Red knots in Europe: a dead end host species or a new niche for highly pathogenic avian influenza?

The 2020/2021 epidemic in Europe of highly pathogenic avian influenza virus (HPAIV) of subtype H5 surpassed all previously recorded European outbreaks in size, genotype constellations and reassortment frequency and continued into 2022 and 2023. The causative 2.3.4.4b viral lineage proved to be highly proficient with respect to reassortment with cocirculating low pathogenic avian influenza viruses and seems to establish an endemic status in northern Europe. A specific HPAIV reassortant of the subtype H5N3 was detected almost exclusively in red knots (Calidris canutus islandica) in December 2020. It caused systemic and rapidly fatal disease leading to a singular and self-limiting mass mortality affecting about 3500 birds in the German Wadden Sea, roughly 1 % of the entire flyway population of islandica red knots. Phylogenetic analyses revealed that the H5N3 reassortant very likely had formed in red knots and remained confined to this species. While mechanisms of virus circulation in potential reservoir species, dynamics of spill-over and reassortment events and the roles of environmental virus sources remain to be identified, the year-round infection pressure poses severe threats to endangered avian species and prompts adaptation of habitat and species conservation practices.

Characterization of Influenza D Virus Reassortant Strain in Swine from Mixed Pig and Beef Farm, France.

Influenza D virus was isolated from pigs on a mixed pig and beef farm in France. Investigation suggested bull-to-pig transmission and spread among pigs. The swine influenza D virus recovered was a reassortant of D/660 and D/OK lineages. Reported mutations in the receptor binding site might be related to swine host adaptation.

Genetic Reassortment in a Child Coinfected with Two Influenza B Viruses, B/Yamagata Lineage and B/Victoria-Lineage Strains.

We identified a child coinfected with influenza B viruses of B/Yamagata and B/Victoria lineages, in whom we analyzed the occurrence of genetic reassortment. Plaque purification was performed using a throat swab specimen from a 9-year-old child, resulting in 34 well-isolated plaques. The genomic composition of eight gene segments (HA, NA, PB1, PB2, PA, NP, M, and NS genes) for each plaque was determined at the lineage level. Of the 34 plaques, 21 (61.8%) had B/Phuket/3073/2013 (B/Yamagata)-like sequences in all gene segments, while the other 13 (38.2%) were reassortants with B/Texas/02/2013 (B/Victoria)-like sequences in 1-5 of the 8 segments. The PB1 segment had the most B/Victoria lineage genes (23.5%; 8 of 34 plaques), while PB2 and PA had the least (2.9%; 1 of 34 plaques). Reassortants with B/Victoria lineage genes in 2-5 segments showed the same level of growth as viruses with B/Yamagata lineage genes in all segments. However, reassortants with B/Victoria lineage genes only in the NA, PB1, NP, or NS segments exhibited reduced or undetectable growth. We demonstrated that various gene reassortments occurred in a child. These results suggest that simultaneous outbreaks of two influenza B virus lineages increase genetic diversity and could promote the emergence of new epidemic strains.

Emergence of Eurasian Avian-Like Swine Influenza A (H1N1) virus in a child in Shandong Province, China.

BACKGROUND: Influenza A virus infections can occur in multiple species. Eurasian avian-like swine influenza A (H1N1) viruses (EAS-H1N1) are predominant in swine and occasionally infect humans. A Eurasian avian-like swine influenza A (H1N1) virus was isolated from a boy who was suffering from fever; this strain was designated A/Shandong-binzhou/01/2021 (H1N1). The aims of this study were to investigate the characteristics of this virus and to draw attention to the need for surveillance of influenza virus infection in swine and humans. METHODS: Throat-swab specimens were collected and subjected to real-time fluorescent quantitative polymerase chain reaction (RT‒PCR). Positive clinical specimens were inoculated onto Madin-Darby canine kidney (MDCK) cells to isolate the virus, which was confirmed by a haemagglutination assay. Then, whole-genome sequencing was carried out using an Illumina MiSeq platform, and phylogenetic analysis was performed with MEGA X software. RESULTS: RT‒PCR revealed that the throat-swab specimens were positive for EAS-H1N1, and the virus was subsequently successfully isolated from MDCK cells; this strain was named A/Shandong-binzhou/01/2021 (H1N1). Whole-genome sequencing and phylogenetic analysis revealed that A/Shandong-binzhou/01/2021 (H1N1) is a novel triple-reassortant EAS-H1N1 lineage that contains gene segments from EAS-H1N1 (HA and NA), triple-reassortant swine influenza H1N2 virus (NS) and A(H1N1) pdm09 viruses (PB2, PB1, PA, NP and MP). CONCLUSIONS: The isolation and analysis of the A/Shandong-binzhou/01/2021 (H1N1) virus provide further evidence that EAS-H1N1 poses a threat to human health, and greater attention should be given to the surveillance of influenza virus infections in swine and humans.

Pathogenicity and escape to pre-existing immunity of a new genotype of swine influenza H1N2 virus that emerged in France in 2020.

In 2020, a new genotype of swine H1N2 influenza virus (H1avN2-HA 1C.2.4) was identified in France. It rapidly spread within the pig population and supplanted the previously predominant H1avN1-HA 1C.2.1 virus. To characterize this new genotype which is genetically and antigenically distant from the other H1avNx viruses detected in France, an experimental study was conducted to compare the outcomes of H1avN2 and H1avN1 infections in pigs and evaluate the protection conferred by the only inactivated vaccine currently licensed in Europe containing an HA 1C (clade 1C.2.2) antigen. Infection with H1avN2 induced stronger clinical signs and earlier shedding than H1avN1. The neutralizing antibodies produced following H1avN2 infection were unable to neutralize H1avN1, and vice versa, whereas the cellular-mediated immunity cross-reacted. Vaccination slightly altered the impact of H1avN2 infection at the clinical level, but did not prevent shedding of infectious virus particles. It induced a cellular-mediated immune response towards H1avN2, but did not produce neutralizing antibodies against this virus. As in vaccinated animals, animals previously infected by H1avN1 developed a cross-reacting cellular immune response but no neutralizing antibodies against H1avN2. However, H1avN1 pre-infection induced a better protection against the H1avN2 infection than vaccination, probably due to higher levels of non-neutralizing antibodies and a mucosal immunity. Altogether, these results showed that the new H1avN2 genotype induced a severe respiratory infection and that the actual vaccine was less effective against this H1avN2-HA 1C.2.4 than against H1avN1-HA 1C.2.1, which may have contributed to the H1avN2 epizootic and dissemination in pig farms in France.

VP4 Mutation Boosts Replication of Recombinant Human/Simian Rotavirus in Cell Culture.

Rotavirus A (RVA) is the leading cause of diarrhea requiring hospitalization in children and causes over 100,000 annual deaths in Sub-Saharan Africa. In order to generate next-generation vaccines against African RVA genotypes, a reverse genetics system based on a simian rotavirus strain was utilized here to exchange the antigenic capsid proteins VP4, VP7 and VP6 with those of African human rotavirus field strains. One VP4/VP7/VP6 (genotypes G9-P[6]-I2) triple-reassortant was successfully rescued, but it replicated poorly in the first cell culture passages. However, the viral titer was enhanced upon further passaging. Whole genome sequencing of the passaged virus revealed a single point mutation (A797G), resulting in an amino acid exchange (E263G) in VP4. After introducing this mutation into the VP4-encoding plasmid, a VP4 mono-reassortant as well as the VP4/VP7/VP6 triple-reassortant replicated to high titers already in the first cell culture passage. However, the introduction of the same mutation into the VP4 of other human RVA strains did not improve the rescue of those reassortants, indicating strain specificity. The results show that specific point mutations in VP4 can substantially improve the rescue and replication of recombinant RVA reassortants in cell culture, which may be useful for the development of novel vaccine strains.

Isolation and genetic characteristics of Novel H4N1 Avian Influenza viruses in ChongQing, China.

BACKGROUND: Avian influenza viruses (AIVs) constitute significant zoonotic pathogens encompassing a broad spectrum of subtypes. Notably, the H4 subtype of AIVs has a pronounced ability to shift hosts. The escalating prevalence of the H4 subtype heightens the concern for its zoonotic potential, signaling an urgent need for vigilance. METHODS: During the period from December 2021 to November 2023, we collected AIV-related environmental samples and assessed them using a comprehensive protocol that included nucleic acid testing, gene sequencing, isolation culture, and resequencing. RESULTS: In this study, a total of 934 environmental samples were assessed, revealing a remarkably high detection rate (43.66%, 289/662) of AIV in the live poultry market. Notably, the H4N1 subtype AIV (cs2301) was isolated from the live poultry market and its complete genome sequence was successfully determined. Subsequent analysis revealed that cs2301, resulting from a reassortment event between wild and domesticated waterfowl, exhibits multiple mutations and demonstrates potential for host transfer. CONCLUSIONS: Our research once again demonstrates the significant role of wild and domesticated waterfowl in the reassortment process of avian influenza virus, enriching the research on the H4 subtype of AIV, and emphasizing the importance of proactive monitoring the environment related to avian influenza virus.

A Potential Nervous Necrosis Virus (NNV) Live Vaccine for Sole Obtained by Genomic Modification.

Viral Encephalopathy and Retinopathy (VER) is a neurological infectious fish disease that causes vacuolization and necrosis in the central nervous system, which lead to swimming abnormalities and, generally, host death in the early stages of development. VER is caused by the Nervous Necrosis Virus (NNV), a non-enveloped virus with a bisegmented and positive-stranded (+) RNA genome. The largest segment (RNA1) codes for viral polymerase while capsid protein is encoded by RNA2. The aim of this study was to explore the potential of a reverse-engineered RGNNV/SJNNV strain that harbors mutations in both 3'NCRs (position 3073 of RNA1 and 1408 and 1412 of RNA2) as an attenuated live vaccine for sole. The attenuation of this strain was confirmed through experimental infections in sole at 22 °C. Vaccination trials were performed by bath, intramuscular, and intraperitoneal injection, at two temperatures (18 and 22 °C). Our results indicate the improved survival of vaccinated fish and delayed and poorer viral replication, as well as an overexpression of immune response genes linked to T cell markers (cd4 and cd8), to an early inflammatory response (tlr7 and tnfα), and to antiviral activity (rtp3 and mx). In conclusion, our study indicates that the attenuated strain is a good vaccine candidate as it favors sole survival upon infection with the wt strain while inducing a significant immune response.

HA N193D substitution in the HPAI H5N1 virus alters receptor binding affinity and enhances virulence in mammalian hosts.

During the 2021/2022 winter season, we isolated highly pathogenic avian influenza (HPAI) H5N1 viruses harbouring an amino acid substitution from Asparagine(N) to Aspartic acid (D) at residue 193 of the hemagglutinin (HA) receptor binding domain (RBD) from migratory birds in South Korea. Herein, we investigated the characteristics of the N193D HA-RBD substitution in the A/CommonTeal/Korea/W811/2021[CT/W811] virus by using recombinant viruses engineered via reverse genetics (RG). A receptor affinity assay revealed that the N193D HA-RBD substitution in CT/W811 increases α2,6 sialic acid receptor binding affinity. The rCT/W811-HA193N virus caused rapid lethality with high virus titres in chickens compared with the rCT/W811-HA193D virus, while the rCT/W811-HA193D virus exhibited enhanced virulence in mammalian hosts with multiple tissue tropism. Surprisingly, a ferret-to-ferret transmission assay revealed that rCT/W811-HA193D virus replicates well in the respiratory tract, at a rate about 10 times higher than that of rCT/W811-HA193N, and all rCT/W811-HA193D direct contact ferrets were seroconverted at 10 days post-contact. Further, competition transmission assay of the two viruses revealed that rCT/W811-HA193D has enhanced growth kinetics compared with the rCT/W811-HA193N, eventually becoming the dominant strain in nasal turbinates. Further, rCT/W811-HA193D exhibits high infectivity in primary human bronchial epithelial (HBE) cells, suggesting the potential for human infection. Taken together, the HA-193D containing HPAI H5N1 virus from migratory birds showed enhanced virulence in mammalian hosts, but not in avian hosts, with multi-organ replication and ferret-to-ferret transmission. Thus, this suggests that HA-193D change increases the probability of HPAI H5N1 infection and transmission in humans.

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.

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.