of the six winter seasons and overall was associated with 61% of NLV outbreaks. MXV was responsible for a single epidemic peak in the winter of 1993/94 but was also observed at other times throughout the study period. NV was only associated with two outbreaks in 1994 that were epidemiologically linked. Strains from the remaining 32% of outbreaks were non‐reactive in all three ELISA. Thus, a single NLV antigenic type seems to have predominated during the period 1992 to 1998 in the UK. J. Med. Virol. 62:99–103, 2000.

Comparison of four RNA extraction methods for the detection of small round structured viruses in faecal specimens

Four methods for extraction of the RNA genome of small round structured viruses (SRSVs) from faecal specimens by reverse transcription polymerase chain reaction (RT-PCR) were evaluated. The efficiency of recovery of viral RNA and removal of amplification inhibitors were compared. RNA extraction using the metal chelating agent Chelex-100 and Sephadex G200 column chromatography were the most sensitive, detecting a 10(-4) dilution of a known SRSV positive specimen. Guanidinium thiocyanate (GTC) with adsorption of viral RNA onto silica was 10-fold less sensitive. The fourth method, based on PEG precipitation followed by phenol/chloroform extraction with the addition of the detergent cetyltrimethylammonium bromide (CTAB), only detected a 1 in 10 dilution of the positive specimen. The CTAB method was 2- to 50-fold less sensitive than the GTC/silica method when dilution series of three further SRSV positive specimens were tested. Thirty-six SRSV negative faecal specimens were spiked with virus and RT-PCR performed following RNA extraction by each of the four techniques in order to assess the ability of these methods to remove amplification inhibitors. The GTC/silica method successfully removed inhibitors from all samples whereas partial or complete inhibition was observed in seven (19%) specimens following extraction by the CTAB method, 17 (47%) by the Sephadex method, and 20 (56%) by the Chelex method. We conclude that, of these four methods, the GTC/silica method is the most appropriate for the extraction of viral RNA from faecal samples prior to RT-PCR for detecting SRSVs.

Generation of recombinant virus-like particles of human and non-human polyomaviruses in yeast Saccharomyces cerevisiae.

Objectives: Non-viral methods of gene transfer have been preferred in gene therapy approaches for several reasons, particularly for their safety, simplicity and convenience in introducing heterologous DNA into cells. Polyomavirus virus-like particles (VLPs) represent a promising carrier for encapsidation of foreign nucleic acids for gene therapy. For the development of such gene delivery systems as well as for providing reagents for improving virus diagnostics, an efficient yeast expression system for the generation of different polyomavirus VLPs was established. Methods: A galactose-inducible Saccharomyces cerevisiae yeast expression system was used. Formation of empty VLPs was confirmed by cesium chloride ultracentrifugation, agarose gel electrophoresis and electron microscopy. Cross-reactivity of the major capsid proteins (VP1) of different polyomaviruses was analyzed by Western blot using rabbit and mice sera raised against the VP1 proteins. Results: VP1 of polyomaviruses from humans (JC polyomavirus and serotypes AS and SB of BK polyomavirus), rhesus monkeys (simian virus 40), hamsters (hamster polyomavirus), mice (murine polyomavirus) and birds (budgerigar fledgling disease virus) were expressed at high levels in yeast. Empty VLPs formed by all yeast-expressed VP1 proteins were dissociated into pentamers and reassociated into VLPs by defined ion and pH conditions. Different patterns of cross-reactivity of the VP1 proteins with heterologous mice and rabbit sera were observed. Conclusion: The developed heterologous yeast expression system is suitable for high-level production of polyomavirus VLPs. Yeast-derived VLPs are generally free of toxins, host cell DNA and proteins. These VLPs might be useful for the generation of new diagnostical tools, gene delivery systems and antiviral vaccines.

Diversity of Noroviruses Cocirculating in the North of England from 1998 to 2001

ABSTRACT A study was undertaken to investigate the diversity of noroviruses (NVs) in fecal samples from patients from 529 outbreaks and 141 sporadic cases of gastroenteritis in the North of England from September 1998 to August 2001. NV strains were detected by electron microscopy and characterized by a combination of the Grimsby virus antigen enzyme-linked immunosorbent assay, reverse transcriptase PCR, the heteroduplex mobility assay, and DNA sequencing. Twenty-one distinct NV strains, including several novel or variant strains not seen previously, were found circulating in the population studied. Genogroup II NVs were responsible for 83% of the outbreaks. Several strains cocirculated at any one time. The Bristol (Grimsby/Lordsdale) and Hawaii (Girlington) genotypes were the most prevalent among the NVs identified, detected in 49 and 20% of the outbreaks, respectively. A limited number of other genogroup II and I strains were cocirculating. The virus populations detected in hospitals and nursing homes were distinct from those found in community-based outbreaks. Outbreaks in hospitals and nursing homes were more likely to be caused by genogroup II strain Grimsby or Girlington (P < 0.0001) than by other genogroup II or I strains.

Diagnostic Accuracy and Analytical Sensitivity of IDEIA Norovirus Assay for Routine Screening of Human Norovirus

ABSTRACT Noroviruses (NoVs) are recognized as the leading cause of epidemic and sporadic acute gastroenteritis. Early detection of NoV is crucial to control the spread of the disease. In this study, we evaluated the diagnostic accuracy, analytical sensitivity, and analytical reactivity of the IDEIA Norovirus assay (an enzyme immunoassay [EIA]) in a prospective and retrospective study design. A total of 557 prospectively collected fecal samples and a panel of 97 archived fecal samples, including 21 different GI and GII genotypes, were tested by conventional reverse transcription-PCR (RT-PCR)/bidirectional sequencing, real-time RT-PCR, and electron microscopy. The sensitivity and specificity of the EIA were 57.6% and 91.9%, respectively. The sensitivity for detecting NoV in fecal samples from outbreaks improved from 44.1% when three samples were tested to 76.9% when five samples per outbreak were tested. The EIA was able to detect strains from 7 GI and 11 GII genotypes. The analytical sensitivity of the EIA was 3.1 × 106 and 1.6 × 107 virus particles g−1 of fecal sample for NoV GI and GII strains, respectively. Most GII samples positive by EIA had a threshold cycle (CT ) of <26.5, and 50% of the GII samples negative by EIA had a CT of >25.6, suggesting that, although strains from genotypes GI.8, GII.10, and GII.16 were not detected, the low sensitivity of the EIA is primarily caused by low virus concentration. In conclusion, the current EIA may be of use as a rapid screening test during a norovirus outbreak investigation when multiple fecal samples are available; however, sporadic samples should be tested by molecular methods.

Homotypic and heterotypic IgG and IgM antibody responses in adults infected with small round structured viruses

Antibody responses to recombinant Norwalk (rNV) and Mexico (rMXV) viral capsid proteins were studied in 39 adults involved in outbreaks of gastroenteritis associated with genogroup 2 small round structured viruses (SRSVs). Nineteen individuals were involved in outbreaks associated with MXV‐like strains and 20 in outbreaks associated with four other genogroup 2 SRSVs. IgG antibodies were measured in acute and convalescent sera using indirect enzyme‐linked immunosorbent assay (ELISA), and IgM was measured by indirect and capture ELISAs. Nineteen (49%) patients demonstrated a significant rise in IgG to rMXV with four (10%) patients also showing anamnestic responses to rNV. Fourteen patients were positive in the rMXV IgM‐capture ELISA, representing 74% of patients demonstrating IgG rises. IgG and IgM responses to rMXV were observed in both groups, although higher levels of responses were seen in adults infected with MXV‐like strains than those infected with non‐MXV genogroup 2 viruses. No significant IgM responses were observed to rNV. These results indicate that, following SRSV infection, adults show a rise in antibody which is broadly reactive to viruses within but not between genogroups, although greater homotypic than heterotypic responses are produced. These findings have implications for interpretation of seroepidemiological studies and serodiagnosis of SRSV infections using recombinant capsids. J. Med. Virol. 54:305–312, 1998.

Epidemiology of Mexico Virus, a Small Round-Structured Virus in Yorkshire, United Kingdom, between January 1992 and March 1995

The epidemiology of the small round-structured virus, Mexico virus (MxV), was investigated in North and West Yorkshire, United Kingdom, between January 1992 and March 1995 using a type-specific antigen ELISA. The results indicate that an epidemic of MxV occurred during the winter of 1993-1994, when this strain was associated with 45 of 99 outbreaks and sporadic childhood cases of gastroenteritis. Only 4 MxV-like isolates were found during the 1992-1993 winter season and none in the 1994-1995 season. This descriptive epidemiologic study suggests that MxV has an epidemic pattern of infection.

Evaluation of an antigen capture ELISA based on recombinant mexico virus capsid protein

BACKGROUND The diagnosis of gastrointestinal infections caused by small round structured viruses (SRSV) has relied upon electron microscopy and antigen/antibody assays based on Norwalk virus. We investigated cases of gastroenteritis associated with SRSVs employing a new sandwich enzyme-linked immunosorbent assay (ELISA) using hyperimmune animal anti-sera against recombinant Mexico virus capsid protein (rMXV). STUDY DESIGN One hundred and thirty-five specimens from 86 episodes of gastroenteritis associated with SRSVs, collected in the UK between October 1993 and September 1994, were tested in the rMXV assay. RESULTS Forty-seven (35%) specimens from 35 of 86 (41%) episodes were positive in the rMXV ELISA and these could further be divided into high and low reactors. Sequencing of a 266-base region of the RNA polymerase gene revealed that strains highly reactive in the rMXV assay demonstrated a high degree of similarity to MXV (97-99% at the nucleotide level), whereas low-reactive strains consist of Mexico-like strains and a heterogeneous group of viruses exhibiting 70-75% similarity to MXV. CONCLUSION Our results indicate that the rMXV ELISA is predominantly a type specific assay, although some cross reactivity with other genogroup 2 SRSVs was observed. MXV was responsible for 26% of SRSV-associated gastrointestinal infections investigated in the UK during one years surveillance.

Prevalence of antibodies to HTLV in antenatal clinic attenders in South East London

The prevalence of antibodies to HTLV in women attending a south east London antenatal clinic between October 1990 and July 1992 was determined using sera referred for routine rubella antibody testing. Samples were screened for HTLV antibody using a modified Fujirebio gel particle agglutination test and reactive sera confirmed by ELISA (Abbott Laboratories, North Chicago, IL) and two commercial Western blots (Cambridge Biotech Inc., Rockville, MD, and Diagnostic Biotechnology, Genelab Diagnostics, Louvaine, Belgium). This strategy confirmed the presence of HTLV‐1 antibodies in 12 out of 6,289 sera (0.19%, 95% confidence limits 0.083% to 0.30%) and HTLV‐2 antibodies in 2 (0.03%) sera. Specimens from 8 of 821 (0.97%, 95% confidence limits 0.42% to 1.9%) Afro‐Caribbean women, three of 1,136 (0.26%, 95% confidence limits 0.055% to 0.78%) African women, and one of 3,049 (0.033%, 95% confidence limits 0.006% to 0.18%) Caucasian women were positive for HTLV‐1 antibodies. Sera from Afro‐Caribbean women born in the Caribbean were 7.6 times more likely to be HTLV‐1 antibody positive than sera from Afro‐Caribbean women born in the UK (P = 0.012). Selective testing of Afro‐Caribbean and African antenatal clinic attenders, in this setting, would have identified 11 of the 12 HTLV‐1 infections at an estimated cost of prevention of HTLV‐1 associated disease of £100,000 per case which is considerably less than the £1.3 million which has been estimated to prevent a case by universal screening of UK blood donors. J. Med. Virol. 52:326–329, 1997.