Serotype 3 human rotavirus strains with subgroup I specificity.

During an epidemiological study on human rotavirus (HRV) infections in Italy, three subgroup I strains not associated with serotype 2 reactivity were detected. All three strains were serotype 3, each with a distinct RNA pattern showing fast-moving tenth and eleventh segments (long electropherotype). Following successful adaptation to growth in cell cultures, the serotype 3 strains (MZ58, PCP5, and PA710) were further characterized by neutralization and by RNA-RNA (Northern blot) hybridization. Antiserum to reference HRV strain YO (subgroup II, serotype 3), as well as a monoclonal antibody to VP7 of YO neutralized, at comparable titers, the homologous virus, the three unusual HRV strains, and two reference simian strains (SA11 and RRV-2, both subgroup I, serotype 3), whereas SA11 antiserum and a monoclonal antibody to VP7 of SA11 neutralized simian strains more efficiently. However, antiserum to PCP5 neutralized the three unusual isolates and the simian strains at significantly higher titers than it did with reference strain YO. With 32P-labeled RNA from MZ58 as a probe, a high degree of homology was detected by Northern blot hybridization with strains PCP5, PA710, SA11, and UK (bovine rotavirus) at the level of several segments and with strain YO only at the level of genes 7 to 9. Conversely, labeled RNA of strain YO hybridized extensively with Wa (subgroup II, serotype 1 HRV strain) but only at the level of genes 7 to 9 with MZ58, PCP5, PA710, SA11, and UK. Finally, the labeled SA11 probe hybridized at the level of RNA segments 1 to 3 and 6 to 11 to the three unusual strains. These findings suggest that the unusual subgroup I, serotype 3, strains isolated from humans are more likely to be animal rotaviruses rather than natural reassortants between different HRV strains.     

Unusual human rotavirus strains having subgroup I specificity and “long” RNA electropherotype

Summary During an epidemiological study of human rotavirus in Metro Manila, Philippines, we found 20 unusual strains which belong to subgroup I but have long RNA pattern typical of subgroup II human rotavirus. The RNA patterns of the 20 strains were classified into four groups though they were very similar to each other. Four strains, designated L4, L26, L27, and L34 were isolated in MA 104 cells from stool specimens. They possessed subgroup I specificity and long RNA pattern identical to that of the viruses in the original stool samples. The serotype specificity of these strains could not be determined by either enzyme-linked immunosorbent assay (ELISA) or neutralization test, while one strain (L27) was neutralized by serotype 2-specific anti-VP4 monoclonal antibody. These strains were suggested to have an unusual antigenicity on VP7.     

A porcine rotavirus strain with dual VP7 serotype specificity

Porcine rotavirus MDR-13, which on original isolation showed a two-way antigenic relationship with human rotavirus RV-3, shows VP7 relationships with serotype G5 as well as G3 viruses upon gene reassortment. Analysis of porcine MDR-13 and the MD-UK reassortant revealed marked nucleotide and amino acid similarity of VP7 genes of these viruses with those of both serotype G3 and G5 viruses. Evolution of such a strain, possibly by sequential mutations in the VP7 gene, is discussed.     

Antigenic and genetic characterization of serotype G2 human rotavirus strains from the African continent

Serotype G2 rotavirus strains were isolated in seven countries on the African continent during 1999 and 2000. To investigate the associated DS-1 genogroup characteristics, subgroup (VP6) enzyme-linked immunosorbent assay, polyacrylamide gel electrophoresis, and P genotyping were performed on 10 G2 strains. The antigenic and genetic variation of the gene encoding the major neutralization glycoprotein (VP7) was also investigated by using G2-specific monoclonal antibodies and sequence analysis. Alterations in the characteristic DS-1 genogroup gene constellations were more likely to occur in the VP4 gene, and three genotypes were observed: P[4], P[6], and a dual P[4]-P[6] type. The failure of G2-specific monoclonal antibodies to type African G2 strains was more likely due to improper storage of the original stool, although G2 monotypes were detected. Phylogenetic analyses revealed clusters of serotype G2 strains that were more commonly associated with seasons during which G2 was predominant. No rotavirus vaccine trials have been conducted in an area where G2 strains were the predominant circulating serotype, and the continued surveillance of rotavirus epidemics in Africa will be preparation for future vaccine implementation in an area that clearly needs these preventative medicines.     

Isolation and characterization of two distinct human rotavirus strains with G6 specificity.

Two new human rotavirus (HRV) strains, PA151 and PA169, with subgroup I specificity and a long RNA pattern, yet with a serotype G (VP7) specificity different from those of any of the six well-established HRV serotypes (G1 to G4, G8, and G9), were isolated 3 months apart from two children with acute gastroenteritis in Sicily, southern Italy, in the winter season of 1987 and 1988. The HRV isolates were adapted to growth in cell cultures and were then characterized by neutralization and RNA-RNA (Northern blot) hybridization. Cross-neutralization studies with type-specific immune sera to RV serotypes 1 to 10 showed the antigenic relatedness of the two strains with serotype 6 bovine strains UK and NCDV. Monoclonal antibodies to VP7 of UK were able to recognize UK and NCDV strains as well as both HRV isolates. Cross-hybridization studies showed a genetic relatedness of PA151 and PA169 to bovine strains for all genes except gene 4. Gene 4 of PA151 appeared to be genetically related to that of AU228 (a human strain of subgroup I and with serotype G3 specificity that belongs to a feline genogroup), whereas gene 4 of PA169 appeared to be unique, yet it was related to gene 4 of two recently reported subgroup I HRV strains, one (PA710) with serotype G3 specificity and the other (HAL1271) with serotype G8 specificity. The new HRV strains must be taken into consideration when deciding strategies for the development of an effective RV vaccine.     

Monoclonal antibodies to subgroup 1 rotavirus.

Subgroup 1-specific monoclones were analyzed and used successfully in an enzyme-linked immunosorbent assay to recognize certain subgroup 1 rotaviruses.     

Antigenic and genetic characterization of serotype G2 human rotavirus strains from South Africa from 1984 to 1998

Within South Africa, cyclic peaks of serotype G2P[4] rotavirus infection have been observed and these strains were prevalent in some locations. To examine the cyclic phenomenon of serotype G2 rotaviruses, historical stool collections from South Africa spanning 15 years were screened for G2 strains. Subgroup (VP6) ELISA, polyacrylamide gel electrophoresis (PAGE), and P genotyping were performed on 43 G2 strains to investigate the associated DS-1 genogroup characteristics. Antigenic variation of the gene encoding the major neutralization glycoprotein (VP7) was also investigated using G2-specific monoclonal antibodies. In addition, the VP7 gene of 14 serotype G2 strains was sequenced to examine genetic variation. Serotype G2 strains from South Africa displayed a 10 year cyclic pattern with major epidemics occurring in 1987 and 1997. Serotype G2 strains were also found co-dominant with G(1) strains in 1984, 1990, and 1993. The G2 strains from the major epidemics appeared to have emerged from community strains in a manner similar to that suggested for G(1) strains The serotype G2 strains displayed subgroup I specificity and short electropherotypes characteristic of DS-1 genogroup rotavirus strains but appeared to differ in the VP4 gene. Genetic analyses revealed three major serotype G2 lineages, i.e., strains isolated prior to 1987, strains isolated between 1988 and 1994, and strains isolated from 1995. The use of monoclonal antibodies and PCR primers designed against older G2 strains has resulted in the failure to serotype G2 strains circulating currently.     

Genetic analysis of a human rotavirus that belongs to subgroup I but has an RNA pattern typical of subgroup II human rotaviruses.

We have previously found (O. Nakagomi, T. Nakagomi, H. Oyamada, and T. Suto, J. Med. Virol. 17:29-34, 1985), during an epidemiological study in Japan, a novel human rotavirus that belongs to subgroup I but has a long RNA pattern typical of subgroup II human rotaviruses. From the stool specimen containing this virus, we successfully isolated in MA104 cells a rotavirus, designated AU-1, which possesses these novel characteristics. The possibility that strain AU-1 was a laboratory contaminant of an animal rotavirus previously adapted to tissue culture cells was ruled out, and the identity of the AU-1 strain was established. Genetic analysis by RNA-RNA hybridization revealed that the AU-1 strain is not a simple reassortant between subgroup I and II human rotaviruses but that it shares a high level of sequence homology only with the gene encoding VP7 (the major neutralization protein) of serotype 3 human rotaviruses. Weak homology of the genomic RNA segments was also observed between the AU-1 strain and animal rotavirus strains, including rhesus rotavirus strain RRV and bovine rotavirus strain NCDV. These results suggest that the AU-1 strain may be an animal rotavirus that infected a human.     

Serotyping of cell culture-adapted subgroup 2 human rotavirus strains by neutralization

Nine human rotavirus strains from stools of infants with gastroenteritis were serially propagated in MA-104 cell cultures. All strains were identified as subgroup 2 rotaviruses by RNA gel electrophoresis, complement fixation, and enzyme-linked immunosorbent assay. The human rotavirus strains were propagated for 15 to 20 passages and then used for immunization of guinea pigs and rabbits. Animal antisera were also raised against a subgroup 1 human strain purified from stools and against the cell culture-adapted Wa strain, a reference subgroup 2 rotavirus of human origin. Cross-neutralization studies revealed the existence of two distinct serotypes within the cell culture-adapted subgroup 2 human rotaviruses: strains related and unrelated to strain Wa were classified as serotypes 1 and 3, respectively. Results with convalescent-phase sera from infants with primary rotavirus infections confirmed the existence of two serotypes within subgroup 2, and the serotypes responsible for primary subgroup 2 infections could be determined on the basis of the neutralizing reactivity of convalescent sera.     

Isolation in Europe of 69 M-like (serotype 8) human rotavirus strains with either subgroup I or II specificity and a long RNA electropherotype

Summary During an epidemiological study on the prevalence of human rotavirus (HRV) serotypes 1–4 in Europe, we found that some strains could not be typed. However, when a monoclonal antibody directed to serotype 8 HRV was included in the typing assay, we detected seven 69 M-like (serotype 8) strains, six from Finland and one from Italy. The previously reported serotype 8 HRV strains, 69 M, B 37, and B 38 isolated in Indonesia, were of subgroup I specificity and presented a peculiar super short RNA electropherotype. In contrast, all the seven European strains possessed a long RNA pattern, and one of them had subgroup II specificity. Three of these strains were adapted to growth in cell cultures and were further characterized by neutralization and by Northern blot hybridization. They appeared to be closely related to serotype 8 HRV strain 69 M by neutralization, but showed partial homology with several human and animal strains by hybridization. The epidemiological importance of these serotype 8 strains circulating in Europe should be investigated, in view of their possible inclusion in a rotavirus vaccine.     

Human serum antibodies reactive with dietary proteins. Antigenic specificity

The antigenic specificity of human serum IgG antibodies reactive with common dietary proteins has been evaluated by competitive binding using a solid-phase immunoassay (ELISA). Antibodies reactive with bovine milk antigens were shown to be reactive predominantly with casein, rather than alpha-lactalbumin, beta-lactoglobulin, gamma-globulin or albumin. Furthermore, sera containing antibodies reactive with bovine casein, wheat gliadin and chicken ovalbumin showed competitive binding only by each respective dietary protein antigen. IgG4 antibodies specifically reactive with ovalbumin, gliadin or casein were also not cross-reactive in competitive binding studies. Furthermore, both IgG2 and IgG4 anti-milk antibodies showed significant inhibition only with bovine casein, and not with alpha-lactalbumin or beta-lactoglobulin. These data are relevant to concepts regarding the immunobiological role of antibodies of the IgG4 isotype.     

Serologic characteristics of a human rotavirus isolate, AU-1, which has a "long" RNA pattern and subgroup I specificity

An unusual human rotavirus (HRV) isolate, designated AU-1, has a "long" RNA electrophoretic pattern and subgroup I specificity, in contrast to properties common to HRVs previously isolated. Hemagglutination activity of the AU-1 strain was demonstrated using erythrocytes of 1-day-old chicken. Neutralization assays revealed that the AU-1 strain was crossneutralized by hyperimmune antisera against the MO strain (serotype 3). However, antiserum directed against a reassortant in which the gene for Vp7 was replaced by the corresponding AU-1 gene neutralized the AU-1 strain to a significantly higher titer than the MO strain.     

Phylogenetic analyses of human rotavirus in central Taiwan in 1996, 2001 and 2002.

BACKGROUND: Rotavirus epidemiology information is required for gastroenteritis disease control and prevention. Information gathered about the serotype distribution of rotaviruses isolated in Taiwan is of crucial significance, before a licensed rotavirus vaccine is introduced. OBJECTIVES: The purpose of the present study is to investigate the epidemiological diversity of rotaviruses in Taiwan. STUDY DESIGN: A total of 51 stool samples taken from cases of acute gastroenteritis were collected from three teaching hospitals in central Taiwan in 1996, 2001 and 2002. The samples were subjected to RT-PCR tests of VP7 gene of the human rotavirus group A, B, C. RESULTS: A total of 16 stool samples were detected positive by RT-PCR and 10 were sequence analyzed and classified into G1, G3, and G9 types. Compared with other HRV strains: the sequences of CS96-40 of G1 are similar to MVD9816 (identity rate 97.15% and 96.09%, respectively, from Uruguay); the sequences of CS02-01 of G3 are similar to 98-B31 (identity rate 98.93% and 98.72%, respectively, from Japan); the sequences of CS01-05, CS01-06, CS01-07, CS01-09, CS01-13, CS02-02, CS02-03, CS02-04 are very similar to other established G9 rotaviruses sequences (identity rate 96.85-99.88%), especially between CS02-04 and SP2737 (from Japan) with an identity rate of 99.88% and 100% nucleotide and amino acid, respectively. Except for CS01-06 strain, it is VR3, but not VR5, VR7 or VR8, that found to be the most frequent mutated amino acid regions of VP7 in these strains. CONCLUSIONS: Our findings are the first to document the high prevalence of G9 HRV strains in Taiwan, and suggest the re-emergence of G3 strains in central Taiwan since 1991. Epidemiological surveys carried out in this study suggest genotype shifts from type G1 before 1996, to G9 in 2001 and 2002 and the re-emergence of G3 type in 2002.     

Antigenic determinants of heavy chain variable regions: immumological typing of the human immunoglobulin VHIII subgroup.

An antigenic determinant of the VHIII variable region subgroup was defined by means of a heterologous specific antiserum using a hemagglutination inhibition procedure. The specificity of this antiserum was established in inhibition experiments with proteins either of known primary structure or belonging to a definite VH subgroup. A series of IgG, IgA, IgM and IgD monoclonal proteins was examined for the presence of this VHIII subgroup antigenic determinant. The data showed that 50% of the IgG, 62% of the IgA, 55% of the IgM and 41% of the IgD were VHIII-positive, and that certain "blocked" monoclonal immunoglobulins belonged to this subgroup. A preferential association of the VHIII antigenic determinant with the IgG1 and IgG3 subclasses was observed among IgG myeloma proteins while the preferential association was only observed with the IgG1 subclass when anti-Rh antibodies were studied. The VHIII subgroup exhibited nonallelic behavior.     

Analysis of the VP6 gene of human and porcine group A rotavirus strains with unusual subgroup specificities

Full‐length VP6 amino acid sequences of human and porcine rotaviruses with subgroup (SG) (I + II) and SG non‐(I + II) were analyzed in comparison with those of SG I and SG II. In human rotaviruses, the strains in the same SG shared a very high degree of amino acid identity, ranging from 97.4% to 99.4% for SG I, 95.9% to 100% for SG II, and 99.4% to100% for SG non‐(I + II), while viruses in different SGs shared somewhat lower sequence identity at 90.4–93.1%. Conserved amino acids that distinguished the strains of SG I from SG II were observed at 21 positions. The viruses with SG non‐(I + II) shared sequence identity with SG II as high as 97.2–99.7%, suggesting that they belonged to genogroup II. Similarly, porcine rotaviruses in the same SG shared 96.4–99.7% for SG I, 98.2–100% for SG II, 97.4–100% for SG (I + II), and 96.2–99.7% for SG non‐(I + II), while strains in different SGs shared sequence identity ranging from 91.9% to 94.4%. Interestingly, the strains with SG (I + II) and SG non‐(I + II) shared a high degree of sequence identity with SG I, at 96.4–100% and 94.7–99.7% respectively, suggesting that they are related to porcine SG I strains. The conserved amino acids which distinguished SG I from SG II were observed at 13 positions. The strains with SG I, SG (I + II), and SG non‐(I + II) showed identical amino acid residues at these positions. Phylogenetic analysis strongly supported the findings of the sequence analysis. J. Med. Virol. 81:183–191, 2009. © 2008 Wiley‐Liss, Inc.     

Antigenic and molecular analyses of different Chlamydia pneumoniae strains.

Chlamydia pneumoniae is an important human respiratory pathogen. Classification of C. pneumoniae isolates into distinguishable serovars or genotypes has not yet been reported. To determine whether antigenic or molecular variants among C. pneumoniae isolates exist, six strains were studied via immunoblot analysis and DNA sequence determination of the entire major outer membrane protein (MOMP) gene omp1. The strains included four prototype strains and two clinical isolates from our laboratory. Immunoblot analysis of sera from patients infected with C. pneumoniae revealed antigenic differences between the C. pneumoniae strains. Strong reactivity of one serum sample with a 65-kDa protein in two C. pneumoniae strains which was not observed with the other strains was the most prominent finding. All sera reacted with the 40-kDa MOMP. Comparison of the omp1 DNA sequences revealed that the omp1 genes of all strains were identical and were 100% identical to the sequence of the omp1 gene of C. pneumoniae AR-39. The results of this study demonstrate that unlike C. trachomatis, the omp1 gene is conserved in C. pneumoniae. Furthermore, it was shown that C. pneumoniae strains are antigenically different. This finding indicates that more than one serovar of C. pneumoniae exist.     

Detection of a human rotavirus with G12 and P[9] specificity in Thailand

G12 rotavirus has not been detected anywhere in the world since the first detection of a human strain, L26 (G12, P1B[4]), in the Philippines in 1990. In this study, we isolated a human rotavirus (strain T152) with a VP7 of G12 specificity from the stool of an 11-month-old diarrheic patient in Thailand. The strain T152 exhibited a long RNA pattern and subgroup I specificity. In the comparison of the nucleotide and amino acid sequences of the VP7 gene of strain T152 with those of rotaviruses with different G type specificities, strain T152 showed the highest identity, 90.9 and 93.9%, respectively, to G12 prototype strain L26. In contrast, the VP4 gene of strain T152 showed the highest identity with P[9] specificity of human strains K8 and AU-1 and feline strains Cat2 and FRV-1, with homologies of 89.3 to 90.6% at the nucleotide level and 93.9 to 95.6% at the amino acid level. Thus, strain T152 was found to be a natural reassortant strain with G12 and P[9] specificities.     

Interspecies hybrid antibody with dual specificity

The preparation of soluble multivalent hybrid antibody by protein A of Staphylococcus aureus (SpA) is limited exclusively to rabbit IgG because other species have SpA-precipitating IgGs. Starting from a soluble complex consisting of one rabbit IgG antibody molecule linked to one SpA molecule (rabbit IgG anti-A/SpA), an interspecies hybrid antibody with dual specificity was prepared using either mouse or human IgG antibody, the molecular formula of this complex being (rabbit IgG anti-A/SpA/mouse or human IgG anti-B)₂. These complexes are useful tools for the investigation of cell surface antigens against which no appropriate antibody can be raised in rabbits.