Seroepidemiological study of human metapneumovirus in New Delhi, India.

PURPOSE: There are a few seroepidemiological studies reported on human metapneumovirus (hMPV) as hMPV was only discovered in the year 2001. This respiratory virus has been reported to be ubiquitous and associated with respiratory tract infections in all age groups. The present study aimed at determining the prevalence of antibodies to hMPV in children and adults of 1 month to 55 years of age. MATERIALS AND METHODS: Serum samples from 100 study subjects were tested for hMPV antibody by an in-house ELISA system that used hMPV-infected cell lysate antigen. RESULT: The prevalence of antibody to hMPV was lowest in children less than 5 years of age (60%) and increased throughout age to > 80%. Similarly, geometric mean titres were 1:180 in children less than 5 years of age and reached a peak of 1:419 in adults over 35 years of age. CONCLUSION: The results show that hMPV infection is acquired early in life and re-infection in later life may maintain the seroprevalence and antibody levels in adult population.

Circulation patterns of group A and B human respiratory syncytial virus genotypes in 5 communities in North America.

Human respiratory syncytial virus (HRSV) is a major cause of serious lower respiratory tract illness in infants, young children, and the elderly. To characterize the circulation patterns of HRSV strains, nucleotide sequencing of the C-terminal region of the G protein gene was performed on 34-53 isolates obtained from 5 communities during 1 epidemic year, representing distinct geographical locations in North America. Phylogenetic analysis revealed that 5-7 HRSV genotypes, including 1 or 2 predominant strains, circulated in each community. The patterns of genotypes were distinct between communities, and less diversity was seen between strains of the same genotype within than between communities. These findings are consistent with HRSV outbreaks' being community based in nature, although transmission of viruses between communities may occur. Several strains are probably introduced or circulate endemically in communities each year, and local factors-possibly immunity induced by previous years' strains-determine which strains predominate during an HRSV season.

Respiratory syncytial virus genetic and antigenic diversity.

Respiratory syncytial virus (RSV) is a major cause of viral lower respiratory tract infections among infants and young children in both developing and developed countries. There are two major antigenic groups of RSV, A and B, and additional antigenic variability occurs within the groups. The most extensive antigenic and genetic diversity is found in the attachment glycoprotein, G. During individual epidemic periods, viruses of both antigenic groups may cocirculate or viruses of one group may predominate. When there are consecutive annual epidemics in which the same group predominates, the dominant viruses are genetically different from year to year. The antigenic differences that occur among these viruses may contribute to the ability of RSV to establish reinfections throughout life. The differences between the two groups have led to vaccine development strategies that should provide protection against both antigenic groups. The ability to discern intergroup and intragroup differences has increased the power of epidemiologic investigations of RSV. Future studies should expand our understanding of the molecular evolution of RSV and continue to contribute to the process of vaccine development.

Mutations of respiratory syncytial virus attachment glycoprotein G associated with resistance to neutralization by primate polyclonal antibodies.

The respiratory syncytial (RS) virus attachment glycoprotein G is a type II transmembrane glycoprotein and an important target of the host immune response. Antigenic variability of the G protein is postulated to contribute to the ability of the virus to evade established immune responses. A glycoprotein G monospecific polyclonal antiserum from a nonhuman primate was used to select for an antibody resistant RS virus. The mutant virus was resistant to neutralization by the selecting antiserum and by the sera from three other G-protein immunized primates. G-protein amino acid changes were found at residues 61 (Phe to Leu), 174 (Ser to Cys), and 183 (Trp to Leu). Thus the mutant protein had amino acid changes in the transmembrane domain (61) and the central ectodomain (174 and 183). The change at amino acid 174 resulted in five rather than the usual four Cys found in the conserved central region of the ectodomain. These data demonstrate that an RS virus with resistance to neutralization by polyclonal antibodies can be selected readily in cell culture. In addition, only a limited number of amino acid changes is required to produce the resistant phenotype.

Respiratory syncytial virus G and/or SH protein alters Th1 cytokines, natural killer cells, and neutrophils responding to pulmonary infection in BALB/c mice.

BALB/c mice sensitized to vaccinia virus expressed G protein of respiratory syncytial virus (RSV) develop a Th2-type cytokine response and pulmonary eosinophilia when challenged with live RSV. In this study, BALB/c mice were immunized or challenged with an RSV mutant lacking the G and SH proteins or with DNA vaccines coding for RSV G or F protein. F or G protein DNA vaccines were capable of sensitizing for pulmonary eosinophilia. The absence of the G and/or SH protein in the infecting virus resulted in a consistent increase both in pulmonary natural killer cells and in gamma interferon and tumor necrosis factor expression, as well as, with primary infection, a variable increase in neutrophils and CD11b(+) cells. The development of pulmonary eosinophilia in formalin-inactivated RSV-vaccinated mice required the presence of the G and/or SH protein in the challenge virus. These data show that G and/or SH protein has a marked impact on the inflammatory and innate immune response to RSV infection.

Genetic variability among group A and group B respiratory syncytial viruses in a children's hospital.

Respiratory syncytial (RS) viruses isolated over three epidemic periods in a children's hospital in the United States were analyzed. The viruses (n = 174) were characterized as to major antigenic group (group A or B) by a PCR-based assay. Group A RS viruses were dominant the first 2 years, followed by a year with group B dominance (ratios of group A to group B viruses for epidemic periods, 56/4 for 1993-1994, 42/3 for 1994-1995, and 19/50 for 1995-1996). Genetic variability within the groups was assessed by restriction fragment analysis of PCR products; 79 isolates were also analyzed by nucleotide sequence determination of a variable region of the glycoprotein G gene. Among the group A RS virus isolates, this G-protein variable region had amino acid differences of as great as 38%. The G-protein amino acids of the group A viruses differed by up to 31% from the G-protein amino acids of a prototype (A2) group A virus. Among the group B RS virus G proteins, amino acid differences were as great as 14%. The G-protein amino acids of the group B viruses differed by up to 27% from the G-protein amino acids of a prototype (18537) group B virus. The group A and group B RS viruses demonstrated genetic variability between years and within individual years. Phylogenetic analysis revealed that there were multiple evolutionary lineages among both the group A and group B viruses. Among the recent group B isolates, variability was less than that seen for the group A viruses. However, comparisons to prototype strains revealed that the group B RS viruses may vary more extensively than was observed over the 3 years studied in the present investigation.

Antigenic and genetic diversity among the attachment proteins of group A respiratory syncytial viruses that have caused repeat infections in children.

Antigenic differences between the two major groups of respiratory syncytial (RS) virus may contribute to reinfections with these viruses. Additional variability occurs within the two major groups; the importance of intra-group variability in reinfections with RS virus has not been defined. Two pairs of group A viruses that had caused sequential infections in children showed G protein amino acid differences of up to 15%. Vaccinia viruses were constructed that expressed the G proteins from 2 of the paired group A isolates. Immunization of cotton rats with the recombinant vaccinia viruses provided equal protection against intranasal challenge by either of the RS viruses. Despite the amino acid differences between the two group A RS virus G proteins, these animal studies did not reveal differences in protection after immunization with the two G proteins. Precise definition of the role of RS virus antigenic variability in the establishment of reinfections in humans will require further investigations in humans.

Monoclonal antibody neutralization escape mutants of respiratory syncytial virus with unique alterations in the attachment (G) protein.

Five monoclonal antibody (MAb) neutralization escape mutants of respiratory syncytial virus (RSV) were produced by growing the Long strain RSV (group A virus) in the presence of a neutralizing, group cross-reactive MAb specific for the attachment protein (G). Four viruses (RSV-2, -6, -14 and -15) had amino acid replacements clustered within a highly conserved centrally located 13 amino acid region (position 164-176). Reactivity with group A-specific MAbs and with polyclonal anti-G serum was maintained and growth kinetics were unaffected. An additional virus (RSV-3) had four amino acid substitutions in the cytoplasmic tail and transmembrane region of G, and had restricted growth and formed small syncytia. Immunofluorescent and Western blot analysis indicated that G protein was not membrane associated and had reduced incorporation into the virion, thereby escaping neutralization by L9 and polyclonal anti-G serum. The predominant form of G produced by RSV-3 was found in infected cell supernatants, consistent with the size of secreted G.

The guinea pig as a model for the study of maternal immunization against respiratory syncytial virus infections in infancy.

Maternal immunization might protect infants from severe disease due to respiratory syncytial virus (RSV). Guinea pigs are susceptible to infections with RSV and transfer antibodies to their offspring prenatally. Pregnant guinea pigs were immunized by infection with RSV and their offspring were challenged intranasally with RSV. Pulmonary viral replication was compared among the pups born to immunized mothers (group A) and the pups from nonimmune mothers (group B) in two studies. Mean (+/-SD) log10 virus titers were, in study 1, group A, 2.3 +/- 0.8 pfu/g of lung (n = 10); group B, 3.6 +/- 1.5 pfu/g (n = 13) (P = .0058); and study 2, group A, < 1.69 pfu/g (n = 8); group B, 3.4 +/- 0.9 pfu/g (n = 6) (P = .0002). Thus, immunization of pregnant guinea pigs resulted in a significant reduction in viral replication in the lungs of their offspring. Guinea pigs should be useful for the study of maternal immunization against RSV.

Detection of antibodies to respiratory syncytial virus attachment and nucleocapsid proteins with recombinant baculovirus-expressed antigens.

The ability to measure antibodies against individual respiratory syncytial virus (RSV) proteins is important in the analysis of immune responses to RSV. We expressed the nucleocapsid (N) protein and the group A and B RSV attachment (G) proteins from recombinant baculoviruses. The three recombinant RSV proteins were used individually in an enzyme-linked immunosorbent assay (ELISA; bac-ELISA for results from assays of all three proteins). The bac-ELISA results were compared to the results obtained by a whole-virus ELISA (RS-ELISA for results from assays of both group A and B viruses). Antibody samples from 113 children were tested. The determination of seronegative or seropositive status by the bac-ELISA was compared to the same determination by the RS-ELISA; the sensitivity of bac-ELISA was 87% (95% confidence interval [CI], 78 to 93%), the specificity was 82% (CI, 59 to 94%), and the positive and negative predictive values were 95% (CI, 86 to 98%) and 60% (CI, 41 to 77%), respectively. The group specificity of the G-protein ELISA was confirmed by testing antibodies from experimentally immunized animals. Thus, the bac-ELISA was shown to be comparable to the whole-virus ELISA in detecting antibody responses to RSV, while it offered the advantage of measuring specific antibody responses to individual RSV proteins.

Antigenic and immunogenic analysis of group A and group B respiratory syncytial virus G proteins expressed from recombinant baculoviruses.

The attachment glycoprotein G plays a major role in the antigenic variability of respiratory syncytial (RS) virus. We have expressed from recombinant baculoviruses antigenic group A and group B RS virus G proteins (designated bacAG for the group A and bacBG for the group B virus G protein). The insect cell-produced G proteins migrated more rapidly in SDS-PAGE as compared to HEp-2 cell derived G proteins owing to glycosylation differences. Antigenicity was tested by immunofluorescence; five or five group cross-reactive, five or six group A-specific, and six of six group B-specific MAbs reacted appropriately with bacAG and/or bacBG. In addition, bacAG and bacBG reacted with human polyclonal antibodies to RS virus. Cotton rats were immunized with bacAG, bacBG or a control lysate and challenged intranasally with a group A RS virus. The bacAG-immunized group had a statistically significant reduction in viral replication in the lungs (lung titres as mean log10 p.f.u./g +/- SD, bacAG = 3.1 +/- 1.2; control = 4.8 +/- 0.6, P = 0.013). The bacBG-immunized group showed less reduction in viral titres (bacBG lung titres = 4.1 +/- 0.6, P = 0.13 for bacBG compared to control). Thus, as expected, homologous protein (bacAG) immunization provided more protection against viral replication than immunization with the heterologous protein (bacBG). The G protein of RS virus expressed in insect cells had antigenic and immunogenic features which were similar to that of the G protein expressed in mammalian cells. The baculovirus-expressed G proteins should be useful for the study of immune responses to RS viruses.

Antigenic analysis of chimeric and truncated G proteins of respiratory syncytial virus.

Extensive antigenic variation occurs on the attachment glycoprotein G between the two major respiratory syncytial (RS) virus groups. Restriction fragment exchanges were performed among regions of the group A and group B G protein cDNAs to produce chimeric molecules to use in a study of the antigenic structure of the G protein. Proteins were expressed from these chimeric cDNAs using the vaccinia virus T7 bacteriophage polymerase system. Region 1 [A1 or B1, G protein amino acids (aa) 1 to 173] included the cytoplasmic and transmembrane domains and part of the ectodomain, region 2 (A2, aa 174 to 214 in the group A; or B2, aa 174 to 215 in the group B G protein) included the central portion of the ectodomain, and region 3 (A3, aa 215 to 298 in the group A; or B3, aa 216 to 292 in the group B G protein) included the C-terminal region of the ectodomain. The chimeric proteins comprised regions A1B2B3, B1A2A3, A1A2B3, and B1B2A3. Truncated proteins were also produced and consisted of regions A1A2, B1B2, A1, and B1. The expressed proteins were tested by immunofluorescence for reactivity with group-cross-reactive or group-specific G protein monoclonal antibodies (MAbs). Most group-cross-reactive MAbs required the presence of G protein regions 1 or 2 for reactivity. G protein MAbs specific for either group A or group B were dependent on the presence of regions 1, 2, or 3 for reactivity. Thus, each of the three regions of the G protein were found to play a role in reactivity with MAbs, including for each region MAbs with neutralizing activity against RS virus.

Analysis of respiratory syncytial virus genetic variability with amplified cDNAs.

Antigenic and genetic heterogeneities exist within the two major antigenic groups of respiratory syncytial (RS) virus. We developed a polymerase chain reaction (PCR)-based assay that not only differentiates the two RS virus groups but allows distinctions within groups on the basis of changes in the nucleotide sequences, as revealed by restriction fragment analysis. In this assay, viral RNA served as a template for cDNA synthesis with extension from a synthetic oligonucleotide primer complementary to bases 164 to 186 in the F protein mRNA. For PCR amplification, two group-specific 5' primers were added. The two primers corresponded to the G protein mRNA sequence of group B (bases 10 to 30) or group A (bases 247 to 267) RS virus. Agarose gel electrophoresis readily discriminated the 1.1-kb group B and the 0.9-kb group A virus amplification products. All 47 viruses tested were assigned to the same group by both PCR and monoclonal antibody reaction pattern analysis. Restriction fragment analysis of the amplified DNAs revealed 12 restriction patterns for group A viruses and 7 restriction patterns for group B viruses, while the monoclonal antibody reaction patterns revealed seven patterns for group A viruses and 3 patterns for group B viruses. Most viruses with the same monoclonal antibody reaction patterns had different restriction patterns, and some viruses with the same restriction patterns had different monoclonal antibody reaction patterns. Thus, the results of the PCR assay concurred with the monoclonal antibody reaction pattern analysis for group classification of RS viruses, while the restriction fragment analysis identified greater diversity within groups than was seen with the monoclonal antibody analysis.

Characterization of two antigenic sites recognized by neutralizing monoclonal antibodies directed against the fusion glycoprotein of human respiratory syncytial virus.

Two antigenic sites recognized by neutralizing monoclonal antibodies (MAbs) directed against the fusion (F) glycoprotein of human respiratory syncytial virus were mapped on the primary structure of the protein by (i) the identification of amino acid substitutions selected in antibody-escape mutants and (ii) the reactivity of synthetic peptides with MAbs. The first site contained several overlapping epitopes which were located within the trypsin-resistant amino-terminal third of the large F1 subunit. Only one of these epitopes was faithfully reproduced by a short synthetic peptide; the others might require specific local conformations to react with MAbs. The second antigenic site was located in a trypsin-sensitive domain of the F1 subunit towards the carboxy-terminal end of the cysteine-rich region. One of these epitopes was reproduced by synthetic peptides. In addition, mutagenized F protein with a substitution of serine for arginine at position 429 did not bind MAbs to the second site. These results are discussed in terms of F protein structure and the mechanisms of virus neutralization.

Genetic diversity of the attachment protein of subgroup B respiratory syncytial viruses.

Respiratory syncytial (RS) virus causes repeated infections throughout life. Between the two main antigenic subgroups of RS virus, there is antigenic variation in the attachment protein G. The antigenic differences between the subgroups appear to play a role in allowing repeated infections to occur. Antigenic differences also occur within subgroups; however, neither the extent of these differences nor their contributions to repeat infections are known. We report a molecular analysis of the extent of diversity within the subgroup B RS virus attachment protein genes of viruses isolated from children over a 30-year period. Amino acid sequence differences as high as 12% were observed in the ectodomains of the G proteins among the isolates, whereas the cytoplasmic and transmembrane domains were highly conserved. The changes in the G-protein ectodomain were localized to two areas on either side of a highly conserved region surrounding four cysteine residues. Strikingly, single-amino-acid coding changes generated by substitution mutations were not the only means by which change occurred. Changes also occurred by (i) substitutions that changed the available termination codons, resulting in proteins of various lengths, and (ii) a mutation introduced by a single nucleotide deletion and subsequent nucleotide insertion, which caused a shift in the open reading frame of the protein in comparison to the other G genes analyzed. Fifty-one percent of the G-gene nucleotide changes observed among the isolates resulted in amino acid coding changes in the G protein, indicating a selective pressure for change. Maximum-parsimony analysis demonstrated that distinct evolutionary lineages existed. These data show that sequence diversity exists among the G proteins within the subgroup B RS viruses, and this diversity may be important in the immunobiology of the RS viruses.

Synthetic oligonucleotide probes differentiate respiratory syncytial virus subgroups in a nucleic acid hybridization assay.

A new approach to respiratory syncytial virus subgroup differentiation has been developed on the basis of the hybridization of subgroup-specific synthetic oligonucleotides with viral mRNA. Two oligonucleotide probes were designed from the nucleotide sequences of an A and a B subgroup respiratory syncytial virus glycoprotein G gene. All 28 virus isolates tested were correctly classified by subgroup with these probes.

The respiratory syncytial virus subgroup B attachment glycoprotein: analysis of sequence, expression from a recombinant vector, and evaluation as an immunogen against homologous and heterologous subgroup virus challenge.

The attachment glycoprotein G of respiratory syncytial (RS) virus is important in both the antigenic and molecular diversity of the RS viruses. Previous work has shown that the glycoprotein G of a subgroup A RS virus expressed from a recombinant vaccinia virus provides significant protection against homologous but not heterologous subgroup virus challenge. We undertook the cDNA cloning and nucleotide sequencing of the G mRNA of a subgroup B RS virus (8/60) to extend molecular comparisons of the G protein both within and between subgroups. We also tested the ability of a subgroup B G protein to provide protection against challenge by A or B subgroup viruses. Sequence analysis showed a deduced amino acid sequence having a single major open reading frame encoding a protein of 292 amino acids with an elevated serine and threonine (30%) and proline (9%) content. The 8/60 G differed from a subgroup A virus (A2) G protein with only a 56% amino acid identity while the 8/60 G shared a 98% amino acid identity with the G protein of another subgroup B virus (18537). The 8/60 G cDNA was placed in a vaccinia virus vector (vvGB) which was shown to express the 8/60 G protein. Cotton rats immunized intradermally with vvGB and later challenged intranasally with 8/60 RS virus had a significant reduction in viral titers in the lungs relative to control animals whereas similarly immunized animals were not protected against heterologous subgroup challenge. Our results indicate that a RS virus subunit vaccine containing the G protein would require both A and B subgroup G proteins to afford protection against viruses of both subgroups.