Molecular epidemiology of rotavirus in cats in the United Kingdom.

Rotaviruses are leading causes of gastroenteritis in the young of many species. Molecular epidemiological studies in children suggest that interspecies transmission contributes to rotavirus strain diversity in people. However, population-based studies of rotaviruses in animals are few. We investigated the prevalence, risk factors for infection, and genetic diversity of rotavirus A in a cross-sectional survey of cats housed within 25 rescue catteries across the United Kingdom. Morning litter tray fecal samples were collected during the winter and summer in 2012 from all pens containing kittens and a random sample of those housing adult cats. Group A rotavirus RNA was detected by real-time reverse transcription-PCR, and positive samples were G and P genotyped using nested VP4 and VP7 PCR assays. A total of 1,727 fecal samples were collected from 1,105 pens. Overall, the prevalence of rotavirus was 3.0% (95% confidence interval [CI], 1.2 to 4.9%). Thirteen out of 25 (52%; 95% CI, 31.3 to 72.2%) centers housed at least one rotavirus-positive cat. The prevalence of rotavirus was associated with season (odds ratio, 14.8 [95% CI, 1.1 to 200.4]; P = 0.04) but not age or diarrhea. It was higher during the summer (4.7%; 95% CI, 1.2 to 8.3%) than in winter (0.8%; 95% CI, 0.2 to 1.5%). Asymptomatic epidemics of infection were detected in two centers. G genotypes were characterized for 19 (33.3%) of the 57 rotavirus-positive samples and P genotypes for 36 (59.7%). Two rotavirus genotypes were identified, G3P[9] and G6P[9]. This is the first population-based study of rotavirus in cats and the first report of feline G6P[9], which questions the previous belief that G6P[9] in people is of bovine origin.

Molecular characterization of rotavirus strains circulating among children with acute gastroenteritis in Madagascar during 2004-2005.

A survey was undertaken of the etiology of acute gastroenteritis in children <16 years of age in Antananarivo, Madagascar, from May 2004 through May 2005. With use of electron microscopy of fecal specimens, 104 (36%) of 285 children were found to be infected with rotavirus. Rotavirus strain characterization was undertaken using enzyme-linked immunosorbent assay, electropherotyping, reverse-transcription polymerase chain reaction genotyping, and nucleotide sequencing. The predominant group A rotavirus strain types identified were P[4]G2 (62%) and P[8]G9 (23%). Nucleotide sequence analysis of the VP7 genes of selected Malagasy G2 and G9 strains demonstrated similarity with those of other recently identified African rotavirus strains belonging to the same genotype.

Cryptosporidium species causing acute diarrhoea in children in Antananarivo, Madagascar.

A 13-month study of children presenting with acute diarrhoeal disease at hospitals and rehydration clinics in Antananarivo, Madagascar, was undertaken between May 2004 and May 2005. Cryptosporidiosis accounted for diarrhoea in 12 (5.6%) of the 215 children investigated. Cases of cryptosporidiosis were detected only in the rainy season, and the median age of cases was 13.5 months (range=1 day-27 months). As 11 of the cases of cryptosporidiosis were caused by Cryptosporidium hominis and only one by C. parvum, most of the cases were probably the result of anthroponotic transmission. GP60/45/15 gene polymorphisms indicated that the causative pathogens were of subtypes Ia, Id, Ie and IIc.

Apparent extinction of non-G2 rotavirus strains from circulation in Recife, Brazil, after the introduction of rotavirus vaccine.

The introduction of a G1P[8] rotavirus vaccine in Recife, Brazil, caused a decrease in rotavirus detection from 27% (March-May, 2006) to 5.0% (March-May, 2007), with all strains becoming G2, against which less protection had been predicted.

Atm is a negative regulator of intestinal neoplasia.

The ataxia telangiectasia-mutated (ATM) gene has been implicated as an early barrier to the growth and progression of incipient solid tumors. Here, we show that germ-line nullizygosity for the mouse Atm gene significantly increases the proliferative index, net growth rate and multiplicity of intestinal adenomas in two distinct models of familial colon cancer: Apc(Min/+) and Apc(1638N/+). These effects of Atm deficiency are quantitatively different from deficiency for either of the genomic stability genes Bloom's syndrome helicase or DNA ligase 4, and the effect of Atm loss on tumor multiplicity is largely independent of the effect of ionizing radiation. Furthermore, the loss of heterozygosity rates at the adenomatous polyposis coli (Apc) locus are unaffected by Atm loss. Taken together, these data implicate the Atm gene product as a barrier to dysplastic growth in the early stages of intestinal tumor progression, independent of its effects on genomic stability.

Lack of shedding of the RIX4414 live attenuated rotavirus vaccine administered to adult volunteers.

Ten adult volunteers were given the RIX4414 rotavirus vaccine; one volunteer shed a small amount of virus antigen but not live virus. This suggests that parents of vaccinated children are unlikely to spread the vaccine.

Epidemiology of rotavirus diarrhoea in Iranian children.

Human rotavirus is the most important cause of severe diarrhoea in infants and young children worldwide. We describe the aetiology of viral diarrhoea and the characteristics of rotavirus infection in Shahrekord, Iran. Two hundred and fifty nine children <5 years old admitted to Hajar Hospital, 245 children with acute diarrhoea attending primary health centres in Shahrekord, and 114 children hospitalised for elective surgery were selected from October 2001 to August 2002. Stool samples were screened for enteric viruses using EM. Rotaviruses were characterised using ELISA, reverse transcription-polymerase chain reaction (RT-PCR), and electropherotyping. One hundred and eighty six viruses were identified, of which 146 (78%) were rotavirus. The second most frequent virus was coronavirus, followed by calicivirus. Rotaviruses exhibited a marked seasonal variation, being most frequently isolated from November to February (50% of rotavirus recovered) and affected mostly children <2 years old. The RT-PCR successfully typed 139 of the 146 (95%) rotavirus G types and 124 (85%) P types. The most frequent P type was, P[8] in 108 (74%), P[4] in 16 (11%), and was P non-typeable in 22 (15%). Among the G types, G1 was identified in 120 (82%), G2 in 19 (13%), and was G-non-typeable in 7 (5%). Our results are the first report of rotavirus genotypes affecting Iranian children. The most frequent G and P types (G1, G2, P[8], and P[4]) are similar to those reported from around the world and will be covered by existing rotavirus vaccines targeting G types G1-G4.

Characterization of rotaviruses causing diarrhoea in Vietnamese children.

Faecal samples from 123 children admitted to the Centre for Tropical Diseases in Ho Chi Minh city, Vietnam, with acute watery diarrhoea were screened by negative-stain electron microscopy for viral enteropathogens. In addition to the 48 children who were found to be infected with rotavirus only, one had both rotavirus and astrovirus, two had adenovirus 40/41, and one had astrovirus only. The rotaviruses were subjected to molecular analysis by electropherotyping, G- and P-genotyping (by reverse-transcriptase PCR), and amplicon sequencing. By use of newly designed PCR primers, all 49 isolates could be G-genotyped and all but one P-genotyped. Novel variants of G1-G1*--were the most commonly detected G-genotype and such variants of P[8]-P[8*]--were the second commonest P-genotype. The P[8*] and G1* amplicons were, respectively, only 92%-93.4% and 88.1%-89% similar to the corresponding sequences from the prototype P[8] G1 rotavirus, Wa. Several unusual P- and G-genotype combinations were detected. Four (8%) of the children investigated were each found to be co-infected with two different rotaviruses. These data add to our knowledge of the continuing evolution and diversity of human rotaviruses, and should help in the rational design of vaccines.

Detection and characterization of rotaviruses in hospitalized neonates in Blantyre, Malawi.

In five separate fecal collections spanning three years, group A rotaviruses were detected by enzyme-linked immunosorbent assay in 35 (25%) of 142 specimens obtained from nondiarrheic, hospitalized neonates in Blantyre, Malawi. Molecular characterization of each strain identified, for the first time in neonates, a short electropherotype, genotype P[6], G8 strain type, similar to the dominant, cocirculating community strain detected in symptomatic infants in Blantyre. Partial sequence analysis of the VP4 and NSP4 genes of neonatal and community strains failed to identify changes which could explain the differences in clinical outcome. Neonatal serotype G8 rotaviruses should be considered as potential rotavirus vaccine candidates for use in Malawi.

Expanding global distribution of rotavirus serotype G9: detection in Libya, Kenya, and Cuba.

Serotype G9 may be the fifth most common human rotavirus serotype, after serotypes G1 to G4. In three cross-sectional studies of childhood diarrhea, we have detected serotype G9 rotaviruses for the first time in Libya, Kenya, and Cuba. Serotype G9 constituted 27% of all rotaviruses identified, emphasizing the reemergence of serotype G9 and suggesting that future human rotavirus vaccines will need to protect against disease caused by this serotype.

Detection of group C rotavirus in children with acute gastroenteritis in Blantyre, Malawi.

Among 606 children who were treated for acute gastroenteritis at the Queen Elizabeth Central Hospital in Blantyre, Malawi, Group C rotavirus (Gp C RV) was detected by enzyme-linked immunosorbent assay in fecal specimens from 16 (3.9%) of 408 inpatients and in 4 (2.0%) of 198 outpatients. Thirteen (65%) children excreting Gp C RV were coinfected with Group A rotavirus.

Effect of concomitant HIV infection on presentation and outcome of rotavirus gastroenteritis in Malawian children.

BACKGROUND: Rotaviruses represent important causes of severe diarrhoea in early childhood. We examined the effect of HIV infection on the presentation and outcome of rotavirus gastroenteritis in Malawian children. METHODS: Children younger than 5 years who were treated for acute gastroenteritis at the Queen Elizabeth Central Hospital in Blantyre from July, 1997, to June, 1999, were enrolled. Children with rotavirus diarrhoea, with and without HIV infection, were followed up for up to 4 weeks after hospital discharge. Rotavirus disease severity (assessed with a 20-point score), duration of rotavirus shedding, and seroresponse to rotavirus were compared between HIV-infected and HIV-uninfected children. FINDINGS: 786 inpatients (median age 8 months, 271 [34%] of whom were HIV-1-infected) and 400 outpatients (median age 9 months, 65 [16%] of whom were HIV-infected) were enrolled. Rotavirus was detected less frequently among HIV-infected children (102 of 336 [30%]) than among HIV-uninfected children (348 of 850 [41%], (relative risk 0.71 [95% CI 0.53-0.87], p=0.0007). There were no differences in rotavirus disease severity for hospitalised children with and without HIV infection, but HIV-infected children were more likely to die during follow-up (11/50 [22%]) than HIV-uninfected children (0/61, p<0.0001). Of 29 HIV-infected and 45 HIV-uninfected children who completed follow-up, six (21%) HIV-infected children shed rotavirus, compared with two (4%) HIV-uninfected children (4.66 [1.01-21.51], p=0.05), but shedding was not associated with diarrhoea. Three-quarters of children exhibited a four-fold rise of serum IgG or IgA to rotavirus, which did not vary by HIV status. INTERPRETATION: Malawian children with concomitant HIV infection resolved acute rotavirus infections. Rotavirus vaccine safety and immunogenicity in HIV-infected infants should now be determined.

Rotavirus strain diversity in Blantyre, Malawi, from 1997 to 1999.

In a 2-year study of viral gastroenteritis in children in Blantyre, Malawi, the diversity of rotavirus strains was investigated by using electropherotyping, reverse transcription-PCR amplification of the VP7 and VP4 genes (G and P genotyping), and nucleotide sequencing. Of 414 rotavirus strains characterized, the following strain types were identified: P[8], G1 (n = 111; 26.8%); P[6], G8 (n = 110; 26.6%); P[8], G3 (n = 93; 22.5%); P[4], G8 (n = 31; 7.5%); P[8], G4 (n = 21; 5.1%); P[6], G3 (n = 12; 2.9%); P[6], G1 (n = 7; 1.7%); P[6], G9 (n = 3; 0.7%); P[6], G4 (n = 3; 0.7%); P[4], G3 (n = 1; 0.2%); and mixed (n = 15; 3.6%). While all strains could be assigned a G type, seven strains (1.7%) remained P nontypeable. The majority of serotype G8 strains and all serotype G9 strains had short electropherotype profiles. All remaining typeable strains had long electropherotypes. Divergent serotype G1 rotaviruses, which contained multiple base substitutions in the 9T-1 primer binding site, were commonly identified in the second year of surveillance. Serotype G2 was not identified. Overall, G8 was the most frequently identified VP7 serotype (n = 144; 34.8%) and P[8] was the most frequently detected VP4 genotype (n = 227; 54.8%). Partial sequence analysis of the VP4 gene of genotype P[8] rotaviruses identified three distinct clusters, which predominantly (but not exclusively) comprised strains belonging to a distinct VP7 serotype (G1, G3, or G4). As a result of mutations in the 1T-1 primer binding site, strains belonging to each cluster required a separate primer for efficient typing. One cluster, represented by P[8], G4 strain OP354, was highly divergent from the established Wa and F45 VP4 P[8] lineages. As is the case for some other countries, the diversity of rotaviruses in Malawi implies that rotavirus vaccines in development will need to protect against a wider panel of serotypes than originally envisioned.

Molecular and serologic characterization of novel serotype G8 human rotavirus strains detected in Blantyre, Malawi.

During a 2-year study of diarrhea among children in Blantyre, Malawi, greater than 50% of rotavirus strains genotyped by using reverse transcription-polymerase chain reaction possessed previously unrecognized combinations of the neutralization proteins VP7 and VP4. Serotype G8 rotaviruses, which have been identified recently in several African countries, were found to possess P[4] or P[6] VP4 genotype specificity. Two of these short electropherotype rotaviruses were further investigated: these comprised a P[6], G8 representative strain (MW23) and a P[4], G8 representative strain (MW333). The VP7 gene sequences of both strains exhibited greatest homology to human and animal serotype G8 rotaviruses. Sequence analysis of the VP4 gene of MW23 indicated closest identity to the P2A[6], G9 strain US1205 from the United States. The VP4 gene of MW333 was most closely related to the P[4], G12 strain L26 isolated in the Philippines and the Australian P[4], G2 strain RV-5. The NSP4 gene sequences of both strains were classified in NSP4 genetic group I. RNA-RNA hybridization demonstrated that each of these two strains is related to the DS-1 genogroup of human rotaviruses. Subgroup analysis and virus neutralization confirmed complete antigenic characterization of MW23 as subgroup I, P2A[6], G8 and MW333 as subgroup I, P1B[4], G8. The similarity of the VP7 gene sequences of the prototype strains described in this report to bovine serotype G8 rotaviruses suggests that they may represent human/bovine reassortant viruses.

Dnmt1N/+ reduces the net growth rate and multiplicity of intestinal adenomas in C57BL/6-multiple intestinal neoplasia (Min)/+ mice independently of p53 but demonstrates strong synergy with the modifier of Min 1(AKR) resistance allele.

Altered patterns of the 5-cytosine methylation of genomic DNA are associated with the development of a wide range of human cancers. We have studied the mechanisms and genetic pathways by which a targeted heterozygous deficiency in the murine 5-cytosine DNA methyltransferase gene (Dnmt1(N/+)) diminishes intestinal tumorigenesis in C57BL/6-multiple intestinal neoplasia (Min)/+ mice. We found that Dnmt1(N/+) retards the net growth rate of intestinal adenomas and reduces tumor multiplicity by approximately 50%. This tumor resistance affects the entire intestinal tract and is independent of the status of modifier of Min 1 and p53, two loci that have been found to confer strong resistance to Min-induced neoplasia Interestingly, Dnmt/(N/+) and modifier of Min 1 resistance interact synergistically, together virtually eliminating tumor incidence. This finding may provide an insight into potential combinatorial therapeutic approaches for treating human colon cancer.

The Mom1AKR intestinal tumor resistance region consists of Pla2g2a and a locus distal to D4Mit64.

The Mom1 (Modifier of Min-1) region of distal chromosome 4 was identified during a screen for polymorphic modifiers of intestinal tumorigenesis in ApcMin/+ mice. Here, we demonstrate that the Mom1AKR allele consists of two genetic components. These include the secretory phospholipase Pla2g2a, whose candidacy as a Mom1 resistance modifier has now been tested with several transgenic lines. A second region, distal to Pla2g2a, has also been identified using fine structure recombinants. Pla2g2aAKR transgenic mice demonstrate a modest resistance to tumorigenesis in the small intestine and a very robust resistance in the large intestine. Moreover, the tumor resistance in the colon of Pla2g2aAKR animals is dosage-dependent, a finding that is consistent with our observation that Pla2g2a is expressed in goblet cells. By contrast, mice carrying the distal Mom1 modifier demonstrate a modest tumor resistance that is confined to the small intestine. Thus, the phenotypes of these two modifier loci are complementary, both in their quantitative and regional effects. The additive effects and tight linkage of these modifiers may have been necessary for the initial identification of the Mom1 region.

Mlh1 deficiency enhances several phenotypes of Apc(Min)/+ mice.

Defects in APC and DNA mismatch repair genes are associated with a strong predisposition to colon cancer in humans, and numerous mouse strains with mutations in these genes have been generated. In this report we describe the phenotype of Min/+ Mlh1-/- mice. We find that these doubly mutant mice develop more than three times the number of intestinal adenomas compared to Min/+ Mlh1+/+ or +/- mice but that these tumors do not show advanced progression in terms of tumor size or histological appearance. Full length Apc protein was not detected in the tumor cells from Min/+ Mlh1-/- mice. Molecular analyses indicated that in many tumors from Min/+ Mlh1-/- mice, Apc was inactivated by intragenic mutation. Mlh1 deficiency in Min/+ mice also led to an increase in cystic intestinal crypt multiplicity as well as enhancing desmoid tumorigenesis and epidermoid cyst development. Thus, Mlh1 deficiency influences the somatic events involved in the development of most of the phenotypes associated with the Min mutation. Oncogene (2000).

Tumorigenesis in the multiple intestinal neoplasia mouse: redundancy of negative regulators and specificity of modifiers.

The interaction between mutations in the tumor-suppressor genes Apc and p53 was studied in congenic mouse strains to minimize the influence of polymorphic modifiers. The multiplicity and invasiveness of intestinal adenomas of Apc(Min/+) (Min) mice was enhanced by deficiency for p53. In addition, the occurrence of desmoid fibromas was strongly enhanced by p53 deficiency. The genetic modifier Mom1 and the pharmacological agents piroxicam and difluoromethylornithine each reduced intestinal adenoma multiplicity in the absence of p53 function. Mom1 showed no influence on the development of desmoid fibromas, whereas the combination of piroxicam and difluoromethylornithine exerted a moderate effect. The ensemble of tumor suppressors and modifiers of a neoplastic process can be usefully analyzed in respect to tissue specificity and synergy.

Identification of three genes expressed primarily during development in Physarum polycephalum.

During the life cycle of Physarum polycephalum, uninucleate amoebae develop into multinucleate syncytial plasmodia. These two cell types differ greatly in cellular organisation, behaviour and gene expression. Classical genetic analysis has identified the mating-type gene, matA, as the key gene controlling the initiation of plasmodium development, but nothing is known about the molecular events controlled by matA. In order to identify genes involved in regulating plasmodium formation, we constructed a subtracted cDNA library from cells undergoing development. Three genes that have their highest levels of expression during plasmodium development were identified: redA, redB (regulated in development) and mynD (myosin). Both redA and redB are single-copy genes and are not members of gene families. Although redA has no significant sequence similarities to known genes, redB has sequence similarity to invertebrate sarcoplasmic calcium-binding proteins. The mynD gene is closely related to type II myosin heavy-chain genes from many organisms and is one of a family of type II myosin genes in P. polycephalum. Our results indicate that many more red genes remain to be identified, some of which may play key roles in controlling plasmodium formation.

Contrasting effects of ENU induced embryonic lethal mutations of the quaking gene.

Multiple alleles of the quaking (qk) gene have a variety of phenotypes ranging in severity from early embryonic death to viable dysmyelination. A previous study identified a candidate gene, QKI, that contains an RNA-binding domain and encodes at least three protein isoforms (QKI-5, -6 and -7). We have determined the genomic structure of QKI, identifying an additional alternative end in cDNAs. Further we have examined the exons and splice sites for mutations in the lethal alleles qkl-1, qkkt1, qkk2, and qkkt3. The mutation in qkl-1 creates a splice site in the terminal exon of the QKI-6 isoform. Missense mutations in the KH domain and the QUA1 domains in qkk2 and qkkt3, respectively, indicate that these domains are of critical functional importance. Although homozygotes for each ENU induced allele die as embryos, their phenotypes as viable compound heterozygotes with qkv differ. Compound heterozygous qkv animals carrying qkkt1, qkk2, and qkkt3 all exhibit a permanent quaking phenotype similar to that of qkv/qkv animals, whereas qkv/qkl-1 animals exhibit only a transient quaking phenotype. The qkl-1 mutation eliminates the QKI-5 isoform, showing that this isoform plays a crucial role in embryonic survival. The transient quaking phenotype observed in qkv/qkl-1 mice indicates that the QKI-6 and QKI-7 isoforms function primarily during myelination, but that QKI-5 may have a concentration-dependent role in early myelination. This mutational analysis demonstrates the power of series of alleles to examine the function of complex loci and suggests that additional mutant alleles of quaking could reveal additional functions of this complex gene.