Human Respiratory Syncytial Research Articles

Different Inflammatory Mechanisms of Human Metapneumovirus and Respiratory Syncytial Virus

S U N D A Y 488 Exosomal Mir-155 Secretion during Rhinovirus Infection in EARLY Childhood Maria J. Gutierrez, MD, Giovanny Perez, MD, Krishna Pancham, Shelanoor Huseni, Gustavo Nino, MD; Section of Asthma, Allergy & Immunology, Pennsylvania State University College of Medicine, Hershey, PA, Division of Pediatric Pulmonology and Sleep Medicine, Children’s National Medical Center. RATIONALE: Innate immune responses are fine-tuned by small noncoding RNA molecules termed microRNAs (miRs) that modify gene expression in response to the environment. During acute infections miRs can be secreted in extracellular vesicles (exosomes) to facilitate cell-to-cell genetic communication. In this study we investigated the potential role of exosomal miRs in modulating airway antiviral immunity during rhinovirus (RV) infection, the most common cause of asthma exacerbations in children. METHODS: Nasal airway secretions were obtained from children (<3 yrs old) during PCR-confirmed RV infections and age-matched controls (n520). Nasal exosomes were isolated with polymer-based precipitation (exoquick method) and miRs profiled using NanoString microarrays. Exosomal miRNA results were contrasted with in vitro data from air-liquid interface (ALI)-differentiated human bronchial epithelium (HBE). RESULTS: Nasal exosomal miR profiling identified miR-155 as the top miR present in childrenwith RV (n510) but not in control subjects (n510). Nasal exosomal miR profiling overlapped significantly with exosomal miRs isolated from in vitro HBE secretions indicating an epithelial secretory origin of the isolated nasal exosomes. Through the use of bioinformatics tools, we identified that miR-155 predicted target genes regulate toll-like receptor (TLR)-mediated signaling. CONCLUSIONS: Our data indicate that acute RV infection in young children is associated with airway secretion of exosomes containing miR 155, which is predicted in silico to regulate antiviral immunity. Further characterization of the potential immune regulatory role of virally-induced exosomal miR secretion will enhance our knowledge about the origins of virally-induced asthma and may identify new strategies to treat and monitor this condition in children.

Human Metapneumovirus and Respiratory Syncytial Virus Are Important Causes of Acute Respiratory Failure and ARDS

Human Metapneumovirus and Respiratory Syncytial Virus Are Important Causes of Acute Respiratory Failure and ARDS

Human HepG2 cells support respiratory syncytial virus and human metapneumovirus replication

Human metapneumovirus (hMPV) and human respiratory syncytial (RSV) virus cause mild to severe infections of the respiratory tract in all age groups. So far, several cell lines derived from respiratory tissues have been identified to support replication of both viruses. Unfortunately, titers attained during replication differ between the both viruses within one cell line despite equal infection conditions, on the one hand giving raise to the assumption that the individual susceptibility may vary in dependence of the virus, and, on the other hand, making it difficult to compare results between both viruses. Low titers may cause problems in experiments such as animal trials, in which high titers in low volumes are a prerequisite for successful experiments. The advantages are described of the use of a human cell line (normally used for hepatitis viruses research) susceptible for RSV and hMPV in which both viruses replicate to comparable and high titers. It is also shown that the cell line can also be used for applications such as cell viability tests. Cell viability tests can be used as reciprocal determination tests of viral titers and therefore offer the opportunity to replace classical virological tests such as TCID 50. The cell line can be also used for high throughput applications like drug screening, making it a useful tool for screening for antiviral compound active against RSV and hMPV.

Nonsegmented Negative-Strand Viruses as Vaccine Vectors

The live-virus vector era began in 1983, when Smith, Mackett, and Moss constructed a recombinant vaccinia virus expressing hepatitis B surface antigen and demonstrated the induction of hepatitis B-specific antibodies in rabbits immunized with the recombinant virus (113). Subsequently, live-virus vectors were developed with other DNA viruses, such as adenoviruses and herpesviruses, and with positive-strand RNA viruses, such as alphaviruses and flaviviruses. The vaccine vector potential of the large group of nonsegmented negative-strand RNA viruses (NNSV) remained unexplored largely due to the lack of infectivity of their genomic RNA in cell culture and the absence of a mechanism for recombinational insertion of foreign genes. The NNSV (order Mononegavirales) comprise four families: Rhabdoviridae, represented by vesicular stomatitis virus (VSV) and rabies virus (RV); Paramyxoviridae, including Sendai virus (SeV), human parainfluenza virus types 1, 2, 3, and 4 (HPIV1 to -4), measles and mumps viruses, Newcastle disease virus (NDV), and human respiratory syncytial and metapneumoviruses (HRSV and HMPV); Filoviridae, containing Ebola and Marburg viruses; and Bornaviridae, containing Borna disease virus. In 1994, Schnell, Mebatsion, and Conzelmann developed a reverse genetic system for RV that allowed the recovery of infectious virus entirely from cloned cDNA (102). This was followed quickly by the development of reverse genetic systems for numerous other NNSV (17, 22, 33, 50, 55, 69, 73, 76, 83, 133). This review will focus on the use of NNSV, in particular members of the Rhabdoviridae and Paramyxoviridae, as live vaccine vectors.

Human Metapneumovirus and Respiratory Syncytial Virus Disease in Children, Yemen

Factors increasing the severity of respiratory infections in developing countries are poorly described. We report factors associated with severe acute respiratory illness in Yemeni children (266 infected with respiratory syncytial virus and 66 with human metapneumovirus). Age, indoor air pollution, and incomplete vaccinations were risk factors and differed from those in industrialized countries.

Bovine respiratory syncytial virus nucleocapsid protein: mRNA sequence analysis and expression from recombinant vaccinia virus vectors

The nucleotide sequence of the mRNA encoding the nucleocapsid (N) protein of bovine respiratory syncytial (BRS) virus, strain 391-2, was determined. Recombinant vectors containing a cDNA of the complete N gene were constructed, and expression of the N protein in eukaryotic cells was demonstrated using two different vector systems. The BRS virus N mRNA was 1197 nucleotides in length, exclusive of poly(A), and had a single major open reading frame that encoded a polypeptide of 391 amino acids with a calculated M(r) of 42.6K. The nucleotide and amino acid sequences of the BRS virus N gene were compared to those of human respiratory syncytial (HRS) virus strains A2 and 18537, and to BRS virus strain A51908. The level of nucleic acid identity between the N mRNA of BRS virus 391-2 and both HRS virus subtypes was 80 to 81%, whereas the identity between the two BRS virus strains was 97%. A 93 to 94% level of identity was observed between the deduced amino acid sequences of the N protein of BRS virus 391-2 and the corresponding sequences of the two HRS virus strains. The two BRS virus N proteins differed in amino acid sequence at only three positions. Recombinant BRS virus N protein was expressed using two different vector systems: in cells from a plasmid using the vaccinia virus/T7 polymerase expression system or from a recombinant vaccinia virus. N proteins synthesized by the two vector systems migrated with an electrophoretic mobility identical to that of authentic BRS virus N protein, and were precipitated by anti-BRS virus antibodies.

Cloning and sequencing of the matrix protein (M) gene of turkey rhinotracheitis virus reveal a gene order different from that of respiratory syncytial virus

Several biochemical properties and the sequence of the fusion glycoprotein (F) have indicated that turkey rhinotracheitis virus (TRTV) is a pneumovirus, subfamily Pneumovirinae of the Paramyxoviridae family. As TRTV was known to generate polycistronic mRNAs, cDNA was generated from TRTV strain U K/3 BV/85-infected Vero cell mRNAs using an oligonucleotide primer corresponding to a region of the F gene. Sequencing of four cDNAs revealed that the gene adjacent to the beginning (3′ end) of the F gene was that for the matrix (M) protein, i.e., that TRTV had the partial gene order 3′-M-F-5′. This was unexpected as human respiratory syncytial (RS) virus, the type species of the genus Pneumovirus, has the partial gene order 3′-M-SH-G-F-5′, where SH and G are the small hydrophobic protein and attachment glycoprotein, respectively. Instead TRTV resembled the Morbillivirus and Paramyxovirus genera of the Paramyxoviridae (subfamily Paramyxoviridae) which have the partial gene order 3′-M-F-5′. Two further oligonucleotides, one corresponding to a sequence near the end of the M gene and the other (oligo B) to a sequence near the beginning of the F gene, with their 5′ ends spaced 300 nucleotides apart on the basis of the cDNA sequence, were used in a polymerase chain reaction (PCR) using genomic RNA as template. Only a PCR product of 0.3 kb was obtained. The same sized product was also obtained using these oligonucleotides and genomic RNA from three other TRTV strains (SA/91 /78, UK/8544/85, and SA/2381/88) which had been grown in chicken tracheal organ cultures. In addition PCR was performed using genomic RNA from TRTV-3BV and SA/2381/88 with oligo B and another oligonucleotide near the 5′ end of the gene upstream from M, spaced 1141 nucleotides apart on the basis of the sequence data. Only a 1.14-kb PCR product was obtained. Larger products would have been expected if another gene had been situated between M and F. The absence of such larger products, plus the demonstration that infected cells contained M-F dicistronic mRNAs, supported the conclusion that in the TRTV genome the M gene is adjacent to the F gene in the order 3'-M-F-5′. The 5′ termini of the M and F mRNAs were confirmed by mRNA mapping. The TRTV M gene encoded a protein of 254 amino acids, very similar to that of RS virus (256 residues; 37% amino acid identity) but very different from that of the morbilliviruses and paramyxoviruses (approximately 350 residues). Thus, on the basis of the sequences of both the M and the F genes, TRTV is more closely related to the pneumoviruses than to the paramyxoviruses and morbilliviruses.

Nucleotide sequence analysis of the bovine respiratory syncytial virus fusion protein mRNA and expression from a recombinant vaccinia virus

Bovine respiratory syncytial (BRS) virus is an important cause of serious respiratory illness in calves. The disease caused in calves is similar to that caused by human respiratory syncytial (HRS) virus in children. The two viruses, however, have distinct host ranges and the attachment glycoproteins, G, have no antigenic cross-reactivity. The fusion glycoproteins, F, of the HRS and BRS viruses, however, have some antigenic cross-reactivity. To further compare the BRS virus and HRS virus fusion proteins, we determined the nucleotide sequence of cDNA clones to the BRS virus F protein mRNA, deduced the amino acid sequence, and compared these sequences with the HRS virus F protein sequences. The BRS virus F mRNA was 1899 nucleotides in length and had a single major open reading frame which could code for a polypeptide of 574 amino acids with an estimated molecular weight of 63.8 kDa. Structural features predicted from the amino acid sequence included an NH 2 terminal signal sequence (residues 1–26), a site for proteolytic cleavage (residues 131–136) to generate the disulfide-linked F, and F2 subunits, and a hydrophobic transmembrane anchor sequence (residues 522–549). The nucleic acid identity between the BRS virus and the HRS virus F mRNA sequences was 71.5%. The predicted BRS virus F protein shared 80.5% overall amino acid identity with the HRS virus F protein with 89% identity in the F, polypeptide but only 68% identity in the F2 polypeptide. The position and number of the cysteine residues in the F, and F2 polypeptides were conserved among all F proteins. However, BRS virus F protein had only three potential N-linked carbohydrate acceptor sites in comparison to four or five for the HRS viruses. A difference in the extent of glycosylation between the BRS and HRS virus F2 polypeptides was shown to be responsible for differences observed in the electrophoretic mobility of these proteins. A cDNA containing the complete open reading frame of the BRS virus F mRNA was inserted into the thymidine kinase gene of vaccinia virus and following homologous recombination, a recombinant virus containing the BRS virus F gene was isolated. The BRS virus F protein was expressed in recombinant virus infected cells as demonstrated by immunoprecipitation and was transported to and expressed on the surface of infected cells as shown by indirect immunofluorescence.

Nucleotide sequence analysis and expression from recombinant vectors demonstrate that the attachment protein G of bovine respiratory syncytial virus is distinct from that of human respiratory syncytial virus

Bovine respiratory syncytial (BRS) virus causes a severe lower respiratory tract disease in calves similar to the disease in children caused by human respiratory syncytial (HRS) virus. While there is antigenic cross-reactivity among the other major viral structural proteins, the major glycoprotein, G, of BRS virus and that of HRS virus are antigenically distinct. The G glycoprotein has been implicated as the attachment protein for HRS virus. We have carried out a molecular comparison of the glycoprotein G of BRS virus with the HRS virus counterparts. cDNA clones corresponding to the BRS virus G glycoprotein mRNA were isolated and analyzed by dideoxynucleotide sequencing. The BRS virus G mRNA contained 838 nucleotides exclusive of poly(A) and had a major open reading frame coding for a polypeptide of 257 amino acid residues. The deduced amino acid sequence of the BRS virus G polypeptide showed only 29 to 30% amino acid identity with the G protein of either the subgroup A or B HRS virus. However, despite this low level of identity, there were strong similarities in the predicted hydropathy profiles of the BRS virus and HRS virus G proteins. A cDNA molecule containing the complete BRS virus G major open reading frame was inserted into the thymidine kinase gene of vaccinia virus by homologous recombination, and a recombinant virus containing the BRS virus G protein gene was isolated. This recombinant virus expressed the BRS virus G protein, as demonstrated by Western immunoblot analysis and immunofluorescence of infected cells. The BRS virus G protein expressed from the recombinant vector was transported to and expressed on the surface of infected cells. Antisera to the BRS virus G protein made by using the recombinant vector to immunize animals recognized the BRS virus attachment protein but not the HRS virus G protein and vice versa, confirming the lack of antigenic cross-reactivity between the BRS and HRS virus attachment proteins. On the basis of the data presented here, we conclude that BRS virus should be classified within the genus Pneumovirus in a group separate from HRS virus and that it is no more closely related to HRS virus subgroup A than it is to HRS virus subgroup B.