Dairy cows inoculated with highly pathogenic avian influenza virus H5N1

Highly pathogenic avian influenza(HPAI)H5N1 hemagglutinin clade 2.3.4.4b was detected in the United States in 2021.These HPAI viruses caused mortality events in poultry,wild birds,and wild mammals.On March 25,2024,HPAI H5N1 clade 2.3.4.4b was confirmed in a dairy cow in Texas in response to a multi-state investigation into milk production losses.1 Over 200 positive herds were identified in 14 U.S.states.The case description included reduced feed intake and rumen motility in lactating cows,decreased milk production,and thick yellow milk.2,3 The diagnostic investigation revealed viral RNA in milk and mammary tissue with alveolar epithelial degeneration and necrosis and positive immunoreactivity of glandular epithelium.A single transmission event,likely from birds,was followed by limited local transmission and onward horizontal transmission of H5N1 clade 2.3.4.4b genotype B3.13.4 We sought to experimentally reproduce infection with genotype B3.13 in Holstein yearling heifers and lactating cows.Heifers were inoculated by aerosol respiratory route and cows by intramammary route.Clinical disease was mild in heifers,but infection was confirmed by virus detection,lesions,and seroconversion.Clinical disease in lactating cows included decreased rumen motility,changes to milk appearance,and production losses.Infection was confirmed by high levels of viral RNA detected in milk,virus isolation,lesions in mammary tissue,and seroconversion.This study provides the foundation to investigate additional routes of infection,pathogenesis,transmission,and intervention strategies.

Cloning and Phylogenetic Analysis of NS1 Genes from Different Isolates of H9N2 Subtype Duck Influenza Virus

[ Objective] The study aimed to lay a foundation for the further studies on function mechanism of NS1 protein in the interspecies transmission of waterfowl influenza virus. [Method] Using the serologic assay and the specific RT-PCR method, some strains of H9 subtype waterfowl influenza virus were isolated from the 12 to 20 day-old muscovy duck flocks without any clinical symptoms in different areas of Guangdong Province. Four of these strains, including A/duck/ZQ/303/2007（H9N2） （A3 for short）, A/Duck/FJ/301/2007 （H9N2） （C1 for short）, A/Duck/NH/306/2007（H9N2） （ D6 for short）, A/duck/SS/402/2007（H9N2） （ E2 for short）, and a strain named A/duck/ZC/2007（H9N2） （L1 for short） from a muscovy duck died of avian influenza virus （AIV）, were used for NSl gene cloning and sequencing. Subsequently, the obtained NSl gene sequences were compared with other NS1 sequences registered in GenBank, and the phylogenetic analysis was also conducted. [Result] When compared with the H9N2 AIV NS1 sequences in GenBank, the NSl genes of the four AIV strains A3, C1, 136 and E2 displayed homologies ranging from 99% to 100% at nucleotide level, and 95% to 100% at amino acid level; while the NSl gene of L1 strain displayed homology ranging from 94% to 97% at nucleotide level, and 93% to 98% at amino acid level. The phylogenetic tree demonstrated that A3, C1, D6 and E2 were highly resemblant, and L1 was closest to AY66473 （chicken, 2003）. By comparison with the NS1 gene sequences of L1, AF523514 （duck）, AY664743 （chicken） and EF155262.1 （quail） using DNAstar, A3, C1, D6 and E.2 presented nucleotide variations at site 21 （ R→Q）, 70, 71 （ KE→EG）, 86 （ A→S）, 124 （V→M） and 225 （ S→N）, and amino acid variations at site 21,70, 71 and 86 in dsRNA- dependent protein kinase （PKR） binding domain of NSl gene, which induced the evident variations of antigenic determinant and surface proba- bility plot of NS1 protein. [ Conclusion] This study suggested that the amino acid sequence variation in PKR binding domain of NS1 protein had something to do with the virus pathogenicity.

H5N1 influenza viruses： outbreaks and biological properties

All known subtypes of influenza A viruses are maintained in wild waterfowl, the natural reservoir of these viruses. Influenza A viruses are isolated from a variety of animal species with varying morbidity and mortality rates. More importantly, influenza A viruses cause respiratory disease in humans with potentially fatal outcome. Local or global outbreaks in humans are typically characterized by excess hospitalizations and deaths. In 1997, highly pathogenic avian influenza viruses of the H5N1 subtype emerged in Hong Kong that transmitted to humans, resulting in the first documented cases of human death by avian influenza virus infection. A new outbreak started in July 2003 in poultry in Vietnam, Indonesia, and Thailand, and highly pathogenic avian H5N1 influenza viruses have since spread throughout Asia and into Europe and Africa. These viruses continue to infect humans with a high mortality rate and cause worldwide concern of a looming pandemic. Moreover, H5N1 virus outbreaks have had devastating effects on the poultry industries throughout Asia. Since H5N1 virus outbreaks appear to originate from Southern China, we here examine H5N1 influenza viruses in China, with an emphasis on their biological properties.

An Overview of the Highly Pathogenic H5N1 Influenza Virus

Since the first human case of H5N1 avian influenza virus infection was reported in 1997, this highly pathogenic virus has infected hundreds of people around the world and resulted in many deaths. The ability of H5N1 to cross species boundaries, and the presence of polymorphisms that enhance virulence, present challenges to developing clear strategies to prevent the pandemic spread of this highly pathogenic avian influenza (HPAI) virus. This review summarizes the current understanding of, and recent research on, the avian influenza H5N1 virus, including transmission, virulence, pathogenesis, clinical characteristics, treatment and prevention.

Antiviral activity of Basidiomycete mycelia against influenza type A(serotype H1N1) and herpes simplex virus type 2 in cell culture

In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A(serotype H1N1) and herpes simplex virus type 2(HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47(H1N1) in MDCK cells reducing the infectious titer by 2.0–6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species—Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes—this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index(324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes(amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.

Molecular Characterization of Avian-like H1N1 Swine Influenza A Viruses Isolated in Eastern China, 2011

Currently, three predominant subtypes of influenza virus are prevalent in pig populations worldwide： H1N1, H3N2, and H1N2. European avian-Hke H1N1 viruses, which were initially detected in European pig populations in 1979, have been circulating in pigs in eastern China since 2007. In this study, six influenza A viruses were isolated from 60 swine lung samples collected from January to April 2011 in eastern China. Based on whole genome sequencing, molecular characteristics of two isolates were determined. Phylogenetic analysis showed the eight genes of the two isolates were closely related to those of the avian-like H1N1 viruses circulating in pig populations, especially similar to those found in China. Four potential glycosylation sites were observed at positions 13, 26, 198, 277 in the HA1 proteins of the two isolates. Due to the presence of a stop codon at codon 12, the isolates contained truncated PB1-F2 proteins. In this study, the isolates contained 591Q, 627E and 701N in the polymerase subunit PB2, which had been shown to be determinants of virulence and host adaptation. The isolates also had a D rather than E at position 92 of the NS1, a marker of mammalian adaptation. Both isolates contained the GPKV motif at the PDZ ligand domain of the 3＇ end of the NS1, a characteristic marker of the European avian-like swine viruses since about 1999, which is distinct from those of avian, human and classical swine viruses. The M2 proteins of the isolates have the mutation （S31N）, a characteristic marker of the European avian-like swine viruses since about 1987, which may confer resistance to amantadine and rimantadine antivirals. Our findings further emphasize the importance of surveillance on the genetic diversity of influenza A viruses in pigs, and raise more concerns about the occurrence of cross-species transmission events.

Antiviral activity of five Asian medicinal pant crude extracts against highly pathogenic H5N1 avian influenza virus

Objective: To study the antiviral properties of the five Asian medicinal plants against in vitro infection by the highly pathogenic avian influenza virus(H5N1).Methods: Crude extracts of Andrographis paniculata, Curcuma longa(C. longa),Gynostemma pentaphyllum, Kaempferia parviflora(K. parviflora), and Psidium guajava obtained by both water and ethanol extractions were investigated for their cytotoxicity in the Madin–Darby canine kidney cells. Thereafter, they were investigated in vitro for antiviral activity and cytokine response upon H5N1 virus infection.Results: The results revealed that both water and ethanol extracts of all the five studied plants showed significant antiviral activity against H5N1 virus. Among these plants,C. longa and K. parviflora showed strong anti-H5N1 activity. Thus, they were selected for further studies on their cytokine response upon virus infection. It was found that ethanol and water crude extracts of C. longa and K. parviflora induced significant upregulation of TNF-a and IFN-b m RNA expressions, suggesting their roles in the inhibition of H5N1 virus replication.Conclusions: To the best of the authors' knowledge, this study is among the earliest reports to illustrate the antiviral property of these Asian medicinal plants against the highly pathogenic avian H5N1 influenza virus. The results of this study shed light on alternative therapeutic sources for treatment of H5N1 influenza virus infection in the future.

Influenza virus H1N1 induced apoptosis of mouse astrocytes and the effect on protein expression

Objective:To investigate the effects of influenza A virus H1N1 infection on the proliferation and apoptosis of mouse astrocytes cells and its protein expression.Methods:After mouse astrocytes was infected with purified influenza A virus H1N1 in vitro,viral integration and replication status of the cells were detected by RT-PCR assay,cell proliferation and apoptosis was determined by MTT method and flow cytometry,respectively.Associated protein expression was delected by Western blotting.Results:Agarose gel electrophoresis showed H1N1 virus can infect astrocytes and can be copied.MTT staining showed H1N1 virus infection can inhibit the proliferation of mouse astrocytes,which makes cell viability decreased significantly.Flow cytometry showed that the proportion of Annein V staining positive vascular endothelial cells in the influenza A virus group was significantly higher than that in the control group.Western blot analysis showed after24 h and 32 h of infection,there were cells caspase-3 protein and the expression of its active form(lysed caspase-3 protein)increased.The proportion of Bax/Bcl-2 also increased.Conclusions:Influenza A virus can infect human vascular endothelial cells and proliferation and it can induce apoptosis of endothelial cells.

Structure and Anti-influenza A(H1N1) Virus Activity of Three Polysaccharides from Eucheuma denticulatum

Three polysaccharides （EW, EH and EA） were prepared from a red alga Eucheuma denticulatum by sequential extraction with cold water, hot water and sodium hydroxide water solution. Their monosaccharide compositions, relative molecular mass and structural characterization were determined by gas chromatography, high performance liquid chromatography, fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy methods. EW was hybrid l/k/v-carrageenan （701/17k/13v-car- rabiose）, EH was mainly t-carrageenan, and EA was mainly α-1,4-Glucan （88%） but mixed with small amount of t-carrageenan （12%）. The relative molecular mass ofEW, EH and EA was 480, 580 and 510kDa, respectively. The anti-influenza A （H1N1） virus activity of these three polysaccharides was evaluated using the Madin-Darby canine kidney cells model. EW showed good anti-H1N1 virus activity, its ICso was 276.5 μg mL-1, and the inhibition rate to H1N1 virus was 52% when its concentration was 250 μgmL-1. The ICs0 of t-carrageenan EH was 366.4 μgmL1, whereas EA showed lower anti-H1N1 virus activity （IC50〉430μgmL-1）. Available data obtained give positive evidence that the hybrid carrageenan EW from Eueheuma denticulatum can be used as potential anti-H1N1 virus inhibitor in future.

Sequence and phylogenetic analysis of hemagglutinin genes of H9N2 influenza viruses isolated from chicken in China from 2013 to 2015

H9N2 avian influenza virus（AIV） infection is a major problem in poultry industry worldwide. In this study, molecular characterizations and phylogenetic relationships of hemagglutinin（HA） gene sequences of H9N2 AIV of 5 Chinese isolates in 2014 recently available in Gen Bank, 3 widely used vaccine strains, and 52 novel isolates in China from 2013 to 2015 were analyzed. The homology analysis showed that the nucleotide sequences of HA gene of these recent Chinese H9N2 AIV isolates shared homologies from 94.1 to 99.9%. Phylogenetic analysis showed that all isolates belonged to AIV lineage h9.4.2.5. Fifty-six out of the 57 recent Chinese H9N2 AIV isolates had the motifs PSRSSR↓GLF at the cleavage sites within the HA protein, while one isolate PWH01 harbored LSRSSR↓GLF. Remarkably, all of the recent Chinese H9N2 AIV strains had the Q216 L substitution in the receptor binding site, which indicated that they had potential to infect humans. Most of recent Chinese H9N2 AIV isolates lost the potential N-linked glycosylation site at residues 200–202 compared with vaccine strains. This present study demonstrated that AIV lineage h9.4.2.5 was more predominant in China than other lineages as it harbored all the H9N2 AIV isolated between 2013 and 2015. Also we showed the importance of continuous surveillance of emerging H9N2 AIV in China and update of vaccine formulation accordingly in order to prevent and control H9N2 AIV.

Cross-neutralizing Anti-hemagglutinin Antibodies Isolated from Patients Infected with Avian Influenza A(H5N1) Virus

Objective To recover broad-neutralizing monoclonal antibodies(Bn Abs)from avian influenza A(H5N1)virus infection cases and investigate their genetic and functional features.Methods We screened the Abs repertoires of expanded B cells circulating in the peripheral blood of H5N1 patients.The genetic basis,biological functions,and epitopes of the obtained Bn Abs were assessed and modeled.Results Two Bn Abs,2-12 D5,and 3-37 G7.1,were respectively obtained from two human H5N1 cases on days 12 and 21 after disease onset.Both Abs demonstrated cross-neutralizing and Ab-dependent cellular cytotoxicity(ADCC)activity.Albeit derived from distinct Ab lineages,i.e.,V^H1-69-D2-15-JH^4(2-12D5)and V^H1-2-D3-9-JH^5(3-32 G7.1),the Bn Abs were directed toward CR6261-like epitopes in the HA stem,and HA2 I45 in the hydrophobic pocket was the critical residue for their binding.Signature motifs for binding with the HA stem,namely,IFY in VH1-69-encoded Abs and LXYFXW in D3-9-encoded Abs,were also observed in 2-12D5 and 3-32 G7.1,respectively.Conclusions Cross-reactive B cells of different germline origins could be activated and re-circulated by avian influenza virus.The HA stem epitopes targeted by the Bn Abs,and the two Ab-encoding genes usage implied the VH1-69 and D3-9 are the ideal candidates triggered by influenza virus for vaccine development.

Genetic Characteristics of 2009 Pandemic H1N1 Influenza A Viruses Isolated from China's Mainland

A total of 100 H1N1 flu real-time-PCR positive throat swabs collected from fever patients in Zhejiang, Hubei and Guangdong between June and November 2009, were provided by local CDC laboratories. After MDCK cell culture, 57 Influenza A Pandemic （H1N1） viruses were isolated and submitted for whole genome sequencing. A total of 39 HA sequences, 52 NA sequences, 36 PB2 sequences, 31 PB1 sequences, 40 PA sequences, 48 NP sequences, 51 MP sequences and 36 NS sequences were obtained, including 20 whole genome sequences. Sequence comparison revealed they shared a high degree of homology （96%-99%） with known epidemic strains （A/Califomia/04/2009（H1N1）. Phylogenetic analysis showed that although the sequences were highly conserved, they clustered into a small number of groups with only a few distinct strains. Site analysis revealed three substitutions at loop 220 （221-228） of the HA receptor binding site in the 39 HA sequences： A/Hubei/86/2009 PKVRDQEG→PKVRDQEA, A/Zhejiang/08/2009 PKVRDQEG→PKVRDQER, A/Hubei/75/2009 PKVRDQEG→PKVRDQGG, the A/Hubei/75/2009 was isolated from an acute case, while the other two were from patients with mild symptoms. Other key sites such as 119, 274, 292 and 294 amino acids of NA protein,627 of PB2 protein were conserved. Meanwhile, all the M2 protein sequences possessed the Ser32Asn mutation, suggesting that these viruses were resistant to adamantanes. Comparison of these sequences with other H1N1 viruses collected from the NCBI database provides insight into H1N1 transmission and circulation patterns.

Gene Expression Profiles Comparison between 2009 Pandemic and Seasonal H1N1 Influenza Viruses in A549 Cells

Objective To perform gene expression profiles comparison so that to identify and understand the potential differences in pathogenesis between the pandemic and seasonal A （H1N1） influenza viruses. Methods A549 cells were infected with A/California/07/09 （H1N1） and A/GuangdongBaoan/51/08 （H1N1） respectively at the same MOI of 2 and collected at 2, 4, 8, and 24 h post infection （p.i.）. Gene expression profiles of A549 cells were obtained using the 22 K Human Genome Oligo Array, and differentially expressed genes were analyzed at selected time points. Results Microarrays results indicated that both of the viruses suppressed host immune response related pathways including cytokine production while pandemic H1N1 virus displayed weaker suppression of host immune response than seasonal H1N1 virus. Observation on similar anti-apoptotic events such as activation of apoptosis inhibitor and down-regulation of key genes of apoptosis pathways in both infections showed that activities of promoting apoptosis were different in later stage of infection. Conclusion The immuno-suppression and anti-apoptosis events of pandemic H1N1 virus were similar to those seen by seasonal H1N1 virus. The pandemic H1N1 virus had an ability to inhibit biological pathways associated with cytokine responses, NK activation and macrophage recognition .

Host markers and correlated mutations in the overlapping genes of influenza viruses: M1, M2;NS1, NS2;and PB1, PB1-F2

The influenza A viruses have three gene segments, M, NS, and PB1, which code for more than one protein. The overlapping genes from the same segment entail their interdependence, which could be reflected in the evolutionary constraints, host distinction, and co-mutations of influenza. Most previous studies of overlapping genes focused on their unique evolutionary constraints, and very little was achieved to assess the potential impact of the overlap on other biological aspects of influenza. In this study, our aim was to explore the mutual dependence in host differentiation and co-mutations in M, NS, and PB1 of avian, human, 2009 H1N1, and swine viruses, with Random Forests, information entropy, and mutual information. The host markers and highly co-mutated individual sites and site pairs (P values < 0.035) in the three gene segments were identified with their relative significance between the overlapping genes calculated. Further, Random Forests predicted that among the three stop codons in the current PB1-F2 gene of 2009 H1N1, the significance of a mutation at these sites for host differentiation was, in order from most to least, that at 12, 58, and 88, i.e., the closer to the start of the gene the more important the mutation was. Finally, our sequence analysis surprisingly revealed that the full-length PB1-F2, if the three stop codons were all mutated, would function more as a swine protein than a human protein, although the PB1 of 2009 H1N1 was derived from human H3N2.

Identification of an H1N1 subtype of swine influenza virus and serological analysis

To investigate the epizootic of swine influenza virus(SIV),60 nasal swabs were collected from a clinical cases of pig farm in Tai’an City,Shandong Province of China in April 2017.SIV was isolated by inoculating into 10-day-old Special Pathogen Free embryonated eggs and the whole genome was sequenced.An H1N1 subtype SIV was isolated and designated as A/swine/Shandong/TA04/2017(H1N1).Phylogenetic analysis showed that apart from the polymerase A(PA) fragment belonging to the 2009 pandemic H1N1 branch,seven genome segments belonged to avian-like H1N1 influenza virus lineage.The cleavage site sequence of the hemagglutinin(HA) protein was PSIQSR↓G,which is a typical molecular biological characteristic.Five potential N-glycosylation sites(N14,N26,N277,N484 and N543) were found in the HA gene.To further investigate the epidemiology of SIV in this farm,the 995 serum samples were assessed with EAH1N1 2009 pandemic H1N1 and H3 N2 antigens.The results showed that the total positive rate was 65.43%.The positive rates of single virus infection detected by EAH1N1,2009 pdmH1N1 and H3 N2 for serum HI(Hemagglutination inhibition) were 48.35,30.85 and 7.47%,respectively.The results showed that SIV in Shandong Province has been reassorted in some segments and the SIV-positive rate was high on the SIV outbreak farm.These data provide evidence of an epizootic of SIV.

New mutational trends in the HA protein of 2009 H1N1 pandemic influenza virus from May 2010 to February 2011

As we enter the year of 2011, the 2009 H1N1 pandemic influenza virus is in the news again. At least 20 people have died of this virus in China since the beginning of 2011 and it is now the predominant flu strain in the country. Although this novel virus was quite stable during its run in the flu season of 2009-2010, a genetic variant of this virus was found in Singapore in early 2010, and then in Australia and New Zealand during their 2010 winter influenza season. Several critical mutations in the HA protein of this variant were uncovered in the strains collected from January 2010 to April 2010. Moreover, a structural homology model of HA from the A/Brisbane/10/2010(H1N1) strain was made based on the structure of A/California/04/2009 (H1N1). The purpose of this study was to investigate mutations in the HA protein of 2009 H1N1 from sequence data collected worldwide from May 2010 to February 2011. A fundamental problem in bioinformatics and biology is to find the similar gene sequences for a given gene sequence of interest. Here we proposed the inverse problem, i.e., finding the exemplars from a group of related gene sequences. With a clustering algorithm affinity propagation, six exemplars of the HA sequences were identified to represent six clusters. One of the clusters contained strain A/Brisbane/12/2010(H1N1) that only differed from A/Brisbane/10/2010 in the HA sequence at position 449. Based on the sequence identity of the six exemplars, nine mutations in HA were located that could be used to distinguish these six clusters. Finally, we discovered the change of correlation patterns for the HA and NA of 2009 H1N1 as a result of the HA receptor binding specificity switch, revealing the balanced interplay between these two surface proteins of the virus.

Distribution of Avian Influenza A Viruses in Poultry-Related Environment and Its Association with Human Infection in Henan, 2016 to 2017

Objective To survey avian influenza A viruses(AIVs) in the environment and explore the reasons for the surge in human H7 N9 cases.Methods A total of 1,045 samples were collected from routine surveillance on poultry-related environments and 307 samples from human H7 N9 cases-exposed environments in Henan from 2016 to2017. The nucleic acids of influenza A(Flu A), H5, H7, and H9 subtypes were detected by real-time polymerase chain reaction.Results A total of 27 H7 N9 cases were confirmed in Henan from 2016 to 2017, 24 had a history of live poultry exposure, and 15 had H7 N9 virus detected in the related live poultry markets(LPMs). About 96%(264/275) Flu A positive-environmental samples were from LPMs. H9 was the main AIV subtype(10.05%) from routine surveillance sites with only 1 H7-positive sample, whereas 21.17% samples were H7-positive in H7 N9 cases-exposed environments. Samples from H7 N9 cases-exposed LPMs(47.56%)had much higher AIVs positive rates than those from routine surveillance sites(12.34%). The H7+H9 combination of mixed infection was 78.18%(43/55) of H7-positive samples and 41.34%(43/104) of H9-positive samples.Conclusion The contamination status of AIVs in poultry-related environments is closely associated with the incidence of human infection caused by AIVs. Therefore, systematic surveillance of AIVs in LPMs in China is essential for the detection of novel reassortant viruses and their potential for interspecies transmission.

Integrated analysis of human influenza A(H1N1)virus infectionrelated genes to construct a suitable diagnostic model

The genome characteristics and structural functions of coding proteins correlate with the genetic diversity of the H1N1 virus,which aids in the understanding of its underlying pathogenic mechanism.In this study,analyses of the characteristic of the H1N1 virus infection-related genes,their biological functions,and infection-related reversal drugs were performed.Additionally,we used multi-dimensional bioinformatics analysis to identify the key genes and then used these to construct a diagnostic model for the H1N1 virus infection.There was a total of 169 differently expressed genes in the samples between 21 h before infection and 77 h after infection.They were used during the protein-protein interaction(PPI)analysis,and we obtained a total of 1725 interacting genes.Then,we performed a weighted gene co-expression network analysis(WGCNA)on these genes,and we identified three modules that showed significant potential for the diagnosis of the H1N1 virus infection.These modules contained 60 genes,and they were used to construct this diagnostic model,which showed an effective prediction value.Besides,these 60 genes were involved in the biological functions of this infectious virus,like the cellular response to type I interferon and in the negative regulation of the viral life cycle.However,20 genes showed an upregulated expression as the infection progressed.Other 36 upregulated genes were used to examine the relationship between genes,human influenza A virus,and infection-related reversal drugs.This study revealed numerous important reversal drug molecules on the H1N1 virus.They included rimantadine,interferons,and shikimic acid.Our study provided a novel method to analyze the characteristic of different genes and explore their corresponding biological function during the infection caused by the H1N1 virus.This diagnostic model,which comprises 60 genes,shows that a significant predictive value can be the potential biomarker for the diagnosis of the H1N1 virus infection.

Nested RT-PCR method for the detection of European avian-like H1 swine influenza A virus

Swine influenza A virus（swine IAV） circulates worldwide in pigs and poses a serious public health threat, as evidenced by the 2009 H1N1 influenza pandemic. Among multiple subtypes/lineages of swine influenza A viruses, European avian-like（EA） H1N1 swine IAV has been dominant since 2005 in China and caused infections in humans in 2010. Highly sensitive and specific methods of detection are required to differentiate EA H1N1 swine IAVs from viruses belonging to other lineages and subtypes. In this study, a nested reverse transcription（RT）-PCR assay was developed to detect EA H1 swine IAVs. Two primer sets（outer and inner） were designed specifically to target the viral hemagglutinin genes. Specific PCR products were obtained from all tested EA H1N1 swine IAV isolates, but not from other lineages of H1 swine IAVs, other subtypes of swine IAVs, or other infectious swine viruses. The sensitivity of the nested RT-PCR was improved to 1 plaque forming unit（PFU） m L^（-1） which was over 10~4 PFU m L^（-1） for a previously established multiplex RT-PCR method. The nested RT-PCR results obtained from screening 365 clinical samples were consistent with those obtained using conventional virus isolation methods combined with sequencing. Thus, the nested RT-PCR assay reported herein is more sensitive and suitable for the diagnosis of clinical infections and surveillance of EA H1 swine IAVs in pigs and humans.