Genes, Viral ; genetics ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; Influenza, Human ; epidemiology ; virology ; Reassortant Viruses ; genetics

Understanding inter-species transmission of influenza viruses is an important research topic. Scientists try to identify and evaluate the functional factors determining the host range of influenza viruses by generating the recombinant viruses through reverse genetics in laboratories, which reveals the viruses' molecular mechanisms of infection and transmission in different species. Therefore, the reverse genetic method is a very important tool for further understanding the biology of influenza viruses and will provide the insight for the prevention and treatment of infections and transmission. However, these recombinant influenza viruses generated in laboratories will become the potential threat to the public health and the environment. In this paper, we discussed the biological safety issues of recombinant influenza viruses and suggested we should set up protocols for risk management on research activities related to recombinant highly pathogenic influenza viruses.
Influenza A Virus, H5N1 Subtype ; genetics ; Laboratories ; Microbiology ; Orthomyxoviridae ; genetics ; Public Health ; Reassortant Viruses ; genetics ; Recombination, Genetic ; Safety

Novel subtypes of Asian-origin (Goose/Guangdong lineage) H5 highly pathogenic avian influenza (HPAI) viruses belonging to clade 2.3.4, such as H5N2, H5N5, H5N6, and H5N8, have been identified in China since 2008 and have since evolved into four genetically distinct clade 2.3.4.4 groups (A–D). Since 2014, HPAI clade 2.3.4.4 viruses have spread rapidly via migratory wild aquatic birds and have evolved through reassortment with prevailing local low pathogenicity avian influenza viruses. Group A H5N8 viruses and its reassortant viruses caused outbreaks in wide geographic regions (Asia, Europe, and North America) during 2014–2015. Novel reassortant Group B H5N8 viruses caused outbreaks in Asia, Europe, and Africa during 2016–2017. Novel reassortant Group C H5N6 viruses caused outbreaks in Korea and Japan during the 2016–2017 winter season. Group D H5N6 viruses caused outbreaks in China and Vietnam. A wide range of avian species, including wild and domestic waterfowl, domestic poultry, and even zoo birds, seem to be permissive for infection by and/or transmission of clade 2.3.4.4 HPAI viruses. Further, compared to previous H5N1 HPAI viruses, these reassortant viruses show altered pathogenicity in birds. In this review, we discuss the evolution, global spread, and pathogenicity of H5 clade 2.3.4.4 HPAI viruses.
Africa ; Animals ; Asia ; Birds ; China ; Disease Outbreaks ; Epidemiology ; Europe ; Influenza in Birds* ; Japan ; Korea ; Poultry ; Reassortant Viruses ; Seasons ; Vietnam ; Virulence*

Hantaviruses are negative-strand RNA viruses that contain three segmented (L/M/S) genome and belong to the genus hantavirus of the family Bunyaviridae. Due to such an unique structure of segmented RNA genome, hantaviruses have a possibility to produce reassortants that containing genomic sets mixed with different segments originated from both parental viruses during the genetic interaction. To investigate whether this phenomenon occurs in vitro, Hantaan (HTN) and Seoul (SEO) viruses were co-infected into Vero-E6 cells and virulent Maaji (MAA) virus was superinfected into avirulent Prospect Hill (PH) virus-infected Vero-E6 cells, respectively. To select only reassortants among progeny viruses, well separated plaque clones were analyzed by multiplex RT-PCR. The putative reassortant viruses detected by 1st multiplex RT-PCR were plaque-purified three times and confirmed by 2nd multiplex RT-PCR. Only 3 reassortants like HTN/HTN/SEO, SEO/HTN/HTN and SEO/HTN/SEO and only 2 reassortants like PH/MAA/MAA, MAA/MAA/PH as designated in order of L/M/S of genomic segments have been identified so far. These results indicate that genetic reassortment can be induced by mixed-infection of two more distantly related serotypes of hantavirus. Interestingly, reassortant SEO/HTN/SEO containing HTN viral M RNA segment is isolated more frequently. This implies that preferential selection of M genome segments occurred when RNA genomes were packaged into virion and also the process of packaging of RNA segments into virion is not random phenomenon. These reassortants would be helpful to know whether genetic reassortment is dependent on genetic distance between hantaviruses and which viral RNA segment plays an important role in coding for virulence marker. Therefore, genetic reassortment can be useful genetic tool to understand genetical, and biological function of hantavirus.
Bunyaviridae ; Clinical Coding ; Clone Cells ; Genome ; Hantavirus* ; Humans ; Parents ; Product Packaging ; Reassortant Viruses ; RNA ; RNA Viruses ; RNA, Viral ; Seoul ; Virion ; Virulence

The prM/E gene of DV2 was cloned into the JEV (SA14-14-2 strain) replicon vector which had been constructed previously, and the resulting recombinant plasmid was named pPartialdeltaprM/E. The constructed chimeric clone was linearized and then was transcripted into RNA in vitro. The produced RNA was transfected into BHK-21 cells. Five to seven days later, CPE could be observed on the transfected BHK-21cells, and then the supernatant containing the chimeric virus was collected. The Supernatant was inoculated to BHK-1 cells and C6/36 cells, respectively. CPE could be observed about 4 days post the infection of C6/36cell with the chimeric virus. The results from RT-PCR, IFA, Western blot showed that the virus contained the chimeric RNA and the envelop protein of DV2. However, the chimeric virus could not be passaged in BHK-21 cell. The successful construction of the infectious clone JE/DEN-2 laid the basis for the further research of the DV vaccine.
Animals ; Blotting, Western ; Cell Line ; Cricetinae ; Dengue Virus ; genetics ; Encephalitis Viruses, Japanese ; genetics ; Genetic Vectors ; genetics ; Reassortant Viruses ; genetics ; Recombination, Genetic ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

From April 2009 onward, a new strain of human H1N1 influenza virus has swept over the world. The genome of influenza virus consists of 8 segments, encoding 10 proteins, respectively. The reassortments among the 8 segments cause the variation of influenza virus. Therefore, phylogenetic analysis of the 8 genes is very important. In this paper, we choose neighboring word frequency as the genomic features, using VC++ programming to analyze evolution of the 8 segments of H1N1 virus. As a result, we found that PB2 genes and PA genes of these three isolated virus were originated from North American avian influenza virus, that PB1 genes were originated from the seasonal influenza virus of human, and that HA genes, NS genes and NP genes came from the North American classical swine influenza A virus. The NA segments and M segments were originated from the European swine influenza virus.
Cloning, Molecular ; Genes, Viral ; Genome, Viral ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; Influenza, Human ; epidemiology ; virology ; Mexico ; epidemiology ; Phylogeny ; Reassortant Viruses ; genetics ; United States ; epidemiology

OBJECTIVETo determine the frequency and characteristics of reassortment among Hantaan and Seoul viruses causing hemorrhagic fever with renal syndrome (HFRS).

METHODSMixed infections were initiated in tissue culture, using Hantaan virus strain 76 - 118 and Seoul virus strain SR-11. Potential reassortant virus plaques were picked out by multiplex RT-PCR, using primers specific for individual genome segments (L, M, S) of each strain.

RESULTSMost of the progeny virus plaques (68.19% of 44) had parental genotype of 76 - 118 strain or SR-11 strain while 2 of 44 plaques had mixed genotypes that yielded RT-PCR bands for the same segment of both parental strains. Reassortant viruses were detected in 68.19% of 44 progeny plaques tested, involving the M and S segments. In addition, approximately 4.55% of the progeny virus plaques appeared to contain S or M segments originating from both parental virus strains, showing that they were diploid.

CONCLUSIONGenetic reassortment can occur between Hantaan virus and Seoul virus strains.

Animals ; Cercopithecus aethiops ; Genome, Viral ; Genotype ; Hantaan virus ; genetics ; RNA, Viral ; genetics ; Reassortant Viruses ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Seoul virus ; genetics ; Vero Cells

We developed a scalable AAV5/5 vector packaging system by using replication competent recombinant herpes simplex type 1 virus as helper virus. The fragment containing rep and cap genes of AAV5 was inserted into the non-necessary gene (UL2) of HSV1 genome, resulting in the helper virus rHSV1-rep5cap5. An AAV5/5 vector pAAV5neo carrying two AAV5 ITRs was constructed by inserting a neo gene expression cassette into the plasmid backbone of pAV5CMV-GFP. pAAV5neo-EGFP was constructed by inserting EGFP gene into pAAV5neo. BHK21 cell was transfected with pAAV5neo-EGFP and cultured in the presence of G418. EGFP expression positive monoclonal cells were picked up, and one that produced rAAV5/5-EGFP with the highest efficiency under the help of rHSV1-rep5cap5 was chosen as the production cell line named as C020. rAAV5/5-EGFP was produced by infecting C020 cells with rHSV1-rep5cap5, and crudely purified by our previous method of 'chloroform treatment-PEG8000/NaCl precipitation- chloroform extract'. rAAV5/5-EGFP preparation with high purity was obtained by ultrafiltration with molecular weight cut-off value of 100 kDa. SDS-PAGE stained with Coomassie brilliant blue R250 showed clearly specific pattern of three bands of AAV capsid proteins. rAAV5/5-EGFP was also assayed using negative stain transmission electron microscopy and the majority of the virus particles were found solid. About 30% green fluorescent cells could be seen after infecting HEK293 cells with rAAV5/5-EGFP 24 h at 1 x 10(5) vg/cell. In conclusion, we have established an efficient AAV5/5 vector production system and could produce recombinant AAV5/5 virus in large amounts for gene therapy research.
Dependovirus ; genetics ; physiology ; Genetic Therapy ; Genetic Vectors ; HEK293 Cells ; Herpesvirus 1, Human ; genetics ; physiology ; Humans ; Reassortant Viruses ; genetics ; Recombination, Genetic ; Viral Proteins ; genetics ; Virus Assembly

OBJECTIVETo investigate the evolution features of whole-genome of influenza virus H3N2 prevalent in Qingdao from year 2007 to 2011.

METHODSThe RNA of 58 strains of influenza virus H3N2 prevalent in Qingdao between 2007 and 2011 was extracted and all segments amplified by RT-PCR. The sequence was then detected and assembled by software Sequencer. A total of 589 strains of influenza virus H3N2 with more than 300 amino acid recorded by GenBank were selected. The phylogeny and molecular features of all gene segments were analyzed by software Mega 5.0, referred by the heavy chain of hemagglutinin (HA1).

RESULTSHemagglutinin (HA) genes of influenza virus H3N2 prevalent in Qingdao between year 2007 and 2011 formed a single trunk of phylogenetic tree. Every prevalent strain originated in last season. The analysis of the evolution of whole genome found that reassortment virus strains were prevalent between year 2009 and 2010, but between 2010 and 2011 there were two series of prevalent strains, which showed complicated reassortment. Compared with the vaccine strains, the variant amino acids of protein of virus HA1 between year 2007 and 2011 were 8, 6, 6, 8 and 11, involving 13 antigenic sites. The sequence analysis of M2 protein showed that the isolated influenza virus H3N2 mutated in amino acid site 31, from serine to asparagine (S31N). HA1 gene of influenza virus H3N2 isolated in Qingdao between 2007 and 2011 shared the similar phylogenetic tree with the globally prevalent strain. The comparison of the sequence and the analysis of the antigenicity found co-infection between H3N2 and A/H1N1 in the strain A/Qingdao/F521/2011.

CONCLUSIONThe evolution features of all segments of influenza virus H3N2 prevalent in Qingdao between year 2007 and 2011 were complicated.

China ; Evolution, Molecular ; Genome, Viral ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Humans ; Influenza A Virus, H3N2 Subtype ; genetics ; Phylogeny ; RNA, Viral ; Reassortant Viruses ; genetics ; Sequence Analysis ; Viral Matrix Proteins ; genetics

In 2013, two episodes of influenza emerged in China and caused worldwide concern. A new H7N9 avian influenza virus (AIV) first appeared in China on February 19, 2013. By August 31, 2013, the virus had spread to ten provinces and two metropolitan cities. Of 134 patients with H7N9 influenza, 45 died. From then on, epidemics emerged sporadically in China and resulted in several victims. On November 30, 2013, a 73-year-old woman presented with an influenza-like illness. She developed multiple organ failure and died 9 d after the onset of disease. A novel reassortant AIV, H10N8, was isolated from a tracheal aspirate specimen that was obtained from the patient 7 d after onset. This case was the first human case of influenza A subtype H10N8. On 4 February, 2014, another death due to H10N8 avian influenza was reported in Jiangxi Province, China.
Aged ; China ; epidemiology ; Female ; Humans ; Influenza A Virus, H10N8 Subtype ; classification ; Influenza A Virus, H7N9 Subtype ; classification ; Influenza A Virus, H9N2 Subtype ; classification ; Influenza, Human ; epidemiology ; virology ; Phylogeny ; Reassortant Viruses ; classification