Genetic Analysis and Rescue of a Triple-reassortant H3N2 Influenza A Virus Isolated From Swine in Eastern China

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 H1N1, NS from classical swine H1N1, 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.

Avian influenza virus surveillance in migratory birds in Egypt revealed a novel reassortant H6N2 subtype

Background:Avian influenza viruses(AIVs)have been identified from more than 100 different species of wild birds around the globe.Wild migratory birds can act as potential spreaders for AIVs to domestic birds between different countries.Egypt is situated on important migratory flyways for wild birds between different continents.While much is known about circulation of zoonotic potential H5N1 and H9N2 AIVs in domestic poultry in Egypt,little is known about the pivotal role of migratory birds in the maintenance and transmission of the viruses in Egypt.Methods:Targeted AIV surveillance has been conducted in 2017 in different wetlands areas in Northern and Eastern Egypt.Results:AIV of subtype H5 was detected in two bird species.In addition,a novel reassortant strain of the H6N2 subtype was identified which reveals the continuous risk of new influenza virus(es)introduction into Egypt.This novel virus possesses a reassortant pattern originating from different AIV gene pools.Conclusions:Intervention control strategies should be performed to minimize the possible contact of domestic birds with wild birds to lower the risk of virus transmission at this interface.In addition,constant monitoring of AIVs in migratory birds is essential in the early detection of influenza virus introduction into Egypt.

A novel mosquito-borne reassortant orbivirus isolated from Xishuangbanna,China

Dear Editor,The genus Orbivirus,within the family Reoviridae,includes 22 virus species(King et al.,2011).They are distributed globally,but are particularly prevalent in Europe,Asia,and Africa.In addition,they can be transmitted by ticks or other hematophagous insect vectors,including Culicoides,mosquitoes,and sandflies(Belaga-

Pathogenic Characterization and Full Length Genome Sequence of a Reassortant Infectious Bursal Disease Virus Newly Isolated in Pakistan

Dear Editor,Infectious bursal disease (IBD) is one of the most important diseases of the poultry. The IBD virus (IBDV), a nonenveloped virus belonging to the Birnaviridae family with a genome consisting of two segments of double-stranded RNA (segments A and B), targets B lymphocytes of bursa of Fabricious leading to immunosuppression. In Pakistan,poultry farming is the second biggest industry and IBD is the second biggest disease threating the poultry sector.However.

Driving forces of continuing evolution of rotaviruses

Rotaviruses are non-enveloped double-stranded RNA virus that causes acute diarrheal diseases in children(<5 years).More than 90%of the global rotavirus infection in humans was caused by Rotavirus group A.Rotavirus infection has caused more than 200000 deaths annually and predominantly occurs in the low-income countries.Rotavirus evolution is indicated by the strain dynamics or the emergence of the unprecedented strain.The major factors that drive the rotavirus evolution include the genetic shift that is caused by the reassortment mechanism,either in the intra-or the inter-genogroup.However,other factors are also known to have an impact on rotavirus evolution.This review discusses the structure and types,epidemiology,and evolution of rotaviruses.This article also reviews other supplemental factors of rotavirus evolution,such as genetic reassortment,mutation rate,glycan specificity,vaccine introduction,the host immune respo-nses,and antiviral drugs.

The origin of a novel kind of reassortant (H1N2) of influenza A virus

Genetic analysis of three H1N2 viruses indicated that only HA genes of H1N2 viruses were similar to that of A/Guangdong/6/91(H1N1) virus (PR8-like strain), while the other seven genes of them were similar to those of H3N2 virus circulating in man in 1995. Therefore, it could be considered that the H1N2 viruses were derived from reassortment between PR8-like strain and H3N2 virus circulating in man in 1995. However, the genomes of H1N2 viruses were very similar to each other. So the H1N2 viruses isolated in 1998 were not derived from new reassortment between PR8-like strain and H3N2 virus circulating in man in 1998, but derived from the evolution of H1N2 virus found in 1995.

Detection of reassortant avian influenza A (H11N9) virus in environmental samples from live poultry markets in China

Background:Avian influenza viruses have caused human infection and posed the pandemic potential.Live poultry markets are considered as a source of human infection with avian influenza viruses.Avian influenza routine surveillance of live poultry markets is taken annually in China.We isolated the 2 H11N9 influenza virus from the surveillance program.To better understand the risk caused by these new viruses,we characterize the genetic and pathogenicity of the two viruses.Methods:Viral isolation was conducted with specific pathogen-free(SPF)embryonated chicken eggs.Whole genome was sequenced,and phylogenetic analysis was conducted.Results:Two H11N9 viruses were identified,with all 8 segments belonging to the Eurasian lineage.The HA,NA,M,NS and PA genes were similar to virus isolates from ducks,and the NP,PB2 and PB1 gene segments were most similar to those viruses from wild birds,indicating that the H11N9 viruses might represent reassortant viruses from poultry and wild birds.The HA receptor binding preference was avian-like,and the cleavage site sequence of HA showed low pathogenic.The NA gene showed 94.6%identity with the novel H7N9 virus that emerged in 2013.There was no drug resistance mutation in the M2 protein.The Asn30Asp and Thr215Ala substitutions in the M1 protein implied a potentially increased pathogenicity in mice.Both viruses were low-pathogenic strains,as assessed by the standards of intravenous pathogenicity index(IVPI)tests.Conclusion:Two reassortant H11N9 avian influenza viruses were detected.These viruses showed low pathogenicity to chickens in the IVPI test.Public health concern caused by the reassortant H11N9 viruses should be emphasized during the future surveillance.

A Review on 2009 Influenza A Virus

[Objective] The paper was to introduce the research progress of 2009 influenza A virus. [Method] 2009 influenza A virus was introduced from the aspects of classification and host, virology, molecular characteristics and vaccine. [Result] A novel influenza A/H1N1 virus emerged in early April 2009 quickly spread worldwide through human-to-human transmission. The virus contained a group of novel gene segments, the nearest known precursor was the virus found in swine. The virus appeared to retain the potential to infect swine again and thus continued reassort with swine viruses. All registered 2009 influenza A vaccines were tested for safety and immunogenicity in clinical trials on human volunteers, and all vaccines were found to be safe, single dose of vaccine could cause protective antibody responses. [Conclusion] The paper provided basis for further study on 2009 influenza A virus.

The circulation of unique reassortment strains of infectious bursal disease virus in Pakistan

Infectious bursal disease(IBD),caused by IBD virus(IBDV),is one of the most devastating and immunosuppressive diseases of the poultry and has been a constraint on the sustainable poultry production around the globe including Pakistan.While the disease is threatening the poulty industry,the nature of predominant strains of IBDV in Pakistan remained l-defined.In this study,an epidemiology survey was conducted in the main chicken-farming regions of Pakistan.The batch of Pakistan IBDVs genes simultaneously covering both VP1 and VP2 were amplified,sequenced,and analyzed.The unique segment-reassortant IBDVs(vv-A/Uniq-B),carrying segmentA from vvIBDV and segment B from one unique ancestor,were identifed as one important type of circulating strains in Pakistan.The data also discovered the characteristic molecular features of Pakistan IBDVs,which will contribute to scientific vaccine selection and effective prevention of the disease.

Generation of Recombinant Equine Influenza Vaccine Candidate RgH3N1 Virus by Reverse Genetics

The antigenic variation of influenza A virus hemagglutinin (HA) glycoproteins requires frequent changes in vaccine formulation. The new strategy of creating influenza seed strains for vaccine production is to generate 7 + 1 reassortants that contain seven genes from a high-yield virus A/Puerto Rico/8/34[A/PR/8/34](H1N1) and the HA gene from the circulating strains. By using this DNA-based cotransfection technique, we generated 7 + 1 reassortants rgH3N1 which had the antigenic determinants of influenza virus A/Songbird/HongKong/102/00[SB/HK/01](H3N8) and 7 other genes from A/PR/ 8/34. The hemagglutinin of A/Songbird/HongKong/102/00 is 96.3% homologous to that of A/Equine/Jilin/98[Eq/Jl/89] (H3N8). The resulting virus rgH3N1 grows to high HA titers in chicken embryonated eggs, allowing vaccine preparation in unconcentrated allantoic fluid. The rgH3N1 is stable after multiple passages in embryonated eggs. The reassortant rgH3N1 virus could be used as vaccine candidate to reduce the reemergence of equine influenza outbreaks.

Generation and characterization of a cold-adapted attenuated live H3N2 subtype influenza virus vaccine candidate

Background H3N2 subtype influenza A viruses have been identified in humans worldwide, raising concerns about their pandemic potential and prompting the development of candidate vaccines to protect humans against this subtype of influenza A virus. The aim of this study was to establish a system for rescuing of a cold-adapted high-yielding H3N2 subtype human influenza virus by reverse genetics, Methods In order to generate better and safer vaccine candidate viruses, a cold-adapted high yielding reassortant H3N2 influenza A virus was genetically constructed by reverse genetics and was designated as rgAA-H3N2. The rgAA-H3N2 virus contained HA and NA genes from an epidemic strain A/Wisconsin/67/2005 （H3N2） in a background of internal genes derived from the master donor viruses （MDV）, cold-adapted （ca）, temperature sensitive （ts）, live attenuated influenza virus strain A/Ann Arbor/6/60 （MDV-A）. Results In this presentation, the virus HA titer of rgAA-H3N2 in the allantoic fluid from infected embryonated eggs was as high as 1：1024. A fluorescent focus assay （FFU） was performed 24-36 hours post-infection using a specific antibody and bright staining was used for determining the virus titer. The allantoic fluid containing the recovered influenza virus was analyzed in a hemagglutination inhibition （HI） test and the specific inhibition was found. Conclusion The results mentioned above demonstrated that cold-adapted, attenuated reassortant H3N2 subtype influenza A virus was successfully generated, which laid a good foundation for the further related research.

Clinical characteristics of human infection with a novel avian-origin influenza A（H10N8） virus

Background Novel influenza A viruses of avian-origin may be the precursors of pandemic strains. This descriptive study aims to introduce a novel avian-origin influenza A （H10N8） virus which can infect humans and cause severe diseases. Methods Collecting clinical data of three cases of human infection with a novel reassortment avian influenza A （H10N8） virus in Nanchang, Jiangxi Province, China. Results Three cases of human infection with a new reassortment avian influenza A（H10N8） virus were described, of which two were fatal cases, and one was severe case. These cases presented with severe pneumonia that progressed to acute respiratory distress syndrome （ARDS） and intractable respiratory failure. Conclusion This novel reassortment avian influenza A （H10N8） virus in China resulted in fatal human infections, and should be added to concerns in clinical practice.

Molecular evolution and genetic diversity analysis of SFTS virus based on next-generation sequencing

SFTS virus(SFTSV)is a novel bunyavirus,which was discovered as the etiological agent of severe fever with thrombocytopenia syndrome(SFTS)in China in 2009,and was now prevalent in at least 25 provinces in China.SFTS was subsequently identified in South Korea and Japan in 2012.To explore themolecular evolution and genetic characteristics of this newly identified pathogen,we reported 72 whole genome sequences of SFTSV,and built a dataset of SFTSV genome sequences containing 292 L-segment,302 M-segment and 502 S-segment.We clearly divided SFTSV into six genotypes,Genotype A-F.It was found that genotype F was the dominant epidemic genotype of Japan,South Korea,and Zhejiang province of China.The coalescent analysis supported that SFTSV originated in the early 18th century from Zhejiang province,and Genotype F was the most primitive one.Henan,Hubei,and Anhui provinces which are located in Dabie Mountain area weremainly epidemic of Genotype A,which emerged relatively late but distributed widely.A total of 37 recombination events were identified,making SFTSV with a high recombination frequency(L segment 5.1%,Msegment 3.6%,S segment 0.8%)among negative-strand segmented RNA viruses.It was identified that 19 reassortant strains belonged to 12 reassortment forms of SFTSV genome containing 6 newly identified forms.The reassortment virus and recombination in tick were both found for the first time.We also found many of genotype-specific mutation sites,7 of which could be considered as potential molecular marker for genotype classification.This study promoted a more comprehensive understanding of the phylogeny and origin,and the genetic diversity of SFTSV,and it could help the studies of other newly discovered tick-borne bunyavirus as reference data and research ideas.

Novel SFTSV Phylogeny Reveals New Reassortment Events and Migration Routes

Severe fever with thrombocytopenia syndrome virus(SFTSV),the causative agent of a febrile human disease,was first identified from central and eastern provinces in China,and later in Japan and South Korea.Hubei Province is one of the major SFTS epidemic areas in the central part of China.This study reported the isolation of 11 new SFTSV strains from patients in Hubei Province collected in 2017.Extensive phylogenetic analyses were conducted based on the complete coding sequences of SFTSV segments including the new strains.It was suggested that five different SFTSV genotypes were circulating in Hubei,and 15 reassortment patterns and migration pathways correlated with each genotype were identified,which was more than previously recognized.Hubei Province was more involved in the evolutionary events of SFTSV than that previously thought in which the evolutionary events of SFTSV were reported to be independent from those in other epidemic regions.Further divergence of SFTSV strains was suggested by pairwise comparison of SFTSV sequences from each genotype and sequence identity normalized to representative strain in genotype C1.Subsequently,amino acid variations specific for genotype(s),strain(s),or cluster(s)were inspected,which may be related to differential biological activity of SFTSV strains/genotypes.In conclusion,we analyzed the current status of SFTSV phylogeny in Hubei Province and discussed the possible events correlated to SFTSV evolution.It provided an in-depth insight into SFTSV evolution,raising concerns for the use of proper SFTSV strains in future studies.

Progress and Challenge in Computational Identification of Influenza Virus Reassortment

Genomic reassortment is an important evolutionary mechanism for influenza viruses.In this process,the novel viruses acquire new characteristics by the exchange of the intact gene segments among multiple influenza virus genomes,which may cause flu endemics and epidemics within or even across hosts.Due to the safety and ethical limitations of the experimental studies on influenza virus reassortment,numerous computational researches on the influenza virus reassortment have been done with the explosion of the influenza virus genomic data.A great amount of computational methods and bioinformatics databases were developed to facilitate the identification of influenza virus reassortments.In this review,we summarized the progress and challenge of the bioinformatics research on influenza virus reassortment,which can guide the researchers to investigate the influenza virus reassortment events reasonably and provide valuable insight to develop the related computational identification tools.