Hospital-based surveillance of rotavirus and other viral agents of diarrhea in children and adults in Russia, 2005-2007.

During a 2-year period in 2005-2007, we conducted surveillance of group A rotaviruses and other enteric agents among patients hospitalized with acute gastroenteritis in 8 different cities of the Russian Federation. Fecal specimens were gathered from 3208 children (including 2848 children aged <5 years) and 1354 adults who were admitted to hospitals in Moscow, St. Petersburg, Chelyabinsk, Nizhnii Novgorod, Tyumen, Khabarovsk, Makhachkala, and Yakutsk. Polymerase chain reaction was performed to detect rotaviruses of groups A and C, noroviruses of genogroups I and II, astrovirus, sapovirus, and enteric adenoviruses (group F). Group A rotavirus was the most common viral pathogen detected among children aged <5 years (43.6%), followed by norovirus (12.5%), whereas norovirus was the pathogen most commonly detected in adults (11.9%). P and G genotypes were determined for 515 rotavirus specimens, and the most prevalent genotypes were G1P[8] (44.9%), G4P[8] (40.0%), G2P[4] (8.5%), and G3P[8] (6.6%). This study is the first multicenter study of rotaviruses in the Russian Federation and documents the important burden of disease caused by this pathogen, which soon may be preventable by vaccination.

Genomic characterization of human rotavirus G8 strains from the African rotavirus network: relationship to animal rotaviruses.

Global rotavirus surveillance has led to the detection of many unusual human rotavirus (HRV) genotypes. During 1996-2004 surveillance within the African Rotavirus Network (ARN), six P[8],G8 and two P[6],G8 human rotavirus strains were identified. Gene fragments (RT-PCR amplicons) of all 11-gene segments of these G8 strains were sequenced in order to elucidate their genetic and evolutionary relationships. Phylogenetic and sequence analyses of each gene segment revealed high similarities (88-100% nt and 91-100% aa) for all segments except for gene 4 encoding VP4 proteins P[8] and P[6]. For most strains, almost all of the genes of the ARN strains other than neutralizing antigens are related to typical human strains of Wa genogroup. The VP7, NSP2, and NSP5 genes were closely related to cognate genes of animal strains (83-99% and 97-99% aa identity). This study suggests that the ARN G8 strains might have arisen through VP7 or VP4 gene reassortment events since most of the other gene segments resemble those of common human rotaviruses. However, VP7, NSP2 (likely), and NSP5 (likely) genes are derived potentially from animals consistent with a zoonotic introduction. Although these findings help elucidate rotavirus evolution, sequence studies of cognate animal rotavirus genes are needed to conclusively determine the specific origin of those genes relative to both human and animal rotavirus strains.

Phylogenetic analysis of novel G12 rotaviruses in the United States: A molecular search for the origin of a new strain.

Rotavirus serotype G12 was initially identified in the Philippines in 1987 and was not described again until it reemerged more than 13 years later. G12 strains were first detected in the United States in 2002 and have recently assumed a worldwide distribution. The high similarity between the sequence of the major outer capsid VP7 gene of human G12 strains and the single porcine G12 isolate raised the prospect that human strains may have arisen through reassortment with porcine strains or, alternatively, that the porcine strain originally came from humans. We sequenced portions of the remaining 10 segments of two human G12 strains (G12P[8] and G12P[6]) and a currently circulating common strain (G1P[8]) identified during the 2005-2006 surveillance season and compared the sequences with those of strains available through GenBank. By comparison, the three strains were all Wa-like and not porcine-like. A newly outlined classification system proposed genotypes for each gene segment based on nucleotide similarity. Using this approach, gene segments VP1-3, VP6 and NSP1-5 grouped within the same genotype, indicating that the three strains analyzed were closely related. These results suggest that the novel G12P[8] strain could have been formed by the solitary introduction of a VP7 gene into a globally common rotavirus strain, G1P[8]. Classifying rotavirus strains based only on VP7 (G) and VP4 (P) genotype potentially underestimates diversity and sequence analysis of the other segments is required to assess the complete genetic relationships between strains.

Rotavirus epidemiology: the Asian Rotavirus Surveillance Network.

Availability of new rotavirus vaccines has highlighted the need to collect local disease and economic burden data to aid decision makers at global, regional and country level. The World Health Organization and the GAVI Alliance recommended that generic protocols be used and that regional surveillance networks be established to collect these data, thereby helping to fast-track the introduction of these new vaccines into developing countries. Nine countries and regions participated in the first phase of the Asian Rotavirus Surveillance Network (ARSN), which collected data over a 2-year period during 2001-2003. Overall 45% of diarrhoea admissions in the region were positive for rotavirus, which was higher than had been anticipated. Significant rotavirus strain diversity was noted during the surveillance period. Data collection for a second phase of the ARSN commenced in 2004 and included a greater proportion of poorer countries that would in future be eligible for funding support for rotavirus immunization from GAVI. Limited economic evaluations in Asia have demonstrated the potential for new rotavirus vaccines to be cost-effective but more local analyses are required. Despite the ARSN's comprehensive data from a mix of developed and developing countries, Asia has lagged the Americas in terms of the introduction of rotavirus vaccines into National Immunization Programmes (NIPs). Lack on rotavirus vaccine efficacy data in Asia, particularly in poorer populations, will have contributed to this delay. Thus ensuring that all global regions are simultaneously involved in the evaluation of new vaccines from the beginning and also encouraging more regional collaborations of Ministry of Health representatives could help to accelerate the introduction of new vaccines into NIPs.

Emergence of G12 rotavirus strains in Delhi, India, in 2000 to 2007.

The prospect that rotavirus diarrhea in children may soon be prevented by vaccines has placed a new priority on understanding the diversity of rotavirus strains and the mechanism by which these strains evolve over time. We have characterized a total of 465 rotavirus strains collected in North India from 2000 to 2007 for G and P types by reverse transcription-PCR and sequencing. The novel G12 rotavirus strains recently detected in other countries were first detected in India in 2001 and have emerged as the predominant strains in Delhi, India, during 2005 to 2007. While the VP7 sequence was highly homologous among G12 strains isolated in Delhi, suggesting recent emergence from a common ancestor, the strains had a diverse constellation of other gene segments, demonstrating substantial reassortment. For the entire period, the common rotavirus G types G1 (26%), G2 (25%), and G9 (14%) comprised 65% of the strains, and common P types, P[4] (19%), P[6] (22%), and P[8] (35%), comprised 76% of the total P types. Of note, we detected a high percentage of unusual (17%) strains and fecal specimens with mixed (12% G and 15% P) rotavirus infections having a variety of genomic constellations. For the first time, we identified two novel rotavirus strains with unusual G/P combinations, G2P[11] and G3P[11], in patients with diarrhea. The study highlights the great diversity among rotaviruses isolated from Indian children, the opportunity for genetic reassortment between strains, and the emergence of a novel G12 strain in our country. Due to the demonstrated effect of antigenic diversity on rotavirus vaccines, it will be important to continue careful monitoring of these strains as rotavirus vaccine programs are implemented in India.

First detection of G12 rotaviruses in newborns with neonatal rotavirus infection at all India Institute of Medical Sciences, New Delhi, India.

Rotavirus genotype G12 strains were detected for the first time among newborns with asymptomatic rotavirus infection (74% of 39 rotavirus strains isolated from the infected infants were genotype G12) in the nursery of the All India Institute of Medical Sciences during a period from 2005 to 2006. Sequence analysis of the VP7 genes from these neonatal strains indicated a high level of homology to other G12 strains reported worldwide, suggesting the recent emergence of these strains in humans. Such nosocomial infections of newborns represent a potential source of introduction of novel rotavirus serotypes into the community.

Rotavirus disease in Uzbekistan: cost-effectiveness of a new vaccine.

We evaluated the cost-effectiveness of rotavirus vaccination in Uzbekistan from the healthcare system and societal perspectives. Disease burden was estimated using national statistics on hospitalizations and deaths, and international estimates of under-five mortality. Without vaccination, the risk for rotavirus hospitalization by age 5 is 10 per 1000 children. Rotavirus hospitalizations cost US$ 406,000 annually, of which US$ 360,000 (89%) is for medical expenses and US$ 46,000 (11%) is for non-medical and indirect costs. Rotavirus mortality rate at 0.7 per 1000 derived from national data was three-fold lower than the same rate calculated from international estimates of under-five mortality. Rotavirus vaccination could reduce hospitalizations and deaths by 91% and avert US$ 370,000 in hospitalization costs alone. Vaccination would be cost-effective with vaccine prices in a range of US$ 2-25 per child. However, the cost-effectiveness is greatly influenced by mortality, vaccine price and vaccine efficacy.

Molecular epidemiology of group A rotavirus diarrhea among children in Buenos Aires, Argentina, from 1999 to 2003 and emergence of the infrequent genotype G12.

To examine the epidemiology of rotaviruses in Buenos Aires, Argentina, we screened 1,212 stool samples from children with diarrhea in the southern district of Buenos Aires from 1999 to 2003. We identified 187 samples (15.4%) that were positive for group A rotavirus by use of antigen enzyme-linked immunosorbent assay. Among these specimens, 112 were available for typing: 93 (83.0%) were single-type infections, 9 (8.0%) were mixed-type infections with more than one G or P type, and 10 (8.9%) were G and/or P nontypeable. In contrast to the findings in our last study, from 1996 to 1998, genotype P[4], G2 strains were almost completely absent and P[8], G1 and P[8], G4 strains were dominant, representing more than 80% of the G and P types found. Genotypes G2 and G9 were detected in few samples, and type G3 was completely absent. We identified several uncommon genotype G12 strains, representing the first detections outside of Asia and the United States, by sequencing. Using a genotype G12-specific reverse transcription-PCR, we identified eight (6.7%) positive samples for the 1999 to 2003 period. The high degree of sequence identity between recent G12 isolates from Argentina, the United States, and Asian countries suggests a relatively recent introduction(s) of these strains into humans from a common progenitor. The Argentinean G12 strains belonged to genotype P[9], similar to most of the recently described Asian G12 strains. The finding of G12 strains in several other regions of the world raises the possibility that G12 may be emerging globally and suggests that surveillance for this strain should be conducted routinely.

Comparative molecular characterization of gene segment 11-derived NSP6 from lamb rotavirus LLR strain used as a human vaccine in China.

Sequence-length polymorphism is known for rotavirus genetic segment 11 (encodes non-structural protein, NSP6). With the exception of 11 strains that have the coding potential for a 98-residue NSP6, majority of the strains have the potential for a 92-residue NSP6. In nine strains, the coding potential for this protein is even shorter. This report focuses on the NSP6 gene nucleotide sequence of Lanzhou Lamb Rotavirus (LLR) strain and its comparative molecular characterization. The LLR strain is a G10 P12 type, which is in use as a licensed human vaccine in China. The LLR NSP6 was compared with 56 other rotaviral NSP6 sequences including a rhesus strain (RRV) available in the database. Analyses indicate that while RRV-NSP6 belongs to the majority (92-residue) group, the LLR NSP6 belongs to the 98-residue group. When the rotavirus NSP6 protein was expressed in cells as GFP fusion protein from human, simian and the LLR strains, they all demonstrated punctate cytoplasmic distribution and, contrary to the computer-aided prediction, the NSP6 did not undergo phosphorylation, which in itself is a novel observation for the rotavirus NSP6.

Serum antibody responses in children with rotavirus diarrhea can serve as proxy for protection.

We examined sera from 42 patients 1 to 30 months of age for rotavirus immunoglobulin M (IgM), IgA, IgG, and IgG subclasses and sought to determine if serum antibody could serve as a reliable marker for prediction of disease severity. Infants in the first few months of life usually had high maternal IgG titers and, when they were infected with rotavirus, had low IgM titers or no IgM in acute-phase sera and poor seroconversions 3 weeks later, suggesting that maternal antibodies had inhibited viral replication and antibody responses. All patients > or =6 months of age had IgM in acute-phase sera, indicating that IgM is a good marker for acute rotavirus infection. IgG was the best overall predictor of an infection, as the convalescent-phase sera of 81% of the patients had a fourfold rise in the IgG titer. IgA titers in convalescent-phase sera and conversion rates were higher among patients > or =12 months of age than among children younger than 12 months. IgG1 was the predominant subclass detected in the acute-phase sera of some children and in all 28 convalescent-phase serum samples examined. Patients with preexisting acute-phase IgG titers of > or =100 or > or =200 had diarrhea that was less severe or of a shorter duration. These results indicate that serum IgG is the most reliable marker for seroconversion and is a consistent proxy for protection against severe disease.

Surveillance of childhood diarrhoeal disease in Hong Kong, using standardized hospital discharge data.

Discharge information for all Hong Kong government hospitals, which is routinely collected through the Clinical Management System (CMS), was used to assess the relative importance of all causes of diarrhoeal illness and to address the issue of under-diagnosis of rotavirus by linking discharge diagnostic codes with actual laboratory results for one hospital. Of all children less than 5 years of age hospitalized in Hong Kong in the 2-year period July 1997 to June 1999, 12,257 (11%) were discharged with a primary diarrhoea diagnosis (74% coded as non-specified, 10.4% as rotavirus, 11% as Salmonella and 5% as other viral or bacterial). Linked laboratory and discharge data for one hospital demonstrated that 15% (n = 1522) of all admissions had a primary diarrhoea diagnosis and that 40% of these had a specimen sent for rotavirus testing, of which 37% were positive. However, 46% (67/145) of children with a diagnosis of rotavirus infection had no virology result, and 69% (172/248) of positive rotavirus results were in children with no diagnosis indicating rotavirus infection. Modification of the CMS to routinely combine existing computerized laboratory data with the CMS discharge diagnoses and to develop mechanisms to enhance reliability of discharge diagnosis coding could produce a powerful resource for disease surveillance, auditing and for monitoring the impact of future vaccination and other prevention programmes.

[Rotavirus surveillance data from Kunming Children's Hospital, 1998 - 2001].

OBJECTIVE: To study the epidemiological status on rotavirus diarrhea in Kunming to improve the rotavirus vaccine immunization program. METHODS: A hospital-based sentinel surveillance program for rotavirus was set up among children less than 5 years old with acute diarrhea in Kunming Children's Hospital. Clinical information and fecal specimens were collected and rotavirus were detected by polyacrylamide gel electrophoresis (PAGE) and/or enzyme linked immunosorbent assay (ELISA). Positive specimens were further serotyped or genotyped by ELISA and/or RT-PCR. RESULTS: During the three years of surveillance, 466 specimens were collected. Rotavirus were detected on 246 (52.8%) specimens. 97% of the rotavirus diarrhea cases occurred among children less than 2 years old. There was a peak of admissions for rotavirus diarrhea cases between October and December which accounted for 48% of all the rotavirus hospitalizations each year. Among 204 specimens with G serotyping, the predominant strain was serotype G1 (47.5%) followed by G2 (17.6%), G3 (15.7%), G9 (4.9%) and G4 (1.0%). Mixed infection (2.5%) were rare and 22 specimens (10.8%) remained non-typeable. P genotyping showed P[4], P[8] and P[6] were the most common strains, accounting for 29.3%, 27.6% and 13.8% respectively. P[4]G2 was the most common strain which accounted for 34.1% (14/41) followed by P[8]G1 (29.3%) and P[6]G9 (12.2%). Another 7 uncommon P-G combinations were also identified. CONCLUSION: Rotavirus was the major cause of acute diarrhea in Kunming. An effective rotavirus vaccine for prevention and control of rotavirus diarrhea should be developed.

[Surveillance finding on rotavirus in Changchun children's hospital during July 1998-June 2001].

OBJECTIVE: To establish baseline patterns of rotavirus diarrhea and to describe its epidemiologic features in Changchun city, prior to rotavirus vaccine immunization. METHODS: Hospital-based surveillance was conducted among children under 5 years old with acute diarrhea in Changchun Children's Hospital. Fecal samples were determined to identify rotavirus by PAGE and/or ELISA. G serotypes of rotavirus were identified by ELISA and/or nested RT-PCR. P genotyping were carried out by RT-PCR. All data were computerized and analysed by "Generic Manual on Rotavirus Surveillance" set by CDC in the USA. RESULTS: In total, 2 343 diarrhea cases were screened and 1 211 fecal samples were collected. Rotavirus was detected in 31.0% among outpatients and 52.9% in inpatients. During the peak of the season (November through March), 58.6% of diarrhea was caused by rotavirus among inpatients. 95.0% of rotavirus diarrhea cases occurred among children aged < 2 years. The predominant strain was serotype G1 (82.4%), followed by G2 (5.0%), G3 (3.3%), G4 (0.9%). P genotyping showed that P[8] and P[4] were the most common ones. Nine different P-G combinations were identified, four strains (P[8]G1, P[4]G2, P[8]G3, and P[8]G4) commonly seen worldwide accounted for 75.6% of the total. Taken together with uncommon strains, including the novel types P[4]G4 and P[8]G2, it highlights the extraordinary diversity of rotaviruses circulating in China. CONCLUSION: Rotavirus is the major cause of severe child diarrhea in Changchun. Developing a rotavirus vaccine for prevention of severe disease and reduction of treatment costs seemed to be necessary.

Cytokines as mediators for or effectors against rotavirus disease in children.

Rotavirus is the most common cause of severe gastroenteritis in young children, but the pathogenesis and immunity of this disease are not completely understood. To examine the host response to acute infection, we collected paired serum specimens from 30 children with rotavirus diarrhea and measured the levels of nine cytokines (interleukin-1beta [IL-1beta], IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) using a microsphere-based Luminex Flowmetrix system. Patients with acute rotavirus infection had elevated median levels of seven cytokines in serum, and of these, the levels of three (IL-6, IL-10, and IFN-gamma) were significantly (P < 0.05) higher than those in serum from control children without diarrhea. Patients with fever had significantly (P < 0.05) higher levels of IL-6 in serum than control children, and those with fever and more episodes of diarrhea had significantly (P < 0.05) higher levels of TNF-alpha than those without fever and with fewer episodes of diarrhea. We further demonstrated a negative association (P < 0.05) between the levels of IL-2 and the number of stools on the day on which the first blood sample was collected. Finally, patients with vomiting had significantly (P < 0.05) lower levels of IFN-gamma than those without vomiting. Our pilot study provides evidence that the types and magnitudes of cytokine responses to rotavirus infection in children influence or reflect the clinical outcome of disease. These findings suggest that certain cytokines may play an important role in the pathogenesis of and the protection against rotavirus disease in children and, consequently, may provide directions and insights that could prove critical to the prevention or treatment of this important disease.

Unexpected detection of animal VP7 genes among common rotavirus strains isolated from children in Mexico.

In the course of characterizing 103 rotaviruses from children in Mexico, we found that the majority of strains were globally common types (55.4% of total), while uncommon types represented 5.7%, mixed infections with common types represented 14.8%, and partially or fully nontypeable isolates represented about 24%. Serotype G9 was detected for the first time in Mexico. We sequenced a subset of strains that were G nontypeable by reverse transcriptase PCR and found surprisingly that two strains having common human rotavirus P genotypes (8 and 6) had serotype G3 and G4 VP7 gene sequences that shared closer homology with canine and porcine strains, respectively, than with human strains, suggesting that these isolates represented reassortants between human and animal rotaviruses.

Characterization of serotype G9 rotavirus strains isolated in the United States and India from 1993 to 2001.

The emergence of rotavirus serotype G9 as a possible fifth globally common serotype in the last decade, together with its increasing detection in association with various genome constellations, raises questions about the origins and epidemiological importance of recent G9 isolates. We examined a collection of 40 G9 strains isolated in the United States from 1996 to 2001 and in India since 1993 to determine their VP7 gene sequences, P types, E types, subgroup specificities, and RNA-RNA hybridization profiles. With the exception of two U.S. strains, all of the study strains shared high VP7 gene sequence homology (<2.5% sequence divergence on both the nucleotide and amino acid levels) and were more closely related to other recent isolates than to the first G9 strains isolated in the 1980s. The VP7 gene sequence and RNA-RNA hybridization profiles of the long-E-type strains showed greater variation than the short-E-type strains, suggesting that the latter strains are the result of a relatively recent reassortment event of the G9 VP7 gene into a short-E-type lineage. No evidence for reassortment of genes other than VP4 and VP7 between major human rotavirus genogroups was observed. Except for Om46 and Om67, which formed a distinct clade, phylogenetic analysis showed that most of the study strains grouped together, with some subgroups forming according to genetic constellation, geographic location, and date of isolation. The high potential of G9 strains to generate different P and G serotype combinations through reassortment suggests that it will be important to determine if current vaccines provide heterotypic protection against these strains and underscores the need for continued surveillance for G9 and other unusual or emerging rotavirus strains.

Detection of human rotavirus serotype G6 in Hungary.

During an ongoing survey of human rotavirus serotypes, we demonstrated for the first time the circulation of serotype G6 in two regions of Hungary. Of five rotavirus seasons surveyed to date (1994-9), serotype G6 was found in all seasons except 1994-5 at an overall prevalence of 1.4% (17 of 1252) and ranging from 0.6 to 4.5%. Children infected with G6 strains were older (mean age, 3.3 years) than children infected with the four (G1-G4) globally common serotypes (mean age, 2.1 years; unpaired Student's t test, P<0.001). Our data indicate that rotavirus serotype G6 may be an epidemiologically important G serotype in Hungary.

First detection of group C rotavirus in children with acute diarrhea in Spain.

Group C rotavirus causes sporadic cases and outbreaks of acute diarrhea in children and adults in many countries, but has never been detected among children in Spain. In a recently conducted surveillance study to screen fecal specimens for bacteria and viruses from a cohort of 822 young children who were treated for acute diarrhea in Madrid, no pathogens were detected in fecal specimens from 238 (29%) children. In this study, we examined 147 of those specimens for group C rotavirus by EIA and PCR and found 22 (15%) were positive. Our findings demonstrate that group C rotavirus is an important cause of childhood diarrhea in Spain.

[Epidemiological study of rotavirus diarrhea in Beijing, China - a hospital-based surveillance from 1998 - 2001].

OBJECTIVE: To provide information on epidemiology of rotavirus infection in Beijing, China. METHODS: An ongoing hospital-based surveillance was conducted among children < 5yr old with acute diarrhea according to WHO generic protocol (CID-98). During a 3-year study (Apr. 1998 to Mar. 2001), a total of 484 stool samples were collected from 1 457 patients, including 275 samples from 1 048 outpatients and 209 samples from 409 inpatients. RESULTS: The overall detection rate of rotavirus infection was 25.4%. Rotavirus was responsible for 27.3% of diarrhea inpatients on a yearly base, and 46.2% during rotavirus season. Two peaks of diarrhea were observed each year, one in the summer (June-Sep.) due to bacterial dysentery (16.7%) and another in fall winter (Oct.-Dec.) due to rotavirus infection (23.0%). The detection rate on rotavirus was the highest in age group of 6 - 11 months (38.2%), followed by 1 - 2 years old (28.5%). Ninety six point eight percentage of children were infected under 3 years of age. The number of deaths, possibly caused by rotavirus diarrhea were accounted for 40% of all diarrhea deaths and 11.1% of the total deaths. Serotyping of 123 rotavirus isolates showed that serotype G1 (55.3%) was predominant, followed by G2 (26.8%), G3 (9.8%), G4 (0.8%), and 10 isolates (8.1%) remained non-typeable. Mixed infections (0.8%) seemed to be rare. CONCLUSION: Rotavirus diarrhea was an important infectious disease among children in Beijing. Safe and effective rotavirus vaccines for the prevention of severe diarrheas and the reduction of treatment costs are of significant importance to China.