Sendai Virus-Vectored Vaccines That Express Envelope Glycoproteins of Respiratory Viruses.

Human respiratory syncytial virus (HRSV), human metapneumovirus (HMPV), and human parainfluenza viruses (HPIVs) are leading causes of respiratory disease in young children, the elderly, and individuals of all ages with immunosuppression. Vaccination strategies against these pneumoviruses and paramyxoviruses are vast in number, yet no licensed vaccines are available. Here, we review development of Sendai virus (SeV), a versatile pediatric vaccine that can (a) serve as a Jennerian vaccine against HPIV1, (b) serve as a recombinant vaccine against HRSV, HPIV2, HPIV3, and HMPV, (c) accommodate foreign genes for viral glycoproteins in multiple intergenic positions, (d) induce durable, mucosal, B-cell, and T-cell immune responses without enhanced immunopathology, (e) protect cotton rats, African green monkeys, and chimpanzees from infection, and (f) be formulated into a vaccine cocktail. Clinical phase I safety trials of SeV have been completed in adults and 3-6-year-old children. Clinical testing of SeVRSV, an HRSV fusion (F) glycoprotein gene recombinant, has also been completed in adults. Positive results from these studies, and collaborative efforts with the National Institutes of Health and the Serum Institute of India assist advanced development of SeV-based vaccines. Prospects are now good for vaccine successes in infants and consequent protection against serious viral disease.

Safety and immunogenicity of an intranasal sendai virus-based vaccine for human parainfluenza virus type I and respiratory syncytial virus (SeVRSV) in adults.

SeVRSV is a replication-competent Sendai virus (SeV)-based vaccine carrying the respiratory syncytial virus (RSV) fusion protein (F) gene. Unmanipulated, non-recombinant SeV is a murine parainfluenza virus type 1 (PIV-1) and serves as a Jennerian vaccine for human PIV-1 (hPIV-1). SeV protects African green monkeys (AGM) from infection after hPIV-1 challenge. The recombinant SeVRSV additionally targets RSV and protects AGM from lower respiratory infections after RSV challenge. The present study is the first to report on the safety, viral genome detection, and immunogenicity following SeVRSV vaccination of healthy adults. Seventeen and four healthy adults received intranasal SeVRSV and PBS, respectively, followed by six months of safety monitoring. Virus genome (in nasal wash) and vaccine-specific antibodies (in sera) were monitored for two and four weeks, respectively, post-vaccination. The vaccine was well-tolerated with only mild to moderate reactions that were also present in the placebo group. No severe reactions occurred. As expected, due to preexisting immunity toward hPIV-1 and RSV in adults, vaccine genome detection was transient. There were minimal antibody responses to SeV and negligible responses to RSV F. Results encourage further studies of SeVRSV with progression toward a clinical trial in seronegative children. Abbreviations: AE-adverse event; SAE-serious adverse event; SeV-Sendai virus; RSV-respiratory syncytial virus; PIV-1-parainfluenza virus-type 1; hPIV-1-human parainfluenza virus-type 1; F-RSV fusion protein; SeVRSV-recombinant SeV carrying the RSV F gene; Ab-antibody; MSW-medically significant wheezing; NOCMC-new onset chronic medical condition, mITT-modified Intent to Treat; ALRI-acute lower respiratory tract infection.

Prevalence of respiratory viruses using polymerase chain reaction in children with wheezing, a systematic review and meta-analysis.

INTRODUCTION: Wheezing is a major problem in children, and respiratory viruses are often believed to be the causative agent. While molecular detection tools enable identification of respiratory viruses in wheezing children, it remains unclear if and how these viruses are associated with wheezing. The objective of this systematic review is to clarify the prevalence of different respiratory viruses in children with wheezing. METHODS: We performed an electronic in Pubmed and Global Index Medicus on 01 July 2019 and manual search. We performed search of studies that have detected common respiratory viruses in children ≤18 years with wheezing. We included only studies using polymerase chain reaction (PCR) assays. Study data were extracted and the quality of articles assessed. We conducted sensitivity, subgroup, publication bias, and heterogeneity analyses using a random effects model. RESULTS: The systematic review included 33 studies. Rhinovirus, with a prevalence of 35.6% (95% CI 24.6-47.3, I2 98.4%), and respiratory syncytial virus, at 31.0% (95% CI 19.9-43.3, I2 96.4%), were the most common viruses detected. The prevalence of other respiratory viruses was as follows: human bocavirus 8.1% (95% CI 5.3-11.3, I2 84.6%), human adenovirus 7.7% (95% CI 2.6-15.0, I2 91.0%), influenza virus6.5% (95% CI 2.2-12.6, I2 92.4%), human metapneumovirus5.8% (95% CI 3.4-8.8, I2 89.0%), enterovirus 4.3% (95% CI 0.1-12.9, I2 96.2%), human parainfluenza virus 3.8% (95% CI 1.5-6.9, I2 79.1%), and human coronavirus 2.2% (95% CI 0.6-4.4, I2 79.4%). CONCLUSIONS: Our results suggest that rhinovirus and respiratory syncytial virus may contribute to the etiology of wheezing in children. While the clinical implications of molecular detection of respiratory viruses remains an interesting question, this study helps to illuminate the potential of role respiratory viruses in pediatric wheezing. REVIEW REGISTRATION: PROSPERO, CRD42018115128.

Structure-based design of a quadrivalent fusion glycoprotein vaccine for human parainfluenza virus types 1-4.

Parainfluenza virus types 1-4 (PIV1-4) are highly infectious human pathogens, of which PIV3 is most commonly responsible for severe respiratory illness in newborns, elderly, and immunocompromised individuals. To obtain a vaccine effective against all four PIV types, we engineered mutations in each of the four PIV fusion (F) glycoproteins to stabilize their metastable prefusion states, as such stabilization had previously enabled the elicitation of high-titer neutralizing antibodies against the related respiratory syncytial virus. A cryoelectron microscopy structure of an engineered PIV3 F prefusion-stabilized trimer, bound to the prefusion-specific antibody PIA174, revealed atomic-level details for how introduced mutations improved stability as well as how a single PIA174 antibody recognized the trimeric apex of prefusion PIV3 F. Nine combinations of six newly identified disulfides and two cavity-filling mutations stabilized the prefusion PIV3 F immunogens and induced 200- to 500-fold higher neutralizing titers in mice than were elicited by PIV3 F in the postfusion conformation. For PIV1, PIV2, and PIV4, we also obtained stabilized prefusion Fs, for which prefusion versus postfusion titers were 2- to 20-fold higher. Elicited murine responses were PIV type-specific, with little cross-neutralization of other PIVs. In nonhuman primates (NHPs), quadrivalent immunization with prefusion-stabilized Fs from PIV1-4 consistently induced potent neutralizing responses against all four PIVs. For PIV3, the average elicited NHP titer from the quadrivalent immunization was more than fivefold higher than any titer observed in a cohort of over 100 human adults, highlighting the ability of a prefusion-stabilized immunogen to elicit especially potent neutralization.

Viral aetiology of wheezing in children under five.

BACKGROUND & OBJECTIVES: Wheezing is a common problem in children under five with acute respiratory infections (ARIs). Viruses are known to be responsible for a considerable proportion of ARIs in children. This study was undertaken to know the viral aetiology of wheezing among the children less than five years of age, admitted to a tertiary care hospital in eastern India. METHODS: Seventy five children, under the age of five years admitted with wheezing, were included in the study. Throat and nasal swabs were collected, and real-time multiplex polymerase chain reaction (PCR) assay was used to screen for influenza 1 and 2, respiratory syncytial virus (RSV), parainfluenza virus (PIV) 1, 2, 3 and 4, rhinovirus, human meta-pneumovirus, bocavirus (HBoV), Coronavirus, adenovirus, Enterovirus and Parechovirus. RESULTS: The total viral detection rate was 28.57 per cent. Viral RNA markers were detected from children diagnosed to be having pneumonia (3 cases), bronchiolitis (9 cases), episodic wheeze (2 cases) and multitrigger wheeze (6 cases). RSV was the most common virus (35%) followed by PIV1, 2 and 3 (20%), HBoV (10%) and rhinovirus (5%). However, mixed infection was observed in 30 per cent of cases. INTERPRETATION & CONCLUSIONS: The study reported the presence of respiratory viral agents in 28.57 per cent of children with wheezing; RSV and PIV were most common, accounting to 55 per cent of the total cases. Mixed infection was reported in 30 per cent of cases. Seasonal variation in the occurrence of these viruses was also noted. Further studies need to be done with a large sample and longer follow up period to verify these findings.

Genetic analyses of the fusion protein genes in human parainfluenza virus types 1 and 3 among patients with acute respiratory infections in Eastern Japan from 2011 to 2015.

PURPOSE: To genetically explore the fusion protein gene (F) in human parainfluenza virus type 1 (HPIV1) and type 3 (HPIV3) strains, we analysed them in patients with acute respiratory infections in Eastern Japan from 2011 to 2015. METHODOLOGY: We constructed phylogenetic trees based on the HPIV and HPIV3 F gene using the maximum likelihood method and conducted P-distance and selective pressure analyses. We also predicted the linear epitopes of the protein in the prototype strains. Furthermore, we mapped the amino acid substitutions of the proteins. RESULTS: Nineteen strains of HPIV1 and 53 strains of HPIV3 were detected among the clinical acute respiratory infection cases. The phylogenetic trees indicated that the HPIV1 and HPIV3 strains were classified into clusters II and III and cluster C, respectively. The P-distance values of the HPIV1 and HPIV3 F genes were <0.03. Two positive selection sites were inferred in the HPIV1 (aa 8 and aa 10), and one positive selection site was inferred in the HPIV3 (aa 108), but over 10 negative selection sites were inferred. Four epitopes were predicted for the HPIV1 prototype strains, while five epitopes were predicted for the HPIV3 prototype strain. A positive selection site (aa 108) or the HPIV3 F protein was involved in the predicted epitope. Additionally, we found that an amino acid substitution (R73K) in the LC76627 HPIV3 strain presumably may affect the resistance to neutralization by antibodies. CONCLUSION: The F gene of HPIV1 and HPIV3 was relatively well conserved in the eastern part of Japan during the investigation period.

Attenuated Human Parainfluenza Virus Type 1 Expressing Ebola Virus Glycoprotein GP Administered Intranasally Is Immunogenic in African Green Monkeys.

The recent 2014-2016 Ebola virus (EBOV) outbreak prompted increased efforts to develop vaccines against EBOV disease. We describe the development and preclinical evaluation of an attenuated recombinant human parainfluenza virus type 1 (rHPIV1) expressing the membrane-anchored form of EBOV glycoprotein GP, as an intranasal (i.n.) EBOV vaccine. GP was codon optimized and expressed either as a full-length protein or as an engineered chimeric form in which its transmembrane and cytoplasmic tail (TMCT) domains were replaced with those of the HPIV1 F protein in an effort to enhance packaging into the vector particle and immunogenicity. GP was inserted either preceding the N gene (pre-N) or between the N and P genes (N-P) of rHPIV1 bearing a stabilized attenuating mutation in the P/C gene (CΔ170). The constructs grew to high titers and efficiently and stably expressed GP. Viruses were attenuated, replicating at low titers over several days, in the respiratory tract of African green monkeys (AGMs). Two doses of candidates expressing GP from the pre-N position elicited higher GP neutralizing serum antibody titers than the N-P viruses, and unmodified GP induced higher levels than its TMCT counterpart. Unmodified EBOV GP was packaged into the HPIV1 particle, and the TMCT modification did not increase packaging or immunogenicity but rather reduced the stability of GP expression during in vivo replication. In conclusion, we identified an attenuated and immunogenic i.n. vaccine candidate expressing GP from the pre-N position. It is expected to be well tolerated in humans and is available for clinical evaluation.IMPORTANCE EBOV hemorrhagic fever is one of the most lethal viral infections and lacks a licensed vaccine. Contact of fluids from infected individuals, including droplets or aerosols, with mucosal surfaces is an important route of EBOV spread during a natural outbreak, and aerosols also might be exploited for intentional virus spread. Therefore, vaccines that protect against mucosal as well as systemic inoculation are needed. We evaluated a version of human parainfluenza virus type 1 (HPIV1) bearing a stabilized attenuating mutation in the P/C gene (CΔ170) as an intranasal vaccine vector to express the EBOV glycoprotein GP. We evaluated expression from two different genome positions (pre-N and N-P) and investigated the use of vector packaging signals. African green monkeys immunized with two doses of the vector expressing GP from the pre-N position developed high titers of GP neutralizing serum antibodies. The attenuated vaccine candidate is expected to be safe and immunogenic and is available for clinical development.

A study of genetic variability of human parainfluenza virus type 1 in Croatia, 2011-2014.

Molecular epidemiology of human parainfluenza viruses type 1 (HPIV1) was investigated. Samples were collected from patients hospitalized in Croatia during the three consecutive epidemic seasons (2011-2014). Results indicated co-circulation of two major genetic clusters of HPIV1. Samples from the current study refer to clades II and III in a phylogenetic tree of haemagglutinin-neuraminidase (HN) gene. Additional phylogenetic trees of fusion (F) and phosphoprotein (P) genes confirmed the topology. Analysis of nucleotide diversity of entire P, F and HN genes demonstrated similar values: 0.0255, 0.0236 and 0.0237, respectively. However, amino acid diversity showed F protein to be the most conserved, while P protein was the most tolerant to mutations. Potential N- and O-glycosylation sites suggested that HPIV1 HN protein is abundantly glycosylated, and a specific N-glycosylation pattern could distinguish between clades II and III. Analysis of potential O-glycosylation sites in F protein indicated that samples from this study have two potential O-glycosylation sites, while publicly available sequences have five potential sites. This study provides data on the molecular characterization and epidemic pattern of HPIV1 in Croatia.

Viral etiology of medically attended influenza-like illnesses in children less than five years old in Suzhou, China, 2011-2014.

Limited information is available on the non-influenza etiology and epidemiology of influenza-like illness (ILI) in China. From April 2011 to March 2014, we collected oropharyngeal swabs from children less than 5 years of age with symptoms of ILI who presented to the outpatient departments of Suzhou University Affiliated Children's Hospital (SCH). We used reverse transcription polymerase chain reaction (rt-PCR) or PCR to detect 11 respiratory viruses. Among 3,662 enrolled ILI patients, 1,292 (35.3%) tested positive for at least one virus. Influenza virus (16.9%) was detected most frequently (influenza A 7.4%, influenza B 9.5%), followed by respiratory syncytial virus (RSV) (5.6%), parainfluenza virus (PIV) types 1-4 (4.8%), human bocavirus (HBoV) (3.8%), human metapneumovirus (HMPV) (3.5%), and adenovirus (ADV) (3.0%). Co-infections were identified in 108 (2.9%) patients. Influenza virus predominantly circulated in January-March and June-July. The 2013-2014 winter peaks of RSV and influenza overlapped. Compared with other virus positive cases, influenza positive cases were more likely to present with febrile seizure, and RSV positive cases were more likely to present with cough and wheezing, and were most frequently diagnosed with pneumonia. These data provide a better understanding of the viral etiology of ILI among children less than 5 years of age in Suzhou, China. Influenza is not only the most frequently identified pathogen but it is also the only vaccine preventable illness among the 11 pathogens tested. Such findings suggest the potential value of exploring value of influenza vaccination among this influenza vaccination target group. J. Med. Virol. 88:1334-1340, 2016. © 2016 Wiley Periodicals, Inc.

Attenuated Human Parainfluenza Virus Type 1 (HPIV1) Expressing the Fusion Glycoprotein of Human Respiratory Syncytial Virus (RSV) as a Bivalent HPIV1/RSV Vaccine.

UNLABELLED: Live attenuated recombinant human parainfluenza virus type 1 (rHPIV1) was investigated as a vector to express the respiratory syncytial virus (RSV) fusion (F) glycoprotein, to provide a bivalent vaccine against RSV and HPIV1. The RSV F gene was engineered to include HPIV1 transcription signals and inserted individually into three gene locations in each of the two attenuated rHPIV1 backbones. Each backbone contained a single previously described attenuating mutation that was stabilized against deattenuation, specifically, a non-temperature-sensitive deletion mutation involving 6 nucleotides in the overlapping P/C open reading frames (ORFs) (C(Δ170)) or a temperature-sensitive missense mutation in the L ORF (L(Y942A)). The insertion sites in the genome were pre-N (F1), N-P (F2), or P-M (F3) and were identical for both backbones. In vitro, the presence of the F insert reduced the rate of virus replication, but the final titers were the same as the final titer of wild-type (wt) HPIV1. High levels of RSV F expression in cultured cells were observed with rHPIV1-C(Δ170)-F1, -F2, and -F3 and rHPIV1-L(Y942A)-F1. In hamsters, the rHPIV1-C(Δ170)-F1, -F2, and -F3 vectors were moderately restricted in the nasal turbinates, highly restricted in lungs, and genetically stable in vivo. Among the C(Δ170) vectors, the F1 virus was the most immunogenic and protective against wt RSV challenge. The rHPIV1-L(Y942A) vectors were highly restricted in vivo and were not detectably immunogenic or protective, indicative of overattenuation. The C(Δ170)-F1 construct appears to be suitably attenuated and immunogenic for further development as a bivalent intranasal pediatric vaccine. IMPORTANCE: There are no vaccines for the pediatric respiratory pathogens RSV and HPIV. We are developing live attenuated RSV and HPIV vaccines for use in virus-naive infants. Live attenuated RSV strains in particular are difficult to develop due to their poor growth and physical instability, but these obstacles could be avoided by the use of a vaccine vector. We describe the development and preclinical evaluation of live attenuated rHPIV1 vectors expressing the RSV F protein. Two different attenuated rHPIV1 backbones were each engineered to express RSV F from three different gene positions. The rHPIV1-C(Δ170)-F1 vector, bearing an attenuating deletion mutation (C(Δ170)) in the P/C gene and expressing RSV F from the pre-N position, was attenuated, stable, and immunogenic against the RSV F protein and HPIV1 in the hamster model and provided substantial protection against RSV challenge. This study provides a candidate rHPIV1-RSV-F vaccine virus suitable for continued development as a bivalent vaccine against two major childhood pathogens.

Preferential Amplification of Pathogenic Sequences.

The application of next generation sequencing (NGS) technology in the diagnosis of human pathogens is hindered by the fact that pathogenic sequences, especially viral, are often scarce in human clinical specimens. This known disproportion leads to the requirement of subsequent deep sequencing and extensive bioinformatics analysis. Here we report a method we called "Preferential Amplification of Pathogenic Sequences (PATHseq)" that can be used to greatly enrich pathogenic sequences. Using a computer program, we developed 8-, 9-, and 10-mer oligonucleotides called "non-human primers" that do not match the most abundant human transcripts, but instead selectively match transcripts of human pathogens. Instead of using random primers in the construction of cDNA libraries, the PATHseq method recruits these short non-human primers, which in turn, preferentially amplifies non-human, presumably pathogenic sequences. Using this method, we were able to enrich pathogenic sequences up to 200-fold in the final sequencing library. This method does not require prior knowledge of the pathogen or assumption of the infection; therefore, it provides a fast and sequence-independent approach for detection and identification of human viruses and other pathogens. The PATHseq method, coupled with NGS technology, can be broadly used in identification of known human pathogens and discovery of new pathogens.

Severe acute respiratory infection in children in a densely populated urban slum in Kenya, 2007-2011.

BACKGROUND: Reducing acute respiratory infection burden in children in Africa remains a major priority and challenge. We analyzed data from population-based infectious disease surveillance for severe acute respiratory illness (SARI) among children <5 years of age in Kibera, a densely populated urban slum in Nairobi, Kenya. METHODS: Surveillance was conducted among a monthly mean of 5,874 (range = 5,778-6,411) children <5 years old in two contiguous villages in Kibera. Participants had free access to the study clinic and their health events and utilization were noted during biweekly home visits. Patients meeting criteria for SARI (WHO-defined severe or very severe pneumonia, or oxygen saturation <90%) from March 1, 2007-February 28, 2011 had blood cultures processed for bacteria, and naso- and oro- pharyngeal swabs collected for quantitative real-time reverse transcription polymerase chain reaction testing for influenza viruses, parainfluenza viruses (PIV), respiratory syncytial virus (RSV), adenovirus, and human metapneumovirus (hMPV). Swabs collected during January 1, 2009 - February 28, 2010 were also tested for rhinoviruses, enterovirus, parechovirus, Mycoplasma pneumoniae, and Legionella species. Swabs were collected for simultaneous testing from a selected group of control-children visiting the clinic without recent respiratory or diarrheal illnesses. RESULTS: SARI overall incidence was 12.4 cases/100 person-years of observation (PYO) and 30.4 cases/100 PYO in infants. When comparing detection frequency in swabs from 815 SARI cases and 115 healthy controls, only RSV and influenza A virus were significantly more frequently detected in cases, although similar trends neared statistical significance for PIV, adenovirus and hMPV. The incidence for RSV was 2.8 cases/100 PYO and for influenza A was 1.0 cases/100 PYO. When considering all PIV, the rate was 1.1 case/100 PYO and the rate per 100 PYO for SARI-associated disease was 1.5 for adenovirus and 0.9 for hMPV. RSV and influenza A and B viruses were estimated to account for 16.2% and 6.7% of SARI cases, respectively; when taken together, PIV, adenovirus, and hMPV may account for >20% additional cases. CONCLUSIONS: Influenza viruses and RSV (and possibly PIV, hMPV and adenoviruses) are important pathogens to consider when developing technologies and formulating strategies to treat and prevent SARI in children.

Population-based incidence of severe acute respiratory virus infections among children aged <5 years in rural Bangladesh, June-October 2010.

BACKGROUND: Better understanding the etiology-specific incidence of severe acute respiratory infections (SARIs) in resource-poor, rural settings will help further develop and prioritize prevention strategies. To address this gap in knowledge, we conducted a longitudinal study to estimate the incidence of SARIs among children in rural Bangladesh. METHODS: During June through October 2010, we followed children aged <5 years in 67 villages to identify those with cough, difficulty breathing, age-specific tachypnea and/or danger signs in the community or admitted to the local hospital. A study physician collected clinical information and obtained nasopharyngeal swabs from all SARI cases and blood for bacterial culture from those hospitalized. We tested swabs for respiratory syncytial virus (RSV), influenza viruses, human metapneumoviruses, adenoviruses and human parainfluenza viruses 1-3 (HPIV) by real-time reverse transcription polymerase chain reaction. We calculated virus-specific SARI incidence by dividing the number of new illnesses by the person-time each child contributed to the study. RESULTS: We followed 12,850 children for 279,029 person-weeks (pw) and identified 141 SARI cases; 76 (54%) at their homes and 65 (46%) at the hospital. RSV was associated with 7.9 SARI hospitalizations per 100,000 pw, HPIV3 2.2 hospitalizations/100,000 pw, and influenza 1.1 hospitalizations/100,000 pw. Among non-hospitalized SARI cases, RSV was associated with 10.8 illnesses/100,000 pw, HPIV3 1.8/100,000 pw, influenza 1.4/100,000 pw, and adenoviruses 0.4/100,000 pw. CONCLUSION: Respiratory viruses, particularly RSV, were commonly associated with SARI among children. It may be useful to explore the value of investing in prevention strategies, such as handwashing and respiratory hygiene, to reduce respiratory infections among young children in such settings.

A single amino acid mutation at position 170 of human parainfluenza virus type 1 fusion glycoprotein induces obvious syncytium formation and caspase-3-dependent cell death.

An escape mutant of human parainfluenza virus type 1 (hPIV1), which was selected by serial passage in the presence of a sialidase inhibitor, 4-O-thiocarbamoylmethyl-2-deoxy-2,3-didehydro-N-acetylneur-aminic acid (TCM-Neu5Ac2en), exhibited remarkable syncytium formation and virus-induced cell death in LLC-MK2 cells but no difference in susceptibility for the sialidase inhibitor TCM-Neu5Ac2en from that of wild-type hPIV1 strain C35 (WT). The mutant virus also had higher replication and plaque formation abilities. The mutant virus acquired two amino acid mutations, Glu to Gly at position 170 and Ala to Glu 442 in fusion (F) glycoprotein, but no mutations in haemaggulutinin-neuraminidase (HN) glycoprotein. Using cells co-expressing F and HN genes with site-specific mutagenesis, we demonstrated that a point mutation of Glu to Gly at position 170, which was estimated to be located in hPIV1 F glycoprotein heptad repeat 1, was required for obvious syncytium formation and caspase-3-dependent cell death. In contrast, wild-type F glycoprotein induced no synctium formation or cell death. The findings suggest that a single amino acid mutation of hPIV1 F glycoprotein promotes syncytium formation that is followed by caspase-3-dependent cell death.

The C proteins of human parainfluenza virus type 1 limit double-stranded RNA accumulation that would otherwise trigger activation of MDA5 and protein kinase R.

Human parainfluenza virus type 1 (HPIV1) is an important respiratory pathogen in young children, the immunocompromised, and the elderly. We found that infection with wild-type (WT) HPIV1 suppressed the innate immune response in human airway epithelial cells by preventing not only phosphorylation of interferon regulatory factor 3 (IRF3) but also degradation of IκBß, thereby inhibiting IRF3 and NF-κB activation, respectively. Both of these effects were ablated by a F170S substitution in the HPIV1 C proteins (F170S) or by silencing the C open reading frame [P(C-)], resulting in a potent beta interferon (IFN-ß) response. Using murine knockout cells, we found that IFN-ß induction following infection with either mutant relied mainly on melanoma-associated differentiation gene 5 (MDA5) rather than retinoic acid-inducible gene I (RIG-I). Infection with either mutant, but not WT HPIV1, induced a significant accumulation of intracellular double-stranded RNA (dsRNA). These mutant viruses directed a marked increase in the accumulation of viral genome, antigenome, and mRNA that was coincident with the accumulation of dsRNA. In addition, the amount of viral proteins was reduced compared to that of WT HPIV1. Thus, the accumulation of dsRNA might be a result of an imbalance in the N protein/genomic RNA ratio leading to incomplete encapsidation. Protein kinase R (PKR) activation and IFN-ß induction followed the kinetics of dsRNA accumulation. Interestingly, the C proteins did not appear to directly inhibit intracellular signaling involved in IFN-ß induction; instead, their role in preventing IFN-ß induction appeared to be in suppressing the formation of dsRNA. PKR activation contributed to IFN-ß induction and also was associated with the reduction in the amount of viral proteins. Thus, the HPIV1 C proteins normally limit the accumulation of dsRNA and thereby limit activation of IRF3, NF-κB, and PKR. If C protein function is compromised, as in the case of F170S HPIV1, the resulting PKR activation and reduction in viral protein levels enable the host to further reduce C protein levels and to mount a potent antiviral type I IFN response.

CFTR delivery to 25% of surface epithelial cells restores normal rates of mucus transport to human cystic fibrosis airway epithelium.

Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl(-) and Na(+) epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%-65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.

The C proteins of human parainfluenza virus type 1 (HPIV1) control the transcription of a broad array of cellular genes that would otherwise respond to HPIV1 infection.

Human parainfluenza virus type 1 (HPIV1) is an important respiratory pathogen in children and the most common cause of viral croup. We performed a microarray-based analysis of gene expression kinetics to examine how wild-type (wt) HPIV1 infection altered gene expression in human respiratory epithelial cells and what role beta interferon played in this response. We similarly evaluated HPIV1-P(C-), a highly attenuated and apoptosis-inducing virus that does not express any of the four C proteins, and HPIV1-C(F170S), a less attenuated mutant that contains a single point mutation in C and, like wt HPIV1, does not efficiently induce apoptosis, to examine the role of the C proteins in controlling host gene expression. We also used these data to investigate whether the phenotypic differences between the two C mutants could be explained at the transcriptional level. Mutation or deletion of the C proteins of HPIV1 permitted the activation of over 2,000 cellular genes that otherwise would be repressed by HPIV1 infection. Thus, the C proteins profoundly suppress the response of human respiratory cells to HPIV1 infection. Cellular pathways targeted by the HPIV1 C proteins were identified and their transcriptional control was analyzed using bioinformatics. Transcription factor binding sites for IRF and NF-kappaB were overrepresented in some of the C protein-targeted pathways, but other pathways were dominated by less-known factors, such as forkhead transcription factor FOXD1. Surprisingly, the host responses to the P(C-) and C(F170S) mutants were very similar, and only subtle differences in the expression kinetics of caspase 3 and TRAIL receptor 2 were observed. Thus, changes in host cell transcription did not reflect the striking phenotypic differences observed between these two viruses.

Loss of the N-linked glycan at residue 173 of human parainfluenza virus type 1 hemagglutinin-neuraminidase exposes a second receptor-binding site.

BCX 2798 (4-azido-5-isobutyrylamino-2,3-didehydro-2,3,4,5-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosic acid) effectively inhibited the activities of the hemagglutinin-neuraminidase (HN) of human parainfluenza viruses (hPIV) in vitro and protected mice from lethal infection with a recombinant Sendai virus whose HN was replaced with that of hPIV-1 (rSeV[hPIV-1HN]) (I. V. Alymova, G. Taylor, T. Takimoto, T. H. Lin., P. Chand, Y. S. Babu, C. Li, X. Xiong, and A. Portner, Antimicrob. Agents Chemother. 48:1495-1502, 2004). The ability of BCX 2798 to select drug-resistant variants in vivo was examined. A variant with an Asn-to-Ser mutation at residue 173 (N173S) in HN was recovered from mice after a second passage of rSeV(hPIV-1HN) in the presence of BCX 2798 (10 mg/kg of body weight daily). The N173S mutant remained sensitive to BCX 2798 in neuraminidase inhibition assays but was more than 10,000-fold less sensitive to the compound in hemagglutination inhibition tests than rSeV(hPIV-1HN). Its susceptibility to BCX 2798 in plaque reduction assays was reduced fivefold and did not differ from that of rSeV(hPIV-1HN) in mice. The N173S mutant failed to be efficiently eluted from erythrocytes and released from cells. It demonstrated reduced growth in cell culture and superior growth in mice. The results for gel electrophoresis analysis were consistent with the loss of the N-linked glycan at residue 173 in the mutant. Sequence and structural comparisons revealed that residue 173 on hPIV-1 HN is located close to the region of the second receptor-binding site identified in Newcastle disease virus HN. Our study suggests that the N-linked glycan at residue 173 masks a second receptor-binding site on hPIV-1 HN.

Papillomavirus 3' UTR regulatory elements.

Papillomaviruses infect epithelial cells causing mainly benign lesions or warts. In some rare instances, these may progress to malignancy. For example, human papillomavirus type 16 (HPV16) is the causative agent of 60 percent of cases of cervical cancer. The replication cycle of papillomaviruses is intimately linked to epithelial differentiation. In particular, late gene expression is completed exclusively in the upper epithelial layers. Regulation of late gene expression is largely by post-transcriptional means. RNAs encoding the late proteins, the virus capsid proteins L1 and L2, can be detected in the lower layers of infected epithelia but protein is detected only in the upper layers. It is clear that cellular factors mediate this gene regulation. RNA regulatory elements that bind RNA processing factors that mediate post-transcriptional control have been identified in the 3' untranslated regions of a number of papillomaviruses. These elements, the proteins they bind and the mechanisms by which they are proposed to act are discussed. Further understanding of such host-virus molecular interactions may lead to development of novel strategies for abrogating virus infection.

Identification of upper respiratory tract pathogens using electrochemical detection on an oligonucleotide microarray.

Bacterial and viral upper respiratory infections (URI) produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment, however, depends on correct identification of the pathogen involved as antibiotics provide little or no benefit with viral infections. Here we describe a rapid and sensitive genotyping assay and microarray for URI identification using standard amplification and hybridization techniques, with electrochemical detection (ECD) on a semiconductor-based oligonucleotide microarray. The assay was developed to detect four bacterial pathogens (Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae and Mycoplasma pneumoniae) and 9 viral pathogens (adenovirus 4, coronavirus OC43, 229E and HK, influenza A and B, parainfluenza types 1, 2, and 3 and respiratory syncytial virus. This new platform forms the basis for a fully automated diagnostics system that is very flexible and can be customized to suit different or additional pathogens. Multiple probes on a flexible platform allow one to test probes empirically and then select highly reactive probes for further iterative evaluation. Because ECD uses an enzymatic reaction to create electrical signals that can be read directly from the array, there is no need for image analysis or for expensive and delicate optical scanning equipment. We show assay sensitivity and specificity that are excellent for a multiplexed format.