Rotavirus genotypes in children under five years hospitalized with diarrhea in low and middle-income countries: Results from the WHO-coordinated Global Rotavirus Surveillance Network.

Rotavirus is the most common pathogen causing pediatric diarrhea and an important cause of morbidity and mortality in low- and middle-income countries. Previous evidence suggests that the introduction of rotavirus vaccines in national immunization schedules resulted in dramatic declines in disease burden but may also be changing the rotavirus genetic landscape and driving the emergence of new genotypes. We report genotype data of more than 16,000 rotavirus isolates from 40 countries participating in the Global Rotavirus Surveillance Network. Data from a convenience sample of children under five years of age hospitalized with acute watery diarrhea who tested positive for rotavirus were included. Country results were weighted by their estimated rotavirus disease burden to estimate regional genotype distributions. Globally, the most frequent genotypes identified after weighting were G1P[8] (31%), G1P[6] (8%) and G3P[8] (8%). Genotypes varied across WHO Regions and between countries that had and had not introduced rotavirus vaccine. G1P[8] was less frequent among African (36 vs 20%) and European (33 vs 8%) countries that had introduced rotavirus vaccines as compared to countries that had not introduced. Our results describe differences in the distribution of the most common rotavirus genotypes in children with diarrhea in low- and middle-income countries. G1P[8] was less frequent in countries that had introduced the rotavirus vaccine while different strains are emerging or re-emerging in different regions.

Aetiology and incidence of diarrhoea requiring hospitalisation in children under 5 years of age in 28 low-income and middle-income countries: findings from the Global Pediatric Diarrhea Surveillance network.

INTRODUCTION: Diarrhoea remains a leading cause of child morbidity and mortality. Systematically collected and analysed data on the aetiology of hospitalised diarrhoea in low-income and middle-income countries are needed to prioritise interventions. METHODS: We established the Global Pediatric Diarrhea Surveillance network, in which children under 5 years hospitalised with diarrhoea were enrolled at 33 sentinel surveillance hospitals in 28 low-income and middle-income countries. Randomly selected stool specimens were tested by quantitative PCR for 16 causes of diarrhoea. We estimated pathogen-specific attributable burdens of diarrhoeal hospitalisations and deaths. We incorporated country-level incidence to estimate the number of pathogen-specific deaths on a global scale. RESULTS: During 2017-2018, 29 502 diarrhoea hospitalisations were enrolled, of which 5465 were randomly selected and tested. Rotavirus was the leading cause of diarrhoea requiring hospitalisation (attributable fraction (AF) 33.3%; 95% CI 27.7 to 40.3), followed by Shigella (9.7%; 95% CI 7.7 to 11.6), norovirus (6.5%; 95% CI 5.4 to 7.6) and adenovirus 40/41 (5.5%; 95% CI 4.4 to 6.7). Rotavirus was the leading cause of hospitalised diarrhoea in all regions except the Americas, where the leading aetiologies were Shigella (19.2%; 95% CI 11.4 to 28.1) and norovirus (22.2%; 95% CI 17.5 to 27.9) in Central and South America, respectively. The proportion of hospitalisations attributable to rotavirus was approximately 50% lower in sites that had introduced rotavirus vaccine (AF 20.8%; 95% CI 18.0 to 24.1) compared with sites that had not (42.1%; 95% CI 33.2 to 53.4). Globally, we estimated 208 009 annual rotavirus-attributable deaths (95% CI 169 561 to 259 216), 62 853 Shigella-attributable deaths (95% CI 48 656 to 78 805), 36 922 adenovirus 40/41-attributable deaths (95% CI 28 469 to 46 672) and 35 914 norovirus-attributable deaths (95% CI 27 258 to 46 516). CONCLUSIONS: Despite the substantial impact of rotavirus vaccine introduction, rotavirus remained the leading cause of paediatric diarrhoea hospitalisations. Improving the efficacy and coverage of rotavirus vaccination and prioritising interventions against Shigella, norovirus and adenovirus could further reduce diarrhoea morbidity and mortality.

Comprehensive surveillance data suggest a prominent role of parvovirus B19 infection in Belarus and the presence of a third subtype within subgenotype 1a.

Human parvovirus B19 (B19V) infection is not notifiable in Belarus and its most common clinical presentation erythema infectiosum (EI) is often difficult to distinguish from other exanthematous diseases. The objective of this study was to provide comprehensive data about EI epidemiology in Belarus based on the serological and molecular investigation of samples from measles and rubella discarded cases collected between 2005 and 2019. Overall, 4919 sera were investigated for IgM antibodies against B19V and the positive cases were analysed according to year, season and age. B19V DNA was amplified by PCR in a total of 238 sera from all over the country, and sequenced for phylogenetic analyses. B19V infection was confirmed in 1377 (27.8%) measles and rubella discarded cases. Two high incidence periods and a seasonal increase of EI between mid-February to mid-July were identified. Children from 4 to 6 and from 7 to 10 years of age represented the largest groups of patients (22.51% and 22.66% of all cases, respectively), followed by adults between 20 and 29 years of age (14.23%). Among the 238 B19Vs sequenced, one belonged to subgenotype 3b and 237 to subgenotype 1a with 81 (34.2%) clustering with subtypes 1a1 and 153 (64.6%) with 1a2. Three strains (1.2%) formed an additional, well-supported cluster suggesting the presence of another subtype of 1a, tentatively named 1a3. The epidemiological and molecular analyses highlighted not only the prominent role of B19V in exanthematous diseases in Belarus, but also suggested a previously underestimated diversity of subgenotype 1a sequences with a third subtype 1a3.

Observations on the epidemiology of rotavirus infection among hospitalized children younger than 5 years in 2 Ukrainian hospitals, 2007-2015.

BACKGROUND: Acute gastroenteritis remains a burden among children under 5 years of age. Ukraine joined the World Health Organization's Global Rotavirus Surveillance Network in 2006, with a goal of providing accurate rotavirus burden data to aid policy makers in planning for rotavirus vaccine introduction. This analysis describes rotavirus epidemiology among Ukrainian children enrolled in Kyiv and Odesa, two large Ukrainian cities. METHODS: Children 0-59 months of age hospitalized for acute gastroenteritis at 2 sentinel sites in Kyiv and Odesa were enrolled into the active, prospective surveillance program. In Odesa, the surveillance period was during 2007-2015 and in Kyiv, it was during 2011-2015. Acute gastroenteritis was defined as 3 or more episodes of diarrhea per day during a 24 h period, with symptom duration before hospitalization not exceeding 7 days. Guardians of enrolled children completed a questionnaire including demographic, clinical and treatment information. Each child provided a stool specimen within 2 days of hospitalization. Stools were tested for rotavirus using ProSpecT™ Rotavirus Kit (Oxoid Ltd., Great Britain), and positive specimens were genotyped. Descriptive data are reported, as well as comparison of demographic, clinical and treatment data among rotavirus positive and negative children. RESULTS: During July 2007-June 2015, 12,350 children were enrolled in the surveillance programs and had stool specimens collected and tested for rotavirus. Overall, rotavirus infection was diagnosed in 5412/12350 (44%) of children, 929/1734 (54%) of those in Kyiv and 4483/10616 (42%) in Odesa. Rotavirus infections peaked during the winter months. Children with rotavirus acute gastroenteritis displayed more severe clinical symptoms than those without rotavirus. Predominant genotypes identified included G1P[8], G2P[4], G3 P[8], G4 P[8] and G9 P[8]. CONCLUSION: Active surveillance of acute gastroenteritis in hospitalized children younger 5 years in two large Ukrainian cities reveals a significant burden of rotavirus infection. These data provide scientific justification for incorporating rotavirus vaccines into the Ukrainian national immunization schedule.

Viral gastroenteritis in rotavirus negative hospitalized children <5 years of age from the independent states of the former Soviet Union.

PURPOSE: Rotavirus causes nearly 40% of all hospitalizations for AGE among children <5 years of age in the NIS of the former Soviet Union. The etiologic role of other established gastroenteritis viruses in this age group is unknown. METHODS: Laboratory-confirmed rotavirus negative fecal specimens (N=495) collected between January and December 2009 from children in 6 NIS (Armenia, Azerbaijan, Belarus, Georgia, Republic of Moldova and Ukraine) were tested for norovirus, sapovirus, enteric adenovirus and astrovirus by real-time RT-PCR. Genotyping was carried out by sequencing and phylogenetic analysis. RESULTS: Norovirus, enteric adenovirus, sapovirus and astrovirus were detected in 21.8%, 4.0%, 3.2%, and 1.4% of the rotavirus negative specimens, respectively. Mixed infections were identified in 4.1% of the specimens. Phylogenetic analysis showed co-circulation of several different genotypes with GII.4 Den Haag (2006b) norovirus, GI.2 sapovirus, adenovirus type 41, and astrovirus type 1 causing majority of the infections. CONCLUSION: Norovirus, enteric adenovirus, sapovirus and astrovirus account for a significant proportion (30.5%) of AGE in hospitalized children <5 years of age in 6 NIS.

Etiology of maculopapular rash in measles and rubella suspected patients from Belarus.

As a result of successful implementation of the measles/rubella elimination program, the etiology of more and more double negative cases remains elusive. The present study determined the role of different viruses as causative agents in measles or rubella suspected cases in Belarus. A total of 856 sera sent to the WHO National Laboratory between 2009 and 2011 were tested for specific IgM antibodies to measles virus (MV), rubella virus (RV) and human parvovirus B19 (B19V). The negatives were further investigated for antibodies to enterovirus (EV) and adenovirus (AdV). Children of up to 3 years were tested for IgM antibodies to human herpesvirus 6 (HHV6). A viral etiology was identified in 451 (52.7%) cases, with 6.1% of the samples being positive for MV; 2.6% for RV; 26.2% for B19V; 9.7% for EV; 4.6% for AdV; and 3.6% for HHV6. Almost all measles and rubella cases occurred during limited outbreaks in 2011 and nearly all patients were at least 15 years old. B19V, EV and AdV infections were prevalent both in children and adults and were found throughout the 3 years. B19V occurred mainly in 3-10 years old children and 20-29 years old adults. EV infection was most common in children up to 6 years of age and AdV was confirmed mainly in 3-6 years old children. HHV6 infection was mostly detected in 6-11 months old infants. Laboratory investigation of measles/rubella suspected cases also for B19V, EV, AdV and HHV6 allows diagnosing more than half of all cases, thus strengthening rash/fever disease surveillance in Belarus.

Rotavirus genotypes in Belarus, 2008-2012.

This study describes group A rotavirus (RVA) genotype prevalence in Belarus from 2008 to 2012. In 2008, data from 3 sites in Belarus (Brest, Mogilev, Minsk) indicated that G4P[8] was the predominant genotype. Data from Minsk (2008-2012) showed that G4P[8] was the predominant RVA genotype in all years except in 2011 when G3P[8] was most frequently detected. Other RVA genotypes common in Europe (G1P[8], G2P[4]) were detected each year of the study. This study reveals the dominance of genotype G4P[8] in Belarus and helps to establish the baseline genotype prevalence prior to RVA vaccine introduction in the country.

Recombination in the evolution of enterovirus C species sub-group that contains types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99.

Genetic recombination is considered to be a very frequent phenomenon among enteroviruses (Family Picornaviridae, Genus Enterovirus). However, the recombination patterns may differ between enterovirus species and between types within species. Enterovirus C (EV-C) species contains 21 types. In the capsid coding P1 region, the types of EV-C species cluster further into three sub-groups (designated here as A-C). In this study, the recombination pattern of EV-C species sub-group B that contains types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99 was determined using partial 5'UTR and VP1 sequences of enterovirus strains isolated during poliovirus surveillance and previously published complete genome sequences. Several inter-typic recombination events were detected. Furthermore, the analyses suggested that inter-typic recombination events have occurred mainly within the distinct sub-groups of EV-C species. Only sporadic recombination events between EV-C species sub-group B and other EV-C sub-groups were detected. In addition, strict recombination barriers were inferred for CVA-21 genotype C and CVA-24 variant strains. These results suggest that the frequency of inter-typic recombinations, even within species, may depend on the phylogenetic position of the given viruses.

The evolution of Vp1 gene in enterovirus C species sub-group that contains types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99.

Genus Enterovirus (Family Picornaviridae,) consists of twelve species divided into genetically diverse types by their capsid protein VP1 coding sequences. Each enterovirus type can further be divided into intra-typic sub-clusters (genotypes). The aim of this study was to elucidate what leads to the emergence of novel enterovirus clades (types and genotypes). An evolutionary analysis was conducted for a sub-group of Enterovirus C species that contains types Coxsackievirus A21 (CVA-21), CVA-24, Enterovirus C95 (EV-C95), EV-C96 and EV-C99. VP1 gene datasets were collected and analysed to infer the phylogeny, rate of evolution, nucleotide and amino acid substitution patterns and signs of selection. In VP1 coding gene, high intra-typic sequence diversities and robust grouping into distinct genotypes within each type were detected. Within each type the majority of nucleotide substitutions were synonymous and the non-synonymous substitutions tended to cluster in distinct highly polymorphic sites. Signs of positive selection were detected in some of these highly polymorphic sites, while strong negative selection was indicated in most of the codons. Despite robust clustering to intra-typic genotypes, only few genotype-specific 'signature' amino acids were detected. In contrast, when different enterovirus types were compared, there was a clear tendency towards fixation of type-specific 'signature' amino acids. The results suggest that permanent fixation of type-specific amino acids is a hallmark associated with evolution of different enterovirus types, whereas neutral evolution and/or (frequency-dependent) positive selection in few highly polymorphic amino acid sites are the dominant forms of evolution when strains within an enterovirus type are compared.

Human parvovirus B19 surveillance in patients with rash and fever from Belarus.

Human parvovirus B19 (B19V) infection in immunocompetent patients usually has a mild clinical course, but during pregnancy it can cause serious and even fatal complications in the fetus. The most common clinical presentation of B19V infection is erythema infectiosum and in this case laboratory confirmation is required for differentiation from other exanthematous diseases. Measles and rubella negative sera collected in Belarus between 2005 and 2008 from 906 patients with a rash and fever were screened for B19V infection by ELISA. More than 35% of the samples (322/906) were positive for B19V. The proportion ranged from 10.1% in 2008 to 53.2% in 2006 when an outbreak took place in Minsk city. All B19V outbreaks and cluster cases occurred during the winter-spring period, but sporadic cases were recorded basically throughout the year. The majority of the cases (56.5%) occurred among the 2 till 10 year old children, and 27.3% of the cases were observed in adults between 19 and 53 years. All 104 B19V strains sequenced in the NS1/VP1u region belonged to genotype 1 with a maximal genetic distance of 1.75%. The two phylogenetic clusters reflected the geographic origins of the viruses within the country. Forty-two unique nucleotide mutations as compared to sequences downloaded from GenBank were found in the VP1u and NS1 regions; most of these changes were nonsynonymous. This report highlights the importance of B19V infection in patients with a rash and fever in Belarus.

Revealing new measles virus transmission routes by use of sequence analysis of phosphoprotein and hemagglutinin genes.

With improved measles virus (MV) control, the genetic variability of the MV-nucleoprotein hypervariable region (NP-HVR) decreases. Thus, it becomes increasingly difficult to determine the origin of a virus using only this part of the genome. During outbreaks in Europe and Africa, we found MV strains with identical NP-HVR sequences. However, these strains showed considerable diversity within a larger sequencing window based on concatenated MV phosphoprotein and hemagglutinin genes (P/H pseudogenes). In Belarus, Germany, Russia, and the Democratic Republic of Congo, the P/H pseudogenes provided insights into chains of transmission, whereas identical NP-HVR provided none. In Russia, for instance, the P/H pseudogene identified temporal clusters rather than geographical clusters, demonstrating the circulation and importation of independent variants rather than large local outbreaks lasting for several years, as suggested by NP-HVR. Thus, by extending the sequencing window for molecular epidemiology, a more refined picture of MV circulation was obtained with more clearly defined links between outbreaks and transmission chains. Our results also suggested that in contrast to the P gene, the H gene acquired fixed substitutions that continued to be found in subsequent outbreaks, possibly with consequences for its antigenicity. Thus, a longer sequencing window has true benefits both for the epidemiological surveillance of measles and for the better monitoring of viral evolution.

Comparison of poliovirus recombinants: accumulation of point mutations provides further advantages.

The roles of recombination and accumulation of point mutations in the origin of new poliovirus (PV) characteristics have been hypothesized, but it is not known which are essential to evolution. We studied phenotypic differences between recombinant PV strains isolated from successive stool specimens of an oral PV vaccine recipient. The studied strains included three PV2/PV1 recombinants with increasing numbers of mutations in the VP1 gene, two of the three with an amino acid change I-->T in the DE-loop of VP1, their putative PV1 parent and strains Sabin 1 and 2. Growth of these viruses was examined in three cell lines: colorectal adenocarcinoma, neuroblastoma and HeLa. The main observation was a higher growth rate between 4 and 6 h post-infection of the two recombinants with the I-->T substitution. All recombinants grew at a higher rate than parental strains in the exponential phase of the replication cycle. In a temperature sensitivity test, the I-->T-substituted recombinants replicated equally well at an elevated temperature. Complete genome sequencing of the three recombinants revealed 12 (3), 19 (3) and 27 (3) nucleotide (amino acid) differences from Sabin. Mutations were located in regions defining attenuation, temperature sensitivity, antigenicity and the cis-acting replicating element. The recombination site was in the 5' end of 3D. In a competition assay, the most mutated recombinant beat parental Sabin in all three cell lines, strongly suggesting that this virus has an advantage. Two independent intertypic recombinants, PV3/PV1 and PV3/PV2, also showed similar growth advantages, but they also contained several point mutations. Thus, our data defend the hypothesis that accumulation of certain advantageous mutations plays a key role in gaining increased fitness.

Evolution of the Sabin vaccine into pathogenic derivatives without appreciable changes in antigenic properties: need for improvement of current poliovirus surveillance.

The Sabin oral polio vaccine (OPV) may evolve into pathogenic viruses, causing sporadic cases and outbreaks of poliomyelitis. Such vaccine-derived polioviruses (VDPV) generally exhibit altered antigenicity. The current paradigm to distinguish VDPV from OPV and wild polioviruses is to characterize primarily those poliovirus isolates that demonstrate deviations from OPV in antigenic and genetic intratypic differentiation (ITD) tests. Here we report on two independent cases of poliomyelitis caused by VDPVs with "Sabin-like" properties in several ITD assays. The results suggest the existence of diverse pathways of OPV evolution and necessitate improvement of poliovirus surveillance, which currently potentially misses this class of VDPV.

High genetic diversity of measles virus, World Health Organization European Region, 2005-2006.

During 2005-2006, nine measles virus (MV) genotypes were identified throughout the World Health Organization European Region. All major epidemics were associated with genotypes D4, D6, and B3. Other genotypes (B2, D5, D8, D9, G2, and H1) were only found in limited numbers of cases after importation from other continents. The genetic diversity of endemic D6 strains was low; genotypes C2 and D7, circulating in Europe until recent years, were no longer identified. The transmission chains of several indigenous MV strains may thus have been interrupted by enhanced vaccination. However, multiple importations from Africa and Asia and virus introduction into highly mobile and unvaccinated communities caused a massive spread of D4 and B3 strains throughout much of the region. Thus, despite the reduction of endemic MV circulation, importation of MV from other continents caused prolonged circulation and large outbreaks after their introduction into unvaccinated and highly mobile communities.

Co-circulation of multiple rubella virus strains in Belarus forming novel genetic groups within clade 1.

Although the WHO recommends a comprehensive genetic characterization, little is known about circulating strains and genotypes of rubella virus (RUBV) for many European countries. Studies investigating the genetic diversity of a sizeable number of strains from a certain location are rare. This study presents the first molecular characterization of isolates from Belarus. Throat swab and urine samples were collected between 2004 and 2005 from patients presenting in two infectious disease hospitals and three outpatient clinics in and around Minsk. In total, 14 isolates were obtained from this clinical material. Phylogenetic analysis of the E1 gene sequence of these isolates showed that three distinct groups of RUBV strains co-circulated. One group of isolates was assigned to genotype 1E, whereas the other two did not group with any of the recognized genotypes but grouped with a strain belonging to the provisional genotype 1g. Detailed analysis showed that the group comprising 1g strains also contained sequences formerly attributed to genotype 1B and could be divided into four subgroups, one of which might represent a putative novel provisional genotype of clade 1. These findings show that three distinct strains with limited variability are present in Belarus, suggesting independent introductory events. As there currently seem to be misattributions of strains to genotypes and unclear phylogenetic relationships, criteria for genotyping of RUBV should be clarified further.

Serotype-specific mucosal immune response and subsequent poliovirus replication in vaccinated children.

A total of 32 unimmunized children (median age 5 months) living in an orphanage in Minsk, Belarus, were vaccinated with three doses of oral poliovirus vaccine (OPV) with a 60-day interval between the doses. Blood samples were drawn before the immunizations and 45-50 days after each vaccine dose. Excretion of the vaccine viruses was followed by examining fecal specimens collected weekly after each vaccine dose. All children seroconverted by the second dose of OPV at the latest to all three serotypes of poliovirus but differences were seen regarding the intestinal responses. The strongest responses in both neutralizing antibodies and virus-binding IgA in fecal suspensions were seen toward poliovirus Type 2. Consistent with this, relatively little poliovirus Type 2 excretion was seen after the second and third doses of OPV as compared to that of poliovirus Type 1 or Type 3. The delayed development of functional intestinal immunity against the latter serotypes was associated with relatively weaker intestinal antibody responses compared to poliovirus Type 2. In the case of poliovirus Type 3, about 10% of children were still excreting the vaccine virus 9 weeks after administering the third dose. These results are consistent with epidemiological observations of the serotype-specific efficacy of OPV immunizations. The proportion of specimens showing nonpolio enteric viruses gradually increased through the 6-month study period. This may reflect the possibility that after the first dose of OPV, intensive replication of the vaccine viruses may out compete the nonpolio viruses in the intestines of the vaccinees or, alternatively, mask nonpolio viruses during the cell culture isolation procedure.

Isolation of an intertypic poliovirus capsid recombinant from a child with vaccine-associated paralytic poliomyelitis.

The isolation of a capsid intertypic poliovirus recombinant from a child with vaccine-associated paralytic poliomyelitis is described. Virus 31043 had a Sabin-derived type 3-type 2-type 1 recombinant genome with a 5'-end crossover point within the capsid coding region. The result was a poliovirus chimera containing the entire coding sequence for antigenic site 3a derived from the Sabin type 2 strain. The recombinant virus showed altered antigenic properties but did not acquire type 2 antigenic characteristics. The significance of the presence in nature of such poliovirus chimeras and the consequences for the current efforts to detect potentially dangerous vaccine-derived poliovirus strains are discussed in the context of the global polio eradication initiative.