Genomic characterization of seven reassortant influenza B viruses in Hangzhou, China / 中华微生物学和免疫学杂志

Objective:To analyze the prevalence of influenza B virus in Hangzhou from 2014 to 2020 and the genetic evolution of seven reassortant strains of influenza B virus.Methods:Influenza viruses were isolated from throat swabs collected from 16 943 patients with influenza-like illness in Hangzhou from January 2014 to December 2020. The subtypes of influenza viruses were identified by real-time RT-PCR. Eight genes ( PB2, PB1, PA, HA, NP, NA, MP and NS) of influenza B viruses were amplified with specific primers and then analyzed with nanopore sequencing and phylogenetic analysis. Results:From January 2014 to December 2020, there were 1 090 influenza B virus-positive samples, including 474 samples of Yamagata lineage and 616 samples of Victoria lineage, were identified in Hangzhou with an overall positive rate of 6.43% (1 090/16 943). Whole genomes of 228 strains of influenza B virus were obtained by nanopore sequencing and seven reassortant strains of influenza B virus were found. There were four reassortant influenza B viruses of Yamagata lineage with NA gene fragments from viruses of Victoria lineage, two strains of Yamagata lineage (H644_BY and H648_BY) with NP and NA gene fragments from Victoria lineage and one strain of Victoria lineage with PB2, PB1, PA and NS gene fragments from Yamagata lineage. Meanwhile, these seven strains possessed several mutations in the antigenic sites of HA and NA genes. Conclusions:Several rare reassortant strains of influenza B virus with epidemic potential were detected in Hangzhou from 2014 to 2020, which indicated that the traditional detection methods should be improved and more attention should be paid to the reassortant influenza B viruses and the match between epidemic and vaccine strains.

Genetic variations and epidemiological characteristics of influenza B virus in Xinxiang from 2012 to 2019 / 中华疾病控制杂志

Objective To investigate the genetic variation and epidemiological characteristics of influenza B virus in Xinxiang to provide policy basis for local influenza vaccination. Methods The influenza surveillance data in Xinxiang from January 2012 to February 2019 was analyzed. 23 isolated influenza B virus were randomly selected for hemagglutinin (HA) and neuraminidase (NA) gene sequencing. Sequence alignment was conducted by using DNAman software and phylogenetic tree analysis was conducted using Neighbor-Joining method. Results Yamagata (BY) and Victoria (BV) strains of influenza B virus circulated alternately every other year in Xinxiang, mainly among people aged 0-15 years (91.4%). The dominant influenza B lineages from 2015 to 2016 and from 2017 to 2018 did not match the corresponding trivalent vaccine strain of the year. The HA phylogenetic tree revealed that 87.5% (7/8) of BV strains coexisted with the vaccine strain in one branch, while 88.98% (8/9) of BY strains from 2013 to 2015 were not in the same branch as the corresponding vaccine strain, with 5 epitope site mutions N116K, S150L, N165Y, D196N and N202S. No drug-resistant site mutation was identified in the NA gene. A total of 6 intra-lineage reassortants were identified. Conclusions The influenza B lineage in the trivalent vaccine recommended by WHO did not match the dominant circulating B lineage of Xinxiang in some epidemic year. Therefore, quadrivalent vaccines are recommended to use in susceptible population, especially under the age of 15. In addition, there are large variations in HA gene of the epidemic BY strain compared with the corresponding vaccine. Then, a vaccine more sutable for the epidemic strains in China is expected to be developed.

Characterisation of a rare, reassortant human G10P[14] rotavirus strain detected in Honduras

BACKGROUND Although first detected in animals, the rare rotavirus strain G10P[14] has been sporadically detected in humans in Slovenia, Thailand, United Kingdom and Australia among other countries. Earlier studies suggest that the strains found in humans resulted from interspecies transmission and reassortment between human and bovine rotavirus strains. OBJECTIVES In this study, a G10P[14] rotavirus genotype detected in a human stool sample in Honduras during the 2010-2011 rotavirus season, from an unvaccinated 30-month old boy who reported at the hospital with severe diarrhea and vomiting, was characterised to determine the possible evolutionary origin of the rare strain. METHODS For the sample detected as G10P[14], 10% suspension was prepared and used for RNA extraction and sequence independent amplification. The amplicons were sequenced by next-generation sequencing using the Illumina MiSeq 150 paired end method. The sequence reads were analysed using CLC Genomics Workbench 6.0 and phylogenetic trees were constructed using PhyML version 3.0. FINDINGS The next generation sequencing and phylogenetic analyses of the 11-segmented genome of the G10P[14] strain allowed classification as G10-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3. Six of the genes (VP1, VP2, VP3, VP6, NSP2 and NSP4) were DS-1-like. NSP1 and NSP5 were AU-1-like and NSP3 was T6, which suggests that multiple reassortment events occurred in the evolution of the strain. The phylogenetic analyses and genetic distance calculations showed that the VP7, VP4, VP6, VP1, VP3, NSP1, NSP3 and NSP4 genes clustered predominantly with bovine strains. NSP2 and VP2 genes were most closely related to simian and human strains, respectively, and NSP5 was most closely related to a rhesus strain. MAIN CONCLUSIONS The genetic characterisation of the G10P[14] strain from Honduras suggests that its genome resulted from multiple reassortment events which were possibly mediated through interspecies transmissions.

Construction of a reassortant influenza B virus with reverse genetics system / 中华微生物学和免疫学杂志

Objective To construct a reverse genetic platform for influenza B virus and to rescue influenza B virus.Methods Eight plasmids carrying the gene segments of B/Florida/4/2006 virus were constructed by using the bidirectional promoter vector pHW2000.293T cells were co-cultured with MadinDarby canine kidney (MDCK) cells and then transfected with the eight plasmids.The supernatants of cell culture and cell debris were collected after transfection and then injected into embryonated chicken eggs and MDCK cells for rescuing the influenza B virus strains.Results This reverse genetic system could be used for the preparation of reassortant influenza B virus strains.The titers of hemagglutination units of the rescued virus achieved 128-256/50μl.Most of the reassortant virus particles were spherical under electron microscope.Conclusion The pHW2000 reverse genetic system could be used for the rescue of influenza B virus.Moreover,it could also be used for the construction of influenza B virus with specific mutations for further in vestigation on the characteristics of influenza B virus and the construction of vaccine strain.

Influenza A H1N1 virus in Indian pigs & its genetic relatedness with pandemic human influenza A 2009 H1N1.

Background & objectives: With the emergence of a new reassortant influenza A H1N1 virus that caused the 2009 pandemic it was felt necessary that pigs should be closely monitored for early detection of any influenza virus infection. Therefore, we investigated disease outbreaks with clinical history suggestive for swine influenza reported to our laboratory by owners of affected pig farms in Uttar Pradesh. Methods: Detection of swine influenza A virus (SIV) was attempted by isolation in embryonated chicken eggs. Presence of virus was detected by haemagglutination (HA) test and RT-PCR for amplification of different gene segments, cloning and sequencing. BLAST analysis of sequence data, phylogenetic analysis and mutation analysis based on HA, NA and matrix genes was done. Results: SIV could be isolated from one farm and all eight gene segments amplified by RT-PCR. BLAST analysis of partial nucleotide sequences and phylogenetic analysis using nucleotide sequence of HA (601 nt), NA (671 nt) and M (1031 nt) genes indicated close genetic relationship of the Indian swine isolate (A/Sw/UP-India-IVRI01/2009) with human pandemic 2009 (H1N1). The HA gene showed close relationship with the viruses of “North American Swine” lineage, whereas the NA and M genes clustered with the viruses of “Eurasian Swine” lineage, indicating a novel HA-NA reassortant. The remaining of 5 genes (NP, PA, PB1, PB2 and NS) belonged to “North American Swine” lineage. Interpretation & conclusions: This is perhaps the first report describing swine influenza among Indian pigs caused by an influenza A H1N1 virus sharing close homology with the human pandemic (H1N1) 2009 virus. Further reassortment with circulating influenza viruses must be closely monitored.

Genetic Analysis and Rescue of a Triple-reassortant H3N2 Influenza A Virus Isolated From Swine in Eastern China / 中国病毒学·英文版

One influenza H3N2 virus, A/swine/Shandong/3/2005 (Sw/SD/3/2005), was isolated from pigs with respiratory disease on a farm in eastern China. Genetic analysis revealed that Sw/SD/3/2005 was a triple-reassortant virus with a PB2 gene from human-like HIN1, NS from classical swine H1NI, and the remaining genes from human-like H3N2 virus. These findings further support the concept that swine can serve as reservoir or mixing vessels of influenza virus strains and maintain genetic and antigenic stability of viruses. Furthermore, we have successfully established a reverse genetics system based on eight plasmids and rescued Sw/SD/3/2005 through cell transfection. HI tests and RT-PCR confirmed that the rescued virus maintained the biological properties of the wild type Sw/SD/3/2005. The successful establishment of the reverse genetics system of Sw/SD/3/2005 will enable us to conduct extensive studies of the molecular evolution of H3N2 influenza viruses in swine.

Rotavirus Vaccine / 한양의대학술지

The development and introduction of rotavirus vaccines into global immunization programs has been a high priority for many international immunization agencies. Vaccine development was started with animal rotaviruses which can be distinguished from human strains on the bases of type specific surface antigens. Bovine (WC3), rhesus (RRV), and lamb (LLR) rotaviruses were the potential candidates for vaccine development. They established the efficacy of animal rotavirus vaccines in infants against severe rotavirus diarrhea, but failed to show consistent efficacy in other trials. Animal rotavirus vaccines were improved by the development of animal-human reassortant vaccines containing the attenuation properties of animal strains and individual genes encoding the outer capsid proteins of human strains. Attenuated human rotavirus vaccine development was designed based on the results of extensive studies on natural rotavirus infections conferring the protection against subsequent infections and reduction of diarrhea severity and heterotypic protections. RIX4414 was a potential candidate for attenuated human rotavirus vaccine. Two live oral rotavirus vaccines are currently licensed in many countries; Rota-Teq(MSD) combines bovine(WC3)-human reassortant strains containing five human serotypes. Rotarix(GSK) is derived from the attenuated human rotavirus strain(RIX4414). Each vaccine has proven highly effective in preventing severe rotavirus diarrhea in children and safe from the possible complication of intussusceptions.

Control of Influenza:Development of Live Vaccine / 감염과화학요법

Although trivalent subunit vaccine has been available, the influenza vaccine has been under-utilized because of cumbersome route of vaccination and low level of protection. Therefore, there has always been a great need to develop live attenuated influenza vaccine which can be administered through nasal route and elicit better immunogenicity. Through conventional repeated passage at low temperature, a live influenza vaccine carrier could be established. By reassortant formation between the 'cold- adapted' vaccine carrier and virulent strains, a prototype of trivalent live influenza vaccine is developed. Influenza A virus was adapted to replicate at low temperature. Serial passage at progressively lower temperature (30degrees C, 27degrees C and 24degrees C)resulted in the generation of cold-adapted (ca), temperature-sensitive (ts) mutant and attenuation (att) phenotype. This strain was evaluated for their ability to protect mice from challenge with same subtype and different subtype of influenza A virus. The study showed that vaccination of mice with live attenuated influenza virus provided complete protection against homologous and heterologous virus challenge. We also evaluated therapeutic potential of ca influenza virus. The mice infected with ca virus before challenge with wild type viruses or infected with simultaneously showed reduced clinical symptoms suggesting potential therapeutic effects.

Control of Influenza:Development of Live Vaccine / 감염과화학요법

Although trivalent subunit vaccine has been available, the influenza vaccine has been under-utilized because of cumbersome route of vaccination and low level of protection. Therefore, there has always been a great need to develop live attenuated influenza vaccine which can be administered through nasal route and elicit better immunogenicity. Through conventional repeated passage at low temperature, a live influenza vaccine carrier could be established. By reassortant formation between the 'cold- adapted' vaccine carrier and virulent strains, a prototype of trivalent live influenza vaccine is developed. Influenza A virus was adapted to replicate at low temperature. Serial passage at progressively lower temperature (30degrees C, 27degrees C and 24degrees C)resulted in the generation of cold-adapted (ca), temperature-sensitive (ts) mutant and attenuation (att) phenotype. This strain was evaluated for their ability to protect mice from challenge with same subtype and different subtype of influenza A virus. The study showed that vaccination of mice with live attenuated influenza virus provided complete protection against homologous and heterologous virus challenge. We also evaluated therapeutic potential of ca influenza virus. The mice infected with ca virus before challenge with wild type viruses or infected with simultaneously showed reduced clinical symptoms suggesting potential therapeutic effects.