NP Genes Research Articles

Development and application of a multiplex PCR method for the simultaneous detection and differentiation of feline panleukopenia virus, feline bocavirus, and feline astrovirus.

A multiplex polymerase chain reaction (mPCR) assay was developed to detect and distinguish feline panleukopenia virus (FPV), feline bocavirus (FBoV) and feline astrovirus (FeAstV). Three pairs of primers were designed based on conserved regions in the genomic sequences of the three viruses and were used to specifically amplify targeted fragments of 237 bp from the VP2 gene of FPV, 465 bp from the NP1 gene of FBoV and 645 bp from the RdRp gene of FeAstV. The results showed that this mPCR assay was effective, because it could detect at least 2.25-4.04 × 104 copies of genomic DNA of the three viruses per μl, was highly specific, and had a good broad-spectrum ability to detect different genotypes of the targeted viruses. A total of 197 faecal samples that had been screened previously for FeAstV and FBoV were collected from domestic cats in northeast China and were tested for the three viruses using the newly developed mPCR assay. The total positive rate for these three viruses was 59.89% (118/197). From these samples, DNA from FPV, FBoV and FeAstV was detected in 73, 51 and 46 faecal samples, respectively. The mPCR testing results agreed with the routine PCR results with a coincidence rate of 100%. The results of this study show that this mPCR assay can simultaneously detect and differentiate FPV, FBoV and FeAstV and can be used as an easy, specific and efficient detection tool for clinical diagnosis and epidemiological investigation of these three viruses.Electronic supplementary materialThe online version of this article (10.1007/s00705-019-04394-8) contains supplementary material, which is available to authorized users.

Variable loci of HA, NA and NP genes as effective RNA targets for genotyping subtypes H1N1 and H7N9

Aim. Influenza viruses are a serious pathogen of humans, animals and birds that regularly cause epidemics. Antigenic variability and reassortment of influenza A virus genes represent a high level of neonatal data, which does not allow to assess the evolutionary stability of proteins. Determination of variable HA, NA and NP gene loci of two different antigenic subtypes of the H1 and H7 influenza virus will allow the establishment of RNA targets for genotyping. Methods. An analysis of substitution of nucleotides in the encoding regions of influenza A subtype genes of various antigenic subtypes obtained from the GenBank database using the MEGA 6.0 program determined the fate of synonymous and non-synonymous substitutions in each position of multiple alignments of the coding regions of the nucleotide sequences using the BLAST algorithm. The analysis of variable locus of proteins was determined by the DISORDER algorithm. Results. Different types of mutations are found in variable locus of the studied genes. The most variable genes are HA and NA, the least NP. In sequences of the NP gene synonymous nucleotide substitutions prevail. The genome of NA is mainly deletions and insertions. Conclusions. The variability of the nucleotide sequences of the HA, NA and NP genes in the subtypes A H1 and H7 was detected. It has been established that the use of variable locus of these genes allows for the identification of influenza A strains and identifies a separate serotype.Keywords: neurominidase, variability, genetic markers, target RNA, genotyping.

Genetic characteristics, pathogenicity and transmission of H5N6 highly pathogenic avian influenza viruses in Southern China.

Since 2014, H5 highly pathogenic avian influenza viruses (HPAIVs) from clade 2.3.4.4 have been persistently circulating in Southern China. This has caused huge losses in the poultry industry. In this study, we analysed the genetic characteristics of seven H5N6 HPAIVs of clade 2.3.4.4 that infected birds in Southern China in 2016. Phylogenetic analysis grouped the HA, PB2, PA, M and NS genes as MIX-like, and the NA genes grouped into the Eurasian lineage. The PB1 genes of the GS24, GS25, CK46 and GS74 strains belonged to the VN 2014-like group and the others were grouped as MIX-like. The NP genes of GS24 and GS25 strains belonged to the ZJ-like group, but the others were MIX-like. Thus, these viruses came from different genotypes, and the GS24, GS25, CK46 and GS74 strains displayed genotype recombination. Additionally, our results showed that the mean death time of all chickens inoculated with 105 EID50 of CK46 or GS74 viruses was 3 and 3.38days, respectively. The viruses replicated at high titers in all tested tissues of the inoculated chickens. They also replicated in all tested tissues of naive contact chickens, but their replication titers in some tissues were significantly different (p<0.05). Thus, the viruses displayed high pathogenicity and variable transmission in chickens. Therefore, it is necessary to focus on the pathogenic variation and molecular evolution of H5N6 HPAIVs in order to prevent and control avian influenza in China.

Development of fast and sensitive protocols for the detection of viral pathogens using a small portable convection PCR platform.

One of the most crucial steps for preventing viral pandemics is the early detection of the causative virus on site. Various molecular and immunological approaches have been developed for virus detection. In this study, we investigated the utility of the recently introduced convection polymerase chain reaction (cPCR) platform for the rapid and sensitive detection of various animal viruses in the field, including the foot-and-mouth disease virus (FMDV) and avian influenza viruses (AIVs). Primer sets were designed to simultaneously detect two highly conserved regions of the FMDV, including the 5' untranslated region (5'-UTR) and 3D gene, and to specifically amplify the NP and hemagglutinin (HA) genes of H5 and H9 subtypes of AIVs. The portable cPCR system was able to amplify from as low as 1 to 10 copies of viral cDNAs in the singleplex mode and 10 to 100 copies of viral cDNAs in the duplex mode within 21min. Thus, our data suggest that the cPCR protocols developed in this study are highly sensitive and enable quick detection of animal viruses in biological samples.

A new reassortant clade 2.3.2.1a H5N1 highly pathogenic avian influenza virus causing recent outbreaks in ducks, geese, chickens and turkeys in Bangladesh.

A total of 15 dead or sick birds from 13 clinical outbreaks of avian influenza in ducks, geese, chickens and turkeys in 2017 in Bangladesh were examined. The presence of H5N1 influenza A virus in the affected birds was detected by RT-PCR. Phylogenetic analysis based on full-length gene sequences of all eight gene segments revealed that these recent outbreaks were caused by a new reassortant of clade 2.3.2.1a H5N1 virus, which had been detected earlier in 2015 during surveillance in live bird markets (LBMs) and wet lands. This reassortant virus acquired PB2, PB1, PA, NP and NS genes from low pathogenic avian influenza viruses mostly of non-H9N2 subtypes but retained HA, NA and M genes of the old clade 2.3.2.1a viruses. Nevertheless, the HA gene of these new viruses was 2.7% divergent from that of the old clade 2.3.2.1a viruses circulated in Bangladesh. Interestingly, similar reassortment events could be traced back in four 2.3.2.1a virus isolates of 2013 from backyard ducks. It suggests that this reassortant virus emerged in 2013, which took two years to be detected at a broader scale (i.e. in LBMs), another two years until it became widely spread in poultry and fully replaced the old viruses. Several mutations were detected in the recent Bangladeshi isolates, which are likely to influence possible phenotypic alterations such as increased mammalian adaptation, reduced susceptibility to antiviral agents and reduced host antiviral response.

Genetic and biological characteristics of avian influenza virus subtype H1N8 in environments related to live poultry markets in China

BackgroundSince 2008, avian influenza surveillance in poultry-related environments has been conducted annually in China. Samples have been collected from environments including live poultry markets, wild bird habitats, slaughterhouses, and poultry farms. Multiple subtypes of avian influenza virus have been identified based on environmental surveillance, and an H1N8 virus was isolated from the drinking water of a live poultry market.MethodsVirus isolation was performed by inoculating influenza A-positive specimens into embryonated chicken eggs. Next-generation sequencing was used for whole-genome sequencing. A solid-phase binding assay was performed to test the virus receptor binding specificity. Trypsin dependence plaque formation assays and intravenous pathogenicity index tests were used to evaluate virus pathogenicity in vitro and in vivo, respectively. Different cell lines were chosen for comparison of virus replication capacity.ResultsAccording to the phylogenetic trees, the whole gene segments of the virus named A/Environment/Fujian/85144/2014(H1N8) were of Eurasian lineage. The HA, NA, PB1, and M genes showed the highest homology with those of H1N8 or H1N2 subtype viruses isolated from local domestic ducks, while the PB2, PA, NP and NS genes showed high similarity with the genes of H7N9 viruses detected in 2017 and 2018 in the same province. This virus presented an avian receptor binding preference. The plaque formation assay showed that it was a trypsin-dependent virus. The intravenous pathogenicity index (IVPI) in chickens was 0.02. The growth kinetics of the A/Environment/Fujian/85144/2014(H1N8) virus in different cell lines were similar to those of a human-origin virus, A/Brisbane/59/2007(H1N1), but lower than those of the control avian-origin and swine-origin viruses.ConclusionsThe H1N8 virus was identified in avian influenza-related environments in China for the first time and may have served as a gene carrier involved in the evolution of the H7N9 virus in poultry. This work further emphasizes the importance of avian influenza virus surveillance, especially in live poultry markets (LPMs). Active surveillance of avian influenza in LPMs is a major pillar supporting avian influenza control and response.

Human bocavirus detection and quantification in fecal and serum specimens from recipients of allogeneic hematopoietic stem cell transplantation: A longitudinal study.

The aim of this study was to evaluate the occurrence of human bocavirus (HBoV) and to determine viral loads in samples of patients admitted for allogeneic hematopoietic stem cell transplantation (allo-HSCT). Fecal and serum samples were collected from 19 patients, during a 24-month period. Samples were screened by quantitative polymerase chain reaction TaqMan assay, with specific probe and primers targeting the NP1 gene of all HBoVs genotypes (HBoV-1 to - 4), and viral loads were determined using serial dilutions of a recombinant plasmid. HBoV DNA was detected in 42.1% (8 of 19) of the patients in at least one type of sample (feces and/or serum) during the study period, with 75% (6 of 8) of the patients being positive in both types of sample. Viral shedding in feces had a median of 26 days (range, 5 to 121) and viremia was detected in 87.5% (7 of 8) of the patients. The HBoV loads in fecal samples were higher than in sera and, in most cases, HBoV was detected earlier in fecal than in sera samples. In six HBoV-positive patients (6 of 8) diarrhea was observed concomitantly to viral detection in fecal samples. A high frequency and loads of HBoV in allo-HSCT recipients was observed, especially in fecal samples. Positivity in fecal samples was an early predictor of HBoV presence.

Swine influenza A viruses with zoonotic potential – PCR HA/NA typing, and differential detection of pandemic09 reassortants in GB and European pigs

Swine influenza A virus (swIAV) causes respiratory disease and productivity loss in pigs. Swine ‘flu viruses have been known to be both zoonotic and reverse zoonotic and they contain genes of swine, avian(av) and human(hu) origin. Surveillance of swIAV subtypes is important as genotypes/phenotypes are fluid and impact with respect to epidemiology, vaccination, pig welfare, veterinary and public health. Three sub-types (H1avN1, H1N1pdm09, H1huN2) are currently found in pigs from Great Britain (GB), plus H3huN2 in Europe and their reassortants. Screening of candidate samples is carried out by RRT-PCR assays – generic detection of swIAV (M gene) followed by a specific RRT-PCR for H1N1pdm09 (HA gene), a suite of RRT-PCR assays for sub-typing (HA and NA genes) and a (differential) RRT-PCR to specifically identify reassortant swIAVs that incorporate the pandemic 2009 internal gene cassette (NP gene). Subtyping assays, conventional and/or molecular, are carried out on virus isolation-positive and –negative (RNA only) samples from clinical material (respiratory tissue and/or nasal swabs). Since 2009, the number of swIAV has expanded with the H1N1pdm09 isolates reassorting with the traditional subtypes. Many European variants arose (&gt;25) of which some have become established – in GB including H1huN2/pdm (since 2010), and H1avN1/pdm (since 2012), and in Belgium the traditional isolates were detected plus H1pdmN1/pdm and H3huN2/pdm reassortants. PCR subtyping (2012 onwards ∼130 from GB and ∼40 from BE/NL), wholegenome sequencing and bioinformatics analysis of these isolates facilitate further diagnostic improvements and assessment of zoonotic pandemic potential (in silico and in vivo).

NP and NS1 proteins of H5N1 virus significantly upregulated IFITM1, IFITM2, and IFITM3 in A549 cells.

Avian influence virus H5N1 causes serious public health concern with significant morbidity and mortality from poultry to humans. Interferon-induced transmembrane (IFITM) proteins usually protect cells from many virus infections by viral entry and replication. The purpose of this study was to investigate whether H5N1 viral proteins involved in regulation IFITM1, IFITM2, and IFITM3 following H5N1 infection. NS1, M1, NP, PB2, HA and NA genes of H5N1 virus were generated by PCR and cloned into pcDNA3.1/myc-His (+) A vector for genes over-expression experiments. Gene expression levels was performed using Real-time PCR. Research displayed that NS1, M1, NP, and PB2 proteins of H5N1 virus increased IFITM1, IFITM2, and IFITM3 expression in A549 cells, only IFITM1 was upregulated by M1 in HEK293T cells. However, our study did not find that HA and NA of H5N1 virus affected IFITM genes family or interferon genes expression. Taken together, our data suggested that IFITM1, IFITM2, and IFITM3 might be directly upregulated via NS1, M1, NP, and PB2 proteins during H5N1 avian influenza virus infection. This study provided new insights into the influence of NS1 and NP proteins on regulation of IFITM1, IFITM2, and IFITM3 expression following H5N1 infection.

Full-genome analysis of the pandemic (H1N1) 2009 influenza viruses isolated in Ukraine during 2009-2017 years

Purpose: New studies increasingly use an analysis of the entire genome of influenza viruses that can shed new light on the evolution of viruses and the process of selecting vaccine strains. The purpose of our study was to carry out molecular-genetic and phylogenetic analysis of all genes of pandemic A (H1N1) influenza viruses which circulated in Ukraine during 2009-2017 years. Methods & Materials: Samples were analyzed using real-time polymerase chain reaction (RT-PCR). The sequences of influenza viruses from other countries were received from web-site GISAID using BLAST analysis. The influenza A (H1N1) pdm09 sequences are characterized in a neighbor-joining phylogenetic tree with reference strains rooted from the current vaccine strain, A/California/07/2009-like virus. Phylogenetic analysis was performed using MEGA 7. 3D structures were constructed in Chimera 1.11.2rc software. Results: We have analyzed viruses isolated in Ukraine in the period of 2009 - 2017 year. Over the last five years the HA genes have evolved and eight genetic groups have been designated, with A/California/7/2009 representing group 1. The viruses were isolated in Ukraine during 2009-2017 fell into genetic groups 1, 2, 6, 7 and 8. This study demonstrated mutation in all five HA antigenic sites. In the NA genes were observed mutations H275Y and D151X caused resistance to antiviral drugs in different years. NP genes had mutations N377S and V425I, which can influence the immune response. In 2015-2016 in NP genes emerged two mutations A22T and M105I. All isolates including the A/California/07/2009 vaccine strain had the D21G mutation in the M2 protein, which causes resistance to antiviral drugs such as amantadine and rimantadine. NS1 genes had few mutations: E55E, E125D and N205S, which can inhibit human immune response and increase virulence of the virus. In PA was mutation P560S that increases virulence. In addition, we found unique changes in all genes of the polymerase complex (PA, PB1, and PB2). Conclusion: According to the literature review, mutations acquired in each genome segment, in combination lead to increased virulence of the pandemic A (H1N1) pdm09 influenza viruses. Their impact on the increasing of influenza viruses’ virulence was demonstrated in the experiments on mice.

Analysis of natural recombination and host-related evolutionary dynamics of avian avulavirus 1 isolates based on positive and negative selection from 1948 to 2017.

Adaptation and evolution of avian avulavirus 1, or Newcastle disease virus (NDV), has led to tremendous economic losses worldwide. The occurrence of natural recombination and selection pressure has been traced for NDV based on a few recent reports, but a dominant pattern based on genomic characteristics is lacking. Here, we used bioinformatics tools to search for evidence of recombination in all of the available complete genome sequence of NDV (462 sequences) using RDP4 software. Geographical linkage and host cell relationships of recombinant viruses were also investigated, and a study of the adaptive evolution of avian avulavirus 1 was performed. The results revealed that recombination events could occur in any gene fragment of the NDV genome. Moreover, class I NDV isolates from wild birds could associate to generate a putative recombinant virus with a class II genome backbone. In addition, not only avirulent-virulent hybrid genotypes but also virulent-virulent natural recombinant NDV viruses were generated. Investigation of geographic relationships of recombinant isolates indicated that the highest rate of recombination occurs in Asia and the Middle East, which can be influenced by vaccination failure, evasion of the immune response, live-bird markets, and the bird trade. The M and NP genes were found to have higher negative selection rates than the other genes, which might lead to the deletion of inadaptable sequences and result in more conserved sequences. Based on our analysis, the highest rate of positive selection was observed in the L, F and HN genes, which we suggest could lead to the occurrence of evolved viruses with high pathogenicity and a better chance of survival under extreme conditions.

Mammalian-adaptive mutation NP-Q357K in Eurasian H1N1 Swine Influenza viruses determines the virulence phenotype in mice

ABSTRACT It has recently been proposed that the Eurasian avian-like H1N1 (EA H1N1) swine influenza virus (SIV) is one of the most likely zoonotic viruses to cause the next influenza pandemic. Two main genotypes EA H1N1 viruses have been recognized to be infected humans in China. Our study finds that one of the genotypes JS1-like viruses are avirulent in mice. However, the other are HuN-like viruses and are virulent in mice. The molecular mechanism underlying this difference shows that the NP gene determines the virulence of the EA H1N1 viruses in mice. In addition, a single substitution, Q357K, in the NP protein of the EA H1N1 viruses alters the virulence phenotype. This substitution is a typical human signature marker, which is prevalent in human viruses but rarely detected in avian influenza viruses. The NP-Q357K substitution is readily to be occurred when avian influenza viruses circulate in pigs, and may facilitate their infection of humans and allow viruses also carrying NP-357K to circulate in humans. Our study demonstrates that the substitution Q357K in the NP protein plays a key role in the virulence phenotype of EA H1N1 SIVs, and provides important information for evaluating the pandemic risk of field influenza strains.

Molecular genetic analysis of variability of HA, NA and NP genes of influenza virus А (compared to H1N1 and H7N9 strains)

Due to the high antigenic variability and properties for reassortment of influenza A virus genes, analyze the variability of genetic markers of two different antigenic subtypes of influenza A virus strains H1N1 and H7N9 isolated from different hosts (humans and avian). The nucleotide sequences for analysis were taken from the database (NCBI). Using the MEGA6 cluster analysis program and calculating genetic distances using the ClustalW algorithm for the coding sequences of the nucleotides of the HA, NA and NP-protein genes, determinations were made on a sample of influenza strains A. Determination of single nucleotide substitutions at positions with mutations was performed using the Flusurver program. The construction of the dendrogram was carried out using the UPGM group pairwise clustering method, the reliability was calculated using the but-strep analysis. The variability of the neurominidase, hemagglutinin, and nucleoprotein genes was determined by local sequence alignment using the Smith – Waterman algorithm of the VectorNTI-11 program. Consensus sequences (СS) for HA, NA and NP-genes were formed; common conservative areas (motives) were found. The analysis of viral nucleic acids on the variability of the genetic markers of the avian influenza virus HA, NA and NP, coding the virulence factors in the H1N1 and H7N9 subtypes, showed genetic variability (variability) of the hemagglutinin and neurominidase genes. A sample of gene sequences showed that the HA genes of the avian influenza virus have more interstitial polymorphism than the NA and NP protein genes. Genetic markers of high variability are the H1N1 subtype hemagglutinin genes and the NA genes in the H7N9 subtype. The article analyzes the structural features of the genes of surface proteins and nucleoproteins of influenza viruses AH1N1 and AH7N9. A certain degree of synonymity of nucleotide substitutions is determined. The relationship between the distribution of nucleotide polymorphism and indicators of synonymous and non-synonymous substitutions is established. The high variability of the NA gene, and somewhat less NA, determines the ability of the avian influenza virus, in particular its highly virulent strain H1N1 and less virulent H7N9, to overcome the interspecific barrier, whereas the replication factor encoded by the NP gene is less important for overcoming the interspecies barrier, which causes its low compared with ON and NA variability.

Toxico-pathological effects of meglumine antimoniate on human umbilical vein endothelial cells

Leishmaniasis is one of the most important parasitic diseases after malaria. The standard treatment of leishmaniasis includes pentavalent antimonials (SbV); however, these drugs are associated with serious adverse effects. There have been very few studies pertaining to their side effects and mechanism of action in the fetus. This investigation examines the effects of meglumine antimoniate (MA) on the survival rate, angiogenesis and cellular apoptosis in the human umbilical vein endothelial cells (HUVECs). HUVECs were treated with varying doses of MA (100–800 μg/ml) for 24, 48 and 72 h and the survival rate was studied by colorimetric assay, flow cytometry, immunocytochemistry, migration (scratch) assay and tube formation assay. The results of quantitative real-time PCR (qPCR) studies indicated that the most important genes involved in presenting angiogenesis included VEGF and its receptors (Kdr and Flt-1), NP1 and Hif-1α genes including the anti-apoptotic gene of Bcl2, were significantly reduced compared to the control group (p < 0.05). In contrast, the most leading genes involved in the phenomenon of apoptosis were P53, Bax, Bak, Apaf-1 and caspases 3, 8 and 9, which were significantly up regulated compared to the control group (p < 0.05).

The evolution and molecular characteristics of H9N2 avian influenza viruses in Jiangxi of China.

To understand the evolution and molecular characteristics of Jiangxi H9N2 viruses, we isolated 17 viruses in 2011 and analyzed their characteristics. Phylogenetic analyses revealed that their hemagglutinin genes originate from JS/1/00‐like sublineage, neuraminidase genes originate from BJ/94‐like sublineage, PB1, PA, NP, and NS genes all come from SH/F/98‐like sublineage, PB2 genes originate from ST/163/04‐like sublineage, while M genes come from G1‐like sublineage. Genotype analysis showed that our isolates were classified as genotype 57. Molecular analyses indicated that our strains contained specific sites characteristic of low‐pathogenic viruses. The current study once again highlights the necessity for continued surveillance of novel H9N2 viruses.

A novel recombinant genome of minute virus of canines in China.

In this study, we determined almost all of the genome sequence of minute virus of canines (MVC) strain CDK47/2017 and performed phylogenetic analysis with this isolate and other MVC isolates. The genome of CDK47/2017 has the following characteristics: 1) The amino acid sequence of the NS1 protein is similar to that of the novel strain 15D009/KT241026.1, which has 17 identical amino acid changes and two identical amino acid insertions compared with other known MVC strains. These two strains clustered in a unique branch in an NSI-based phylogenetic tree. 2) Phylogenetic analysis based on the NP1 protein showed that strain CDK47/2017clustered in an independent branch with strains 15D009/KT241026.1 and HM-6/AB158475.1, both of which has 10 identical amino acid changes compared with other known MVC strains. 3) Eight unique amino acid substitutions of the CDK47/2017 capsid protein resulted in it forming a unique branch in the phylogenetic tree. 4) Recombination events were identified between the 3' end of the NS1 gene and 5' end of NP1 gene. Together, these characteristics suggest that strain CDK47/2017 represents a novel MVC strain that is distinct from all known MVC strains with sequences in the GenBank database. This contributes to a greater understanding of the genetic evolution of MVC.

THE SPANISH INFLUENZA VIRUS: TREATS TO THE PORTRAIT AFTER 100 YEARS

The purpose of the study was to compare molecular characteristics of genes and proteins of pandemic influenza strains and find features of the 1918 Spanish influenza virus. Computer analysis has shown that the genes of the Spanish influenza virus in contrast to other pandemic strains contain optimal quantity of long complementary sequences that allow to obtain a supramolecular assembly of 8 virus RNA in according to a model ensuring selective packing of one copy of each virus RNA by the only possible scheme and high transmission to induce infection by single virions. Other pandemic strains contain redundant or insufficient quantity of complementary sequences that allow an assembly of its genome by means of some models including a stochastic one and occurrence of virions with incomplete genome, that is influenza virusts can exist primarily as a swarm of complementation-dependent semi-infectious virions. Analysis of an HA gene of the Spanish influenza virus found out exclusion from its translation code four triplets (CGG, CGA, CGC и CGU) coding arginine. This exclusion is observed in all the HlNl strains isolated during 100 years. Coding arginine in an HA gene of HlNl strains is provided by only triplets AGG and AGA. A NP gene of the Spanish influenza virus in contrast to other pandemics strains is avian-like and its NP protein is characterized by elevated quantity of arginine and decreased quantity of lysine that is considered as viral adaptation to avian body temperature. Prevalence of arginine provides more high positive charge for the Spanish influenza NP protein and its more powerful interaction with RNA and consequently more high thermal stability of the its RNP in comparison with the RNP of other pandemic strains. Potential consequence of existence of the avian-like NP in the Spanish influenza virus could be its high pathogenicity as infection fever creates optimal temperature for virus replication. These new data obtained by computer analysis of genomes in the Spanish influenza virus and other pandemic strains (altogether information about its proteins) can potentially be used to track pre-pandemic strains among circulating influenza A viruses and detect the formation of a possible trajectory of pandemic alert.

Development of a Novel Recombinant Adeno-Associated Virus Production System Using Human Bocavirus 1 Helper Genes

Human bocavirus 1 (HBoV1), an autonomous parvovirus, is a helper virus supporting replication of wild-type adeno-associated virus 2 (AAV2). In this study, we compared the helper functions from HBoV1 with those from adenovirus (Ad) for the production of recombinant AAV (rAAV) vector in HEK293 cells. We demonstrated that triple plasmids transfection of (1) a cloned HBoV1 helper minigenome (pBocaHelper) that expresses HBoV1 genes NP1, NS2, and BocaSR, (2) pAAV transfer plasmid, and (3) pAAVRepCap supports rAAV production in HEK293 cells. Despite a production yield of 1–2 log lower than that using pAdHelper (expressing Ad genes E2A, E4, and VA), rAAV vector produced using pBocaHelper transduced cells as efficiently as that produced using pAdHelper. The low vector production is largely due to the inefficient expression of the AAV Rep52 and capsid proteins, as well as reduced rAAV genome replication. When the AAV capsid proteins and Rep52 were ectopically expressed under strong promoters, the enhanced protein expression significantly improved the rAAV production using pBocaHelper, approaching a level of 50%–70% of that produced using pAdHelper. Through further dissection of the helper functions from pAdHelper in a five-plasmid transfection system, we found that the addition of the Ad E2A gene to the above HBoV1 helper system significantly increased rAAV DNA replication, which increased the rAAV vector production to a level of 3–7 times higher than that using pAdHelper. We finally combined HBoV1 NP1 and NS2 genes with Ad helper genes to create a novel dual helper plasmid (pABHelper) for rAAV vector production in the conventional three-plasmid transfection system. The pABHelper facilitated rAAV production at a yield ∼2 times higher than that using the pAdHelper.

Peste des Petits Ruminants outbreaks in Tunisia in 2016.

In Tunisia, 86 outbreaks of Peste des Petits Ruminants (PPR) were reported in ovine and caprine herds in 2016. Molecular characterization of PPRV strains was carried out by partial sequencing of nucleoprotein (Np) gene from diagnostic specimens. The results showed that disease outbreaks were caused by virus strains closely related to PPRV strains collected in Egypt in 2014 and 2015.

Inhibition of influenza A virus by mixed siRNAs, targeting the PA, NP, and NS genes, delivered by hybrid microcarriers

In the present study, a highly effective carrier system has been developed for the delivery of antiviral siRNA mixtures. The developed hybrid microcarriers, made of biodegradable polymers and SiO2 nanostructures, more efficiently mediate cellular uptake of siRNA than commercially available liposome-based reagents and polyethyleneimine (PEI); they also demonstrate low in vitro toxicity and protection of siRNA from RNase degradation.A series of siRNA designs (targeting the most conserved regions of three influenza A virus (IAV) genes: NP, NS, and PA) were screened in vitro using RT-qPCR, ELISA analysis, and hemagglutination assay. Based on the results of screening, the three most effective siRNAs (PA-1630, NP-717, and NS-777) were selected for in situ encapsulation into hybrid microcarriers. It was revealed that pre-treatment of cells with a mixture of PA-1630, NP-717, and NS-777 siRNAs, delivered by hybrid microcarriers, provided stronger inhibition of viral M1 mRNA expression and control of NP protein level, after viral infection, than single pre-treatment by any of three encapsulated siRNAs used in the study. Moreover, the effective inhibition of replication in several IAV subtypes (H1N1, H1N1pdm, H5N2, and H7N9) using a cocktail of the three selected siRNAs, delivered by our hybrid capsules to the cells, was achieved. In conclusion, we have developed a proof-of-principle which shows that our hybrid microcarrier technology (utilizing a therapeutic siRNA cocktail) may represent a promising approach in anti-influenza therapy.