Respiratory syncytial virus epidemic periods in an equatorial city of Brazil.

BACKGROUND: Characterization of the human respiratory syncytial virus (HRSV) season at the local level has important implications for appropriate decisions on the time period for administration of specific prophylaxis. OBJECTIVES: (1) To describe five consecutive epidemic periods of HRSV in an equatorial city of Brazil and (2) to show preliminary data on genomic diversity of circulating HRSV. PATIENTS/METHODS: Nasopharyngeal aspirates of 2885 children attending the emergency room and wards of a public hospital were collected and screened by indirect immunofluorescence for HRSV infections during five consecutive years (from January 2004 to December 2008). In addition, the genetic and antigenic variability of the HRSV strains isolated was evaluated by partial nucleotide sequencing of the protein G gene. RESULTS: HRSV was detected in 15·8% of the analyzed samples. HRSV seasons occurred in a restricted period of each year. The onset of each HRSV season was variable (February to May), but the end always occurred in July. From the 456 HRSV infections found, 86 cases with bronchiolitis were genotyped. Both HRSV subgroups (A and B) cocirculated during the five epidemic periods. The 58 HRSV-A strains grouped into two clades, GA2 and GA5. In respect of the HRSV-B strains, the 28 samples grouped into two clades: GB3 and BA. CONCLUSIONS: HRSV accounts for a substantial proportion of ARI in the study population. As in temperate countries, HRSV infections in this equatorial area of Brazil also cause seasonal yearly epidemics, and this has implications for prophylaxis strategies. The city of Fortaleza follows the same worldwide trend of circulation of genotypes of HRSV.

Comparative dynamics, morbidity and mortality burden of pediatric viral respiratory infections in an equatorial city.

BACKGROUND: Although acute respiratory infections (ARIs) are the global leading cause of pediatric morbidity and mortality, the relative impact of viral pathogens on pediatric ARIs is still poorly understood, especially in equatorial settings. Long-term studies of multiple viruses concurrently circulating in these regions are still lacking. Here, we report the results of a systematic prospective surveillance of multiple respiratory viruses conducted every weekday for nearly a decade in an equatorial city in Brazil. METHODS: We analyzed the relative burden of influenza, parainfluenza, respiratory syncytial virus (RSV), adenovirus, and metapneumovirus, their seasonality, and their association with climatic and demographic factors, ARI diagnosis, and pediatric mortality. RESULTS AND CONCLUSIONS: RSV was the primary driver of severe childhood respiratory infections, including pneumonia. RSV was also the virus most strongly associated with respiratory-associated deaths, with RSV circulation and pediatric mortality being in phase. Annual circulation of influenza peaked much earlier than annual mortality due to respiratory causes. The results also show that viral circulation can be strongly seasonal even in equatorial regions, which lack seasons with low temperatures: RSV and influenza were concentrated in the rainy season, whereas parainfluenza predominantly circulated in the dry season. The consistent epidemiologic patterns observed can be used for an effective adjustment of the timing of therapeutic and prophylactic interventions in this and potentially in other equatorial regions.

Design of primers and use of RT-PCR assays for typing European bluetongue virus isolates: differentiation of field and vaccine strains.

Bluetongue virus (BTV) is the causative agent of bluetongue, a disease of ruminant livestock that occurs almost worldwide between latitudes 3 degrees S and 5 degrees N. There are 24 serotypes of BTV (currently identified by serum neutralization assays). Since 1998, eight strains of six BTV serotypes (1, 2, 4, 8, 9 and 16) have invaded Europe. The most variable BTV protein is major outer-capsid component VP2, encoded by segment 2 (Seg-2) of the double-stranded RNA virus genome. VP2 represents the major target for neutralizing (and protective) antibodies that are generated in response to BTV infection, and is therefore the primary determinant of virus serotype. RT-PCR primers and assays targeting Seg-2 have been developed for rapid identification (within 24 h) of the six European BTV types. These assays are sensitive, specific and show perfect agreement with the results of conventional virus-neutralization methods. Previous studies have identified sequence variations in individual BTV genome segments that allow different isolates to be grouped on the basis of their geographical origins (topotypes). The assays described in this paper can detect any of the BTV isolates of the homologous serotype that were tested from different geographical origins (different Seg-2 topotypes). Primers were also identified that could be used to distinguish members of these different Seg-2 topotypes, as well as field and vaccine strains of most of the European BTV serotypes. The serotype-specific assays (and primers) showed no cross-amplification when they were evaluated with multiple isolates of the most closely related BTV types or with reference strains of the remaining 24 serotypes. Primers developed in this study will be updated periodically to maintain their relevance to current BTV distribution and epidemiology (http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm).

Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes.

The outer capsid protein VP2 of Bluetongue virus (BTV) is a target for the protective immune response generated by the mammalian host. VP2 contains the majority of epitopes that are recognized by neutralizing antibodies and is therefore also the primary determinant of BTV serotype. Full-length cDNA copies of genome segment 2 (Seg-2, which encodes VP2) from the reference strains of each of the 24 BTV serotypes were synthesized, cloned and sequenced. This represents the first complete set of full-length BTV VP2 genes (from the 24 serotypes) that has been analysed. Each Seg-2 has a single open reading frame, with short inverted repeats adjacent to conserved terminal hexanucleotide sequences. These data demonstrated overall inter-serotype variations in Seg-2 of 29 % (BTV-8 and BTV-18) to 59 % (BTV-16 and BTV-22), while the deduced amino acid sequence of VP2 varied from 22.4 % (BTV-4 and BTV-20) to 73 % (BTV-6 and BTV-22). Ten distinct Seg-2 lineages (nucleotypes) were detected, with greatest sequence similarities between those serotypes that had previously been reported as serologically 'related'. Fewer similarities were observed between different serotypes in regions of VP2 that have been reported as antigenically important, suggesting that they may play a role in the neutralizing antibody response. The data presented form an initial basis for BTV serotype identification by sequence analyses and comparison of Seg-2, and for development of molecular diagnostic assays for individual BTV serotypes (by RT-PCR).

Incidence and isolation of bluetongue virus infection in cattle of the Santo Tomé Department, Corrientes Province, Argentina.

Sentinel herds were monitored for the detection of bluetongue (BT)-specific antibodies and virus over two periods, namely: June 1999 to August 2000 and September 2000 to April 2001. Herds were located in Santo Tomé (Herds 1 and 2) where BTV activity was known to occur. From June 1999 to August 2000, the cumulative incidence (CI) of bluetongue virus (BTV) infection was 0% and 35% in Herds 1 and 2, respectively. In the second period, the CI of BTV infection was 10% and 97% in Herds 1 and 2, respectively. The virus was isolated from red blood cells of animals that seroconverted and was identified as serotype 4. Averages of the monthly maximal temperatures were always above 19 degrees C. However, averages of the monthly median temperatures were below 19 degrees C and averages of the monthly minimal temperatures were below 15 degrees C from May 2000 to August 2000. There was no viral activity detected at that time. Culicoides insignis was identified as the predominant potential vector species (99%) trapped near sentinel herds. Although clinical disease has never been reported in Argentina, viral activity was detected and the virus has been isolated in sentinel herds.

Bluetongue virus replication, molecular and structural biology.

The icosahedral bluetongue virus (BTV) particle (~80 nm diameter) is composed of three distinct protein layers. These include the subcore shell (VP3), core-surface layer (VP7) and outer capsid layer (VP2 and VP5). The core also contains ten dsRNA genome segments and three minor proteins (VP1[Pol], VP4[CaP]and VP6[Hel]), which form transcriptase complexes. The atomic structure of the BTV core has been determined by X-ray crystallography, demonstrating how the major core proteins are assembled and interact. The VP3 subcore shell assembles at an early stage of virus morphogenesis and not only determines the internal organisation of the genome and transcriptase complexes, but also forms a scaffold for assembly of the outer protein layers. The BTV polymerase (VP1) and VP3 have many functional constraints and equivalent proteins have been identified throughout the Reoviridae, and even in some other families of dsRNA viruses. Variations in these highly conserved proteins can be used to identify members of different genera (e.g. by comparing the polymerase) and different virus species (serogroups) within the genus Orbivirus (e.g. by comparison of VP3). This has helped to identify three new genera within the Reoviridae and two new Orbivirus species. In contrast, sequences of the BTV outer capsid proteins (involved in interactions with neutralising antibodies) are much more variable (particularly VP2) and comprehensive sequence analyses for the 24 types demonstrate that they can be used to identify BTV serotype. The 21 species (158 serotypes) currently recognised within the genus Orbivirus are listed, along with 11 unassigned viruses.

Phylogenetic analysis of bluetongue virus genome segment 6 (encoding VP5) from different serotypes.

Full-length cDNAs were sequenced for genome segment 6 from representative isolates of all 24 bluetongue virus (BTV) serotypes. Segment 6 is 1635 to 1645 nucleotides in length with a single open reading frame in all isolates. The deduced VP5 protein sequence is 526 amino acids long for most BTVs, except BTV-15 (isolate number RSArrrr/15), which is 527 amino acids long, and BTV-12 (isolate number RSArrrr/12) and BTV-22 (isolate number Nig1982/11), which were 529 amino acids long. Sequence comparisons have revealed the level of genetic diversity in segment 6, between different BTV serotypes and between isolates within a single serotype. The resulting sequences can be used to design RT-PCR primers for amplification and identification of segment 6 from new BTV isolates, providing potentially valuable diagnostic and research tools. These data are available on the international databases and accession numbers are listed on the website of the Institute for Animal Health (iah.bbsrc.ac.uk/dsRNA_virus_proteins/ btv_sequences.htm).

Completion of the sequence analysis and comparisons of genome segment 2 (encoding outer capsid protein VP2) from representative isolates of the 24 bluetongue virus serotypes.

Bluetongue (BT) is a non-contagious, arthropod-transmitted viral disease of domestic and wild ruminants. It is caused by bluetongue virus (BTV), a double-stranded (ds) RNA virus that is classified within the genus Orbivirus, family Reoviridae. There are at least twenty-four serotypes of BTV worldwide, five of which (1, 2, 4, 9 and 16) have been identified recently in Europe. BTV infects ruminants and its distribution throughout temperate and tropical regions of the world is dependent on the activity and abundance of certain vector-competent species of Culicoides midge. The outer capsid protein VP2 of BTV is a major protective antigen and the primary determinant of virus serotype. For the first time, the authors have completed the sequence analysis of full-length VP2 genes from the reference strains of each of the 24 BTV serotypes and their amino acid sequences were deduced. Multiple alignment of the VP2 gene (protein) sequences revealed that the level of nucleotide (amino acid) sequence variation between serotypes ranged from 29% (23%) to 59% (73%), confirming that segment 2/VP2 is the most variable BTV gene/protein. Phylogenetic analysis of VP2 grouped together the BTV types that are known to cross-react serologically. Low identity between types was demonstrated for specific regions within the VP2 amino acid sequences that have been shown to be antigenic and play a role in virus neutralisation. The sequence data represent the completion of an important step in the creation of a comprehensive BTV sequence database, which will support more rapid molecular methods for diagnosis and identification of BTV 'types', as well as continuing molecular epidemiology and surveillance studies of BTV.

Molecular epidemiology of bluetongue viruses from disease outbreaks in the Mediterranean Basin.

Bluetongue virus (BTV) serotype is primarily controlled by the variable outer coat protein VP2, encoded by genome segment 2. Phylogenetic analyses of segment 2 show that recent Mediterranean isolates of BTV-2 have a similar genetic lineage to those from sub-Saharan Africa and North America but are distinct from Asian strains. In contrast, isolates of BTV-9, from the eastern Mediterranean, are related to a genetic lineage from Asia. BTV-1 from Greece 2001 is also more closely related to Indian isolates, suggesting (in both cases) virus movement from east to west. Recent BTV-4 field isolates from Greece and Turkey are similar to each other, but differ from the Turkish type 4 vaccine strain. These sequencing studies are being used to establish a database for molecular epidemiological studies which is available on the website of the Institute for Animal Health. This resource will support and improve BTV serotype identification methods, by using sequence comparisons (via the Web) rather than by conventional serological techniques that require standardised (and therefore expensive) serological reagents. Phylogenetic trees for BTV genome segment 2 are available on the website.

Development of reverse transcriptase-polymerase chain reaction-based assays and sequencing for typing European strains of bluetongue virus and differential diagnosis of field and vaccine strains.

Bluetongue virus (BTV) is a double-stranded (ds) RNA virus, classified within the genus Orbivirus, family Reoviridae, which causes bluetongue (BT), an infectious, non-contagious disease of ruminants. The virus exists as 24 distinct serotypes, which are currently identified by virus isolation and serum neutralisation assays. The most variable outer capsid protein VP2 (encoded by genome segment 2), is the primary determinant of BTV serotype. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays, based on amplification of segment 2, have been developed for identification of the five European BTV types (BTV-1, BTV-2, BTV-4, BTV-9 and BTV-16). Primer pairs were designed that are specific for each BTV serotype. The resulting RT-PCR assay was both sensitive and specific, providing BTV typing within 24 h. Perfect agreement was recorded between the RT-PCR and virus neutralisation assays. The primers for each serotype could successfully amplify the BTV isolates of that serotype from different regions and showed no cross-amplification of the most closely related BTV serotypes. RT-PCR primers were also developed for the discrimination of field and vaccine strains of BTV serotypes currently circulating in Europe. The primer pairs which could amplify field and vaccine strains of BTV-1, BTV-2, BTV-4 and BTV-9 were validated with several isolates of each serotype from various geographic origins around the world and their type specificity was again tested with the most closely related serotypes. Overall, these RT-PCR assays provide a rapid and reliable method for the identification and differentiation of field and vaccine strains of different BTV types. The primers used in this study are listed on the website of the Institute for Animal Health, Pirbright.

Differential diagnosis of bluetongue virus using a reverse transcriptase-polymerase chain reaction for genome segment 7.

Bluetongue virus (BTV) has persisted within Europe for the past five years, highlighting a need for rapid and reliable virus detection and identification methods. Various RT-PCR protocols and strategies, which target genome segment 7, were evaluated for their ability to detect all members of the BTV species (serogroup), with the aim of developing a fully validated reverse transcriptase-polymerase chain reaction- (RT-PCR) based diagnostic assay. A nested PCR strategy, using near terminal and internal segment 7 primers, detected all 24 BTV serotypes, but also cross-reacted with some other related Orbivirus species. In an attempt to circumvent these problems, conventional PCR and touch-down PCR methods, using similar primers were also investigated. Both methods were able to amplify cDNA from only 21 of the 24 BTV types. Further sequence analyses of the VP7 gene from the remaining isolates (types 7, 15 and 19) will permit the design of additional and more effective virus-species specific primers and RT-PCR-based assays. This may include the introduction of a multiplex PCR system.

Molecular epidemiology of foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is the most economically important veterinary pathogen due to its highly infectious nature, ability to cause persistent infections and long term effects on the condition and productivity of the many animal species it affects. Countries which have the disease have many trade restrictions placed upon them. In the last 15 years there have been significant advances in the understanding of FMD epidemiology. These have largely been due to the application of the molecular biological techniques of polymerase chain-reaction amplification and nucleotide sequencing. In the World Reference Laboratory for FMD (Pirbright, UK), a large sequence database has been built up. This database has been used to aid in the global tracing of virus movements. It has been possible to genetically group many FMDV's based on their geographic origin and this has led to their being referred to as topotypes. The implications of this are that inter-regional spread of viruses can often be easily recognised and any evolutionary changes which subsequently occur can be monitored. Using these techniques, for the first time, we have been able to unequivocally show the recent pandemic spread of a FMDV type O strain through the whole of Asia and into Africa and Europe. This type of surveillance will become increasingly important as further globalisation of markets occurs. An increased understanding of how FMDV strains move between geographic regions will play a pivotal role in the development of future disease control strategies.

The generation and persistence of genetic variation in foot-and-mouth disease virus.

Genetic variation in foot-and-mouth disease virus (FMDV) is of interest for at least two reasons. First, changes to the genes encoding capsid proteins results in antigenic variation, and affects vaccine efficiency and effectiveness of vaccination programs; second, genetic changes can lead to important insights into the transport of virus between countries, regions, herds, and even possibly individuals. Current estimates of RNA virus mutation rates suggest that an average of about one base mis-incorporation is likely to occur each time a single FMDV genome replicates. This should result in the introduction of every possible 1-step mutation from the progenitor genotype into the viraemia of a single infected animal many times a day. In the absence of purifying selection, a single infected animal should therefore generate a genetically very diverse population of virus.Viral-capsid sequences obtained from infected animals sampled over long-term FMDV epidemics suggest that these genetic changes accrue in a remarkably linear 'clock-like' fashion and at rates of around 1% change per year. While such a rate is generally regarded as quite high, it is actually somewhat lower than one might expect based on the rate at which viral diversity could be generated within a single animal. The difference might be explained in a variety of possible ways: (1) the mutation rate has been overestimated; (2) purifying selection is stronger than predicted; (3) only a restricted subset of excreted virus is actually infectious; (4) infected animals only excrete virus from a small partitioned subset of amplified virus, and that most of the generated viral diversity is unable to exit the animal; or (5) only a small fraction of all infected animals participate in the actual disease-transmission process.

Foot-and-mouth disease virus: cause of the recent crisis for the UK livestock industry.

The recent outbreak of foot-and-mouth disease (FMD) in the United Kingdom is a stark reminder of the economic devastation that this disease can wreak. Tracing the origin of such an outbreak is an essential part of disease control. Modern molecular methods have been in place for a number of years to enable scientists to identify unambiguously the strain of virus responsible. However, tracing the precise origin of such a strain is not so straightforward because the virus can move rapidly around the world with legal and illegal trade in animals and animal products. This short review describes the virus, its control and epidemiology.

The suitability of the 'emergency' foot-and-mouth disease antigens held by the International Vaccine Bank within a global context.

The International Vaccine Bank (IVB) based at the Institute of Animal Health (IAH) in Pirbright, United Kingdom (UK), routinely monitors the suitability of the currently held strains of foot-and-mouth disease (FMD) vaccine virus, in anticipation that vaccine may be required to control FMD outbreaks that pose a threat to member countries. Using primarily the two-dimensional micro-neutralisation test (VNT), bovine polyclonal sera raised against each of the seven current 'emergency' antigens were utilised to measure the relationship of IVB stocks to selected field isolates. The 'O' serotypes, Manisa and Lausanne, exhibited adequate levels of cross-protection against most of the type 'O' field isolates examined. A(22) Iraq 24/64 showed the broadest spectrum of reactivity against the type 'A' field isolates examined and was supplemented by A(15) Thailand 1/60. Some type 'Asia1' field isolates, particularly those from South East Asia, showed antigenic difference to the Asia1 India 8/79 vaccine strain by VNT, but in-vivo testing in the guinea pig model indicated this to be insignificant. The only 'C' serotype representative, C(1) Oberbayern, may be one of the least antigenically diverse of the current portfolio of bank antigens. Comparison of the serological and sequence data shows that despite significant genetic variation between the field isolates examined the antigens held by the IVB should still prove efficacious in the field.

Foot-and-mouth disease type O viruses exhibit genetically and geographically distinct evolutionary lineages (topotypes).

Serotype O is the most prevalent of the seven serotypes of foot-and-mouth disease (FMD) virus and occurs in many parts of the world. The UPGMA method was used to construct a phylogenetic tree based on nucleotide sequences at the 3' end of the VP1 gene from 105 FMD type O viruses obtained from samples submitted to the OIE/FAO World Reference Laboratory for FMD. This analysis identified eight major genotypes when a value of 15% nucleotide difference was used as a cut-off. The validity of these groupings was tested on the complete VP1 gene sequences of 23 of these viruses by bootstrap resampling and construction of a neighbour-joining tree. These eight genetic lineages fell within geographical boundaries and we have used the term topotype to describe them. Using a large sequence database, the distribution of viruses belonging to each of the eight topotypes has been determined. These phylogenetically based epidemiological studies have also been used to identify viruses that have transgressed their normal ecological niches. Despite the high rate of mutation during replication of the FMD virus genome, the topotypes appear to represent evolutionary cul-de-sacs.

Evidence for positive selection in foot-and-mouth disease virus capsid genes from field isolates.

The nature of selection on capsid genes of foot-and-mouth disease virus (FMDV) was characterized by examining the ratio of nonsynonymous to synonymous substitutions in 11 data sets of sequences obtained from six different serotypes of FMDV. Using a method of analysis that assigns each codon position to one of a number of estimated values of nonsynonymous to synonymous ratio, significant evidence of positive selection was identified in 5 data sets, operating at 1-7% of codon positions. Evidence of positive selection was identified in complete capsid sequences of serotypes A and C and in VP1 sequences of serotypes SAT 1 and 2. Sequences of serotype SAT-2 recovered from a persistently infected African buffalo also revealed evidence for positive selection. Locations of codons under positive selection coincide closely with those of antigenic sites previously identified with the use of monoclonal antibody escape mutants. The vast majority of codons are under mild to strong purifying selection. However, these results suggest that arising antigenic variants benefit from a selective advantage in their interaction with the immune system, either during the course of an infection or in transmission to individuals with previous exposure to antigen. Analysis of amino acid usage at sites under positive selection indicates that this selective advantage can be conferred by amino acid substitutions that share physicochemically similar properties.

Diagnosis of foot-and-mouth disease by RT-PCR: use of phylogenetic data to evaluate primers for the typing of viral RNA in clinical samples.

The results of type-specific RT-PCR diagnostic assays on foot-and-mouth disease (FMD) viruses in clinical samples were mapped onto serotype-specific dendrograms representing the degree of nucleotide sequence variation between the FMD virus isolates. This novel approach assisted the selection of suitable PCR primer sets for the diagnosis of FMD virus isolates belonging to different topotypes within each serotype. These interpretations were qualified by using a universal (FMD virus group) specific primer to confirm that FMD virus RNA had been extracted from the samples under investigation. The analyses showed that the design of primer sets for the detection of FMD virus serotypes O, A, Asia 1, SAT 1 and SAT 3 were generally satisfactory, as most virus isolates within the major virus sub-groupings were successfully detected. However, the FMD virus serotype C and SAT 2 specific primers were less efficient as certain virus sub-groups were not detected. This identified the need for additional or alternative primers to improve RT-PCR procedures for more comprehensive detection of divergent virus strains within these serotypes. There were some examples where not all virus isolates from the same outbreak reacted with particular type-specific primers which suggested that either further minor refinements may be necessary in the primer design or that there were shortcomings in the RT-PCR methodology.

Primary diagnosis of foot-and-mouth disease by reverse transcription polymerase chain reaction.

Universal and serotype-specific primer sets were used in simple reverse transcription polymerase chain reaction (RT-PCR) assays on field samples of epithelium and vesicular fluid to determine their suitability for primary diagnosis of all seven serotypes of foot-and-mouth disease (FMD). The specificity of reactions was confirmed by using other vesicular disease viruses, namely: swine vesicular disease virus, vesicular stomatitis virus and three vesiviruses. This resulted in the identification of a universal O/A/C/Asia 1 primer set (1F/1R) located in the 5' untranslated region (UTR) of the FMD virus genome for the successful detection of virus of these serotypes in clinical samples although this primer set detected FMD virus of the SAT1/2/3 serotypes less efficiently. The 5' UTR universal primer set could be used for the primary diagnosis of FMD in conjunction with the routine diagnostic methods of virus isolation in cell culture and ELISA, although a more favourable reaction would be expected with FMD viruses of the O/A/C/Asia 1 group than with those of the SAT serotypes. The other examined universal and serotype-specific primer sets, located principally in the P1 capsid-coding region, were generally inferior to the 5' UTR universal primer set. It is envisaged that this evaluation of primers will lead to the development of alternative PCR strategies, for example nested PCR formats, with concomitant improvement in the speed of primary diagnosis of FMD which under present procedures can be lengthy.