Detailed Evolutionary Analyses of the F Gene in the Respiratory Syncytial Virus Subgroup A

We performed evolution, phylodynamics, and reinfection-related antigenicity analyses of respiratory syncytial virus subgroup A (RSV-A) fusion (F) gene in globally collected strains (1465 strains) using authentic bioinformatics methods. The time-scaled evolutionary tree using the Bayesian Markov chain Monte Carlo method estimated that a common ancestor of the RSV-A, RSV-B, and bovine-RSV diverged at around 450 years ago, and RSV-A and RSV-B diverged around 250 years ago. Finally, the RSV-A F gene formed eight genotypes (GA1‑GA7 and NA1) over the last 80 years. Phylodynamics of RSV-A F gene, including all genotype strains, increased twice in the 1990s and 2010s, while patterns of each RSV-A genotype were different. Phylogenetic distance analysis suggested that the genetic distances of the strains were relatively short (less than 0.05). No positive selection sites were estimated, while many negative selection sites were found. Moreover, the F protein 3D structure mapping and conformational epitope analysis implied that the conformational epitopes did not correspond to the neutralizing antibody binding sites of the F protein. These results suggested that the RSV-A F gene is relatively conserved, and mismatches between conformational epitopes and neutralizing antibody binding sites of the F protein are responsible for the virus reinfection.

Molecular Characterization and Pathology of Virulent Newcastle Disease Virus Isolated from Broiler Chickens in District Lahore, Pakistan

Background: This study elucidated the molecular detection and pathological alterations in broiler chickens naturally infected with field circulating NDV strains along with their phylogenomic dynamics. Methods: Morbid tissue samples of diseased/dead chickens were collected from 100 poultry flocks presented to poultry disease diagnostic laboratories from September 2018 to August 2019. Samples were subjected to molecular detection of NDV along with phylogenetic analysis and subsequent gross and histopathological examination. Result: Based on RT-PCR results, the positivity of NDV was 04/100 (4%). Genetic analysis of the NDV Fusion (F) gene revealed 98.92% and 98.74% similarity with Iranian and Pakistani isolates, respectively. The evolutionary tree showed that present study isolates were placed in a clade belongs to genotype Vll sub-genotype i and l. Necropsy examinations revealed the petechial haemorrhages associated with multifocal necrosis in gastrointestinal and respiratory organs. Besides these pathological findings, amino acid sequence of F gene revealed that study isolates are having pathogenic potential similar to the velogenic strains of NDV. Based on all essential analyses, the present study concluded that the evolution and distribution of the Newcastle disease virus of various genotypes VIIi and VIIl in Pakistan are having significant pathogenic potential. Therefore, it emphasizes developing ND vaccine from indigenous strains for better protection of commercial poultry in Pakistan.

Molecular typing and epidemiologic profiles of human metapneumovirus infection among children with severe acute respiratory infection in Huzhou, China

Background Human metapneumovirus (hMPV) is one of the important pathogens in infant respiratory tract infection. However, the molecular epidemiology of hMPV among children < 14 years of age hospitalized with severe acute respiratory infection (SARI) is unclear. We investigated the hMPV infection status and genotypes of children hospitalized with SARI from January 2016 to December 2020 in Huzhou, China. Methods A nasopharyngeal flocked swab, nasal wash, or nasopharyngeal swab/or opharyngeal swab combination sample was collected from children with SARI in Huzhou from January 2016 to December 2020. Quantitative reverse transcription-polymerase chain reaction was performed to detect hMPV RNA. The hMPV F gene was amplified and sequenced, followed by analysis using MEGA software (ver. 7.0). Epidemiological data were analyzed using Microsoft Excel 2010 and SPSS (ver. 22.0) software. Results A total of 1133 children with SARI were recruited from 2016 to 2020. Among them, 56 (4.94%) were positive for hMPV-RNA. Children < 5 years of age accounted for 85.71% of the positive cases. The hMPV incidence was high in spring and winter, especially in December and January to March. Phylogenetic analysis of the F-gene sequences of 28 hMPV strains showed that the A1, B1, and B2 genotypes were prevalent in Huzhou, and the dominant hMPV genotype varied according to surveillance year. Conclusions HMPV is an important respiratory pathogen in children in Huzhou, with a high incidence in winter and spring in children < 5 years of age. In this study, genotypes A1, B1, and B2 were the most prevalent.

The Prevalence and Genetic Characterization of Human Metapneumovirus in Bulgaria, 2016–2019

<b><i>Introduction:</i></b> We investigated the prevalence of human metapneumovirus (hMPV) among patients with acute respiratory infections in Bulgaria, and performed genetic characterization of the F gene of these strains. <b><i>Methods:</i></b> Nasopharyngeal swabs collected from patients of a range of ages were tested by using real-time PCR for 12 respiratory viruses. The F gene was sequenced, and phylogenetic and amino acid analyses of the F gene/protein were performed. <b><i>Results:</i></b> A total of 1,842 patients were examined during a 3-year period; 1,229 patients (66.7%) were positive for at least one respiratory virus. hMPV was identified in 83 (4.5%) patient samples. Eleven (13%) of hMPV-positive patients were coinfected with another respiratory virus. The hMPV incidence rate in the 2016/2017, 2017/2018, and 2018/2019 winter seasons was 5.4, 5.4, and 3.1%, respectively. hMPV was mainly detected in specimens collected between January and May (89.2% of cases). The incidence of hMPV infection was highest (5.1%) among the youngest age-group (0–4 years), where hMPV was a causative agent in 8.1 and 4.8% of bronchiolitis and pneumonia cases, respectively. Among the patients aged ≥5 years, hMPV was detected in 2.2 and 3.2% of cases of pneumonia and central nervous system infections, respectively. Phylogenetic analysis of the F gene showed that the sequenced hMPV strains belonged to the A2b, B1, and B2 genotypes. Numerous amino acid substitutions were identified compared with the NL00/1 prototype strain. <b><i>Conclusion:</i></b> This study revealed the significant role of hMPV as a causative agent of serious respiratory illnesses in early childhood, and also demonstrated year-to-year changes in hMPV prevalence and genetic diversity in circulating strains.

Pathotyping of Newcastle disease virus: A novel single BsaHI digestion method of detection and differentiation of avirulent strains (lentogenic and mesogenic vaccine strains) from virulent virus

We provide a novel single restriction enzyme (RE) (BsaHI) digestion approach for detecting distinct pathotypes of the Newcastle disease virus (NDV). After scanning 4000 F gene nucleotide sequences in the NCBI database, a single RE (BsaHI) digesting site was discovered in the cleavage site. APMV-I "F gene" Class II specific primer-based reverse transcriptase PCR (RT-PCR) was utilized to amplify a 535 bp fragment, which was then digested with a single RE (BsaHI) for pathotyping avian NDV field isolates and pigeon paramyxovirus-1 isolates. The avirulent (lentogenic and mesogenic strains) produce 189 and 346 bp fragments, respectively, but the result in velogenic strains remains undigested with 535 bp fragments. In addition, 45 field NDV isolates and 8 vaccine strains were used to confirm the approach. The sequence-based analysis also agrees with the data obtained utilizing the single RE (BsaHI) digestion approach. The proposed technique had the potential to distinguish between avirulent and virulent strains in a short space of time, making it valuable in NDV surveillance and monitoring research.

Molecular Typing and Epidemiology Profiles of Human Metapneumovirus Infection Among Children With Severe Acute Respiratory Infection in Huzhou, China

Background: Human metapneumovirus (hMPV) is one of the important pathogens of infant respiratory tract infection. However, the molecular epidemiological information relating to hMPV among children hospitalized patients with severe acute respiratory infection (SARI) have not been thoroughly studied as yet. To investigate the infection status and genotypes of hMPV among children hospitalized patients with SARI from January 2016 to December 2020 in Huzhou, China. Methods: A nasopharyngeal flocked swab, nasal wash or combination of nasopharyngeal swab and oropharyngeal swab samples were collected from children with SARI in Huzhou from January 2016 to December 2020. RT-PCR was used to detect the nucleic acid of hMPV. F gene was amplified and sequenced for the positive nucleic acid samples of hMPV. The obtained gene sequences were analyzed by MEGA software (version 7.0). Epidemiological data were analyzed using Microsoft Excel 2010 and service solutions (SPSS) 21.0 software.Results: A total of 1133 children with SARI were collected from 2016 to 2020. Among them, 56 cases were positive for hMPV nucleic acid, with a positive rate of 4.94%. Children under 5 years old accounted for 85.71% of the total positive cases. Higher activity of hMPV infection could be seen in the period in Spring and Winter. and the main epidemic months were December and January-March. The F gene sequences of 28 strains of hMPV were obtained by co-sequencing. Phylogenetic analysis showed that there were A1, B1 and B2 genotypes of hMPV prevalent in Huzhou, and the dominant genotype of hMPV during our study period varied according to surveillance year.Conclusions: HMPV is one of the important pathogens causing acute respiratory virus infection in children in Huzhou, with high incidence in winter and spring seasons and children under the age of 5，A1, B1 and B2 are three prevalent genotypes.

Epitope Peptide-Based Predication and Other Functional Regions of Antigenic F and HN Proteins of Waterfowl and Poultry Avian Avulavirus Serotype-1 Isolates From Uganda

Uganda is a Newcastle disease (ND) endemic country where the disease is controlled by vaccination using live LaSota (genotype II) and I2 (genotype I) vaccine strains. Resurgent outbreak episodes call for an urgent need to understand the antigenic diversity of circulating wild Avian Avulavirus serotype-1 (AAvV-1) strains. High mutation rates and the continuous emergence of genetic and antigenic variants that evade immunity make non-segmented RNA viruses difficult to control. Antigenic and functional analysis of the key viral surface proteins is a crucial step in understanding the antigen diversity between vaccine lineages and the endemic wild ND viruses in Uganda and designing ND peptide vaccines. In this study, we used computational analysis, phylogenetic characterization, and structural modeling to detect evolutionary forces affecting the predicted immune-dominant fusion (F) and hemagglutinin-neuraminidase (HN) proteins of AAvV-1 isolates from waterfowl and poultry in Uganda compared with that in LaSota vaccine strain. Our findings indicate that mutational amino acid variations at the F protein in LaSota strain, 25 poultry wild-type and 30 waterfowl wild-type isolates were distributed at regions including the functional domains of B-cell epitopes or N-glycosylation sites, cleavage site, fusion site that account for strain variations. Similarly, conserved regions of HN protein in 25 Ugandan domestic fowl isolates and the representative vaccine strain varied at the flanking regions and potential linear B-cell epitope. The fusion sites, signal peptides, cleavage sites, transmembrane domains, potential B-cell epitopes, and other specific regions of the two protein types in vaccine and wild viruses varied considerably at structure by effective online epitope prediction programs. Cleavage site of the waterfowl isolates had a typical avirulent motif of 111GGRQGR'L117 with the exception of one isolate which showed a virulent motif of 111GGRQKR'F117. All the poultry isolates showed the 111GRRQKR'F117 motif corresponding to virulent strains. Amino acid sequence variations in both HN and F proteins of AAvV-1 isolates from poultry, waterfowl, and vaccine strain were distributed over the length of the proteins with no detectable pattern, but using the experimentally derived 3D structure data revealed key-mapped mutations on the surfaces of the predicted conformational epitopes encompassing the experimental major neutralizing epitopes. The phylogenic tree constructed using the full F gene and partial F gene sequences of the isolates from poultry and waterfowl respectively, showed that Ugandan ND aquatic bird and poultry isolates share some functional amino acids in F sequences yet do remain unique at structure and the B-cell epitopes. Recombination analyses showed that the C-terminus and the rest of the F gene in poultry isolates originated from prevalent velogenic strains. Altogether, these could provide rationale for antigenic diversity in wild ND isolates of Uganda compared with the current ND vaccine strains.

Molecular Epidemiology of Human Metapneumovirus Infection among Children with Severe Acute Respiratory Infection in Huzhou from 2016 to 2020

Background: Human metapneumovirus (hMPV) is one of the important pathogens of infant respiratory tract infection. However, the molecular epidemiological information relating to hMPV among children hospitalized patients with severe acute respiratory infection (SARI) have not been thoroughly studied as yet. To investigate the infection status and genotypes of hMPV among children hospitalized patients with SARI from January 2016 to December 2020 in Huzhou, China. Methods: A nasopharyngeal flocked swab, nasal wash or combination of nasopharyngeal swab and oropharyngeal swab samples were collected from children with SARI in Huzhou from January 2016 to December 2020. RT-PCR was used to detect the nucleic acid of hMPV. F gene was amplified and sequenced for the positive nucleic acid samples of hMPV. The obtained gene sequences were analyzed by MEGA software (version 7.0). Epidemiological data were analyzed using Microsoft Excel 2010 and service solutions (SPSS) 21.0 software.Results: A total of 1133 children with SARI were collected from 2016 to 2020. Among them, 56 cases were positive for hMPV nucleic acid, with a positive rate of 4.94%. Children under 5 years old accounted for 85.71% of the total positive cases. Higher activity of hMPV infection could be seen in the period in Spring and Winter. and the main epidemic months were December and January-March. The F gene sequences of 28 strains of hMPV were obtained by co-sequencing. Phylogenetic analysis showed that there were A1, B1 and B2 genotypes of hMPV prevalent in Huzhou, and the dominant genotype of hMPV during our study period varied according to surveillance year.Conclusions: HMPV is one of the important pathogens causing acute respiratory virus infection in children in Huzhou, with high incidence in winter and spring seasons and children under the age of 5，A1, B1 and B2 are three prevalent genotypes.

Isolation and Molecular Characterization of Newcastle Disease Virus in Layers

Background: Newcastle disease (ND) in spite of the availability of vaccines remains a constant threat to poultry producers worldwide. It is prevalent in Indian subcontinent and leads to economic losses. The present study was aimed with isolate and identify virulent Newcastle disease virus (NDV) in layer poultry from field outbreaks.Methods: Total 47 samples consisting of nasal (05), oropharyngeal (13) and cloacal swabs (11) and tissue samples consisting of trachea (07), lungs (06), larynx (05) were collected from layer birds. For isolation of NDV swab and tissue samples were inoculated in 9-11 days old embryonated eggs via allantoic cavity route. After preparing the viral inoculum, 47 suspected samples (29 swab and 18 tissue samples) were inoculated in 141 embryonated eggs to isolate the virus.Result: Out of 47 samples 10 (21.27%) samples were positive for HA activity. All the 10 isolates showing HA activity subjected to Reverse-Transcriptase PCR of F gene and 6 were found positive in RT-PCR for F1 gene. The PCR amplified product showed amplicon at 356 bp and 254 bp positive for F1 and F2 gene, respectively. On basis of F gene, 06 (50%) isolates were considered as virulent Newcastle Disease Virus. One isolate sequence was submitted at NCBI with accession MT890653 On phylogenetic analysis MT890653 designated as Class II/ genotype II/ virulent strain and had the motif 112R-R-R-K-R-F117 at the cleavage site of the fusion protein.