Introduction of a Newcastle disease virus challenge strain (sub-genotype VII.1.1) isolated in Iran.

Newcastle disease virus (NDV) sub-genotype VII.1.1 is the most common circulating NDV in Iran. In this study, a velogenic NDV isolate was plaque purified and then characterized according to Office International des Epizooties (OIE) standard protocols. The biological properties of the purified isolate named CH/RT40/IR/2011 were characterized using sequencing and phylogenetic analysis, measurement of pathogenicity indexes and challenge studies. The isolate was plaque purified on chicken embryo fibroblast cells for three rounds and then characterized using molecular and biological approaches. Phylogenetic and evolutionary distance analysis of fusion and hemagglutinin-neuraminidase genes classified the virus in sub-genotype VII.1.1. No mutation was observed in the glycosylation and neutralizing epitope sites of the fusion and hemagglutinin-neuraminidase proteins compared to other reported Iranian NDV VII.1.1 isolates. The presence of the 112RRQKRF117 motif in the fusion protein cleavage site together with mean death time, intracerebral pathogenicity index and intravenous pathogenicity index of 57 hr, 1.80 and 2.50 respectively, revealed that the RT40 isolate was a velogenic NDV. In the challenge study, all chickens were inoculated via eye drop, and intranasal route with RT40 isolate died within a week. While all chickens in the vaccinated and challenged group survived and showed no clinical signs. In conclusion, according to genetic analysis, pathotyping and challenge study, the RT40 isolate was similar to virulent NDVs in Iran and was a suitable candidate for a national standard challenge strain, vaccine trials and vaccine production in commercial levers.

Genetic Characteristics of Human Parainfluenza Virus Types 1-4 From Patients With Clinical Respiratory Tract Infection in China.

Human parainfluenza viruses (HPIV1-4) cause acute respiratory tract infections, thereby impacting human health worldwide. However, there are no current effective antivirals or licensed vaccines for infection prevention. Moreover, sequence information for human parainfluenza viruses (HPIVs) circulating in China is inadequate. Therefore, to shed light on viral genetic diversity and evolution, we collected samples from patients infected with HPIV1-4 in China from 2012 to 2018 to sequence the viruses. We obtained 24 consensus sequences, comprising 1 for HPIV1, 2 for HPIV2, 19 for HPIV3, and 2 for HPIV4A. Phylogenetic analyses classified the 1 HPIV1 into clade 2, and the 2 HPIV4 sequences into cluster 4A. Based on the hemagglutinin-neuraminidase (HN) gene, a new sub-cluster was identified in one of the HPIV2, namely G1c, and the 19 HPIV3 sequences were classified into the genetic lineages of C3f and C3a. The results indicated that HPIV1-4 were co-circulated in China. Further, the lineages of sub-cluster C3 of HPIV3 were co-circulated in China. A recombination analysis indicated that a putative recombination event may have occurred in the HN gene of HPIV3. In the obtained sequences of HPIV3, we found that two amino acid substitution sites (R73K in the F protein of PUMCH14028/2014 and A281V in the HN protein of PUMCH13961/2014) and a negative selection site (amino acid position 398 in the F protein) corresponded to the previously reported neutralization-related sites. Moreover, amino acid substitution site (K108E) corresponded to the negative selection site (amino acid position 108) in the 10 F proteins of HPIV3. However, no amino acid substitution site corresponded to the glycosylation site in the obtained HPIV3 sequences. These results might help in studying virus evolution, developing vaccines, and monitoring HPIV-related respiratory diseases.

Cross-neutralization between vaccine and circulating wild-type mumps viruses in Korea.

Mumps is a contagious disease caused by the mumps virus. It can be prevented using mumps vaccines, administered as a measles-mumps-rubella (MMR) vaccine. For first and second dose immunization, children aged 12-15 months and 4-6 years have been administered this vaccine since 1997 in Korea. Nevertheless, mumps outbreaks still occur in vaccinated populations worldwide. Hence, immunity against these diseases may be attenuated, or there are antigenic differences between currently available vaccine strains and circulating wild-type viruses. After the introduction of national immunization programs in Korea, mumps cases became sporadic. Viral genotypes F, H, and I have emerged since 1998 whereas the vaccine strains belong to genotype A. Here, we compared the amino acid sequences of the haemagglutinin-neuraminidase (HN) gene from wild-type viruses and the mumps vaccine and measured the cross-neutralization titers between them. We selected the F, H, and I wild-type mumps strains circulating in Korea from 1998 to 2016 and analyzed changes in the amino acid sequence of the protein encoded by the HN gene. We measured mumps virus-specific IgG and rapid focus reduction neutralization test (FRNT) titers in Korean isolates and sera obtained from 50 children aged 1-2 years who had been administered a single dose of MMR vaccine. Analysis of the HN protein sequences disclosed no changes in the glycosylation sites but did reveal 4-5 differences between the Korean isolates and the genotype A vaccine strain in terms of the neutralizing epitope sites on their HN proteins. Post-vaccination FRNT titers were significantly lower against genotypes F, H, and I than they were against genotype A. This finding highlights the possibility of a recurrence of mumps outbreaks in vaccinated populations depending on the degree of genetic conservation of the HN gene. Further research into this issue is needed to prevent the resurgence of mumps.

Development of a real-time RT-qPCR assay for the detection of porcine respirovirus 1.

Porcine respirovirus 1 (PRV1) was first reported in the pig nasopharyngeal samples in Hong Kong in 2013. It has been widespread in US swine herds. Recently, PRV1 was also detected in South America and European countries. Currently, there is no validated diagnostic assay available for the detection of this virus. In this study, we developed a real-time reverse transcriptase quantitative PCR (RT-qPCR) assay targeting the hemagglutinin-neuraminidase (HN) gene for molecular diagnosis. The analytical sensitivity of this RT-qPCR assay was evaluated using in vitro transcribed RNA standard, and the limit of detection was 10 copies of viral RNA in a 20 µl reaction. No cross-reactivity was observed with nucleic acid prepared from common swine respiratory pathogens. The diagnostic performance of this assay was determined with 114 pig nasal swabs and 19 oral fluid samples with known PRV1 infection status. The RT-qPCR results were consistent with conventional RT-PCR and DNA sequencing of the HN gene, demonstrating a 100 % sensitivity and 100 % specificity. This assay was further applied to field samples. Among 310 nasal swab samples that were tested, 201 samples from 8 swine farms were PRV1 positive. No viremia was detected in PRV1 infected pigs using the available field samples. Nasal swab and oral fluid samples appear to be reliable for PRV1 detection with the RT-qPCR assay. Taken together, we developed and validated an RT-qPCR assay for accurate detection of PRV1 in nasal swab and oral fluid samples. It will be a useful tool for the rapid diagnosis of PRV1 infection and in aid of PRV1 epidemiological surveillance.

Molecular characteristics of the hemagglutinin 1 and neuraminidase genes of influenza B viruses isolated in Yancheng city from 2015 to 2017 / 中华疾病控制杂志

Objective To analyze the genetic characteristics of the hemagglutinin （HA) and neuraminidase （NA) genes of influenza B viruses isolated in Yancheng City from 2015 to 2017. Methods The throat swab specimens of influenza-like illness( ILI) from sentinel surveillance hospital and outbreak sites were collected and sent to Yancheng CDC for virus nucleic acids and virus isolation testing. After validation with serological tests, eighteen strains of influenza B virus isolates were selected to amplify their HA1 and NA genes through RT-PCR assay. Their molecular characteristics of the obtained viral HA1 and NA gene sequences were analyzed using bioinformation software from three aspects, including nucleic acid level, amino acid level and molecular evolution level. Results Basically, the clustering relationships and the branche patterns between HA1 and NA genes from the 18 Yancheng influenza B virus strains were similar. The Yamagata lineage strains in 2015 were distributed in the Yamagata Clade 3 branch, belonging to Phuket/3073 strains. The Victoria lineage strains in 2016-2017 were distributed in the Victoria Clade 1A branch, belonging to Brisbane/60 strains. D196N substitution was detected on HA1 protein in all of Yamagata lineage strains at 190-helix epitope; Amino acid substitutions of victoria lineage strains involved two antigenic epitopes, 117 and 129 sites of 120-loop epitope and 197 and 199 sites of 190-helix epitope. No Intra-lineage or inter-lineage rearrangements occurred in Yancheng strains. Eighteen influenza B strains had no mutations in catalytic residues and drug resistant sites of NA genes. Conclusion The Yamagata strains well matched with vaccine strain B/Phuket/3073/2013. The HA1 and NA genes of victoria lineage strains circulated in Yancheng City during 2016 to 2017 are changing gradually. The accumulation of these mutations will result in antigenic drift of victoria lineage strains and increase the mismatch of the IFV field stains with the available vaccine strains, which may reduce the protective effect of flu vaccine.

Molecular characterization of haemagglutinin-neuraminidase gene among virulent Newcastle disease viruses isolated in Iran.

BACKGROUND: Virulent Newcastle disease virus (vNDV) causes great economic losses to the poultry industry throughout the world. Despite the endemicity of Newcastle disease (ND) and occurrence of recurrent outbreaks, the nature and genetic features of circulating NDV strains in Iran are largely unknown. Aims: This study was conducted to characterize 13 NDV isolates obtained from different outbreaks in various regions of Iran during 1999-2000 by sequencing and phylogenetic analysis of complete coding sequences of haemagglutinin-neuraminidase (HN) gene. METHODS: All isolates were analyzed based on the previously determined in vivo pathogenicity indices and amino acid (aa) sequences of fusion (F) protein cleavage site (FPCS). RESULTS: Phylogenetic analysis based on the HN gene coding region revealed a very close relationship of these viruses with the recently defined genotype XIII, and more specifically, subgenotype XIIIa viruses. Analysis of HN gene nucleotide (nt) sequences revealed that all studied isolates encode for a protein length of 571 aa and there is no C-terminal extension on HN aa sequences. Sequence analysis revealed multiple aa residue substitutions at antigenic sites or neutralizing epitopes on the HN glycoprotein of studied viruses compared with commonly used vaccinal strains. CONCLUSION: In this study, molecular characterization of vNDV isolates, obtained from commercial poultry farms in Iran, were conducted through complete sequencing and analysis of HN gene. Isolation and molecular characterization of further NDV isolates from other parts of Iran and from neighboring countries in the region will be helpful to identify the nature and origin of indigenous viruses.

Molecular characteristic analysis of neuraminidase genes of avian influenza virus H9N2 in environments in Weining, Guizhou Province during 2015-2017 / 中华传染病杂志

Objective To understand the genetic variations of neuraminidase (NA) genes of avian influenza virus H9N2 in Weining,Guizhou Province,and to provide the scientific evidence for the prevention and control of avian influenza virus.Methods Ribonucleic acids (RNA) were extracted and NA genes were amplified and sequenced from 13 randomly selected H9N2 positive samples from the live poultry market (LPM)environments in north of Weining Yi and Hui and Miao autonomous county (Weining),Guizhou Province during 2015 to 2017.Then the homology,genetic evolution,and sites of stalk deletion areas,potential N-glycosylation,receptor binding regions and drug resistance of H9N2 subtype avian influenza viruses were analyzed by a series of bioinformation software.Results Homology analysis revealed that there were 93.0％-100.0％ and 92.1％-100.0％ similarity among 13 strains H9N2 avian influenza viruses in nucleotide and amino acid of the NA gene,respectively.All strains belonged to DK/HK/Y280/97 sub-lineage,but their genetic sources were complex and diverse.Thirteen strains had a stalk deletion of 3 amino acid residues TEI at positions 63-65 and 3 isolates had mutation QN to QK at positions 39-40.The potential N-glycosylation sites at amino acid residues 86,146,200,and 234 of the NA protein of all strains were highly conserved,while other N-glycosylation sites had quantity and site mutations.There were different mutation types at the three sialic acid binding site areas,especially at 399-404 area.All NA protease activity sites and key sites of the 13 strains had no mutations associated with resistance to the neuraminidase inhibitor drugs.Conclusions All 13 strains H9N2 viruses belongs to DK/HK/Y280/97 sub-lineage in Weining,Guizhou Province during 2015-2017,and their genetic sources are complex and diverse.The mutations on sites of stalk areas,potential N-glycosylation and sialic acid binding site areas are presented at different degrees.Hence,enhancing surveillance and controlling H9N2 avian influenza virus is necessary.

[Genetic characteristics of hemagglutinin and neuraminidase of avian influenza A (H7N9) virus in Guizhou province, 2014-2017].

Objective: To understand the molecular characteristics of hemagglutinin (HA) and neuraminidase (NA) as well as the disease risk of influenza virus A H7N9 in Guizhou province. Methods: RNAs were extracted and sequenced from HA and NA genes of H7N9 virus strains obtained from 18 cases of human infection with H7N9 virus and 6 environmental swabs in Guizhou province during 2014-2017. Then the variation and the genetic evolution of the virus were analyzed by using a series of bioinformatics software package. Results: Homology analysis of HA and NA genes revealed that 2 strains detected during 2014-2015 shared 98.8%-99.2% and 99.2% similarities with vaccine strains A/Shanghai/2/2013 and A/Anhui/1/2013 recommended by WHO, respectively. Two strains detected in 2016 and 14 strains detected in 2017 shared 98.2%-99.3% and 97.6%-98.8% similarities with vaccine strain A/Hunan/02650/2016, respectively. Other 6 stains detected in 2017 shared 99.1%-99.4% and 98.9%-99.3% similarities with strain A/Guangdong/17SF003/2016, respectively. Phylogenetic analysis showed that all the strains were directly evolved in the Yangtze River Delta evolution branch, but they were derived from different small branch. PEVPKRKRTAR↓GLF was found in 6 of 24 strains cleavage site sequences of HA protein, indicating the characteristic of highly pathogenic avian influenza virus. Mutations A134V, G186V and Q226L at the receptor binding sites were found in the HA. All the strains had a stalk deletion of 5 amino acid residue "QISNT" in NA protein, and drug resistance mutation R294K occurred in strain A/Guizhou-Danzhai/18980/2017. In addition, potential glycosylation motifs mutations NCS42NCT were found in the NA of 9 of 24 strains. Conclusions: HA and NA genes of avian influenza A (H7N9) virus showed genetic divergence in Guizhou province during 2014-2017. The mutations of key sites might enhance the virulence of the virus, human beings are more susceptible to it. Hence, the risk of infection is increasing.

Analysis of the characteristics of hemagglutinin and neuraminidase genes of influenza A/H1N1(09pdm) viruses isolated in Yancheng area in 2014-2017 / 中华实验和临床病毒学杂志

Objective@#To analyze the genetic characteristics of the hemagglutinin(HA) and neuraminidase(NA) genes of the influenza A/H1N1(09pdm) viruses isolated in the city of Yancheng in 2014-2017.@*Methods@#The throat swab specimens of patients with influenza-like illness (ILI) from sentinel surveillance hospitals and outbreak sites were detected using the method of real time RT-PCR. The influenza A/H1N1(09pdm) viruses were isolated using MDCK cell culture method in 2014-2017. The strains in 2014-2017 were selected randomly and their sequences of the HA1 and NA genes were amplified through one step RT -PCR method and the PCR products were sequenced. The mutations of genes and acid locus were analyzed and the evolutional trees were generated using bioinformatics software.@*Results@#The clustering relationships of the respective branches of HA1 and NA genes of seventeen A/H1N1(09pdm) strains isolated in Yancheng area were basically the same and the phylogenetic trees of HA1 and NA genes were respectively clustered into four evolutionary branches. Compared with the vaccine strain A/California/07/2009(H1N1pdm)in the Northern Hemisphere, a total of three antigen epitopes (Ca, Sa, Sb) in HA1 genes of strains in Yancheng area were involved in six antigenic sites (K154R, S162N, K163Q, S185T, L191I, S203T); there were three mutations (D222G/N, G223R, E224K) in the 220 ring and one locus (L191I) in the 190 helix of the receptor binding sites; the two strains (A/Jiangsu-YC/SWL1540/2017, A/Jiangsu-YC/SWL1545/2017) isolated in 2017 increased the 162NQS glycosylation site. Because the strains of the antigen epitopes, receptor binding sites and glycosylation sites in the HA1 genes had a certain degree of variations in Yancheng area in 2014-2017, the protective effects of vaccine strain A/California/07/2009 (H1N1pdm) was limited at the gene level. The two strains (A/Jiangsu-YC/SWL1540/2017 and A/Jiangsu-YC/SWL1545/2017) isolated in 2017 were clustered with vaccine strain A/Michigan/45/2015(H1N1pdm) and had better protective effects. Seventeen A/H1N1(09pdm) strains had no mutations in catalytic residues and drug resistant sites of NA genes, but a part of strains had a certain degree of variations in glycosylation sites of NA genes.@*Conclusions@#These results indicated the HA1 and NA genes of influenza A/H1N1(09pdm) viruses circulated in Yancheng area in 2014-2017 changed gradually. The accumulation of these mutations would result in antigenic drift of influenza A/H1N1(09pdm) viruses.

Genetic characteristics of hemagglutinin and neuraminidase of avian influenza A (H7N9) virus in Guizhou province, 2014-2017 / 中华流行病学杂志

Objective To understand the molecular characteristics of hemagglutinin (HA) and neuraminidase (NA) as well as the disease risk of influenza virus A H7N9 in Guizhou province.Methods RNAs were extracted and sequenced from HA and NA genes of H7N9 virus strains obtained from 18 cases of human infection with H7N9 virus and 6 environmental swabs in Guizhou province during 2014-2017.Then the variation and the genetic evolution of the virus were analyzed by using a series of bioinformatics software package.Results Homology analysis of HA and NA genes revealed that 2 strains detected during 2014-2015 shared 98.8％-99.2％ and 99.2％ similarities with vaccine strains A/Shanghai/2/2013 and A/Anhui/1/2013 recommended by WHO,respectively.Two strains detected in 2016 and 14 strains detected in 2017 shared 98.2％-99.3％ and 97.6％-98.8％ similarities with vaccine strain A/Hunan/02650/2016,respectively.Other 6 stains detected in 2017 shared 99.1％-99.4％ and 98.9％-99.3％ similarities with strain A/Guangdong/17SF003/2016,respectively.Phylogenetic analysis showed that all the strains were directly evolved in the Yangtze River Delta evolution branch,but they were derived from different small branch.PEVPKRKRTAR ↓ GLF was found in 6 of 24 strains cleavage site sequences of HA protein,indicating the characteristic of highly pathogenic avian influenza virus.Mutations A134V,G186V and Q226L at the receptor binding sites were found in the HA.All the strains had a stalk deletion of 5 amino acid residue "QISNT" in NA protein,and drug resistance mutation R294K occurred in strain A/Guizhou-Danzhai/ 18980/2017.In addition,potential glycosylation motifs mutations NCS42NCT were found in the NA of 9 of 24 strains.Condusions HA and NA genes of avian influenza A (H7N9) virus showed genetic divergence in Guizhou province during 2014-2017.The mutations of key sites might enhance the virulence of the virus,human beings are more susceptible to it.Hence,the risk of infection is increasing.

Genetic characteristics of hemagglutinin and neuraminidase of avian influenza A (H7N9) virus in Guizhou province, 2014-2017 / 中华流行病学杂志

Objective To understand the molecular characteristics of hemagglutinin (HA) and neuraminidase (NA) as well as the disease risk of influenza virus A H7N9 in Guizhou province.Methods RNAs were extracted and sequenced from HA and NA genes of H7N9 virus strains obtained from 18 cases of human infection with H7N9 virus and 6 environmental swabs in Guizhou province during 2014-2017.Then the variation and the genetic evolution of the virus were analyzed by using a series of bioinformatics software package.Results Homology analysis of HA and NA genes revealed that 2 strains detected during 2014-2015 shared 98.8％-99.2％ and 99.2％ similarities with vaccine strains A/Shanghai/2/2013 and A/Anhui/1/2013 recommended by WHO,respectively.Two strains detected in 2016 and 14 strains detected in 2017 shared 98.2％-99.3％ and 97.6％-98.8％ similarities with vaccine strain A/Hunan/02650/2016,respectively.Other 6 stains detected in 2017 shared 99.1％-99.4％ and 98.9％-99.3％ similarities with strain A/Guangdong/17SF003/2016,respectively.Phylogenetic analysis showed that all the strains were directly evolved in the Yangtze River Delta evolution branch,but they were derived from different small branch.PEVPKRKRTAR ↓ GLF was found in 6 of 24 strains cleavage site sequences of HA protein,indicating the characteristic of highly pathogenic avian influenza virus.Mutations A134V,G186V and Q226L at the receptor binding sites were found in the HA.All the strains had a stalk deletion of 5 amino acid residue "QISNT" in NA protein,and drug resistance mutation R294K occurred in strain A/Guizhou-Danzhai/ 18980/2017.In addition,potential glycosylation motifs mutations NCS42NCT were found in the NA of 9 of 24 strains.Condusions HA and NA genes of avian influenza A (H7N9) virus showed genetic divergence in Guizhou province during 2014-2017.The mutations of key sites might enhance the virulence of the virus,human beings are more susceptible to it.Hence,the risk of infection is increasing.

Mutation signature in neuraminidase gene of avian influenza H9N2/G1 in Egypt.

The low pathogenic avian influenza (LPAI) H9N2 subtype has become the most prevalent and widespread in many Asian and Middle Eastern countries. It causes an enzootic situation in commercial poultry and known as a potential facilitator virus that can be transmitted to human from birds. The neuraminidase (NA) gene plays an important role the release and spread of the virus from infected cells and throughout the bird. The complete nucleotide sequences of the NA gene of seven H9N2 viruses collected from apparent healthy chicken and quail flocks in Egypt during 2014-2015, were amplified and sequenced. The phylogenetic relationships were investigated and all viruses were belonging to the A/Q/HK/G1/97 strain (G1-like). There were no insertions or deletions or shortening in NA stalk regions when compared to Y280-lineage and the human H9N2 isolates. No obvious changes NA interactions with antiviral drugs. We found that the Egyptian H9N2 viruses have seven glycosylation sites like the most recorded H9N2 viruses in the country, except A/Q/Egypt/14864V/2014 virus which has only six. The NA has four amino acid substitutions distributed in different parts of the hemadsorbing site. The most characteristic substitutions in this site were S372A and W403R these substitutions were a distinctive feature resembling to human H9N2, H2N2 and H3N2 viruses but differs from the other avian influenza viruses. These Special features of surface glycoproteins of LPAI-H9N2 viruses refer to the tendency for enhanced introductions into humans and ensuring the importance of poultry in the transfer influenza viruses.

Neuraminidase Gene Variations in Influenza A(H1N1)pdm09 Virus among Patients Admitted to Refferal Pulmonary Hospital, Tehran, Iran in 2009-2013.

BACKGROUND: Neuraminidase (NA) is one of the surface proteins of influenza A virus, which plays an important role in immunization against influenza infection and is recognized as an important therapeutic target. Genetic and antigenic changes and substitutions can influence the efficacy of vaccine and change viral sensitivity to NA inhibitors (NAIs). In this study, we performed phylogenetic and molecular analyses of NA changes in influenza A(H1N1)pdm09 virus, compared them with the corresponding vaccine strain, and examined drug resistance mutations in isolates from patients. MATERIALS AND METHODS: The complete sequence of NA genes from 34 pandemic H1N1 isolates (identified in 2009-2010, 2010-2011, and 2013) was determined and analyzed both genetically and antigenically. The phylogenetic tree was plotted relative to the corresponding vaccine strain, using MEGA6 software package, based on the maximum likelihood method and JTT matrix (bootstrap value of 1000). RESULTS: The phylogenetic analysis of pandemic isolates showed 31 amino acid substitutions in NA genes, compared to the vaccine strain. Some of these substitutions (N248D, V241I, N369K, N44S, and N200S) were important in terms of phylogenetic relationship, while the rest (D103N, V106I, R130T, N200S, G201E, and G414R) influenced the antigenic indices of B-cell epitopes. The catalytic sites, framework sites, and N-glycosylation remained unchanged in the studied samples. Meanwhile, H275Y substitution, related to oseltamivir resistance, was detected in 3 isolates. The average nucleotide identity of NAs with the corresponding vaccine strain was 99.415%, 98.607%, and 98.075% in 2009-2010, 2010-2011, and 2012-2013, respectively. CONCLUSION: In this study, we provided basic information on the genetic and antigenic changes of NA genes in influenza A(H1N1)pdm09 virus from patients in 3 different seasons in Tehran, Iran. Considering the viral NAI resistance and changes in NA gene sequences of the isolates in comparison with the vaccine strain, further studies should be performed to monitor genetic changes in Iran. Moreover, the efficacy of vaccines should be examined.

Detection of D151G/N mutations in the neuraminidase gene of influenza A (H3N2) viruses by real-time RT-PCR allelic discrimination assay.

Single nucleotide polymorphisms (SNPs) at D151 position of neuraminidase (NA) gene of influenza A (H3N2) virus has been associated with drug resistance and increased binding affinity. NA-D151G/N-substitutions of influenza A (H3N2) viruses are frequently induced and selected by culturing in Madin-Darby canine kidney (MDCK) cell lines. It is important to consider and exclude D151G/N mutants after isolation of influenza virus in MDCK cell line; since, the substitutions can highly influence the results of experimental research. The study aims to develop an allelic discrimination real-time reverse transcriptase polymerase chain reaction (RT-PCR) for the screening of D151G/N mutants. Thirty-six influenza A (H3N2) virus isolates were included and screened for D151G/N mutants using allelic discrimination assay. Out of the 36 isolates, 11 isolates (30.5%) were detected as heterozygous for D and G/N substitutions. Twenty-one (58.3%) isolates were identified as homozygous wild type and four isolates (11.1%) were undetermined. Isolates with substitutions at D151 position were sequenced by Sanger sequencing method. The present study demonstrates a rapid and convenient method for primary screening of the mutation after culturing of the influenza virus in MDCK cell lines in order to avoid potential misinterpretations of results and improve the quality of experimental research.

Neuraminidase gene profiling of influenza B virus in Shangrao / 中国感染与化疗杂志

Objective To characterize the neuraminidase (NA) genes of influenza B virus in Shangrao.Methods The specimens of nasopharyngeal swabs were collected from patients with influenza-like symptoms in influenza sentinel hospital.Seven strains of influenza B virus were randomly selected for culture and isolation in Madin-Darby Canine Kidney Epithelial Cells (MDCK Line).Viral RNA was extracted.Fragments of NA genes were amplified by one-step RT-PCR and then were sequenced.The data obtained were analyzed with software DNAStar 6.0 and Mage 5.0.The deduced amino acid sequences were examined to explore the features ofNA gene.Results The NA gene showed high homology ofnucleotides between the 7 strains of influenza B virus.No amino acid substitution was found in catalytic or framework residues of the deduced amino acid sequences of NA gene.Conclusions All the 7 strains of influenza B virus were sensitive to neuraminidase inhibitors.However,ongoing resistance surveillance is necessary for control and prevention of influenza.

Genomic characterization of avian influenza A(H7N9) virus in Zhaoqing, China, 2014-2016 / 中国人兽共患病学报

We analyzed genetic evolution characteristics of avian influenza A (H7N9) virus isolated in Zhaoqing,China,2014-2016.Nucleic acid were extracted and sequenced from 17 samples of H7N9 positive cases in Zhaoqing.Genetic characteristics of homology and important amino acid sites were analyzed by using BioEdit5.0 and MEGA6.0.The evolutionary trees were constructed by Neighbor-Joining and the referenced sequences were downloaded from GenBank,Eight nucleic acid fragments from 7 strains of H7N9 viruses were successfully generated.The highest homology was found in HA gene with A/chicken/Dongguan/695/2014(H7N9),and NA gene with A/chicken/Dongguan/1075/2014(H7N9).The internal genes were high homology with avian H7N9 and H9N2 virus from Dongguan and Shenzhen in Guangdong,China.The HA and NA genes were directly evolved in the Pearl River Delta evolution branch with the H7N9 sequences from the cities of Dongguan,Guangzhou and Shenzhen,while the sequences from the provinces of Anhui,Zhejiang,and Jiangsu were in the Yangtze River Delta evolution branch.There were 2 alkaline amino acids in cleavage site of HA,2 mutations (G186V and Q226L) in the crucial sites related with the receptor of HA protein,1 mutation (E627K) in PB2 protein,and 1 drug resistance mutation (S31N) in M2 protein.And no evidence of neuraminidase resistance in NA protein was found.In conclusion,the H7N9 virus for human infection in Zhaoqing may originate from avian H7N9 and H9N2 viruses,which circulated in the Pearl River Delta region of Guangdong from 2013 to 2014.The mutations of G186V,Q226L and E627 K might be related with high susceptibility to human beings.

Development and application of dual real time RT-PCR for avian influenza H5N6 virus / 中华实验和临床病毒学杂志

Objective@#To establish a TaqMan-MGB probe-based real-time fluorescence RT-PCR assay for avian influenza H5N6 virus used in rapid diagnosis for suspected cases and surveillance for outer environment of live poultry markets.@*Methods@#Based on the conservative sequences of avian influenza H5N6 virus for HA and NA gene published on GenBank, specific primers and TaqMan-MGB probes were designed to develop and optimize for the dual real-time RT-PCR assay. Specificity, sensitivity, repeatability and comparison tests were carried out.@*Results@#This dual real-time RT-PCR detection can be completed within 80 minutes. There was no cross-reaction with other subtypes of influenza virus and common respiratory pathogens. The minimum detection limit could be up to 10 copies/reaction. The correlation coefficient of standard curve for the gene of H5 and N6 were 0.999 and 0.993, and the coefficients of variation for cycle threshold were range from 0.151%-0.549%and 0.213%-0.575%, respectively. The positive and negative coincidence rates of the validation test were 100%.@*Conclusions@#This TaqMan-MGB probe-based dual real-time RT-PCR for avian influenza H5N6 virus was rapid, specific and sensitive. It will have a good use in early emergency detection of suspected cases and continuous monitoring of external environment in live poultry trade market.

Therapeutic targeting of liver cancer with a recombinant DNA vaccine containing the hemagglutinin-neuraminidase gene of Newcastle disease virus via apoptotic-dependent pathways.

A total of ~38.6 million mortalities occur due to liver cancer annually, worldwide. Although a variety of therapeutic methods are available, the efficacy of treatment at present is extremely limited due to an increased risk of malignancy and inherently poor prognosis of liver cancer. Gene therapy is considered a promising option, and has shown notable potential for the comprehensive therapy of liver cancer, in keeping with advances that have been made in the development of cancer molecular biology. The present study aimed to investigate the synergistic effects of the abilities of the hemagglutinin neuraminidase protein of Newcastle disease virus (NDV), the pro-apoptotic factor apoptin from chicken anaemia virus, and the interferon-γ inducer interleukin-18 (IL-18) in antagonizing liver cancer. Therefore, a recombinant DNA plasmid expressing the three exogenous genes, VP3, IL-18 and hemagglutinin neuraminidase (HN), was constructed. Flow cytometry, acridine orange/ethidium bromide staining and analysis of caspase-3 activity were performed in H22 cell lines transfected with the recombinant DNA plasmid. In addition, 6-week-old C57BL/6 mice were used to establish a H22 hepatoma-bearing mouse model. Mice tumor tissue was analyzed by immunohistochemistry and scanning electron microscopy. The results of the present study revealed that the recombinant DNA vaccine containing the VP3, IL-18 and HN genes inhibited cell proliferation and induced autophagy via the mitochondrial pathway in vivo and in vitro.

Mumps virus F gene and HN gene sequencing as a molecular tool to study mumps virus transmission.

Various mumps outbreaks have occurred in the Netherlands since 2004, particularly among persons who had received 2 doses of measles, mumps, and rubella (MMR) vaccination. Genomic typing of pathogens can be used to track outbreaks, but the established genotyping of mumps virus based on the small hydrophobic (SH) gene sequences did not provide sufficient resolution. Therefore, we expanded the sequencing to include fusion (F) gene and haemagglutinin-neuraminidase (HN) gene sequences in addition to the SH gene sequences from 109 mumps virus genotype G strains obtained between 2004 and mid 2015 in the Netherlands. When the molecular information from these 3 genes was combined, we were able to identify separate mumps virus clusters and track mumps virus transmission. The analyses suggested that multiple mumps virus introductions occurred in the Netherlands between 2004 and 2015 resulting in several mumps outbreaks throughout this period, whereas during some local outbreaks the molecular data pointed towards endemic circulation. Combined analysis of epidemiological data and sequence data collected in 2015 showed good support for the phylogenetic clustering.

Genetic diversity among human parainfluenza virus type 2 isolated in Croatia between 2011 and 2014.

The dynamics and evolution of the human parainfluenza virus type 2 (HPIV2) in Croatia, and also globally, are largely unknown. Most HPIV2 infections are treated symptomatically outside the hospital setting. Thus, the diagnosis is missing making it difficult to follow the genetic variation and evolution of the HPIV2. This study explores hospitalized HPIV2 cases in Croatia during 4-year period (2011-2014). Most cases in this period were reported in October or November (68.75%) and most of patients were under 2 years of age (81.25%). For molecular analyses, we used the F and HN gene sequences and showed that although both regions are equally suitable for phylogenetic analyses it would be advantageous to use regions longer than 2 kb for HPIV2 analyses of isolates which are spatially and temporally closely related. We show here that the dominant cluster in this area was cluster G3 while only one strain isolated in this period was positioned in the distant cluster G1a. Further monitoring of the HPIV2 will determine whether cluster G3 will remain dominant or it will be overruled by cluster G1a. This will be important for the surveillance of virus circulation in population and significance of the viral infection. J. Med. Virol. 88:1733-1741, 2016. © 2016 Wiley Periodicals, Inc.