Assessment of the Utility of Whole Genome Sequencing of Measles Virus in the Characterisation of Outbreaks.

BACKGROUND: Measles is a highly infectious disease caused by measles virus (MeV). Despite the availability of a safe and cost-effective vaccine, measles is one of the world-leading causes of death in young children. Within Europe, there is a target for eliminating endemic measles in 2015, with molecular epidemiology required on 80% of cases for inclusion/exclusion of outbreak transmission chains. Currently, MeV is genotyped on the basis of a 450 nucleotide region of the nucleoprotein gene (N-450) and the hemagglutinin gene (H). However, this is not sufficiently informative for distinguishing endemic from imported MeV. We have developed an amplicon-based method for obtaining whole genome sequences (WGS) using NGS or Sanger methodologies from cell culture isolates or oral fluid specimens, and have sequenced over 60 samples, including 42 from the 2012 outbreak in the UK. RESULTS: Overall, NGS coverage was over 90% for approximately 71% of the samples tested. Analysis of 32 WGS excluding 3' and 5' termini (WGS-t) obtained from the outbreak indicates that the single nucleotide difference found between the two major groups of N-450 sequences detected during the outbreak is most likely a result of stochastic viral mutation during endemic transmission rather than of multiple importation events: earlier strains appear to have evolved into two distinct strain clusters in 2013, one containing strains with both outbreak-associated N-450 sequences. Additionally, phylogenetic analysis of each genomic region of MeV for the strains in this study suggests that the most information is acquired from the non-coding region located between the matrix and fusion protein genes (M/F NCR) and the N-450 genotyping sequence, an observation supported by entropy analysis across genotypes. CONCLUSIONS: We suggest that both M/F NCR and WGS-t could be used to complement the information from classical epidemiology and N-450 sequencing to address specific questions in the context of measles elimination.

Emergence of the GII-4 Norovirus Sydney2012 strain in England, winter 2012-2013.

Norovirus is the commonest cause of acute gastrointestinal disease and is the main aetiological agent of outbreaks of gastroenteritis, particularly in semi-closed environments. Norovirus infections in England typically peak between December and March each year. The most commonly detected norovirus strains belong to the genetically diverse genogroup-II genotype-4 (GII-4) genocluster and in the previous two norovirus winter seasons the majority of GII-4 strains in circulation worldwide have been genetically similar to the GII-4 strain New Orleans 1805/2009/USA. At the beginning of the 2012/13 season a genetically distinct GII-4 strain (Sydney 2012/NSW0514/2012/AU) was described which emerged worldwide during the winter of 2012/13. Here we describe the emergence of norovirus strains genetically related to Sydney2012 in England during the 2012/13 season to replace NewOrleans2009 strains as the most commonly detected variant of GII-4 norovirus in England. Furthermore, we demonstrate that whilst the emergence of Sydney2012 coincided with an early peak in the number of norovirus outbreaks, there was not an overall increase in norovirus activity compared to the previous season. Finally, we show that the Sydney2012 strain is associated with distinct genetic changes compared to the NewOrleans2009 strain, and these changes may have contributed to the emergence of the Sydney2012 strain.

Rice-based oral antibody fragment prophylaxis and therapy against rotavirus infection.

Rotavirus-induced diarrhea is a life-threatening disease in immunocompromised individuals and in children in developing countries. We have developed a system for prophylaxis and therapy against rotavirus disease using transgenic rice expressing the neutralizing variable domain of a rotavirus-specific llama heavy-chain antibody fragment (MucoRice-ARP1). MucoRice-ARP1 was produced at high levels in rice seeds using an overexpression system and RNAi technology to suppress the production of major rice endogenous storage proteins. Orally administered MucoRice-ARP1 markedly decreased the viral load in immunocompetent and immunodeficient mice. The antibody retained in vitro neutralizing activity after long-term storage (>1 yr) and boiling and conferred protection in mice even after heat treatment at 94°C for 30 minutes. High-yield, water-soluble, and purification-free MucoRice-ARP1 thus forms the basis for orally administered prophylaxis and therapy against rotavirus infections.

Comparison of age-stratified seroprevalence of antibodies against norovirus GII in India and the United Kingdom.

Noroviruses are a common cause of gastroenteritis worldwide, but outbreaks appear to be more common in industrialized countries than in developing countries, possibly reflecting differences in exposure and immunity. In this study, age-stratified sera from India and UK populations were analysed for the presence of norovirus-genogroup II specific IgG by a time resolved immunofluorescence assay and relative levels of antibodies in the two populations were compared. Antibody levels were higher among all age groups in India than in UK and increased with age in India, whereas in the UK, levels of antibody decreased in adulthood. These results indicate different patterns of exposure to noroviruses in the two countries.

Human bocaviruses are not significantly associated with gastroenteritis: results of retesting archive DNA from a case control study in the UK.

Gastroenteritis is a common illness causing considerable morbidity and mortality worldwide. Despite improvements in detection methods, a significant diagnostic gap still remains. Human bocavirus (HBoV)s, which are associated with respiratory infections, have also frequently been detected in stool samples in cases of gastroenteritis, and a tentative association between HBoVs, and in particular type-2 HBoVs, and gastroenteritis has previously been made. The aim of this study was to determine the role of HBoVs in gastroenteritis, using archived DNA samples from the case-control Infectious Intestinal Disease Study (IID). DNA extracted from stool samples from 2,256 cases and 2,124 controls were tested for the presence of HBoV DNA. All samples were screened in a real time PCR pan-HBoV assay, and positive samples were then tested in genotype 1 to 3-specific assays. HBoV was detected in 7.4% but no significantly different prevalence was observed between cases and controls. In the genotype-specific assays 106 of the 324 HBoV-positive samples were genotyped, with HBoV-1 predominantly found in controls whilst HBoV-2 was more frequently associated with cases of gastroenteritis (p<0.01). A significant proportion of HBoV positives could not be typed using the type specific assays, 67% of the total positives, and this was most likely due to low viral loads being present in the samples. However, the distribution of the untyped HBoV strains was no different between cases and controls. In conclusion, HBoVs, including HBoV-2 do not appear to be a significant cause of gastroenteritis in the UK population.

In vitro neutralisation of rotavirus infection by two broadly specific recombinant monovalent llama-derived antibody fragments.

Rotavirus is the main cause of viral gastroenteritis in young children. Therefore, the development of inexpensive antiviral products for the prevention and/or treatment of rotavirus disease remains a priority. Previously we have shown that a recombinant monovalent antibody fragment (referred to as Anti-Rotavirus Proteins or ARP1) derived from a heavy chain antibody of a llama immunised with rotavirus was able to neutralise rotavirus infection in a mouse model system. In the present work we investigated the specificity and neutralising activity of two llama antibody fragments, ARP1 and ARP3, against 13 cell culture adapted rotavirus strains of diverse genotypes. In addition, immunocapture electron microscopy (IEM) was performed to determine binding of ARP1 to clinical isolates and cell culture adapted strains. ARP1 and ARP3 were able to neutralise a broad variety of rotavirus serotypes/genotypes in vitro, and in addition, IEM showed specific binding to a variety of cell adapted strains as well as strains from clinical specimens. These results indicated that these molecules could potentially be used as immunoprophylactic and/or immunotherapeutic products for the prevention and/or treatment of infection of a broad range of clinically relevant rotavirus strains.

Identification of G8 rotavirus strains determined as G12 by rotavirus genotyping PCR: updating the current genotyping methods.

BACKGROUND: Rotaviruses are classified into G- and P-types, which are determined by the reactivity with antibodies to the outer viral proteins, VP7 and VP4, respectively, or sequence variation in the genes encoding these proteins. There are presently a number of different rotavirus strains co-circulating within the UK, with the common human strains G1P[8], G2P[4] and G9P[8] being the most prevalent. As part of strain surveillance for the European Rotavirus Network (EuroRotaNet) a cluster (n=29) of G8 strains was detected in the UK between February and May 2009. OBJECTIVES: G8 strains were initially mistyped as G12 through multiplex RT-PCR, therefore further investigation was performed to ascertain the reasons behind this mistyping. STUDY DESIGN: The genes encoding the VP7 of these G8 strains were sequenced and aligned with the existing G8- and G12-specific oligonucleotide primers. RESULTS: Multiple alignment of sequences derived from these strains and the G8- and G12-specific oligonucleotide primers revealed a series of point mutations which resulted in mismatches at the 3' end of the G8-specific primer binding site that prevented amplification with the G8-specific primer, whilst a close homology with the G12-specific primer allowed mis-priming. Both the G8 and G12 primers were redesigned and their ability to correctly identify G8 and G12 strains was evaluated and confirmed. CONCLUSION: These findings highlight the importance of monitoring the specificity and sensitivity of the genotyping methods in order to detect changes in the genotype distribution and changes associated with genetic drift of common or uncommon genotypes.

Targeting tumour cells with defects in the MHC Class I antigen processing pathway with CD8+ T cells specific for hydrophobic TAP- and Tapasin-independent peptides: the requirement for directed access into the ER.

It is becoming increasingly apparent that the majority of tumours display defects in the MHC class I antigen processing pathway, particularly low levels of the transporters-associated with antigen processing (TAP) and tapasin. Thus, immunotherapy approaches targeting such tumours with CD8+ cytotoxic T lymphocytes (CTL) requires strategies to overcome these defects. Previously we had identified an antigen processing pathway by which cytosolically derived hydrophobic peptides could be presented in the absence of TAP. Here we show in the tapasin-negative cell line 721.220 that a number of these hydrophobic TAP-independent peptides can also be presented in a tapasin-independent manner. Yet when these experiments were extended to tumour cell lines derived from small cell lung cancer (SCLC), which we show to be tapasin deficient in addition to TAP-negative, the TAP-, tapasin-independent peptides were not presented. This lack of presentation could be rectified by pre-treatment of SCLC cells with IFNgamma. Alternatively, by directing the TAP-, tapasin-independent peptides into the endoplasmic reticulum (ER) via an ER signal sequence, these peptides were presented efficiently by SCLC cells. We infer from this data that the TAP-independent pathway for presentation of hydrophobic peptides generates a low concentration of peptide in the ER and, for tumour cells which also lack tapasin, this concentration of antigenic peptide is insufficient to load onto MHC class I molecules. Thus, for immunotherapeutic approaches to target SCLC and other tumours with defects in the MHC class I antigen processing pathway it will be important to consider strategies that address tapasin-defects.