Correlates of protection against human rotavirus disease and the factors influencing protection in low-income settings.

Rotaviruses (RV) are the leading cause of gastroenteritis in infants and children worldwide and are associated with high mortality predominately in low-income settings. The virus is classified into G and P serotypes and further into P genotypes based on differences in the surface-exposed proteins VP7 and VP4, respectively. Infection results in a variable level of protection from subsequent reinfection and disease. This protection is predominantly homotypic in some settings, whereas broader heterotypic protection is reported in other cohorts. Two antigenically distinct oral RV vaccines are licensed and are being rolled out widely, including in resource-poor setting, with funding provided by the GAVI alliance. First is a monovalent vaccine derived from a live-attenuated human RV strain, whereas the second is a pentavalent bovine-human reassortment vaccine. Both vaccines are highly efficacious in high-income settings, but greatly reduced levels of protection are reported in low-income countries. Here, the current challenges facing mucosal immunologists and vaccinologists aiming to define immunological correlates and to understand the variable levels of protection conferred by these vaccines in humans is considered. Such understanding is critical to maximize the public health impact of the current vaccines and also to the development of the next generation of RV vaccines, which are needed.

Structured surveillance of infantile gastroenteritis in East Anglia, UK: incidence of infection with common viral gastroenteric pathogens.

The aim of this study was to investigate the burden of disease associated with gastroenteric viruses (rotavirus, norovirus, sapovirus, astrovirus and enteric adenovirus) using structured surveillance of children aged <6 years in the community. Faecal samples were collected between 2000 and 2003 from 685 children with symptoms of gastroenteritis. The children comprised three groups; 223 in the structured surveillance cohort, 203 in a community cohort and 259 in a cohort of hospitalized children. All samples were tested for the presence of viral pathogens using molecular methods. Questionnaires were sent to the parents/carers of the children recruited to the structured surveillance cohort in order to collect data that would allow an estimation of the severity of illness by means of the Vesikari score, and of the cost associated with gastrointestinal disease in this age group. A viral aetiological agent was detected in 53.5% of samples tested. Rotavirus was the most common pathogen found in all three cohorts followed by norovirus and enteric adenoviruses. Multiple viruses were found in 8% of the samples, and commonly involved rotavirus and any other virus. G1P[8] was the most commonly detected rotavirus strain and there was no significant difference in the distribution of rotavirus genotypes among the three cohorts. Analysis of the questionnaires indicated that rotavirus infections were likely to be more severe than any other virus infection, and children from whom a viral pathogen was identified were more likely to require rehydration therapy.

Interaction of rotavirus polymerase VP1 with nonstructural protein NSP5 is stronger than that with NSP2.

Rotavirus morphogenesis starts in intracellular inclusion bodies called viroplasms. RNA replication and packaging are mediated by several viral proteins, of which VP1, the RNA-dependent RNA polymerase, and VP2, the core scaffolding protein, were shown to be sufficient to provide replicase activity in vitro. In vivo, however, viral replication complexes also contain the nonstructural proteins NSP2 and NSP5, which were shown to be essential for replication, to interact with each other, and to form viroplasm-like structures (VLS) when coexpressed in uninfected cells. In order to gain a better understanding of the intermediates formed during viral replication, this work focused on the interactions of NSP5 with VP1, VP2, and NSP2. We demonstrated a strong interaction of VP1 with NSP5 but only a weak one with NSP2 in cotransfected cells in the absence of other viral proteins or viral RNA. By contrast, we failed to coimmunoprecipitate VP2 with anti-NSP5 antibodies or NSP5 with anti-VP2 antibodies. We constructed a tagged form of VP1, which was found to colocalize in viroplasms and in VLS formed by NSP5 and NSP2. The tagged VP1 was able to replace VP1 structurally by being incorporated into progeny viral particles. When applying anti-tag-VP1 or anti-NSP5 antibodies, coimmunoprecipitation of tagged VP1 with NSP5 was found. Using deletion mutants of NSP5 or different fragments of NSP5 fused to enhanced green fluorescent protein, we identified the 48 C-terminal amino acids as the region essential for interaction with VP1.

Infantile viral gastroenteritis: on the way to closing the diagnostic gap.

A total of 305 faecal specimens collected from children under the age of 5 who presented with symptoms of acute gastroenteritis either as inpatients at Addenbrooke's Hospital (N = 100) or to General Practitioners in East Anglia (N = 205) during 1999-2001 were tested for the presence of rotavirus, norovirus, sapovirus, enteric adenoviruses (Group F, serotypes 40 and 41), and astrovirus. An aetiologic agent was found in 184 specimens (60.3%). The most commonly found single viral pathogen was rotavirus (27.9%), followed by norovirus (13.4%), enteric adenoviruses (7.9%), astrovirus (2.3%), and sapovirus (1%). Mixed infections were observed in 27 specimens (8.9%), and no aetiologic agent was found in over a third of the specimens tested. These data demonstrate that the diagnostic gap can be reduced considerably through the use of molecular amplification and detection techniques. However, additional work is required to reduce this deficit further by optimising sampling algorithms and by identifying other agents associated with viral gastroenteritis.

Rotavirus gastroenteritis and central nervous system (CNS) infection: characterization of the VP7 and VP4 genes of rotavirus strains isolated from paired fecal and cerebrospinal fluid samples from a child with CNS disease.

Rotavirus RNA was detected in the cerebrospinal fluid (CSF) of a child with central nervous system disease symptoms associated with rotavirus gastroenteritis. The rotavirus isolates from the fecal and CSF samples were genotyped as G1P[8]. Sequence analysis of the VP7 and VP4 proteins derived from the fecal and CSF samples were remarkably similar to each other and to G1P[8] rotavirus strains commonly circulating in the community and associated with gastroenteritis.

Amino acid substitution within the VP7 protein of G2 rotavirus strains associated with failure to serotype.

Rotavirus strains collected in the United Kingdom during the 1995-1996 season and genotyped as G2 by reverse transcription-PCR failed to serotype in enzyme-linked immunosorbent assays using three different G2-specific monoclonal antibodies. The deduced amino acid sequences of the antigenic regions A (amino acids 87 to 101), B (amino acids 142 to 152), and C (amino acids 208 to 221) of VP7 revealed that a substitution at position 96 (Asp-->Asn) correlated with the change in ability to serotype these G2 strains.

Rotavirus epidemiology and surveillance.

There is extensive antigenic and genomic diversity among co-circulating human rotaviruses. They are differentiated into groups, subgroups and types. There are at least 7 groups (A-G) and 4 subgroups within group A. To distinguish types within group A, a dual classification system has been established with the glycoprotein VP7 defining G types, and the protease-sensitive protein VP4 defining P types. At least 14 G types and more than 20 P types have been distinguished, of which at least 10 G types and at least 11 P types have been found in humans. Using the typing system, the complex molecular epidemiology of rotaviruses was investigated. Rotaviruses of different G and P types co-circulate. The main types found are G1P1A[8], G2P1B[4], G3P1A[8], G4P1A[8]; their relative incidence rates change over time in any one location and are different at the same time between different locations. Viruses with G/P constellations such as G1P1B[4] and G2P1A[8] are mostly natural reassortants of the co-circulating main virus types emerging after double infection of hosts. Viruses carrying G and or P types not represented in the four most common types, e.g. G8P[8], G1P[6] or G9P[6], could be introduced into the population by reassortment with animal viruses, or directly from animals or exotic human sources. Naturally circulating rotaviruses constantly undergo point mutations which can be used to classify lineages and sublineages within types. The full significance of human infections with group B and C rotaviruses remains to be established. Surveillance of rotavirus types in different parts of the world is essential to monitor the emergence of new types or of new G/P constellations which may predominate over time. The efficacy and effectiveness of any future rotavirus vaccine may differ depending on the predominant natural strain types. Detailed epidemiological and molecular surveillance data should be utilized to study the transmission dynamics of rotaviruses.

Reassortment in vivo: driving force for diversity of human rotavirus strains isolated in the United Kingdom between 1995 and 1999.

The G and P genotypes of 3,601 rotavirus strains collected in the United Kingdom between 1995 and 1999 were determined (M. Iturriza-Gómara et al., J. Clin. Microbiol. 38:4394-4401, 2000). In 95.4% of the strains the most common G and P combinations, G1P[8], G2P[4], G3P[8], and G4P[8], were found. A small but significant number (2%) of isolates from the remaining strains were reassortants of the most common cocirculating strains, e.g., G1P[4] and G2P[8]. Rotavirus G9P[6] and G9P[8] strains, which constituted 2.7% of all viruses, were genetically closely related in their G components, but the P components of the G9P[8] strains were very closely related to those of cocirculating strains of the more common G types (G1, G3, and G4). In conclusion, genetic interaction by reassortment among cocirculating rotaviruses is not a rare event and contributes significantly to their overall diversity.

Rapid screening technique for class 1 integrons in Enterobacteriaceae and nonfermenting gram-negative bacteria and its use in molecular epidemiology.

A screening technique for integrons in members of the family Enterobacteriaceae and nonfermenting gram-negative bacteria by real-time PCR is reported. A total of 226 isolates of gram-negative bacteria obtained from a variety of clinical specimens were screened for class 1 integrons by real-time PCR performed on a LightCycler instrument. This technique used a primer pair specific for a 300-bp conserved region at the 5' ends of class 1 integrons. The screening assay was evaluated by comparison with results obtained by the conventional, thermal-block PCR (long PCR) by using established conditions and primers for the detection of class 1 integrons, and the real-time PCR technique was thus shown to be both sensitive and specific. DNA from 50 of 226 (22%) isolates screened was identified as containing an integron by the screening PCR, and sequence data were obtained across the integron for 34 of 50 (68%) of these isolates. In an attempt to study the molecular epidemiology of antimicrobial resistance genes carried within integrons, a comparison of the types of gene cassettes carried by isolates from different patients was made. Adenyltransferase genes conferring resistance to streptomycin and spectinomycin were the predominant gene cassettes amplified in the study. Resistance to trimethoprim was also frequently found to be encoded within integrons. Furthermore, multiple bacterial isolates obtained from one patient over a 5-month period were all shown to carry an integron containing the same single adenyltransferase gene cassette, suggesting that these elements were relatively stable in this case.

Molecular epidemiology of human group A rotavirus infections in the United Kingdom between 1995 and 1998.

The G and P types of 2,912 rotavirus-positive fecal specimens collected from eight geographical areas of the United Kingdom between 1995 and 1998 were determined by reverse transcription-PCR. Although 15 different G-P combinations were identified, G1P[8], G2P[4], G3P[8], and G4P[8] strains constituted 95% of all the rotaviruses typed. Other genotypes included G9P[6] and G9P[8], which were first identified in the United Kingdom in 1995, or other uncommon G and/or P types of strains that may have had an animal origin. Unusual combinations of G1 or G4 with P[4] and G2 with P[8] which may have arisen by reassortment between human strains were also identified. G1P[8] was the genotype most frequently found (57 to 87%) in each season, followed by G2P[4] in the 1995-1996 (18%) and 1997-1998 (16%) seasons, although the incidence of infection with this virus decreased significantly to 2% during the 1996-1997 season. Significant differences were seen in the distributions of G1P[8], G2P[4], and G9P[8] strains between children and adults, in the temporal distributions of G4P[8] and G9P[8] strains within a season, and in the geographical distributions of each of the four most common genotypes from one season to the next.

Single-tube real-time nested polymerase chain reaction for detecting human papillomavirus DNA.

A single-tube real-time nested polymerase chain reaction (PCR) was developed to detect human Papillomavirus (HPV) DNA in a closed tube system. The oligonucleotide primers MY09/MY11 and GP5+/GP6+ were included in contiguous reactions, thus eliminating the need to transfer first round PCR product into a second tube. The sensitivity and specificity of the optimized single-tube nested PCR were comparable with that achieved by two separate reactions on a conventional thermal block system using serial dilutions derived from plasmids containing DNA of 20 HPV types. A minimum of 10 copies of HPV types 11 and 16 DNA could be detected by both systems. In clinical samples, HPV types 1A, 2, 3, 5, 6-8, 10, 11, 14, 16, 17, 18, 20, 31, 33, 35, 39, 45, 49, 50, 52-54, 57, 62, 66, 70, CP8304 and LVX82/MM7 could be detected by both PCR methods. A total of 145 samples collected from patients were tested for the presence of HPV DNA with the two PCR systems; 124 (86.1%) of 144 samples gave concordant results in both assays. The HPV DNA positive PCR amplicons were typed and concordant results were obtained in 47 of 67 positive samples tested in both amplicons. In samples containing multiple HPV types at least one type was common to both amplicons.

Characterisation of rotavirus G9 strains isolated in the UK between 1995 and 1998.

G9P[6] and G9P[8] rotavirus strains were identified during 1995/96 through the molecular epidemiological surveillance of rotavirus strains circulating in the UK between 1995 and 1998. An increase in the incidence and spread of sporadic infections with rotavirus genotype G9P[8] across the UK was detected in the two following seasons. Partial sequencing of the VP7 gene showed that all the UK strains shared a high degree of homology and were related very closely to G9 strains from the US and from symptomatic infections in India (> or =96% homology). The UK strains were related more distantly to the apathogenic Indian strain 116E (85-87.8% homology). Phylogenetic analysis revealed clustering of the UK strains into 3 different lineages (I to III) and into two sub-lineages within lineage I. There were correlations between VP7 sequence clustering, the P type and the geographical origin of the G9 strains. Partial sequencing of the VP4 gene showed high degree of homology (>98%) among all the P[6] strains, and the sequences obtained from the P[8] strains clustered into 2 of the 3 global lineages described for P[8] strains associated with other G types. These data suggest that G9 strains may be a recent importation into the UK, and that G9P[8] strains may have emerged through reassortment in humans between G9P[6] strains introduced recently and the more prevalent cocirculating G1, G3 and G4 strains that normally carry VP4 genes of P[8] type.

Emerging and re-emerging infectious diseases.

Microbial pathogens discovered as aetiological agents of human disease over the last 25 years are reviewed. Strengthening of laboratory and public health surveillance is of paramount importance for early detection and management of emerging infectious diseases.

Diversity within the VP4 gene of rotavirus P[8] strains: implications for reverse transcription-PCR genotyping.

A degenerate version (1T1-D) of the rotavirus P[8]-specific primer (1T-1) allowed strains previously untypeable due to the accumulation of point mutations at the primer binding site to be P typed by reverse transcription-PCR. Sequencing of the cDNA followed by sequence alignment and phylogenetic analysis identified lineages and sublineages within the rotavirus P[8] types, while the use of 1T-1 or 1T-1D primers did not yield viral clusters in any particular lineage.

Virus inactivation in a proportion of human T-cell leukaemia virus type I-infected T-cell clones arises through naturally occurring mutations.

Human T-cell leukaemia virus type I (HTLV-I) is the aetiological agent of adult T-cell leukaemia/lymphoma and tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). The trans-activating protein (Tax) of HTLV-I is strongly implicated in cellular proliferation. We examined the tax gene and 5' long terminal repeat (LTR) sequences in eight naturally infected T-cell clones derived from TSP/HAM-affected individuals who were either productively (proliferate spontaneously) or silently (do not proliferate spontaneously) infected. In two silently infected clones point mutations within the proviruses resulted in truncation of the Tax protein. One clone harboured both a deleterious tax gene mutation and a point mutation in an enhancer element of the 5' LTR. Sequence changes, immunological escape mutation, integration site context and host cell phenotype may all contribute to the high proportion of latently or silently infected T-cells found in vivo in virus carriers.