Complete genome analysis of coxsackievirus A2 and A5 strains in Changsha / 中国热带医学

@#Abstract: Objective　To investigate the molecular characteristic and evolutionary trends of full-genome sequences of coxsackievirus A2 (CV-A2) and A5 (CV-A5) in Changsha City. Methods　The CV-A2 and CV-A5 strains were isolated and detected from patients with hand, foot and mouth disease (HFMD) cases. The full-genome sequences of CV-A2 and CV-A5 strains were obtained using NGS sequencing. Homology and phylogenetic tree analysis were performed, and the recombination regions of the strains were examined by SimPlot software. Results　The full-genome sequences of CV-A2 and CV-A5 strains were obtained from routine surveillance cases of HFMD in Changsha in 2019. The CV-A2 strain was named S281/Changsha/CHN/2019 with the full-genome sequence of 7 422 bp long; the CV-A5 strain was named S272/Changsha/CHN/2019 with the full-genome sequence of 7 425 bp long. Homology analysis of the isolates by comparison with the nucleic acid sequences of CV-A2 and other CV-A2 strains in China showed that the non-structural protein region shared lower similarity than that of structural protein region. The CV-A2 showed 79.20% similarity with Fleetwood strain (NC038306), showed the highest similarity 95.60% with MN419014 strain from Hubei Province. The non-structural protein 3C and 3D region shared the lowest similarity with MN419014, 90.51 and 92.06%, respectively. Phylogenetic tree analysis showed that 3C and 3D regions were located in the CV-A4 branch. Amino acid mutation sites were found in non-structural protein region, and the amino acid sequence in structural protein region was conserved. SimPlot analysis showed that genetic recombination was found in the 3C and 3D region of CV-A2 strains. The full-genome sequence of CV-A5 showed 80.7% similarity with the Swartz (AY421763) and 97.43% similarity with the strain (MH111030) from Australian. Homology analysis showed that the non-structural protein region shared lower similarity than that of structural protein region, based on full-genome of CV-A5. Phylogenetic tree analysis showed that CV-A5 and MH111030 were in the same branch, indicating that CV-A5 strain not from local. The amino acid sequence of CV-A5 strain was conserved. Conclusions　The CV-A2 strain in Changsha City shared genome sequence information with CV-A4, and the CV-A5 strain was imported from abroad. Our findings are expected to understand the molecular and recombination characteristics of CV-A2 and CV-A5, provided the data of evolution and genetic features of the coxsackievirus, and interrupt disease transmission in a timely and effective manner.

Construction of influenza B virus vero cell adapted strain by genetic reassortment / 中华实验和临床病毒学杂志

Objective@#To construct influenza B virus Vero cell adapted strain by genetic recombination technology by using the influenza B virus Vero cell adapted strain as the parent strain.@*Methods@#The chick embryo and Vero cell were co-infected with influenza virus Vero cell adapted strain B/Malaysia/2506/2004 Va (Bv) and the vaccine strain B/massachusetts/2/2012 (BX-51B) recommended by WHO. The reassortants were screened with the anti-Bv serum. Plaque-purified reassortants were used to screen for Vero cell-adapted influenza B virus strains containing the surface antigen of the epidemic strain.@*Results@#A Vero cell-adapted influenza B virus strain was obtained with successive passage in Vero cells. The hemagglutination inhibition test and the one-way immunogold agar diffusion test both showed that the reassortant virus was homologous to NYMC BX-51B, and sequence analysis result showed that the reassortment virus has the same HA and NA gene with the vaccine strain.@*Conclusion@#B/Malaysia/2506/2004Va (Bv) can be used as a parent strain to prepare Vero cell vaccine against influenza B virus.

Novel reassortants of clade 2.3.4.4 H5N6 highly pathogenic avian influenza viruses possessing genetic heterogeneity in South Korea in late 2017

Novel H5N6 highly pathogenic avian influenza viruses (HPAIVs) were isolated from duck farms and migratory bird habitats in South Korea in November to December 2017. Genetic analysis demonstrated that at least two genotypes of H5N6 were generated through reassortment between clade 2.3.4.4 H5N8 HPAIVs and Eurasian low pathogenic avian influenza virus in migratory birds in late 2017, suggesting frequent reassortment of clade 2.3.4.4 H5 HPAIVs and highlighting the need for systematic surveillance in Eurasian breeding grounds.

G26P[19] rotavirus A strain causing acute gastroenteritis in the American continent

In Brazil, the rotavirus A genotype G26 was first identified in suckling piglets, while the P[19] genotype has not been identified in any animal species so far. This report details the genetic characterisation of a G26P[19] RVA strain detected from an eight year-old child, vaccinated with Rotarix®, hospitalised with acute diarrhoeal disease in Rio de Janeiro in 2015. Most likely, the genome constellation (I5-R1-C1-M1-A8-N1-T1-E1-H1) observed in the G26P[19] Brazilian strain was a result of interspecies transmission events between humans and pigs. In addition, a rearrangement in the NSP5 gene was observed downstream of the 3' non-coding region.

Preparation and identification of influenza H1N1 subtype vaccine candidate strain in China / 中华实验和临床病毒学杂志

Objective@#Influenza H1N1 subtype vaccine candidate strains from a 2015—2016 year epidemic strain in China were prepared and identified by themethod of classical reassortment.@*Methods@#The influenza H1N1 epidemic strain and H3N2 high-yield reassortant parental strain (X-157) were mixed and inoculated into embryonated chicken eggs by the classical reassortmentmethod . The negative selection of mixed culture virus was carried out with the antiserum of H3 protein and the antiserum of X-157 strain. Real-time PCRmethod was used to test the HA and NA genes. Restriction enzyme digestionmethod was used to identify the internal genes. HA and NA genes of selected strains were sequenced. The strain which HA and NA genes possessed the same amino acid constitution with the wild type virus was selected and immunized to ferret. Two-way test was carried out.@*Results@#Five strains with expected HA and NA genes were selected by real-time PCR. Internal genes were identified, with 4 strains had 6+ 2 constitution, 1 strain had 5+ 3 constitution. Comparing with the wild type virus, HA and NA genes of the 5 strains had no mutation. HA titer of reassortant strains was above 1 024. HI titer of the selected NO.12 reassortment strain reached 5 120, and two-way test was passed. The yield of reassortant strain was 64 times that of the wild type strain.@*Conclusions@#A circulating influenza A (H1N1) strain of influenza A (2015—2016) was successfully prepared in China and laid the foundation for vaccine storage and disease prevention and control.

Evolutionary dynamics of highly pathogenic avian influenza A/H5N1 HA clades and vaccine implementation in Vietnam

Based on hemagglutinin (HA) and neuraminidase (NA), influenza A virus is divided into 18 different HA (H1 to H18) and 11 NA types (N1 to N11), opening the possibility for reassortment between the HA and NA genes to generate new HxNy subtypes (where x could be any HA and y is any NA, possibly). In recent four years, since 2010, highly pathogenic avian influenza (HPAI) viruses of H5N1 subtype (HPAI A/H5N1) have become highly enzootic and dynamically evolved to form multiple H5 HA clades, particularly in China, Vietnam, Indonesia, Egypt, Cambodia, and Bangladesh. So far, after more than 10 years emerged in Vietnam (since late 2003), HPAI A/H5N1 is still posing a potential risk of causing outbreaks in poultry, with high frequency of annual endemics. Intragenic variation (referred to as antigenic drift) in HA (e.g., H5) has given rise to form numerous clades, typically marking the major timelines of the evolutionary status and vaccine application in each period. The dominance of genetically and antigenically diversified clade 2.3.2.1 (of subgroups a, b, c), clade 1.1 (1.1.1/1.1.2) and re-emergence of clade 7.1/7.2 at present, has urged Vietnam to the need for dynamically applied antigenicity-matching vaccines, i.e., the plan of importing Re-6 vaccine for use in 2014, in parallel use of Re-1/Re-5 since 2006. In this review, we summarize evolutionary features of HPAI A/H5N1 viruses and clade formation during recent 10 years (2004-2014). Dynamic of vaccine implementation in Vienam is also remarked.

Evidence for Genetic Reassortment between Human Rotaviruses by Full Genome Sequencing of G3P[4] and G2P[4] Strains Co-circulating in India

<i>Rotavirus A</i> causes severe diarrhoea in infants and young children worldwide. Many unusual combinations of G and P genotypes have been observed in rotaviruses circulating in developing countries. Mixed infection of a single individual with more than one strain is a mechanism by which genetic reassortants are formed with unusual G and P combinations. However, few studies have provided direct evidence for the formation of such unusual strains as a result of co-infection of co-circulating strains. Here, we used full-genome sequencing to re-analyze a G3P[4] strain (107E1B) and a G2P[4] strain (116E3D) detected in India in 1993 and showed that 107E1B had virtually an identical nucleotide sequence with 116E3D, except the VP7 gene. Phylogenetic analysis revealed that the 107E1B VP7 gene was of typical human rotavirus origin, with a 99.3% nucleotide sequence identity with another Indian G3 VP7 gene. Thus, this study provided robust evidence for the formation of the G3P[4] strain through genetic reassortment in which a G2P[4] strain with a typical DS-1 genogroup background acquired the VP7 gene from a co-circulating G3 human rotavirus strain. This study established a basis on which to facilitate full genome sequence analysis of an increasing number of G3P[4] strains in China and elsewhere in the world.

Molecular characterization of G and P-types bovine rotavirus strains from Goiás, Brazil: high frequency of mixed P-type infections

In this study, 331 samples from calves less than one month old from a dairy herd in the district of Piracanjuba, state of Goiás, Brazil were tested for rotavirus. Thirty-three samples (9.9 percent) tested positive for rotavirus. Out of those, 31 were submitted to G and P characterization by reverse transcription followed by semi-nested polymerase chain reaction. Two samples were characterized as G6P[1], three as G10P[11] and five as G6P[11]. The majority of the samples (51.6 percent) displayed multiple P genotypes (P-genotype mixtures), including typical human genotypes P[4] and P[6M], suggesting the occurrence of co-infections and genetic reassortment. Also, the detection of human genotypes in bovine samples may be considered evidence of the zoonotic potential of rotaviruses. To our knowledge, this is the first report of such a high frequency of P genotype mixtures in bovine rotavirus samples. It also increases data on G and P rotavirus genotypes circulating in dairy herds in Brazil and can help in the development of more efficient immunization approaches, thereby controlling infection and reducing economical losses.

Recombination analysis of full-length genomic sequences of novel influenza virus A/H1N1 in 2009 pandemic / 第二军医大学学报

Objective: To analyze the recombination of full-length genomic sequences of novel influenza virus A/H1N1 in 2009 pandemic. Methods: The full-length sequences of the novel A/H1N1 and reference sequences were downloaded from NCBI database. MEGA4.0 software was used to connect, align sequences, and analyze the similarity between the full-length sequences of the novel virus and each of the reference strains. Recombination was analyzed by Simplot software (version 3.5.1). Results: Simplot analysis indicated that the PB1 genes (polymerase B1, PB1) of the novel A/H1N1 viruses might evolve from human H3N2 virus (identity: 93.7%); the PB2 genes (polymerase B2, PB2) and the PA genes (polymerase A, PA) might evolve from avian H5N1 viruses (identity: 89.0%, 89.9%, respectively); the HA genes (hemagglutinin, HA), the NP genes (nucleoprotein, NP) and the NS genes (non-structural protein, NS) showed high similarities with those of swine H1N1 viruses isolated in North America (identity: 91.7%, 93.1%, and 93.1%, respectively); and the NA genes (neuraminidase, NA) and the MP genes (matrix protein, MP) might evolve from European swine H1N1 viruses (identity: 90.5%, 95.5%, respectively). The full-length sequence of the novel A/H1N1 viruses had a highest similarities with swine H1N1 viruses isolated in North America (identity: 83.9%). Conclusion: The novel influenza virus A/H1N1 is a recombinant virus evolving from human H3N2 viruses, swine H1N1 from North America, swine H1N1 from Europe, and swine H5N1 from Asia.

Threat of an influenza panzooty: a review based on conservation medicine

Among reemerging illnesses, influenza constitutes one of the main concerns. The avian influenza has recently demonstrated the strong transmission capacity of the etiological agent -a virus from the Orthomyxoviridae family - associated to high pathogenic manifestations of the illness. The strong mutation capacity of this virus, through different hosts, reveals how important integrated actions aiming at monitoring its presence in different species are. The swine infection represents an additional concern not only in relation to that species but also in relation to the possibility of the virus to mutate and adapt to humans. The elements that determine the pathogenicity of the various viral subtypes must be well understood, for the tools used to control the illness - such as vaccination - may promote viral mutation and thus render the control even more difficult instead of favoring it. The present review aims at characterizing various components involved in the virus maintenance in different species as well as the determinant elements involved in its evolution, from the point of view of Conservation Medicine, which is the branch of science that deals exactly with the interaction among the environment, human beings, and animals, thus creating a holistic vision not only of the problem but also of the coherent and effective actions involved in their solution.

A influenza representa um dos principais temores dentre as doenças re-emergentes. A gripe aviária tem demonstrado atualmente a grande capacidade de transmissão do agente etiológico, um vírus da família Orthomyxoviridae, associada a manifestações da enfermidade com alta patogenicidade. A grande capacidade de mutação deste vírus utilizando diferentes hospedeiros, denota a importância de ações integradas que visam monitorar sua presença em diferentes espécies. A infecção dos suínos determina uma preocupação adicional não apenas para a espécie mas, com possibilidades de mutação e adaptação do vírus aos seres humanos. Os fatores que determinam a patogenicidade dos diferentes subtipos virais devem ser bem compreendidos, pois as ferramentas utilizadas no controle da enfermidade, como vacinação, podem fomentar a mutação viral e com isto dificultar o controle ao invés de favorecê-lo. Esta revisão tem por objetivo caracterizar vários componentes envolvidos na manutenção do vírus em diferentes espécies, bem como os fatores envolvidos em sua evolução, sob a ótica da medicina da conservação, que é um capítulo da ciência que trata justamente das interações entre o ambiente, o ser humano e os animais, criando assim uma visão holística tanto do problema, como das ações coerentes e efetivas envolvidas na resolução do mesmo.

Molecular characteristics and its evolution of the complete genome of avian influenza H5N1 virus isolated in Zhejiang province from 2002 to 2006 / 中华流行病学杂志

Objective To analyze the molecular characteristics and evolution reassortment of the complete genome of avian influenza H5N1 virus isolated in Zhejiang province in recent years. Methods Complete genomes of avian influenza H5N1 viruses isolated in Zhejiang province from 2002 to 2006 were sequenced. Molecular and evolution reassortment characterization of these virus strains were analyzed by Mega 3.0 bioinformatics software. Results Through study on the HA genes from all these strains,our data revealed that there were multiple basic amino acids at the cleavage site, which was typical for HPAIV.Compared with Gs/Guangdong/1/96,all these strains had a 20 amino acid deletion in the stalk of the NA, except for Dk/Zhejiang/2/02 and Ck/Zhejiang/8/03. Results from phylogenetic analysis showed that from2002 to 2003 the H5N1 viruses belonged to the genotype of B,W,X,Y,Z, and other genotypes were prevailed in corresponding year. However, different gene fragments of several strains belonged to different genotypes. Conclusion Recombinant might be widespread among the poultry in Zhejiang province. The genetic genes of viruses isolated after 2005, the Ck/Zhejiang/24/05 and Zhejiang/16/06 strains were almost all originated from the FJ-like genotype stably.

Origins and Views of The 2009 A/H1N1 Influenza Pandemic / 生物化学与生物物理进展

The pandemic outbreak of influenza has been started from Mexico in 2009 to 70 countries during 2 months. On 11th of June , WHO announced influenza pandemic alert level rose to the highest level 6, which means the first influenza pandemic in 21st century is coming. Till 6th of July, 94 512 confirmed cases from more than 120 countries and areas were reported, including 429 cases were died. The genetic fragment of swine, poultry sources and human influenza viruses are contained in this strain, A/H1N1 influenza virus, of the pandemic. It is of great significance of studying the genetic reassortment, evolution and its biological characteristics of this virus strain to prevent and control the pandemic. At present, the genetic evolution of strain has been identified, and the potential biological characteristics have been analyzed by genetic traits, however, clinical manifestation should be further concerned, and the tendency of influenza pandemic and genetic changes need to be monitored closely. The complexity of influenza virus ecosystems, mutation of genome, and easy to preserve in "Nature Gene Pool" and reassortment, make the influenza pandemic inevitable. We should face the threat of influenza pandemic, enhance the surveillance of influenza virus in ecosystems, strengthen the epidemiological investigation, develop the vaccines and drugs, and establish an effective public health security system, in order to reduce the destruction of the influenza pandemic.

The Generation of Reassortants by Genetic Reassortment between Different Serotypes of Hantaviruses

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.

Rescue of Synthetic Hantavirus-Like Foreign RNA by Hantavirus as a Helper Virus

A system for the expression of synthetic hantavirus-like luciferase RNA was developed using Hantaan (HTN) virus as a helper virus. The hantavirus-like luciferase RNA was constructed by the deletion of the coding region in HTN virus (76~118) S genome and by replacement of a luciferase gene. PCR was performed using primers designed to amplify the whole region of hantavirus-like foreign gene. The resulting PCR product was placed under the control of the T7 RNA polymerase promoter for in vitro transcription. The produced hantavirus-like luciferase RNA was transfected into Vero-E6 cell infected with HTN virus using lipofectin. The level of expression was measured using a luminometer. The hantavirus-like luciferase RNA was allowed to amplify and express. And also, the hantavirus-like luciferase RNA was packaged into HTN virions. The 5' terminal and 3' terminal conserved sequences of HTN virus genome were sufficient to provide the signals for RNA amplification and packaging. This suggests that the RNA promoter region for hantavirus RNA synthesis is located in 3' terminal region. The luciferase activity was analyzed from the progeny virus-infected cells in order to examine if the 5' and 3' terminal sequences play a role in regulating a packaging pathway while genome of HTN virus is packaged. The luciferase activity was detectable in every cell passage. However, the activity of luciferase was decreased gradually after each passage. The fact that the hantavirus-like luciferase RNA can be packaged into progeny virus suggests that the 5' and 3' terminal sequences of HTN virus genome play an important role in regulating a packaging pathway.