H5N1 Isolates Research Articles

Genetic and Phylogenetic Characterization of the M Gene of Influenza A Virus Isolated from Iranian Patients

A few studies have been done on the molecular analysis of Iranian influenza A isolates M gene. In 2014, nasal swabs collected from outpatients with clinical symptoms in the hospital clinics of Tehran, Iran were subjected for influenza detection and subtyping using Real-Time RT-PCR. Sequence and phylogenetic analysis performed on four randomly selected isolates from each subtype (H1N1 and H3N2) using neighbor-joining method. Phylogenetic dendrograms drawn based on M nucleotide sequence of H1N1 isolates showed close relatedness with Omanian isolates while the most isolates of H3N2 have clustered with Kuwait isolates and isolates from outside of geographical location. Amino acid sequence analysis showed S31N substitution in all isolates rendering the virus resistant to adamantanes. This study determined the sequence identity and phylogenetic relatedness of M gene sequence got from Iranian influenza A isolates to elucidate the modality of relationship of this gene in comparison with its counterparts from other regions.

Phylogenetic analysis of the hemagglutinin gene of influenza A(H1N1)pdm09 and A(H3N2) virus isolates from Haryana, India.

Influenza A viruses are highly adaptable and are the main pathogen behind winter time morbidity. The present study reports the molecular and phylogenetic characterization of A(H1N1)pdm09 and H3N2 isolates from Haryana, India during 2015 influenza outbreak. A total of 144 nasopharyngeal samples were collected from Post Graduate Institute of Medical Sciences, Rohtak, Haryana, India form September 2014 to February 2016. The samples were screened for influenza A subtypes; A(H1N1)pdm09 and H3N2 by using real-time RT-PCR. Virus isolation and hemagglutinin gene sequencing studies were performed for selected positive samples. Out of 24 (16.6%) Influenza A positive samples, 13 (54.2%) and 11 (45.8%) were subtyped into A(H1N1)pdm09 and H3N2, respectively by real-time RT-PCR. Genetic analysis of A(H1N1)pdm09 isolates revealed the presence of key mutations (P100S, S202T and S220T) in HA gene as compare to reference strain A/California/07/2009 and these isolates were grouped in clade 6B.1 and 6B.2. All A(H3N2) isolates were clustered in clade 3C.2a and revealed specific amino acid substitutions of N161S and P214S in their HA genes in comparison to the reference strain A/Texas/50/2012. The HA gene sequences of all isolates showed 97-98% of nucleotide sequence similarity with their respective reference strains. Influenza A(H1N1)pdm09 and H3N2 isolates were drifted significantly from their respective vaccines strains of 2015-2016 and were more closely related to recommended vaccine strains for flu season 2017-2018. The study supports the need of routine influenza surveillance and continuous monitoring of the genetic changes in the major antigenic sites of these viruses.

Studying the mammalian adaptation potential of influenza A viruses in a single infection cycle

Various influenza A viruses (AIVs) have infected humans in the last decade. These infections result from spill over into humans at times of AIV poultry outbreaks with severe clinical consequences for those infected. These AIVs do not have the ability to be efficiently spread among humans via respiratory droplets or aerosols and subsequently cause a pandemic. However, adaptation of these avian influenza viruses to humans by mutation or reassortment may change this. We know that AIVs require adaptation of several critical characteristics in order to effectively spread between humans. Therefore, in order for human pandemic emergence, AIV that crosses into a human host must already be sufficiently able, or must rapidly adapt to overcome these constraints to transmit to a subsequent human host before infection is cleared by an immune response. In this study, we used mice and analysed the mutations acquired by avian origin H7N9 and H9N2 isolates in a temporal fashion during a single infection cycle in individual hosts. Viruses recovered from infected mice lungs were subjected to Next Generation sequencing (NGS) to identify mammalian adaptation enhancing mutations. Moreover, the timing of these mutations was correlated to the host response, with particular emphasis on cytokine profile and innate immune responses during the time prior to and following viral sequence changes to help understand the virus-host dynamics and the host environment favouring adaptation. Furthermore, understanding of the determinants and mechanisms of adaptation and transmission may aid in assessing the risks posed by avian influenza A viruses to human health.

Molecular characterization and receptor binding specificity of H9N2 avian influenza viruses based on poultry-related environmental surveillance in China between 2013 and 2016

H9N2 avian influenza viruses (AIVs) have become panzootic and caused sporadic human cases since 1998. Based on the poultry-related environmental surveillance data in mainland China from 2013 to 2016, a total of 68 representative environment isolates were selected and further investigated systematically.Phylogenetic analysis indicated that Y280-like H9N2 viruses have been predominant during 2013–2016 and acquired multiple specific amino acid substitutions that might favor viral transmission from avian to mammalians. Additionally, the viruses have undergone dramatic evolution and reassortment, resulting in an increased genetic diversity or acting as the gene contributors to new avian viruses. Receptor-binding tests indicated that most of the H9N2 isolates bound to human-type receptor, making them easily cross the species barrier and infect human efficiently. Our results suggested that the H9N2 AIVs prevalent in poultry may pose severe public health threat.

Genetic and antigenic characterization of avian influenza H9N2 viruses during 2016 in Iraq.

BackgroundLittle is known about the antigenic and genetic characteristics of influenza A viruses circulating in poultry in Iraq.ObjectiveThis study describes the genetic and antigenic characteristics of the detected avian influenza H9N2 viruses in Iraq during 2016.MethodsFull genome sequences of two H9N2 viruses isolated from chickens in Iraq during 2016 were assembled. Antigenic analyses of Iraqi H9N2 viruses and contemporary H9N2 isolates from Lebanon and Egypt were performed by hemagglutination inhibition assay.ResultsPhylogenetic analysis of surface glycoproteins and internal segments (PB2, PA, NP, M, and NS) indicated that the Iraqi H9N2 viruses were closely related to G1-like lineage of H9N2 viruses isolated from Pakistan and Iran indicating possible epidemiological links. The PB1 segments of the current characterized H9N2 viruses were not related to any of the previously characterized H9N2 viruses and closely similar to H7N7 virus detected in chickens in Germany in 2015. Multiple genetic determinants for virulence and mammalian transmission were characterized in the characterized H9N2 viruses in Iraq. The antigenic analysis showed a close relationship between H9N2 viruses in Iraq and contemporary H9N2 viruses in Egypt and Lebanon. Like H9N2 viruses, Iraqis H9N2 virus bound to human-like receptor rather than avian-like receptor thus represent a public health risk.ConclusionActive surveillance of avian influenza virus in poultry and migratory birds should be adopted to monitor the genesis and emergence of new viruses in Iraq.

Active surveillance and genetic evolution of avian influenza viruses in Egypt, 2016–2018

ABSTRACT Egypt is a hotspot for avian influenza virus (AIV) due to the endemicity of H5N1 and H9N2 viruses. AIVs were isolated from 329 samples collected in 2016–2018; 48% were H9N2, 37.1% were H5N8, 7.6% were H5N1, and 7.3% were co-infections with 2 of the 3 subtypes. The 32 hemagglutinin (HA) sequences of the H5N1 viruses formed a well-defined lineage within clade 2.2.1.2. The 10 HA sequences of the H5N8 viruses belonged to a subclade within 2.3.4.4. The 11 HA of H9N2 isolates showed high sequence homology with other Egyptian G1-like H9N2 viruses. The prevalence of H5N8 viruses in ducks (2.4%) was higher than in chickens (0.94%). Genetic reassortment was detected in H9N2 viruses. Antigenic analysis showed that H9N2 viruses are homogenous, antigenic drift was detected among H5N1 viruses. AI H5N8 showed higher replication rate followed by H9N2 and H5N1, respectively. H5N8 was more common in Southern Egypt, H9N2 in the Nile Delta, and H5N1 in both areas. Ducks and chickens played a significant role in transmission of H5N1 viruses. The endemicity and co-circulation of H5N1, H5N8, and H9N2 AIV coupled with the lack of a clear control strategy continues to provide avenues for further virus evolution in Egypt.

Diverse biological characteristics and varied virulence of H7N9 from Wave 5

ABSTRACT There was a substantial increase with infections of H7N9 avian influenza virus (AIV) in humans during Wave 5 (2016-2017). To investigate whether H7N9 had become more infectious/transmissible and pathogenic overall, we characterized the receptor binding and experimentally infected ferrets with highly pathogenic (HP)- and low pathogenic (LP)-H7N9 isolates selected from Wave 5, and compared their pathogenicity and transmissibility with a Wave 1 isolate from 2013. Studies show that A/Anhui/1/2013 (LP) and A/Chicken/Heyuan/16876/2016 (HP) were highly virulent in ferrets, A/Guangdong/Th008/2017 (HP) and A/Chicken/Huizhou/HZ-3/2017 (HP) had moderate virulence and A/Shenzhen/Th001/2016 (LP) was of low virulence in ferrets. Transmission was observed only in ferrets infected with A/Anhui/1/2013 and A/Chicken/Heyuan/16876/2016, consistent with the idea that sicker ferrets had a higher probability to transmit virus to naive animals. Given the Varied virulence and transmissibility observed in circulating H7N9 viruses from Wave 5, we conclude that the current public health risk of H7N9 has not substantially increased compared to 2013 and the circulating viruses are quite diverse.

The Pathogenesis of H7 Highly Pathogenic Avian Influenza Viruses in Lesser Scaup (Aythya affinis).

Waterfowl are the natural hosts of avian influenza virus (AIV), and through migration spread the virus worldwide. Most AIVs carried by wild waterfowl are low pathogenic strains; however, Goose/Guangdong/1996 lineage clade 2.3.4.4 H5 highly pathogenic (HP) AIV now appears to be endemic in wild birds in much of the Eastern Hemisphere. Most research efforts studying AIV pathogenicity in waterfowl thus far have been directed toward dabbling ducks. In order to better understand the role of diving ducks in AIV ecology, we previously characterized the pathogenesis of clade 2.3.4.4 H5 HPAIV in lesser scaup (Aythya affinis). In an effort to further elucidate AIV infection in diving ducks, the relative susceptibility and pathogenesis of two North American lineage H7 HPAIV isolates from the most recent outbreaks in the United States was investigated. Lesser scaup were inoculated with either A/turkey/IN/1403-1/2016 H7N8 or A/chicken/TN/17-007147-2/2017 H7N9 HPAIV by the intranasal route. The approximate 50% bird infectious dose (BID50) of the H7N8 isolate was determined to be 103 50% egg infectious doses (EID50), and the BID50 of the H7N9 isolate was determined to be <102 EID50, indicating some variation in adaptation between the two isolates. No mortality or clinical disease was observed in either group except for elevated body temperatures at 2 and 4 days postinoculation (DPI). Virus shedding was detected up to 14 DPI from both groups, and there was a trend for shedding to have a longer duration and at higher titer levels from the cloacal route. These results demonstrate that lesser scaup are susceptible to both H7 lineages of HPAIV, and similar to dabbling duck species, they shed virus for long periods relative to gallinaceous birds and don't present with clinical disease.

Apoptotic and Early Innate Immune Responses to PB1-F2 Protein of Influenza A Viruses Belonging to Different Subtypes in Human Lung Epithelial A549 Cells.

PB1-F2 is a multifunctional protein and contributes to the pathogenicity of influenza A viruses. PB1-F2 is known to have strain and cell specific functions. In this study we have investigated the apoptotic and inflammatory responses of PB1-F2 protein from influenza viruses of diverse pathogenicities in A549 lung epithelial cells. Overexpression of PB1-F2 resulted in apoptosis and heightened inflammatory response in A549 cells. Comparison revealed that the response varied with each subtype. PB1-F2 protein from highly pathogenic H5N1 virus induced least apoptosis but maximum inflammatory response. Results indicated that apoptosis was mediated through death receptor ligands TNFα and TRAIL via Caspase 8 activation. Significant induction of cytokines/chemokines CXCL10, CCL5, CCL2, IFNα, and IL-6 was noted in A549 cells transfected with PB1-F2 gene construct of 2008 West Bengal H5N1 virus (H5N1-WB). On the contrary, PB1-F2 construct from 2007 highly pathogenic H5N1 isolate (H5N1-M) with truncated N-terminal region did not evoke as exuberant inflammatory response as the other H5N1-WB with full length PB1-F2, signifying the importance of N-terminal region of PB1-F2. Sequence analysis revealed that PB1-F2 proteins derived from different influenza viruses varied at multiple amino acid positions. The secondary structure prediction showed each of the PB1-F2 proteins had distinct helix-loop-helix structure. Thus, our data substantiate the notion that the contribution of PB1-F2 to influenza pathogenicity is greatly strain specific and involves multiple host factors. This data demonstrates that PB1-F2 protein of influenza A virus, when expressed independently is minimally apoptotic and strongly influences the early host response in A549 cells.

The phylogenetic analysis and molecular characterization of H9N2 avian influenza viruses isolated in Jiangsu province from 2014 to 2016

H9N2 avian influenza virus (AIV) circulates widely in China, causing a large economic loss to poultry industry and posing a big potential threat to public health. From 2014 to 2016, the positive ratio of H9N2 AIV isolation from chicken in the living market of Jiangsu province is 8.39%–13.74%. Ten H9N2 virus isolates were selected for whole genome sequencing and phylogenetic analysis. The result demonstrated that the HAs of those H9N2 virus isolates belonged to the same lineage and had a high homology with earlier Jiangsu isolates. In contrast, NA genes of those isolates evolved far away from the earlier Jiangsu isolates. Compared to earlier isolates, the nucleotide homology of NA genes and amino acid homology of NA protein were ranged from 91.4% to 93.9% and 91.2% to 93.8%, respectively. All those H9N2 viruses belonged to genotype B69 which predominates at present in China. The result of MegAlign analysis showed that 226 site in the hemagglutinins of those H9N2 isolates were Leucine. Further, it was proved that those H9N2 virus isolates preferred to bind to human type α2,6 sialic acid receptor in solid-phase receptor binding assay.

Structure-function analysis of neutralizing antibodies to H7N9 influenza from naturally infected humans.

Little is known about the specificities and neutralization breadth of the H7-reactive antibody repertoire induced by natural H7N9 infection in humans. We have isolated and characterized 73 H7-reactive monoclonal antibodies from peripheral B cells from four donors infected in 2013 and 2014. Of these, 45 antibodies were H7-specific, and 17 of these neutralized the virus, albeit with few somatic mutations in their variable domain sequences. An additional set of 28 antibodies, isolated from younger donors born after 1968, cross-reacted between H7 and H3 haemagglutinins in binding assays, and had accumulated significantly more somatic mutations, but were predominantly non-neutralizing in vitro. Crystal structures of three neutralizing and protective antibodies in complex with the H7 haemagglutinin revealed that they recognize overlapping residues surrounding the receptor-binding site of haemagglutinin. One of the antibodies, L4A-14, bound into the sialic acid binding site and made contacts with haemagglutinin residues that were conserved in the great majority of 2016-2017 H7N9 isolates. However, only 3 of the 17 neutralizing antibodies retained activity for the Yangtze River Delta lineage viruses isolated in 2016-2017 that have undergone antigenic change, which emphasizes the need for updated H7N9 vaccines.

Pathogenicity and Full Genome Sequencing of the Avian Influenza H9N2 Moroccan Isolate 2016.

In Morocco in early 2016, a low pathogenic avian influenza virus serotype H9N2 caused large economic losses to the poultry industry, with specific clinical symptoms and high mortality rates on infected farms. Subsequent to the H9N2 outbreak, the causal agent was successfully isolated from chicken flocks with high morbidity and mortality rates, propagated on embryonated eggs, and fully sequenced. The phylogenetic analysis suggested that the Moroccan isolate could have derived from the Middle East isolate A/chicken/Dubai/D2506.A/2015. This study was designed to assess the pathogenicity of the Moroccan isolate H9N2 in experimentally infected broiler and specific-pathogen-free (SPF) chickens. At 22 days of age, one broiler and two SPF chicken groups were inoculated by dropping 0.2 ml of the H9N2 isolate (107.5 EID50/ml) in both nostrils and eyes. Clinically inoculated chickens with H9N2 displayed mild lesions, low mortality rates, and an absence of clinical signs. The H9N2 virus was more pathogenic in broiler chickens and produced more severe tissue lesions compared to SPF chickens. The viral shedding was detected up to 6 days postinoculation (pi) in oropharyngeal and cloacal swabs in infected birds with a maximum shedding in the oropharynges of the broiler group. All experimental chickens seroconverted and registered high hemagglutination inhibition titers as early as day 7 pi. The present study indicates that the H9N2 virus isolated from a natural outbreak was of low pathogenicity under experimental conditions. However, under field conditions infection with other pathogens might have aggravated the disease.

Broad neutralizing activity of a human monoclonal antibody against H7N9 strains from 2013 to 2017

H7N9 influenza virus has been circulating among humans for five epidemic waves since it was first isolated in 2013 in China. The recent increase in H7N9 infections during the fifth outbreak in China has caused concerns of a possible pandemic. In this study, we describe a previously characterized human monoclonal antibody, HNIgGA6, obtained by isolating rearranged heavy-chain and light-chain genes from patients who had recovered from H7N9 infections. HNIgGA6 recognized multiple HAs and neutralized the infectivity of 11 out of the 12 H7N9 strains tested, as well as three emerging HPAI H7N9 isolates. The only resistant strain was A/Shanghai/1/2013 (H7N9-SH1), which carries the avian receptor alleles 186V and 226Q in the sialic acid-binding pocket. The mAb broadly neutralized divergent H7N9 strains from 2013 to 2017 and represents a potential alternative treatment for H7N9 interventions.

Genetic characteristics of H9N2 avian influenza viruses isolated from free-range poultry in Eastern China, in 2014-2015.

Circulating H7N9 influenza viruses in live poultry markets continue to pose a threat to human health. Free-range poultry, one of the sources for these markets, are common in China as well as in many developing countries. Because the H9N2 virus could be a source of internal genes for the H7N9 virus, we conducted surveillance in free-range poultry and live poultry markets to study the evolution of H7N9 and H9N2 viruses in Eastern China. We found 28 samples positive for the H9N2 virus (a rate of 3.2%), but no positive samples for the H7N9 virus. Six representative H9N2 isolates were sequenced and analyzed, and the results showed that these viruses shared high nucleotide identities (99.0 to 100%) and were in a same branch in the phylogenetic trees. All these 6 viruses are closely clustered with Zhejiang H9N2 chicken isolates, and belonged to genotype G57, along with some novel H7N9 strains and H9N2 strains circulating in humans in China. We hope that surveillance of AIVs in free-range poultry will be strengthened for further identification more genetic diversity.

Studies on pathogenicity of local Newcastle disease genotype VII and Avian influenza H9N2 isolates to commercial vaccinated male layer chickens.

This study was carried out to compare pathogenicity of single and co-infection of Newcastle disease virus (NDV) genotype VIIj and Avian Influenza (AI) H9N2 virus in commercial male layer chicken vaccinated with live ND (Hitchner B1, La Sota) vaccines from different commercial producers in two vaccination schemes. NDV haemaglutination inhibition (HI (log2) mean titers in group received vaccines 1 (4.8 ± 0.8, 2.4 ± 1.2 and 3.9 ± 0.5) were lower than group given vaccine 2 (4.5 ± 1.2, 3.6 ± 1.1 and 4.0 ± 0.4) at 14, 21 and 29 days of age, respectively. Chicken group received ND vaccine 2 and inactivated H9 showed relatively higher HI titres (5.0 ± 1.5, 3.5 ± 1.4 and 4.5 ± 1.3). HI titres against H9N2 in group given H9N2 inactivated vaccine are 3.7 ± 1.6, 2.5 ± 1.1 and 5.1 ± 1.3 at 14, 21 and 29 days, respectively. Challenge with Newcastle disease virus (NDV) genotype VIIj at 33 days of age resulted in 100% mortality with severe signs as well as in non-vaccinated control sub-group challenged and 100% mortalities. Sub-groups (2a, 3a and 4a) vaccinated with ND vaccine 1, ND vaccine 2 and ND vaccine 2+ H9N2 showed signs of depression, off food and moderate respiratory signs with protection rates is 70, 70 and 75%, respectively. Control group challenged with H9N2 showed general signs with mild respiratory signs and 10% mortalities while, sub-groups given ND vaccine 2+ H9N2 and challenged with H9N2 showed 100% survival. Chicken sub-groups vaccinated with ND vaccine 1, ND vaccine 2 or ND vaccine 2+ H9N2 and challenged with NDV+ H9N2 (co-infection) showed signs form the 3rd s post infection (dpi) with moderate respiratory signs and protection rate of 50, 70 and 70%, respectively. Post-mortem lesions in Velogenic NDV (vNDV) challenged birds were septicemia, intestinal and respiratory lesion, while those challenged with both NDV and H9N2 showed more prominent lesions. The vaccinated groups showed unsatisfactory protection rates (50- 75%). Vaccine 2 showed higher protection and HI titers than vaccine 1. Simultaneous chicken challenge with H9N2 and vNDV pointed out that co-infection increased severity of clinical signs, mortality and gross lesions. Histological changes were reported in lung, intestine, and spleen of challenged non-vaccinated and vaccinated groups. The severity of tissue alteration was remarkably high in the non-vaccinated group and slightly mild tissue alteration in the vaccinated group. The histological changes ranged from severe congestion in blood vessels in tested organ with lymphoid depletion in spleen and hyperplasia of mucosal-associated lymphoid tissue in the intestine, with partial limited tissue change in the challenged vaccinated group. The local NDV strain genotype VIIj is highly pathogenic to male layer commercial chickens and the inoculation of NDV with H9N2 at the same time didn›t increase its severity. Also, it was notable that commercial Hitchner B1 and La Sota vaccines conferred partial protection for experimentally used chickens against challenge this ND field isolate.

Molecular identification of avian influenza virus subtypes H5N1 and H9N2 in birds from farms and live bird markets and in respiratory patients.

BackgroundAvian influenza viruses (AIVs) have been endemic in Egypt since 2006, and the co-circulation of high-pathogenic avian influenza H5N1 and low-pathogenic avian influenza H9N2 subtypes in poultry has been reported; therefore, Egypt is considered a hotspot for the generation of new subtypes and genotypes. We aimed to characterize AIVs circulating on commercial farms and in live bird markets (LBMs) during the winters of 2015 and 2016 in the study area and to identify H5N1 and H9N2 viruses in respiratory patients.MethodsIn total, 159 samples were collected from ducks, pigeons and quails on farms (n = 59) and in LBMs (n = 100) and screened by real-time RT-PCR for H5N1 and H9N2 subtypes. Clinical and postmortem examination was carried out on birds from the farms. Positive H5N1 samples were sequenced and analysed for mutations. Tracheal swabs were also collected from 89 respiratory patients admitted to respiratory hospitals in the same study area.ResultsOverall, H5N1 was identified in 13.6% of birds from farms, while it was detected in 17% of birds in LBMs. Subtype H9N2 was only identified from pigeons on farms (6.5%) and LBMs (11.4%). Sequencing of the haemagglutination gene (HA) in nine representative H5N1 isolates revealed a multi-basic amino acid motif at the cleavage site (321-PQGEKRRKKR/GLF-333), which is characteristic of highly pathogenic AIV, in five of our isolates, while the other four isolates showed an amino acid substitution (Q322K) at this cleavage site to make it (321-P K GEKRRKKR/GLF-333). All the isolates belonged to clade 2.2.1.2, and a comparison of HA sequences at the amino acid level showed 98.8–100% homology among the nine isolates, while they showed 94.1–96.1% identity with reference strains and the commonly used vaccine strain in Egypt. Out of 89 respiratory patients, 3.4% were positive for H5N1 and no patients were positive for H9N2.DiscussionOur results indicated the circulation of the endemic H5N1 and H9N2 viruses among poultry in 2015 and 2016. Birds on farms and in LBMs are reservoirs playing a role in the dissemination of the virus and producing a public health risk. The application of proper hygienic measures in farms and LBMs to control the exposure of birds and humans to the source of infection along with continuous monitoring of the circulating viruses will provide information on understanding the evolution of the viruses for vaccine studies.

Development of a quadruple qRT-PCR assay for simultaneous identification of highly and low pathogenic H7N9 avian influenza viruses and characterization against oseltamivir resistance

BackgroundDuring the fifth wave of human H7N9 infections, a novel highly pathogenic (HP) H7N9 variant emerged with an insertion of multiple basic amino acids in the HA cleavage site. Moreover, a neuraminidase inhibitor (NAI) resistance (R292K in NA) mutation was found in H7N9 isolates from humans, poultry and the environment. In this study, we set out to develop and validate a multiplex quantitative reverse transcript polymerase chain reaction (qRT-PCR) to simultaneously detect the presence of H7N9 and further identify the HP and NAI-resistance mutations.MethodsA quadruple qRT-PCR to simultaneously detect the presence of H7N9 and further identify the HP and NAI-resistance mutations was designed based on the analyses of the HA and NA genes of H7N9. This assay was further tested for specificity and sensitivity, and validated using clinical samples.ResultsThe assay was highly specific and able to detect low pathogenic (LP)- or HP-H7N9 with/without the NAI-resistance mutation. The detection limit of the assay was determined to be 50 genome-equivalent copies and 2.8 × 10− 3 50% tissue culture infectious doses (TCID50) of live H7N9 per reaction. Clinical validation was confirmed by commercial kits and Sanger sequencing with ten clinical samples.ConclusionsWe developed and validated a rapid, single-reaction, one-step, quadruple real-time qRT-PCR to simultaneously detect the presence of H7N9 and further identify the HP- and NAI-resistance strains with excellent performance in specificity and sensitivity. This assay could be used to monitor the evolution of H7N9 viruses in the laboratory, field and the clinic for early-warning and the prevention of H7N9 infections.

Co-infection of highly pathogenic avian influenza and duck hepatitis viruses in Egyptian backyard and commercial ducks

Highly pathogenic avian influenza (HPAI) H5N1 virus poses a major challenge to the poultry industry and human health in Egypt. Twenty one households and eight duck farms in Sharkia Province, Egypt were investigated for the presence of avian influenza virus (AIV) and/or duck hepatitis virus 1 (DHV-1). Mortality rates among the investigated farms and yards were, 18.9% (69/365) of native ducks, 60.9% (25/41) of Pekin ducks, 60.2% (6306/10473) of Muscovy ducks and 44.9% (1353/3015) of Mallard ducks. The RT-PCR revealed the circulation of HPAI-H5N1 virus (81/104) among the examined birds with a high percentage in Muscovy (83.7%) and Pekin (83.4%) ducks. Interestingly, co-infection of HPAI and DHV-1 viruses in three ducklings with age of 4–19 days was detected. Severe neurological signs with high mortality were observed in ducklings as early as 4 days of age. Influenza virus antigen was detected in the neurons and glial cells of the brain, hepatocytes, and the intestinal submucosal plexus. Although, genetic characterization of H5N1 isolates revealed HPAIV of clade 2.2.1.2, such increased mortalities and neurological signs regardless of the duck age might imply the natural selection of HPAI in ducks. Crucial monitoring of the disease situation in ducks is essential for the implementation of an effective prevention and control program.

Protective efficacy of a high-growth reassortant H1N1 influenza virus vaccine against the European Avian-like H1N1 swine influenza virus in mice and pigs

Swine influenza A viruses (SIVs) causing outbreaks of acute, highly contagious respiratory disease in pigs also pose a potential threat to public health. European avian-like H1N1 (EA H1N1) SIVs are the predominant circulating viruses in pigs in China and also occasionally cause human infection. In this study, a high-growth reassortant virus (SH1/PR8), with HA and NA genes from a representative EA H1N1 isolate A/Swine/Shanghai/1/2014 (SH1) in China and six internal genes from the high-growth A/Puerto Rico/8/34 (PR8) virus, was generated by plasmid-based reverse genetics and tested as a candidate seed virus for the preparation of inactivated vaccine. The protective efficacy of inactivated SH1/PR8 was evaluated in mice and pigs challenged with wild-type SH1 virus. After primer and boost vaccination, the SH1/PR8 vaccine induced high-level hemagglutination inhibiting (HI) antibodies, IgG antibodies, and neutralization antibodies in mice and pigs. Mice and pigs in the vaccinated group showed less clinical phenomena and pathological changes than those in the unvaccinated group. In conclusion, the inactivated high-growth reassortant vaccine SH1/PR8 could induce high antibody levels and complete protection is expected against SH1 wild type SIV, and protection against heterologous EA H1N1 SIV needs further evaluation.

Experimental infection of pigs with H1 and H3 influenza A viruses of swine by using intranasal nebulization

BackgroundExperimental infection of pigs via direct intranasal or intratracheal inoculation has been mainly used to study the infectious process of influenza A viruses of swine (IAVs-S). Nebulization is known to be an alternative method for inoculating pigs with IAVs-S, because larger quantities of virus potentially can be delivered throughout the respiratory tract. However, there is very little data on the experimental infection of pigs by inhalation using nebulizer. In the current study, we used intranasal nebulization to inoculate pigs with 9 different IAVs-S—3 H1N1, 2 H1N2, and 4 H3N2 strains. We then assessed the process of infection by evaluating the clinical signs, nasal and oral viral shedding, and seroconversion rates of the pigs inoculated.ResultsLethargy and sneezing were the predominant clinical signs among pigs inoculated with 7 of the 9 strains evaluated; the remaining 2 strains (1 H1N1 and 1 H1N2 isolate) failed to induce any clinical signs throughout the experiments. Significantly increased rectal temperatures were observed with an H1N1 or H3N2 strains between 1 and 3 days post-inoculation (dpi). In addition, patterns of nasal viral shedding differed among the strains: nasal viral shedding began on 1 dpi for 6 strains, with all 9 viruses being shed from 2 to 5 dpi. The detection of viral shedding was less sensitive from oral samples than nasal secretions. Viral shedding was not detected in either nasal or oral swabs after 10 dpi. According to hemagglutination–inhibition assays, all inoculated pigs had seroconverted to the inoculating virus by 14 dpi, with titers ranging from 10 to 320.ConclusionsOur current findings show that intranasal nebulization successfully established IAV-S infections in pigs and demonstrate that clinical signs, viral shedding, and host immune responses varied among the strains inoculated.