Evaluation of protective efficacy of respiratory syncytial virus vaccine against A and B subgroup human isolates in Korea

Human respiratory syncytial virus (HRSV) is a significant cause of upper and lower respiratory tract illness mainly in infants and young children worldwide. HRSV is divided into two subgroups, HRSV-A and HRSV-B, based on sequence variation within the G gene. Despite its importance as a respiratory pathogen, there is currently no safe and effective vaccine for HRSV. In this study, we have detected and identified the HRSV by RT-PCR from nasopharyngeal aspirates of Korean pediatric patients. Interestingly, all HRSV-B isolates exhibited unique deletion of 6 nucleotides and duplication of 60 nucleotides in the G gene. We successfully amplified two isolates ('KR/A/09-8' belonging to HRSV-A and 'KR/B/10-12' to HRSV-B) on large-scale, and evaluated the cross-protective efficacy of our recombinant adenovirus-based HRSV vaccine candidate, rAd/3xG, by challenging the immunized mice with these isolates. The single intranasal immunization with rAd/3xG protected the mice completely from KR/A/09-8 infection and partially from KR/B/10-12 infection. Our study contributes to the understanding of the genetic characteristics and distribution of subgroups in the seasonal HRSV epidemics in Korea and, for the first time, to the evaluation of the cross-protective efficacy of RSV vaccine against HRSV-A and -B field-isolates.     

Circulating strains of human respiratory syncytial virus in central and south America

Human respiratory syncytial virus (HRSV) is a major cause of viral lower respiratory tract infections among infants and young children. HRSV strains vary genetically and antigenically and have been classified into two broad subgroups, A and B (HRSV-A and HRSV-B, respectively). To date, little is known about the circulating strains of HRSV in Latin America. We have evaluated the genetic diversity of 96 HRSV strains by sequencing a variable region of the G protein gene of isolates collected from 2007 to 2009 in Central and South America. Our results show the presence of the two antigenic subgroups of HRSV during this period with the majority belonging to the genotype HRSV-A2.     

Molecular Characterization of Human Respiratory Syncytial Virus in the Philippines, 2012-2013

Human respiratory syncytial virus (HRSV) is a major cause of acute lower respiratory tract infections in infants and children worldwide. We performed molecular analysis of HRSV among infants and children with clinical diagnosis of severe pneumonia in four study sites in the Philippines, including Biliran, Leyte, Palawan, and Metro Manila from June 2012 to July 2013. Nasopharyngeal swabs were collected and screened for HRSV using real-time polymerase chain reaction (PCR). Positive samples were tested by conventional PCR and sequenced for the second hypervariable region (2^nd HVR) of the G gene. Among a total of 1,505 samples, 423 samples were positive for HRSV (28.1%), of which 305 (72.1%) and 118 (27.9%) were identified as HRSV-A and HRSV-B, respectively. Two genotypes of HRSV-A, NA1 and ON1, were identified during the study period. The novel ON1 genotype with a 72-nucleotide duplication in 2^nd HVR of the G gene increased rapidly and finally became the predominant genotype in 2013 with an evolutionary rate higher than the NA1 genotype. Moreover, in the ON1 genotype, we found positive selection at amino acid position 274 (p<0.05) and massive O- and N-glycosylation in the 2^nd HVR of the G gene. Among HRSV-B, BA9 was the predominant genotype circulating in the Philippines. However, two sporadic cases of GB2 genotype were found, which might share a common ancestor with other Asian strains. These findings suggest that HRSV is an important cause of severe acute respiratory infection among children in the Philippines and revealed the emergence and subsequent predominance of the ON1 genotype and the sporadic detection of the GB2 genotype. Both genotypes were detected for the first time in the Philippines.     

Genetic variability and molecular evolution of the human respiratory syncytial virus subgroup B attachment G protein

Human respiratory syncytial virus (HRSV) is the most important cause of acute respiratory disease in infants. Two major subgroups (A and B) have been identified based on antigenic differences in the attachment G protein. Antigenic variation between and within the subgroups may contribute to reinfections with these viruses by evading the host immune responses. To investigate the circulation patterns and mechanisms by which HRSV-B viruses evolve, we analyzed the G protein genetic variability of subgroup B sequences isolated over a 45-year period, including 196 Belgian strains obtained over 22 epidemic seasons (1982 to 2004). Our study revealed that the HRSV-B evolutionary rate (1.95 x 10(-3) nucleotide substitutions/site/year) is similar to that previously estimated for HRSV-A (1.83 x 10(-3) nucleotide substitutions/site/year). However, natural HRSV-B isolates appear to accommodate more drastic changes in their attachment G proteins. The most recent common ancestor of the currently circulating subgroup B strains was estimated to date back to around the year 1949. The divergence between the two major subgroups was calculated to have occurred approximately 350 years ago. Furthermore, we have identified 12 positively selected sites in the G protein ectodomain, suggesting that immune-driven selective pressure operates in certain codon positions. HRSV-A and -B strains have similar phylodynamic patterns: both subgroups are characterized by global spatiotemporal strain dynamics, where the high infectiousness of HRSV permits the rapid geographic spread of novel strain variants.     

Chimeric bovine respiratory syncytial virus with glycoprotein gene substitutions from human respiratory syncytial virus (HRSV): effects on host range and evaluation as a live-attenuated HRSV vaccine

We recently developed a system for the generation of infectious bovine respiratory syncytial virus (BRSV) from cDNA. Here, we report the recovery of fully viable chimeric recombinant BRSVs (rBRSVs) that carry human respiratory syncytial virus (HRSV) glycoproteins in place of their BRSV counterparts, thus combining the replication machinery of BRSV with the major antigenic determinants of HRSV. A cDNA encoding the BRSV antigenome was modified so that the complete G and F genes, including the gene start and gene end signals, were replaced by their HRSV A2 counterparts. Alternatively, the BRSV F gene alone was replaced by that of HRSV Long. Each antigenomic cDNA directed the successful recovery of recombinant virus, yielding rBRSV/A2 and rBRSV/LongF, respectively. The HRSV G and F proteins or the HRSV F in combination with BRSV G were expressed efficiently in cells infected with the appropriate chimeric virus and were efficiently incorporated into recombinant virions. Whereas BRSV and HRSV grew more efficiently in bovine and human cells, respectively, the chimeric rBRSV/A2 exhibited intermediate growth characteristics in a human cell line and grew better than either parent in a bovine line. The cytopathology induced by the chimera more closely resembled that of BRSV. BRSV was confirmed to be highly restricted for replication in the respiratory tract of chimpanzees, a host that is highly permissive for HRSV. Interestingly, the rBRSV/A2 chimeric virus was somewhat more competent than BRSV for replication in chimpanzees but remained highly restricted compared to HRSV. This showed that the substitution of the G and F glycoproteins alone was not sufficient to induce efficient replication in chimpanzees. Thus, the F and G proteins contribute to the host range restriction of BRSV but are not the major determinants of this phenotype. Although rBRSV/A2 expresses the major neutralization and protective antigens of HRSV, chimpanzees infected with this chimeric virus were not significantly protected against subsequent challenge with wild-type HRSV. This suggests that the growth restriction of rBRSV/A2 was too great to provide adequate antigen expression and that the capacity of this chimeric vaccine candidate for replication in primates will need to be increased by the importation of additional HRSV genes.     

Phylodynamics and Dispersal of HRSV Entails Its Permanence in the General Population in between Yearly Outbreaks in Children

Background

Human respiratory syncytial virus (HRSV) is one of the major etiologic agents of respiratory tract infections among children worldwide.

Methodology/Principal Findings

Here through a comprehensive analysis of the two major HRSV groups A and B (n = 1983) which comprise of several genotypes, we present a complex pattern of population dynamics of HRSV over a time period of 50 years (1956–2006). Circulation pattern of HRSV revealed a series of expansions and fluctuations of co-circulating lineages with a predominance of HRSVA. Positively selected amino acid substitutions of the G glycoprotein occurred upon population growth of GB3 with a 60-nucleotide insertion (GB3 Insert), while other genotypes acquired substitutions upon both population growth and decrease, thus possibly reflecting a role for immune selected epitopes in linkage to the traced substitution sites that may have important relevance for vaccine design. Analysis evidenced the co-circulation and predominance of distinct HRSV genotypes in Brazil and suggested a year-round presence of the virus. In Brazil, GA2 and GA5 were the main culprits of HRSV outbreaks until recently, when the GB3 Insert became highly prevalent. Using Bayesian methods, we determined the dispersal patterns of genotypes through several inferred migratory routes.

Conclusions/Significance

Genotypes spread across continents and between neighboring areas. Crucially, genotypes also remained at any given region for extended periods, independent of seasonal outbreaks possibly maintained by re-infecting the general population.     

一株引起严重急性呼吸道感染病例的A亚型人呼吸道合胞病毒全基因组基因特征分析

人呼吸道合胞病毒(Human respiratory syncytial virus,HRSV)是导致儿童急性呼吸道感染的最重要的呼吸道病毒之一。根据对单克隆抗体的反应,HRSV分为A、B两个亚型。为探讨严重急性呼吸道感染(Severe acute respi-ratory infection,SARI)病例中HRSV全基因组基因特征,本研究对2017年河南省漯河市住院SARI病例中检测到的1株HRSV A亚型病毒通过Sanger测序方法对其全基因组序列进行了测定和分析。通过Sequencher 5.4.5、MEGA 5.05、BioEdit 7.0.5等生物信息学软件进行序列拼接和比对,进行了基因亲缘性关系分析、氨基酸变异和糖基化位点分析。基于HRSV全基因组序列和11个单个蛋白基因序列构建的亲缘性关系分析结果提示本研究中检测到的这株HRSVA病毒(RSVAs/Luohe.Henan/CHN/42.17)属于ON1基因型,该型是我国近年流行的优势基因型。该病毒全基因组序列与35条全球代表株的核苷酸和氨基酸同源性分别为92.69%～99.82%和93.63%～99.67%;G蛋白编码区氨基酸变异最高,而F蛋白相对保守。糖基化位点分析发现,该病毒的F蛋白有6个N-糖基化位点,未发现O-糖基化位点,此结果与原型株long株相同;G蛋白N-糖基化位点有6个,O-糖基化位点为82个,而原型株long株有11个N-糖基化位点,15个O-糖基化位点。本研究对2017年河南省漯河市SARI病例中一株HRSVA病毒全基因组序列进行了测定,与世界其他地区报道的HRSVA亚型病毒全基因组序列进行了对比分析,揭示了SARI病例中我国HRSV优势流行ON1基因型病毒全基因组的核苷酸和氨基酸变异特征,以及G蛋白和F蛋白编码区糖基化情况,丰富了我国HRSV基因数据库,也为HRSV的核酸检测方法的建立、疫苗研发和预防性单克隆抗体的评价提供了核苷酸和氨基酸的基础数据。     

Respiratory syncytial virus (RSV) fusion protein subunit F2, not attachment protein G,determines the specificity of RSV infection

Human respiratory syncytial virus (HRSV) and bovine RSV (BRSV) infect human beings and cattle in a species-specific manner. We have here analyzed the contribution of RSV envelope proteins to species-specific entry into cells. In contrast to permanent cell lines, primary cells of human or bovine origin, including differentiated respiratory epithelia, peripheral blood lymphocytes, and macrophages, showed a pronounced species-specific permissiveness for HRSV and BRSV infection, respectively. Recombinant BRSV deletion mutants lacking either the small hydrophobic (SH) protein gene or both SH and the attachment glycoprotein (G) gene retained their specificity for bovine cells, whereas corresponding mutants carrying the HRSV F gene specifically infected human cells. To further narrow the responsible region of F, two reciprocal chimeric F constructs were assembled from BRSV and HRSV F1 and F2 subunits. The specificity of recombinant RSV carrying only the chimeric F proteins strictly correlated with the origin of the membrane-distal F2 domain. A contribution of G to the specificity of entry could be excluded after reintroduction of BRSV or HRSV G. Virus with F1 and G from BRSV and with only F2 from HRSV specifically infected human cells, whereas virus expressing F1 and G from HRSV and F2 from BRSV specifically infected bovine cells. The introduction of G enhanced the infectiousness of both chimeric viruses to equal degrees. Thus, the role of the nominal attachment protein G is confined to facilitating infection in a non-species-specific manner, most probably by binding to cell surface glycosaminoglycans. The identification of the F2 subunit as the determinant of RSV host cell specificity facilitates identification of virus receptors and should allow for development of reagents specifically interfering with RSV entry.     

Bovine respiratory syncytial virus protects cotton rats against human respiratory syncytial virus infection.

Human respiratory syncytial virus (HRSV) is the most frequent cause of severe respiratory infections in infancy. No vaccine against this virus has yet been protective, and antiviral drugs have been of limited utility. Using the cotton rat model of HRSV infection, we examined bovine respiratory syncytial virus (BRSV), a cause of acute respiratory disease in young cattle, as a possible vaccine candidate to protect children against HRSV infection. Cotton rats were primed intranasally with graded doses of BRSV/375 or HRSV/Long or were left unprimed. Three weeks later, they were challenged intranasally with either BRSV/375, HRSV/Long (subgroup A), or HRSV/18537 (subgroup B). At intervals postchallenge, animals were sacrificed for virus titration and histologic evaluation. Serum neutralizing antibody titers were determined at the time of viral challenge. BRSV/375 replicated to low titers in nasal tissues and lungs. Priming with 10(5) PFU of BRSV/375 effected a 500- to 1,000-fold reduction in peak nasal HRSV titer and a greater than 1,000-fold reduction in peak pulmonary HRSV titer upon challenge with HRSV/Long or HRSV/18537. In contrast to priming with HRSV, priming with BRSV did not induce substantial levels of neutralizing antibody against HRSV and was associated with a delayed onset of clearance of HRSV upon challenge. Priming with BRSV/375 caused mild nasal and pulmonary pathology and did not cause exacerbation of disease upon challenge with HRSV/Long. Our findings suggest that BRSV may be a potential vaccine against HRSV and a useful tool for studying the mechanisms of immunity to HRSV.     

Molecular epidemiology and evolution of human respiratory syncytial virus and human metapneumovirus

Human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) are ubiquitous respiratory pathogens of the Pneumovirinae subfamily of the Paramyxoviridae. Two major surface antigens are expressed by both viruses; the highly conserved fusion (F) protein, and the extremely diverse attachment (G) glycoprotein. Both viruses comprise two genetic groups, A and B. Circulation frequencies of the two genetic groups fluctuate for both viruses, giving rise to frequently observed switching of the predominantly circulating group. Nucleotide sequence data for the F and G gene regions of HRSV and HMPV variants from the UK, The Netherlands, Bangkok and data available from Genbank were used to identify clades of both viruses. Several contemporary circulating clades of HRSV and HMPV were identified by phylogenetic reconstructions. The molecular epidemiology and evolutionary dynamics of clades were modelled in parallel. Times of origin were determined and positively selected sites were identified. Sustained circulation of contemporary clades of both viruses for decades and their global dissemination demonstrated that switching of the predominant genetic group did not arise through the emergence of novel lineages each respiratory season, but through the fluctuating circulation frequencies of pre-existing lineages which undergo proliferative and eclipse phases. An abundance of sites were identified as positively selected within the G protein but not the F protein of both viruses. For HRSV, these were discordant with previously identified residues under selection, suggesting the virus can evade immune responses by generating diversity at multiple sites within linear epitopes. For both viruses, different sites were identified as positively selected between genetic groups.     

Molecular evolution and circulation patterns of human respiratory syncytial virus subgroup a: positively selected sites in the attachment g glycoprotein

Human respiratory syncytial virus (HRSV) is the most common etiological agent of acute lower respiratory tract disease in infants and can cause repeated infections throughout life. In this study, we have analyzed nucleotide sequences encompassing 629 bp at the carboxy terminus of the G glycoprotein gene for HRSV subgroup A strains isolated over 47 years, including 112 Belgian strains isolated over 19 consecutive years (1984 to 2002). By using a maximum likelihood method, we have tested the presence of diversifying selection and identified 13 positively selected sites with a posterior probability above 0.5. The sites under positive selection correspond to sites of O glycosylation or to amino acids that were previously described as monoclonal antibody-induced in vitro escape mutants. Our findings suggest that the evolution of subgroup A HRSV G glycoprotein is driven by immune pressure operating in certain codon positions located mainly in the second hypervariable region of the ectodomain. Phylogenetic analysis revealed the prolonged cocirculation of two subgroup A lineages among the Belgian population and the possible extinction of three other lineages. The evolutionary rate of HRSV subgroup A isolates was estimated to be 1.83 x 10(-3) nucleotide substitutions/site/year, projecting the most recent common ancestor back to the early 1940s.     

The envelope proteins from purified respiratory syncytial virus protect mice from intranasal virus challenge

A lyophilized subunit vaccine prepared from purified respiratory syncytial virus, which contained the envelope glycoproteins F and G and the nonglycosylated matrix protein VPM, was tested in SJL mice for its ability to protect the lungs of mice from intranasal viral challenge. Initially, the mice were injected subcutaneously with one, two, or three doses of 5 or 25 micrograms of vaccine in 50% complete Freund's adjuvant or with complete Freund's adjuvant or phosphate-buffered saline only. Although none of the mice produced neutralizing serum antibody, three doses of 25 micrograms elicited antibodies to F, G, and VPM. Despite the absence of detectable neutralizing antibodies, the lungs of 93% of the vaccinated mice were protected from intranasal viral challenge. Because the initial protocol did not elicit neutralizing antibodies and a few single-dose animals were not protected, a second vaccine trial was carried out. For these studies the priming dose was increased to 50 micrograms, which was followed, in half the vaccine recipients, by a second dose of 25 micrograms. Mice given the priming dose of vaccine produced antibody to G and showed no neutralizing activity, whereas the mice given two doses of vaccine produced antibodies to G, F, and VPM and also displayed neutralizing activity for respiratory syncytial virus. The lungs of 100% of the vaccine recipients in this trial were protected from intranasal challenge. Although the vaccine elicited antibody to VPM, this response did not correlate with protection. In addition, examination of the sera from unimmunized mice recovering from respiratory syncytial virus infection revealed a serum antibody profile similar to that noted for humans, lacking antibody to VPM. Thus, the data show that a combined glycoprotein subunit vaccine affords complete protection to viral challenge and offers an approach to develop a multivalent subunit vaccine.     

Variable Immune-Driven Natural Selection in the Attachment (G) Glycoprotein of Respiratory Syncytial Virus (RSV)

A maximum-likelihood analysis of selection pressures acting on the attachment (G) glycoprotein gene of respiratory syncytial virus (RSV) from humans (HRSV) and bovines (BRSV) is presented. Six positively selected sites were identified in both group A and group B of HRSV, although only one site was common between them, while no positively selected sites were detected in BRSV. All positively selected sites were located within the ectodomain of the G protein and showed some association with positions of immunoglobulin (Ig) epitopes and sites of O-glycosylation. These results suggest that immune (antibody)-driven natural selection is an important determinant of RSV evolution and that this selection pressure differs among strains. The passage histories of RSV strains were also shown to affect the distribution of positively selected sites, particularly in HRSV B, and should be considered whenever retrospective analysis of adaptive evolution is undertaken. Received: 15 August 2000 / Accepted: 2 November 2000     

Natural history of human respiratory syncytial virus inferred from phylogenetic analysis of the attachment (G) glycoprotein with a 60-nucleotide duplication

A total of 47 clinical samples were identified during an active surveillance program of respiratory infections in Buenos Aires (BA) (1999 to 2004) that contained sequences of human respiratory syncytial virus (HRSV) with a 60-nucleotide duplication in the attachment (G) protein gene. This duplication was analogous to that previously described for other three viruses also isolated in Buenos Aires in 1999 (A. Trento et al., J. Gen. Virol. 84:3115-3120, 2003). Phylogenetic analysis indicated that BA sequences with that duplication shared a common ancestor (dated about 1998) with other HRSV G sequences reported worldwide after 1999. The duplicated nucleotide sequence was an exact copy of the preceding 60 nucleotides in early viruses, but both copies of the duplicated segment accumulated nucleotide substitutions in more recent viruses at a rate apparently higher than in other regions of the G protein gene. The evolution of the viruses with the duplicated G segment apparently followed the overall evolutionary pattern previously described for HRSV, and this genotype has replaced other prevailing antigenic group B genotypes in Buenos Aires and other places. Thus, the duplicated segment represents a natural tag that can be used to track the dissemination and evolution of HRSV in an unprecedented setting. We have taken advantage of this situation to reexamine the molecular epidemiology of HRSV and to explore the natural history of this important human pathogen.     

Comparison of Mutation Patterns in Full-Genome A/H3N2 Influenza Sequences Obtained Directly from Clinical Samples and the Same Samples after a Single MDCK Passage

Human influenza viruses can be isolated efficiently from clinical samples using Madin-Darby canine kidney (MDCK) cells. However, this process is known to induce mutations in the virus as it adapts to this non-human cell-line. We performed a systematic study to record the pattern of MDCK-induced mutations observed across the whole influenza A/H3N2 genome. Seventy-seven clinical samples collected from 2009-2011 were included in the study. Two full influenza genomes were obtained for each sample: one from virus obtained directly from the clinical sample and one from the matching isolate cultured in MDCK cells. Comparison of the full-genome sequences obtained from each of these sources showed that 42% of the 77 isolates had acquired at least one MDCK-induced mutation. The presence or absence of these mutations was independent of viral load or sample origin (in-patients versus out-patients). Notably, all the five hemagglutinin missense mutations were observed at the hemaggutinin 1 domain only, particularly within or proximal to the receptor binding sites and antigenic site of the virus. Furthermore, 23% of the 77 isolates had undergone a MDCK-induced missense mutation, D151G/N, in the neuraminidase segment. This mutation has been found to be associated with reduced drug sensitivity towards the neuraminidase inhibitors and increased viral receptor binding efficiency to host cells. In contrast, none of the neuraminidase sequences obtained directly from the clinical samples contained the D151G/N mutation, suggesting that this mutation may be an indicator of MDCK culture-induced changes. These D151 mutations can confound the interpretation of the hemagglutination inhibition assay and neuraminidase inhibitor resistance results when these are based on MDCK isolates. Such isolates are currently in routine use in the WHO influenza vaccine and drug-resistance surveillance programs. Potential data interpretation miscalls can therefore be avoided by careful exclusion of such D151 mutants after further sequence analysis.     

The baculovirus GP64 protein mediates highly stable infectivity of a human respiratory syncytial virus lacking its homologous transmembrane glycoproteins

Baculovirus GP64 is a low-pH-dependent membrane fusion protein required for virus entry and cell-to-cell transmission. Recently, GP64 has generated interest for practical applications in mammalian systems. Here we examined the membrane fusion function of GP64 from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressed in mammalian cells, as well as its capacity to functionally complement a mammalian virus, human respiratory syncytial virus (HRSV). Both authentic GP64 and GP(64/F), a chimeric protein in which the GP64 cytoplasmic tail domain was replaced with the 12 C-terminal amino acids of the HRSV fusion (F) protein, induced low-pH-dependent cell-cell fusion when expressed transiently in HEp-2 (human) cells. Levels of surface expression and syncytium formation were substantially higher at 33 degrees C than at 37 degrees C. The open reading frames (ORFs) encoding GP64 or GP(64/F), along with two marker ORFs encoding green fluorescent protein (GFP) and beta-glucuronidase (GUS), were used to replace all three homologous transmembrane glycoprotein ORFs (small hydrophobic SH, attachment G, and F) in a cDNA of HRSV. Infectious viruses were recovered that lacked the HRSV SH, G, and F proteins and expressed instead the GP64 or GP(64/F) protein and the two marker proteins GFP and GUS. The properties of these viruses, designated RSDeltaSH,G,F/GP64 or RSDeltaSH,G,F/GP(64/F), respectively, were compared to a previously described HRSV expressing GFP in place of SH but still containing the wild-type HRSV G and F proteins (RSDeltaSH [A. G. Oomens, A. G. Megaw, and G. W. Wertz, J. Virol., 77:3785-3798, 2003]). By immunoelectron microscopy, the GP64 and GP(64/F) proteins were shown to incorporate into HRSV-induced filaments at the cell surface. Antibody neutralization, ammonium chloride inhibition, and replication levels in cell culture showed that both GP64 proteins efficiently mediated infectivity of the respective viruses in a temperature-sensitive, low-pH-dependent manner. Furthermore, RSDeltaSH,G,F/GP64 and RSDeltaSH,G,F/GP(64/F) replicated to higher levels and had significantly higher stability of infectivity than HRSVs containing the homologous HRSV G and F proteins. Thus, GP64 and a GP64/HRSV F chimeric protein were functional and efficiently complemented an unrelated human virus in mammalian cells, producing stable, infectious virus stocks. These results demonstrate the potential of GP64 for both practical applications requiring stable pseudotypes in mammalian systems and for studies of viral glycoprotein requirements in assembly and pathogenesis.     

Antiviral activity and structural characteristics of the nonglycosylated central subdomain of human respiratory syncytial virus attachment (G) glycoprotein

Segments of the cystine noose-containing nonglycosylated central subdomain, residues 149-197, of the attachment (G) glycoprotein of human respiratory syncytial virus (HRSV) have been assessed for impact on the cytopathic effect (CPE) of respiratory syncytial virus (RSV). Nalpha-acetyl residues 149-197-amide (G149-197), G149-189, and G149-177 of the A2 strain of HRSV protected 50% of human epithelial HEp-2 cells from the CPE of the A2 strain at concentrations (IC(50)) between 5 and 80 microm. Cystine noose-containing peptides G171-197 and G173-197 did not inhibit the CPE even at concentrations above 150 microm. Systematic C- and N-terminal truncations from G149-189 and G149-177 and alanine substitutions within G154-177 demonstrated that residues 166-170 (EVFNF), within a sequence that is conserved in HRSV strains, were critical for inhibition. Concordantly, G154-177 of bovine RSV and of an antibody escape mutant of HRSV with residues 166-170 of QTLPY and EVSNP, respectively, were not inhibitory. Surprisingly, a variant of G154-177 with an E166A substitution had an IC(50) of 750 nm. NMR analysis demonstrated that G149-177 adopted a well-defined conformation in solution, clustered around F168 and F170. G154-170, particularly EVFNF, may be important in binding of RSV to host cells. These findings constitute a promising platform for the development of antiviral agents for RSV.     

Cloning and characterization of variable-sized gypsy mobile elements in Drosophila melanogaster

A cosmid genomic library from a known gypsy-induced forked mutation, f1, was screened by 32P-labeled gypsy transposable element. Of more than 250 positive clones we randomly selected 21 for in situ hybridization to wild-type polytene chromosomes. Two clones hybridized to region 15F on the X-chromosome, the cytological position of forked. A third clone hybridized to at least 17 sites on the chromosomes indicating the presence of repetitive sequences in the gypsy flanking DNA. All clones labeled the centromeric regions heavily. Ten clones, including the two hybridizing at 15F, were chosen for further analysis, and restriction mapping allowed us to place them into three groups: (1) full-length, (2) slightly diverging, and (3) highly diverging gypsy elements. Group (2) is missing the XbaI site in both their long terminal repeats (LTRs) as well as the middle HindIII site; four of these gypsy elements also have a approximately 100-bp deletion at the 5' LTR. The group (3) gypsy transposons are missing one LTR and also have highly diverging DNA sequences. The restriction analyses further imply that most of these different gypsy elements are present in more than one copy in the genome of the f1 stock used in this study. The results raise intriguing questions regarding the significance of transposable elements in evolution and biological functions.