Wayne M. Sullender

Low Risk of Herpes Simplex Virus Infections in Neonates Exposed to the Virus at the Time of Vaginal Delivery to Mothers with Recurrent Genital Herpes Simplex Virus Infections

We studied the risk of herpes simplex virus (HSV) infections in neonates exposed to HSV at the time of vaginal delivery to mothers with a history of recurrent genital HSV infections. None of 34 infants exposed to HSV type 2 acquired an HSV infection. On the basis of this sample, the 95 percent confidence limit for the theoretical maximum infection rate is 8 percent. Cord blood or blood obtained during the first two weeks of life was available from 33 of the 34 exposed, uninfected neonates. All 33 of the samples possessed demonstrable neutralizing antibody to HSV type 2, and 79 percent had titers above 1:20. These results were compared with those in a previously studied group of neonates with HSV infections; the latter infants were significantly less likely at the onset of symptoms to have demonstrable neutralizing antibody to HSV type 2 (P = 0.000148) or to have titers above 1:20 (P less than 0.00001). We conclude that given the low attack rate, empirical antiviral therapy is not warranted in all infants of mothers with recurrent genital HSV infection who are exposed to the virus in the birth canal. Our findings suggest that the presence and titer of neutralizing antibody to HSV contribute to the low attack rate.

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.

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.

A Prospective Three-Year Cohort Study of the Epidemiology and Virology of Acute Respiratory Infections of Children in Rural India

Background Acute respiratory infection (ARI) is a major killer of children in developing countries. Although the frequency of ARI is similar in both developed and developing countries, mortality due to ARI is 10–50 times higher in developing countries. Viruses are common causes of ARI among such children, yet the disease burden of these infections in rural communities is unknown. Methodology/Principal Findings A prospective longitudinal study was carried out in children enrolled from two rural Indian villages at birth and followed weekly for the development of ARI, classified as upper respiratory infection, acute lower respiratory infection (ALRI), or severe ALRI. Respiratory syncytial virus (RSV), influenza, parainfluenza viruses and adenoviruses in nasopharyngeal aspirates were detected by direct fluorescent antibody testing (DFA) and, in addition, centrifugation enhanced culture for RSV was done. 281 infants enrolled in 39 months and followed until 42 months. During 440 child years of follow-up there were 1307 ARIs, including 236 ALRIs and 19 severe ALRIs. Virus specific incidence rates per 1000 child years for RSV were total ARI 234, ALRI 39, and severe ALRI 9; for influenza A total ARI 141, ALRI 39; for INF B total ARI 37; for PIV1 total ARI 23, for PIV2 total ARI 28, ALRI 5; for parainfluenza virus 3 total ARI 229, ALRI 48, and severe ALRI 5 and for adenovirus total ARI 18, ALRI 5. Repeat infections with RSV were seen in 18 children. Conclusions/Significance RSV, influenza A and parainfluenza virus 3 were important causes of ARI among children in rural communities in India. These data will be useful for vaccine design, development and implementation purposes.

Genetic variability in the G protein gene of group A and B respiratory syncytial viruses from India.

ABSTRACT Respiratory syncytial virus (RSV) is the most commonly identified viral agent of acute respiratory tract infection (ARI) of young children and causes repeat infections throughout life. Limited data are available on the molecular epidemiology of RSV from developing countries, including India. This study reports on the genetic variability in the glycoprotein G gene among RSV isolates from India. Reverse transcription-PCR for a region of the RSV G protein gene was done with nasopharyngeal aspirates (NPAs) collected in a prospective longitudinal study in two rural villages near Delhi and from children with ARI seen in an urban hospital. Nucleotide sequence comparisons among 48 samples demonstrated a higher prevalence of group A (77%) than group B (23%) RSV isolates. The level of genetic variability was higher among the group A viruses (up to 14%) than among the group B viruses (up to 2%). Phylogenetic analysis revealed that both the GA2 and GA5 group A RSV genotypes were prevalent during the 2002-2003 season and that genotype GA5 was predominant in the 2003-2004 season, whereas during the 2004-2005 season both genotype GA5 and genotype BA, a newly identified group B genotype, cocirculated in almost equal proportions. Comparison of the nonsynonymous mutation-to-synonymous mutation ratios (dN/dS) revealed greater evidence for selective pressure between the GA2 and GA5 genotypes (dN/dS, 1.78) than within the genotypes (dN/dS, 0.69). These are among the first molecular analyses of RSV strains from the second most populous country in the world and will be useful for comparisons to candidate vaccine strains.

Respiratory viral infections detected by multiplex PCR among pediatric patients with lower respiratory tract infections seen at an urban hospital in Delhi from 2005 to 2007

BackgroundAcute lower respiratory tract infections (ALRI) are the major cause of morbidity and mortality in young children worldwide. Information on viral etiology in ALRI from India is limited. The aim of the present study was to develop a simple, sensitive, specific and cost effective multiplex PCR (mPCR) assay without post PCR hybridization or nested PCR steps for the detection of respiratory syncytial virus (RSV), influenza viruses, parainfluenza viruses (PIV1–3) and human metapneumovirus (hMPV). Nasopharyngeal aspirates (NPAs) were collected from children with ALRI ≤ 5 years of age. The sensitivity and specificity of mPCR was compared to virus isolation by centrifugation enhanced culture (CEC) followed by indirect immunofluorescence (IIF).ResultsFrom April 2005–March 2007, 301 NPAs were collected from children attending the outpatient department or admitted to the ward of All India Institute of Medical Sciences hospital at New Delhi, India. Multiplex PCR detected respiratory viruses in 106 (35.2%) of 301 samples with 130 viruses of which RSV was detected in 61, PIV3 in 22, PIV2 in 17, hMPV in 11, PIV1 in 10 and influenza A in 9 children. CEC-IIF detected 79 viruses only. The sensitivity of mPCR was 0.1TCID50 for RSV and influenza A and 1TCID50 for hMPV, PIV1, PIV2, PIV3 and Influenza B. Mixed infections were detected in 18.8% of the children with viral infections, none detected by CEC-IIF. Bronchiolitis was significantly associated with both total viral infections and RSV infection (p < 0.05). History of ARI in family predisposed children to acquire viral infection (p > 0.05).ConclusionMultiplex PCR offers a rapid, sensitive and reasonably priced diagnostic method for common respiratory viruses.

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.

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.

In Vivo Selection of Respiratory Syncytial Viruses Resistant to Palivizumab

ABSTRACT Palivizumab (PZ) is the only monoclonal antibody currently available for use in humans against an infectious disease. PZ is administered prophylactically for respiratory syncytial virus (RSV) infections. RSV selected in cell culture for growth in the presence of PZ develops F gene mutations and can be resistant to PZ prophylaxis in cotton rats. Here, we evaluated the potential for PZ-resistant RSV mutants to arise in vivo. Cotton rats were immunosuppressed with cyclophosphamide, administered PZ, and inoculated intranasally with RSV. Lungs were harvested 12 weeks after RSV infection, reverse transcription-PCR-amplified F gene fragments were cloned into plasmids, and the nucleotide sequences of the cloned cDNAs were determined. Three of the five animals had mixed populations of lung virus, and over 50% of the clones from the three animals revealed F gene mutations associated with resistance to PZ. A virus completely resistant to PZ neutralization was recovered from the lung homogenate of a rat that had received PZ. Thus, prolonged pulmonary replication of RSV in the presence of PZ was followed by the appearance of viruses resistant to PZ. The potential for the development of resistance is a consideration as the antibody is used prophylactically against RSV and as passively administered antibodies are under development for other infections, including emerging viruses and agents of biodefense.