VP7 and VP4 sequence analyses of rotavirus strains from Italian children with viraemia and acute diarrhoea.

Information on genotypes or variants associated with systemic rotavirus infection is lacking. Seven viraemic children with acute rotavirus diarrhoea were evaluated. All faecal strains were genotyped (5 G1P[8] type, 1 G9P[8], 1 G1-G4P[8]). Only 3 of 7 blood strains were typeable (G1P[8], G4P8, and G1P[ND]). A discrepancy between faecal and blood VP7 amino acid sequences (Ser-->Asp at position 94) was found in the first child. Two amino acid substitutions (Ile-->Val and Asn-->Ser at position 106 and 113) in the VP4 sequences were demonstrated in the second child. Complete correspondence was found in the third child. All of the observed amino acid substitutions may be involved in rotavirus neutralisation. We speculate that during co-infection by multiple strains, a preferential extraintestinal dissemination of some variants may occur, possibly because of differential viral characteristics.

Genetic characterization of G3 rotaviruses detected in Italian children in the years 1993-2005.

In recent years an apparent increase in the frequency of detection of G3P[8] rotaviruses has been observed worldwide. Similarly, in Italy G3P[8] strains have been detected sporadically and in a scattered fashion over 20 years, whereas in 2003 and 2005 G3P[8] rotavirus activity increased markedly. By analysis of the VP7, VP4, VP6, and NSP4 genes of a selection of G3P[8] rotaviruses detected between 1993 and 2005, a remarkable sequence conservation was observed in the VP7, VP4, and VP6 genes. By converse, after 2002 the Italian G3P[8] strains were found to possess unique mutations in significant regions of the NSP4 protein.

Assignment of the group A rotavirus NSP4 gene into genotypes using a hemi-nested multiplex PCR assay: a rapid and reproducible assay for strain surveillance studies.

The rotavirus non-structural protein NSP4 has been implicated in a number of biological functions during the rotavirus cellular cycle and pathogenesis, and has been addressed as a target for vaccine development. The NSP4 gene has been classified into six genotypes (A-F). A semi-nested triplex PCR was developed for genotyping the major human NSP4 genotypes (A-C), which are common in human rotavirus strains but are also shared among most mammalian rotavirus strains. A total of 192 previously characterized human strains representing numerous G and P type specificities (such as G1P[8], G1P[4], G2P[4], G3P[3], G3P[8], G3P[9], G4P[6], G4P[8], G6P[4], G6P[9], G6P[14], G8P[10], G8P[14], G9P[8], G9P[11], G10P[11], G12P[6] and G12P[8]) were tested for NSP4 specificity by the collaborating laboratories. An additional 35 animal strains, including the reference laboratory strains SA11 (simian, G3P[2]), NCDV (bovine, G6P[1]), K9 and CU-1 (canine, G3P[3]), together with 31 field isolates (canine, G3P[3]; feline, G3P[9]; porcine, G2P[23], G3P[6], G4P[6], G5P[6], G5P[7], G5P[26], G5P[27], G9P[6] and G9P[7]) were also successfully NSP4-typed. Four human G3P[9] strains and one feline G3P[9] strain were found to possess an NSP4 A genotype, instead of NSP4 C, suggesting a reassortment event between heterologous strains. Routine NSP4 genotyping may help to determine the genomic constellation of rotaviruses of man and livestock, and identify interspecies transmission of heterologous strains.

Are human P[14] rotavirus strains the result of interspecies transmissions from sheep or other ungulates that belong to the mammalian order Artiodactyla?

A limited number of human G6P[14] rotavirus strains that cause gastroenteritis in humans have been isolated in Europe and Australia. The complete genome sequences were determined for five of these human strains--B10925-97 (isolated in Belgium in 1997), 111/05-27 (Italy, 2005), PA169 (Italy, 1987), MG6 (Australia, 1993), and Hun5 (Hungary, 1997)--and their genetic relatedness to animal rotavirus strains was evaluated by sequencing the complete genome of the sheep rotavirus OVR762 (G8P[14]; Spain, 2002), the guanaco (Lama guanicoe) rotavirus strains Arg/Chubut/99 and Arg/Río Negro/98 (G8P[14] and G8P[1], respectively; Argentina, 1999 and 1998), the sable antelope strain RC-18/08 (G6P[14]; South Africa, 2008), and the bovine rotavirus strain Arg/B383/98 (G15P[11]; Argentina, 1998). These analyses revealed an overall consensus genomic constellation (G6/G8)-P[14]-I2-(R2/R5)-C2-M2-(A3/A11)-N2-T6-(E2/E12)-H3, together with a few gene reassortments, and the phylogenetic analyses confirmed that the P[14] human strains evaluated in this study were closely related to rotavirus strains isolated from sheep, cattle, goats, guanacos, and antelopes and to rabbits (albeit to a lesser extent), suggesting that one (or more) of these animal species might be the source of the human G6P[14] strains. The main feature of the genotype and phylogenetic analyses was the close overall genomic relatedness between the five human G6P[14] rotavirus strains and the ovine and antelope rotavirus strains. Taken together, these data strongly suggest a common origin for the human P[14] strains and those of the even-toed ungulates belonging to the mammalian order Artiodactyla, with sheep probably playing a key role in the interspecies transmission responsible for the introduction of P[14] rotavirus strains into the human population.

Emerging GII.4 norovirus variants affect children with diarrhea in Palermo, Italy in 2006.

Although the genetic/antigenic heterogeneity of human noroviruses (NoVs) is impressive, a few genogroup II strains of genotype 4 (GII.4) are dominant worldwide. GII.4 NoVs evolve rapidly and in the last 15 years six epidemic variants have been identified. In 2005-2006, surveillance of sporadic viral gastroenteritis in children in Palermo, Italy, resulted in the detection of NoV strains in 20.9% of the patients admitted to hospital. By restriction fragment length polymorphism (RFLP) and sequence analysis of region A in the RNA-dependent RNA-polymerase (RdRp) gene, 59 NoV strains were successfully characterized. Eighty-one percent of the strains were characterized as GII.4, 14% as GIIb/Hilversum and 5% as GI.1. Phylogenetic analysis of region A and of the ORF1/ORF2 overlapping region of the GII.4 strains recovered in Palermo in the years 2002-2006 revealed the sequential emergence of four variants, GII.4 2002, 2004, 2006a, and 2006b. The variant GII.4 2006a was detected in June and July, 2006, while the variant 2006b first appeared in August, 2006, becoming predominant thereafter. Based on these findings, the dynamics of replacement and circulation of the GII.4 NoV variants in Italy in 2005-2006 appear to have matched the temporal pattern observed in Europe during the same period.

Genotyping of GII.4 and GIIb norovirus RT-PCR amplicons by RFLP analysis.

GII.4 and GIIb/Hilversum norovirus (NoV) strains appear to have a prominent epidemiological role in outbreaks or sporadic cases of human gastroenteritis. Sequence analysis, although laborious, is the reference method used for characterization of noroviruses. In this study a screening test is proposed to characterize GIIb and GII.4 NoVs based on restriction fragment length polymorphism (RFLP) analysis of amplicons obtained from the RNA-dependent RNA polymerase (RdRp) region. Virtual analysis of 793 RdRp sequences of GGI and GGII NoVs, retrieved from GenBank, and representative of global geographical origins on a long-time period, permitted the selection of four restriction enzymes, XmnI, AhdI, BstXI, and AcuI, suitable for correct identification of GIIb and GII.4 NoV genotypes. Experimental analysis by the RT-PCR RFLP analysis of 41 NoV strains detected in Palermo during the years 2002-2005 allowed to recognize all the Italian strains as belonging to GIIb/Hilversum or GII.4, and sequence analysis confirmed these results. The PCR-RFLP protocol developed in this study proved to be a simple and reliable proxy for sequence-based classification of the GIIb/Hilversum and GII.4 NoV variants displaying high specificity (100%) and sensitivity (94%).

Genetic heterogeneity in the VP7 of group C rotaviruses.

Evidence for a possible zoonotic role of group C rotaviruses (GCRVs) has been recently provided. To gain information on the genetic relationships between human and animal GCRVs, we sequenced the VP7 gene of 10 porcine strains detected during a large surveillance study from different outbreaks of gastroenteritis in piglets. Four GCRV strains were genetically related to the prototype GCRV porcine Cowden strain. A completely new VP7 genotype included 4 strains (344/04-7-like) that shared 92.5% to 97.0% aa identity to each other, but <83% to human GCRVs and <79% to other porcine and bovine GCRVs. A unique 4-aa insertion (SSSV or SSTI), within a variable region at the carboxy-terminus of VP7, represented a distinctive feature for these 4 unique strains. An additional strain, 134/04-18, was clearly different from all human and animal GCRVs (<85% aa identity) and likely accounts for a distinct VP7 genotype. The VP7 of a unique strain, 42/05-21, shared similar ranges of aa sequence identities with porcine and human strains (88.0-90.7% to porcine GCRVs and 85.2-88.2% to human GCRVs). Plotting the VP7 gene of strain 42/05-21 against the VP7 of human and porcine strains revealed discontinuous evolution rates throughout the VP7 molecule, suggesting different mutational pressure or a remote intragenic recombination event. These findings provide the need for future epidemiological surveys and warrant studies to investigate the pathogenic potential of these novel GCRVs in pigs.

Prevalence of group C rotaviruses in weaning and post-weaning pigs with enteritis.

Diarrheic fecal specimens collected from porcine herds were screened for the presence of group C rotaviruses using a reverse transcription-polymerase chain reaction (RT-PCR) assay. A total of 188 samples were tested and 54 were positive. When compiled these data with diagnostic results on group A rotaviruses and enteric caliciviruses we found that all but 5 group C rotavirus positive samples contained at least one additional virus. A subset of samples were subjected to nucleotide sequencing. The selected strains showed an unexpectedly wide range of nucleotide sequence heterogeneity (88.6-100%) to each other and to the reference porcine group C rotavirus strain, Cowden. The nucleotide sequence identity to the genuine bovine and human strains were, respectively, 86.8 and 87.2% or less. In conclusion, our study demonstrates that infection with group C rotavirus is frequent in Italian piggeries. The considerable rate of multiple infections requires further studies to investigate the pathogenic potential of group C rotaviruses in pigs, alone or in mixed infection, and raises challenges in the laboratory diagnosis of porcine enteric infections.

Rotavirus and not age determines gastroenteritis severity in children: a hospital-based study.

BACKGROUND: The severity of childhood gastroenteritis is generally believed to be age-related rather than aetiology-related. Rotavirus-induced gastroenteritis is more severe than gastroenteritis caused by other enteric pathogens and is also age-related. We thus addressed the question of whether the increased severity of rotavirus-induced gastroenteritis is related to age or to features intrinsic to the agent. STUDY DESIGN: In this multicentre, hospital-based, prospective survey, we evaluated the severity of diarrhoea in rotavirus-positive and rotavirus-negative children up to 4 years of age. Severity was assessed with a score in four groups of age-matched children. RESULTS: Rotavirus was detected in 381 of 911 children. Disease severity was evaluated in 589 cases for which clinical data were complete. The rotavirus-positive and rotavirus-negative groups differed with regards to diarrhoea duration, hospital stay, degree of dehydration and the number of episodes of vomiting. Gastroenteritis was more severe in rotavirus-positive than in rotavirus-negative children. In contrast, none of the main severity parameters differed in the four age groups, irrespective of the presence of rotavirus. CONCLUSIONS: These data provide the evidence that aetiology and not age determines diarrhoeal severity. The demonstration that diarrhoea was more severe in rotavirus-positive children supports the need for a rotavirus vaccine and for studies that address the duration of vaccine protection.

Norovirus and gastroenteritis in hospitalized children, Italy.

Noroviruses were detected in 48.4% of 192 children (<3 years of age) hospitalized for gastroenteritis in Palermo, Italy, during 2004; predominant genotypes were GGIIb/Hilversum and GGII.4 Hunter. Of children with viral enteritis, 19.6% had a mixed norovirus-rotavirus infection. The severity of infection was lower for norovirus than for rotavirus but increased in co-infection.

Norovirus in captive lion cub (Panthera leo).

African lions (Panthera leo) are susceptible to viral diseases of domestic carnivores, including feline calici-virus infection. We report the identification of a novel enteric calicivirus, genetically related to human noroviruses of genogroup IV, in a lion cub that died of severe hemorrhagic enteritis.

Canine-origin G3P[3] rotavirus strain in child with acute gastroenteritis.

Infection by an animal-like strain of rotavirus (PA260/97) was diagnosed in a child with gastroenteritis in Palermo, Italy, in 1997. Sequence analysis of VP7, VP4, VP6, and NSP4 genes showed resemblance to a G3P[3] canine strain identified in Italy in 1996. Dogs are a potential source of human viral pathogens.

Detection of the norovirus variants GGII.4 hunter and GGIIb/hilversum in Italian children with gastroenteritis.

Noroviruses (NoVs) are important enteric pathogens of humans. Although they exhibit an impressive genetic diversity, few NoV strains appear to predominate worldwide. Limited epidemiological data are available on NoV gastroenteritis in Italy. In this study, we assessed the prevalence of human NoV in Italian children with gastroenteritis by using a reverse-transcription nested polymerase chain reaction (RT-PCR) assay specific for the RNA-dependent RNA polymerase (RdRp) on faecal samples collected throughout the 2004 surveillance activity in Palermo, Italy. NoVs were detected in 47% of the stool samples obtained from children <5 years age, admitted to hospital with acute non-bacterial gastroenteritis. A selection of strains was further analyzed by partial sequence analysis of the RdRp gene. The strains were characterized as genogroup (GG) II and clustered into two distinct virus populations that resembled the emerging European GGIIb/Hilversum strains and the Australian Hunter GGII.4 strains. A temporal pattern of distribution of the two NoV strains was observed which was consistent with an independent circulation of two separate strains in the local population. Based on this 1-year study we concluded that NoVs were a diffuse cause of sporadic cases of acute childhood gastroenteritis and that strains of global epidemiological relevance were circulating in Palermo, Italy in 2004.

Heterogeneity and temporal dynamics of evolution of G1 human rotaviruses in a settled population.

A rotavirus sample collection from 19 consecutive years was used to investigate the heterogeneity and the dynamics of evolution of G1 rotavirus strains in a geographically defined population. Phylogenetic analysis of the VP7 gene sequences of G1P[8] human rotavirus strains showed the circulation of a heterogeneous population comprising three lineages and seven sublineages. Increases in the circulation of G1 rotaviruses were apparently associated with the introduction of novel G1 strains that exhibited multiple amino acid changes in antigenic regions involved in rotavirus neutralization compared to the strains circulating in the previous years. The emergence and/or introduction of G1 antigenic variants might be responsible for the continuous circulation of G1 rotaviruses in the local population, with the various lineages and sublineages appearing, disappearing, or cocirculating in an alternate fashion under the influence of immune-pressure mechanisms. Sequence analysis of VP4-encoding genes of the G1 strains revealed that the older strains were associated with a unique VP4 lineage, while a novel VP4 lineage emerged after 1995. The introduction of human rotavirus vaccines might alter the forces and balances that drive rotavirus evolution and determine the spread of novel strains that are antigenically different from those included in the vaccine formulations. The continuous emergence of VP7-VP4 gene combinations in human rotavirus strains should be taken into consideration when devising vaccination strategies.

Relationships among porcine and human P[6] rotaviruses: evidence that the different human P[6] lineages have originated from multiple interspecies transmission events.

Porcine rotavirus strains (PoRVs) bearing human-like VP4 P[6] gene alleles were identified. Genetic characterization with either PCR genotyping or sequence analysis allowed to determine the VP7 specificity of the PoRVs as G3, G4, G5 and G9, and the VP6 as genogroup I, that is predictive of a subgroup I specificity. Sequence analysis of the VP8* trypsin-cleavage product of VP4 allowed PoRVs to be characterized further into genetic lineages within the P[6] genotype. Unexpectedly, the strains displayed significantly higher similarity (up to 94.6% and 92.5% at aa and nt level, respectively) to human M37-like P[6] strains (lineage I), serologically classifiable as P2A, or to the atypical Hungarian P[6] human strains (HRVs), designated as lineage V (up to 97.0% aa and 96.1% nt), than to the porcine P[6] strain Gottfried, lineage II (<85.1% aa and 82.2 nt), which is serologically classified as P2B. Interestingly, no P[6] PoRV resembling the original prototype porcine strain, Gottfried, was detected, while Japanase P[6] PoRV clustered with the atypical Japanase G1 human strain AU19. By analysis of the 10th and 11th genome segments, all the strains revealed a NSP4B genogroup (Wa-like) and a NSP5/6 gene of porcine origin. These findings strongly suggest interspecies transmission of rotavirus strains and/or genes, and may indicate the occurrence of at least 3 separate rotavirus transmission events between pigs and humans, providing convincing evidence that evolution of human rotaviruses is tightly intermingled with the evolution of animal rotaviruses.

G2 rotavirus infections in an infantile population of the South of Italy: variability of viral strains over time.

Rotavirus positive samples collected in Palermo, Italy, during 2002-2004 did not react with the G2 type-specific RV5:3 monoclonal antibodies (MAbs) and could be identified as G2 only by RT-PCR genotyping. The genetic variation of VP7 and VP4 antigenic proteins was studied in 14 G2 samples including a selection of both those successfully characterized by serotyping and those failing to be serotyped. The phylogenetic analysis performed on partial VP7 sequences showed a temporal clustering of these strains, with those isolated in Palermo in 2003 belonging to the same lineage of G2 MAbs-unreactive strains identified in UK in 1996-1997 and in Bari, Italy, in 2003-2004. A single amino acid substitution in VP7 antigenic region A, at position 96 (Asp-->Asn), was consistently associated with the loss of antigenic reactivity. Five of the G2 strains were further characterized by sequencing of VP4-encoding genes as belonging to the P[4] type, and separate lineages clustering the strains according to a temporal distribution could be described. VP7 and VP4 antigenic proteins analysis provided evidence that over the last 11 years, at least two different populations of G2P[4] rotavirus strains have been infecting the infant population in Palermo. Considering the role of anti-VP7 and anti-VP4 neutralizing antibodies in rotavirus immunity, the emergence of new VP7-VP4 gene combinations might influence rotavirus circulation in the infant population and should be taken into consideration when devising vaccination strategies.

Nucleotide variation in the VP7 gene affects PCR genotyping of G9 rotaviruses identified in Italy.

A modified (aFT9m) and a degenerate (aFT9d) version of the rotavirus G9-specific primer (aFT9) allowed strains that were previously untypable, because of point mutations accumulating at the primer binding site, to be G typed by reverse transcription-polymerase chain reaction. The strains were collected during 2001-2002 in Italy in hospitals of the Apulia region, from children affected by severe rotavirus-associated enteritis. Using a wide selection of G9 rotaviruses detected worldwide, sequencing of the G9 untypable strains, sequence comparison, and phylogenetic analysis showed that the Italian strains have strong genetic similarity (< or =99.4%) to G9 rotaviruses identified recently in many parts of the world and different from the old G9 strains identified during the 1980s (less than 90%). Genetic variation of G9 rotaviruses explains the constraints encountered in the typing assays and presumably accounts, together with genetic reassortment events, for the emergence on a global scale of the G9 serotype.