Human Rhinovirus C Research Articles

In silico immunoinformatics based prediction and designing of multi-epitope construct against human rhinovirus C

Human rhinovirus C (HRV-C) is an RNA virus infecting human respiratory tract. It is associated with complexities like asthma, chronic obstructive pulmonary disease, and respiratory damage. HRV-C has many serotypes. Till date there is no vaccine. Despite some limitations, corticosteroids, bronchodilators, and common cold medicines are used to treat HRV-C infections. Here, we have used immunoinformatics approach to predict suitable cytotoxic T-cell, helper T-cell and linear B-cell epitopes from the most antigenic protein. VP2 protein of Rhinovirus C53 strain USA/CO/2014-20993 was found to be most antigenic. The multi-epitope construct was designed using the best CTL, HTL and linear B-cell epitopes and attaching them with adjuvant and linkers. Interferon-gamma inducing epitopes and conformational B-cell epitopes were also predicted from the construct. Physicochemical and structural properties of the construct were satisfactory. Binding pockets were identified that could be the targets for designing effective inhibitors. Molecular docking revealed strong binding affinity of the construct with human Toll-like receptors 2 and 4. Normal mode analysis divulged stability of the docked complex. Codon optimization, in silico cloning and immune simulation analysis demonstrated suitability of the construct. These findings are likely to aid in vitro studies for developing vaccine against HRV-C.

Temperature controlled high-throughput magnetic tweezers show striking difference in activation energies of replicating viral RNA-dependent RNA polymerases.

RNA virus survival depends on efficient viral genome replication, which is performed by the viral RNA dependent RNA polymerase (RdRp). The recent development of high throughput magnetic tweezers has enabled the simultaneous observation of dozens of viral RdRp elongation traces on kilobases long templates, and this has shown that RdRp nucleotide addition kinetics is stochastically interrupted by rare pauses of 1–1000 s duration, of which the short-lived ones (1–10 s) are the temporal signature of a low fidelity catalytic pathway. We present a simple and precise temperature controlled system for magnetic tweezers to characterize the replication kinetics temperature dependence between 25°C and 45°C of RdRps from three RNA viruses, i.e. the double-stranded RNA bacteriophage Φ6, and the positive-sense single-stranded RNA poliovirus (PV) and human rhinovirus C (HRV-C). We found that Φ6 RdRp is largely temperature insensitive, while PV and HRV-C RdRps replication kinetics are activated by temperature. Furthermore, the activation energies we measured for PV RdRp catalytic state corroborate previous estimations from ensemble pre-steady state kinetic studies, further confirming the catalytic origin of the short pauses and their link to temperature independent RdRp fidelity. This work will enable future temperature controlled study of biomolecular complex at the single molecule level.

Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections

ObjectiveNewly identified human rhinovirus C (HRV-C) and human bocavirus (HBoV) cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses. MethodsA platform for culturing human airway epithelia in a three-dimensional (3D) pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction (PCR). Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA. ResultsProperties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-1, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial (HAE) cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system. ConclusionOur data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.

Detection and genotyping of rhinovirus from exacerbated asthmatic patients in Baghdad, Iraq

Asthma is a chronic disease with multiple environmental and genetic causes. Determining the role of rhinovirus in asthma incidence and exacerbation could improve the controlling measures for this disease. This study aims to detect and genotype human rhinovirus (HRV) in asthmatic patients in Iraqi population. A total of 45 patients with asthma participated in this study. Viral RNA was extracted from nasopharyngeal swabs (NPS) and cDNA was created using reverse transcriptase-polymerase chain reaction (PCR). Specific primers for HRV were used with two rounds nested-PCR to amplify the 5’-noncoding region of the viral genome. PCR products of the second round nested-PCR underwent direct sequencing and the resultant sequences were aligned with reference sequences in GenBank. MEGA 5 software was used to construct phylogenetic tree between eight successfully sequenced isolates and eight reference isolates. Alignment of viral sequence revealed highly genetic diversity between these sequences and the reference isolates. Phylogenetic tree showed that five isolates belong to Human Rhinovirus- A (HRV-A), while three isolate belong to Human Rhinovirus-C (HRV-C). The HRV-C was detected and genotyped for the first time in Iraq. The results of the current study suggest the significant role of HRV infection among patients with exacerbated asthma in Iraq. Key words: Human rhinovirus, genotyping, asthma exacerbation.

Comparative Genetic Analyses of Human Rhinovirus C (HRV-C) Complete Genome from Malaysia.

Human rhinovirus-C (HRV-C) has been implicated in more severe illnesses than HRV-A and HRV-B, however, the limited number of HRV-C complete genomes (complete 5′ and 3′ non-coding region and open reading frame sequences) has hindered the in-depth genetic study of this virus. This study aimed to sequence seven complete HRV-C genomes from Malaysia and compare their genetic characteristics with the 18 published HRV-Cs. Seven Malaysian HRV-C complete genomes were obtained with newly redesigned primers. The seven genomes were classified as HRV-C6, C12, C22, C23, C26, C42, and pat16 based on the VP4/VP2 and VP1 pairwise distance threshold classification. Five of the seven Malaysian isolates, namely, 3430-MY-10/C22, 8713-MY-10/C23, 8097-MY-11/C26, 1570-MY-10/C42, and 7383-MY-10/pat16 are the first newly sequenced complete HRV-C genomes. All seven Malaysian isolates genomes displayed nucleotide similarity of 63–81% among themselves and 63–96% with other HRV-Cs. Malaysian HRV-Cs had similar putative immunogenic sites, putative receptor utilization and potential antiviral sites as other HRV-Cs. The genomic features of Malaysian isolates were similar to those of other HRV-Cs. Negative selections were frequently detected in HRV-Cs complete coding sequences indicating that these sequences were under functional constraint. The present study showed that HRV-Cs from Malaysia have diverse genetic sequences but share conserved genomic features with other HRV-Cs. This genetic information could provide further aid in the understanding of HRV-C infection.

Molecular epidemiology of human rhinovirus in children with acute respiratory diseases in Chongqing, China.

Human rhinovirus-C (HRV-C) has been increasingly detected in patients with acute respiratory diseases (ARDs). Prolonged surveillance was performed on children with ARD to investigate the molecular epidemiology and clinical characteristics of HRV in Chongqing, China. Nasopharyngeal aspirates (NPA) were collected from hospitalized children with ARD during 2009–2012. HRV-C was genotyped by sequencing the VP4/VP2 coding region. Among the 1,567 NPAs obtained, 223 (14.2%) were HRV positive, and 75.3% of these 223 NPAs were co-infected with other viruses. HRV-A (54.7%) and HRV-C (39.9%) accounted for the majority of HRV infections. Logistic regression models demonstrated significant associations between HRV-A, HRV-C, and asthma attacks, as well as between HRV-C and wheezing. A phylogenetic tree showed that HRV-C2 was the predominant type of HRV-C, followed by HRV-C43, HRV-C1, and HRV-C17. Three novel genotypes were proposed on the basis of a low identity with the known HRVs. Our results showed that HRV-A and HRV-C were the predominant types of HRV infection, and HRV-C showed a high genetic variation in Chongqing, China. HRV infection was associated with asthma attacks and wheezing; furthermore, HRV infections played a minor role in causing severe pneumonia. This knowledge provides information for the prevention and control of HRV associated with ARDs.

Infection in Severe Asthma Exacerbations and Critical Asthma Syndrome

In chronic persistent asthma and severe acute exacerbations of bronchial asthma, infectious agents are the predominant triggers that drive disease and airway pathobiology. In acute exacerbations of bronchial asthma (AEBA) including near fatal and fatal asthma, viral agents, particularly human rhinovirus-C, respiratory syncytial virus and influenza A appear to be the more prevalent and recurring threats. Both viral, and to a lesser extent bacterial agents, can play a role, and co-infection may also be present and worsen prognosis in hospitalized patients, placing a portion at risk for critical asthma syndrome. During severe acute exacerbations, infectious agents must be treated empirically, but the initial treatment regimens can vary and viral coverage may also vary based on seasonality and patient age. Early treatment with ceftriaxone and azithromycin, along with oseltamivir in winter months, should be initiated with all cases of severe exacerbations where infection is suspected, and definitely in critical asthma syndrome until infection is excluded by appropriate diagnostic testing. In this manuscript we will outline the impact of the major viral agents on severe asthma including the data from the 2009 H1N1 influenza pandemic. The role of bacterial infections in acute exacerbations of asthma will also be reviewed as well as the benefit of empiric antibiotics and the role of macrolides in both acute and chronic asthma.

Biological characteristics and propagation of human rhinovirus-C in differentiated sinus epithelial cells

Information about the basic biological properties of human rhinovirus-C (HRV-C) viruses is lacking due to difficulties with culturing these viruses. Our objective was to develop a cell culture system to grow HRV-C. Epithelial cells from human sinuses (HSEC) were differentiated at air–liquid interface (ALI). Differentiated cultures supported 1–2 logs growth of HRV-C15 as detected by quantitative RT-PCR. Two distinguishing features of HRVs are acid lability and optimal growth at 33–34 °C. We used this system to show that HRV-C15 is neutralized by low pH (4.5). In contrast to most HRV types, replication of HRV-C15 and HRV-C41 was similar at 34 and 37°C. The HSEC ALI provides a useful tool for quantitative studies of HRV-C replication. The ability of HRV-C to grow equally well at 34°C and 37°C may contribute to the propensity for HRV-C to cause lower airway illnesses in infants and children with asthma.

Update on Rhinovirus and Coronavirus Infections

Rhinoviruses and coronaviruses cause significant morbidity in immunocompetent people of all ages and in patients with underlying chronic medical or immunosuppressed conditions. Newer diagnostic tests, such as polymerase chain reaction (PCR), have expanded our understanding of these respiratory viruses in clinical infections. These sensitive diagnostic tests have been used to describe new members of these virus families, such as human rhinovirus C (HRVC) and human coronavirus NL-63 (HCoV-NL63). The epidemiology of these newly described viruses will help us develop better intervention strategies.

Advances of human rhinovirus C

Recently,human rhinovirus C(HRV-C) was identified as a new species of HRV.HRV-C was also identified in more clinical samples worldwide,especially in those obtained from children with respiratory illnesses.In children,HRV-C was most often associated with wheezing and asthma.It was increasingly shown to be associated with asthma,compared with those of HRV-A and HRV-B.A large diversity of recombination in HRV has become obvious,the occurrence of novel HRV may become highly virulent.The purpose of this article was to review recent progresses of the prevalence,clinical feature and possible therapy of HRV-C. Key words: Rhinovirus; Human rhinovirus C

Epidemiology of human rhinovirus C (HRV-C) in Hong Kong reveals a potential HRV-C subgroup

Epidemiology of human rhinovirus C (HRV-C) in Hong Kong reveals a potential HRV-C subgroup

Clinical severity and molecular typing of human rhinovirus C strains during a fall outbreak affecting hospitalized patients

Background The circulation rate and the clinical severity of infections caused by members of the new human rhinovirus C (HRV-C) species remain to be defined. Objectives To investigate the epidemiologic and clinical impact of HRV-C strains in a fall outbreak interesting hospitalized patients. Study design HRV species (A–C) were determined by phylogenetic analysis following amplification of two genome regions (5′NCR and VP4/VP2) by RT-PCR. HRV species were correlated with age, respiratory tract involvement, clinical symptoms, and HRV load in respiratory secretions. Results During the first week of the period October–November 2008, single HRV infections were associated with 95% of all respiratory syndromes affecting hospitalized patients. Then, HRV infections (single + coinfections) interested about 90% of positive samples until the end of October, when they declined in frequency until reaching about 30% at the end of November. Overall, 104 HRV strains were detected and, of these, 90 could be classified by phylogenetic analysis, as follows: 45 HRV-A, 12 HRV-B, 28 HRV-C, and 5 human enterovirus D strains. HRV-C identity was confirmed by detection of cis-acting replication elements ( cre) in 23/23 strains. As for severity of respiratory syndromes, unlike HRV-A and HRV-B strains, HRV-C strains were responsible for a significantly higher rate ( p < 0.05) of lower respiratory tract infections in the pediatric as compared to adult patient population. Conclusions HRV-C strains have been shown to circulate at a rate intermediate between HRV-A and HRV-B strains, showing a greater degree of clinical severity in the pediatric population.

5′ Noncoding Region Alone Does Not Unequivocally Determine Genetic Type of Human Rhinovirus Strains

During the last couple of years there has been a delightful increase in interest in genetic typing of human rhinoviruses. This is to a large extent due to the discovery of a proposed novel clade, human rhinovirus C (HRV-C). As a consequence, new methods have been reported aiming at unequivocal distinction of traditional HRV prototype strains as well as the newly found uncultivable HRV-C strains. We read with interest the article by Kiang and coworkers (2) describing reverse transcription-PCR-sequencing applications for genetic typing of human rhinoviruses targeting the 5′noncoding region (5′ NCR). A similar approach with largely similar results was published earlier (6). Relatively conserved areas within this region enable broad-spectrum primer design for sensitive methods. However, there are several issues to consider when using the 5′ NCR for genetic typing of HRV. Current taxonomy and classification of picornaviruses are based on capsid region coding sequences. On the basis of this region, a group of previously characterized novel HRV strains form one distinct clade (5, 7) (Fig. ​(Fig.1A),1A), a fact that has also been the basis of the proposal of the Picornavirus Study Group to form a new species, HRV-C, within the Enterovirus genus (4). However, in the article by Kiang et al., on the basis of the partial 5′ NCR sequences, the designated HRV-C strains clustered within the HRV-A clade (2). In contrast, the strains labeled HRV-C in this article formed a clade of their own. As a consequence, because 5′ NCR sequences do not segregate the designated HRV-C from HRV-A (Fig. ​(Fig.1B),1B), they should not be used for typing of new strains. Nevertheless, this region is quite suitable for selected topics of molecular epidemiology, such as analysis of short-term transmission routes (1, 8) or tentative prediction of genetic type as in human enteroviruses (HEV) (9). The sequences nominated as HRV-C by Kiang et al. (2) and by Lee et al. (6) form a new clade in the 5′ NCR. The exact taxonomic position of this clade should be determined according to the clustering of these strains in the capsid region. Clearly, it is divergent from all known HRV and HEV clades in the 5′ NCR, but the decision on whether the strains represent HRV-C or some other picornavirus group cannot be made on the basis of the 5′ NCR alone. FIG. 1. Phylogenetic trees in the VP4/VP2 capsid coding region (A) and in the 5′ NCR (B) of different species of the enterovirus genus showing different clustering of the species in the two regions. Trees were constructed with MEGA4 using the neighbor-joining ... The area close to the beginning of the open reading frame in the 5′ NCR is known to be a recombination hot spot in HEV. Although frequent recombination has not yet been shown for HRV, the analysis of the complete genome sequence data of all HRV prototype strains has not yet been published. Furthermore, the number of completely sequenced genomes of circulating HRV strains has remained low and is too low to conclude that the evolution in the 5′ NCR is always congruent with that of the capsid. Therefore, we would see phylogenetic analysis of the 5′ NCR of HRV as a welcome addition to HRV research, but not a surrogate of capsid coding sequence-based typing.