Correction: Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication.

[This corrects the article DOI: 10.1371/journal.ppat.1006851.].

PIAS1-mediated SUMOylation of influenza A virus PB2 restricts viral replication and virulence.

Host defense systems employ posttranslational modifications to protect against invading pathogens. Here, we found that protein inhibitor of activated STAT 1 (PIAS1) interacts with the nucleoprotein (NP), polymerase basic protein 1 (PB1), and polymerase basic protein 2 (PB2) of influenza A virus (IAV). Lentiviral-mediated stable overexpression of PIAS1 dramatically suppressed the replication of IAV, whereas siRNA knockdown or CRISPR/Cas9 knockout of PIAS1 expression significantly increased virus growth. The expression of PIAS1 was significantly induced upon IAV infection in both cell culture and mice, and PIAS1 was involved in the overall increase in cellular SUMOylation induced by IAV infection. We found that PIAS1 inhibited the activity of the viral RNP complex, whereas the C351S or W372A mutant of PIAS1, which lacks the SUMO E3 ligase activity, lost the ability to suppress the activity of the viral RNP complex. Notably, the SUMO E3 ligase activity of PIAS1 catalyzed robust SUMOylation of PB2, but had no role in PB1 SUMOylation and a minimal role in NP SUMOylation. Moreover, PIAS1-mediated SUMOylation remarkably reduced the stability of IAV PB2. When tested in vivo, we found that the downregulation of Pias1 expression in mice enhanced the growth and virulence of IAV. Together, our findings define PIAS1 as a restriction factor for the replication and pathogenesis of IAV.

SUMOylation of Matrix Protein M1 and Filamentous Morphology Collectively Contribute to the Replication and Virulence of Highly Pathogenic H5N1 Avian Influenza Viruses in Mammals.

The matrix protein (M1) of influenza A virus plays an important role in replication, assembly, and budding. A previous study found that aspartic acid (D) at position 30 and alanine (A) at position 215 of M1 contribute to the high pathogenicity of H5N1 viruses in mice, and double mutations of D to asparagine (N) at position 30 (D30N) and A to threonine (T) at position 215 (A215T) in M1 dramatically attenuate H5N1 viruses in mice. However, the underlying mechanisms by which these M1 mutations attenuate the virulence of H5N1 viruses are unknown. Here, we found that the amino acid mutation A215T eliminates the SUMOylation of M1 by reducing its interaction with the host SUMO1 protein, significantly reducing the stability of M1, slowing the export of the M1-vRNP complex from the nucleus to the cytoplasm, and reducing viral replication in MDCK cells. We further found that the D30N mutation in M1 alters the shape of progeny viruses from filamentous to spherical virions. Our findings reveal an essential role for M1 215A SUMOylation and M1 30D-related filamentous morphology in the pathogenesis of avian influenza viruses, which could be targeted in novel antiviral drug designs. IMPORTANCE Identification of the pathogenic mechanism of highly pathogenic avian influenza viruses in mammals is helpful to develop novel anti-influenza virus strategies. Two amino acid mutations (D30N and A215T) in M1 were found to collectively attenuate H5N1 influenza viruses in mice, but the underlying mechanism remained unknown. This study found that the A215T mutation significantly decreases the SUMOylation of M1, which in turn attenuates the replication of H5N1 virus in mammalian cells. The D30N mutation in M1 was found to change the virion shape from filamentous to spherical. These findings are important for understanding the molecular mechanism of virulence of highly pathogenic avian influenza viruses in mammals.

Genetic and biological properties of H7N9 avian influenza viruses detected after application of the H7N9 poultry vaccine in China.

The H7N9 avian influenza virus (AIV) that emerged in China have caused five waves of human infection. Further human cases have been successfully prevented since September 2017 through the use of an H7N9 vaccine in poultry. However, the H7N9 AIV has not been eradicated from poultry in China, and its evolution remains largely unexplored. In this study, we isolated 19 H7N9 AIVs during surveillance and diagnosis from February 2018 to December 2019, and genetic analysis showed that these viruses have formed two different genotypes. Animal studies indicated that the H7N9 viruses are highly lethal to chicken, cause mild infection in ducks, but have distinct pathotypes in mice. The viruses bound to avian-type receptors with high affinity, but gradually lost their ability to bind to human-type receptors. Importantly, we found that H7N9 AIVs isolated in 2019 were antigenically different from the H7N9 vaccine strain that was used for H7N9 influenza control in poultry, and that replication of these viruses cannot, therefore, be completely prevented in vaccinated chickens. We further revealed that two amino acid mutations at positions 135 and 160 in the HA protein added two glycosylation sites and facilitated the escape of the H7N9 viruses from the vaccine-induced immunity. Our study provides important insights into H7N9 virus evolution and control.

Viral RNA-binding ability conferred by SUMOylation at PB1 K612 of influenza A virus is essential for viral pathogenesis and transmission.

Posttranslational modifications, such as SUMOylation, play specific roles in the life cycle of invading pathogens. However, the effect of SUMOylation on the adaptation, pathogenesis, and transmission of influenza A virus (IAV) remains largely unknown. Here, we found that a conserved lysine residue at position 612 (K612) of the polymerase basic protein 1 (PB1) of IAV is a bona fide SUMOylation site. SUMOylation of PB1 at K612 had no effect on the stability or cellular localization of PB1, but was critical for viral ribonucleoprotein (vRNP) complex activity and virus replication in vitro. When tested in vivo, we found that the virulence of SUMOylation-defective PB1/K612R mutant IAVs was highly attenuated in mice. Moreover, the airborne transmission of a 2009 pandemic H1N1 PB1/K612R mutant virus was impaired in ferrets, resulting in reversion to wild-type PB1 K612. Mechanistically, SUMOylation at K612 was essential for PB1 to act as the enzymatic core of the viral polymerase by preserving its ability to bind viral RNA. Our study reveals an essential role for PB1 K612 SUMOylation in the pathogenesis and transmission of IAVs, which can be targeted for the design of anti-influenza therapies.

Genetic analysis and biological characterization of H10N3 influenza A viruses isolated in China from 2014 to 2021.

The H10 subtypes of avian influenza viruses pose a continual threat to the poultry industry and human health. The sporadic spillover of H10 subtypes viruses from poultry to humans is represented by the H10N8 human cases in 2013 and the recent H10N3 human infection in 2021. However, the genesis and characteristics of the recent reassortment H10N3 viruses have not been systemically investigated. In this study, we characterized 20 H10N3 viruses isolated in live poultry markets during routine nationwide surveillance in China from 2014 to 2021. The viruses in the recent reassortant genotype acquired their hemagglutinin (HA) and neuraminidase (NA) genes from the duck H10 viruses and H7N3 viruses, respectively, whereas the internal genes were derived from chicken H9N2 viruses as early as 2019. Receptor-binding analysis indicated that two of the tested H10N3 viruses had a higher affinity for human-type receptors than for avian-type receptors, highlighting the potential risk of avian-to-human transmission. Animal studies showed that only viruses belonging to the recent reassortant genotype were pathogenic in mice; two tested viruses transmitted via direct contact and one virus transmitted by respiratory droplets in guinea pigs, though with limited efficiency. These findings emphasize the need for enhanced surveillance of H10N3 viruses.

Rapid detection of avian influenza virus based on CRISPR-Cas12a.

BACKGROUND: Avian influenza (AI) is a disease caused by the avian influenza virus (AIV). These viruses spread naturally among wild aquatic birds worldwide and infect domestic poultry, other birds, and other animal species. Currently, real-time reverse transcription polymerase chain reaction (rRT-PCR) is mainly used to detect the presence of pathogens and has good sensitivity and specificity. However, the diagnosis requires sophisticated instruments under laboratory conditions, which significantly limits point-of-care testing (POCT). Rapid, reliable, non-lab-equipment-reliant, sensitive, and specific diagnostic tests are urgently needed for rapid clinical detection and diagnosis. Our study aimed to develop a reverse transcription recombinase polymerase amplification (RT-RPA)/CRISPR method which improves on these limitations. METHODS: The Cas12a protein was purified by affinity chromatography with Ni-agarose resin and observed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Specific CRISPR RNA (crRNA) and primers targeting the M and NP genes of the AIV were designed and screened. By combining RT-RPA with the Cas12a/crRNA trans-cleavage system, a detection system that uses fluorescence readouts under blue light or lateral flow strips was established. Sensitivity assays were performed using a tenfold dilution series of plasmids and RNA of the M and NP genes as templates. The specificity of this method was determined using H1-H16 subtype AIVs and other avian pathogens, such as newcastle disease virus (NDV), infectious bursal disease virus (IBDV), and infectious bronchitis virus (IBV). RESULTS: The results showed that the method was able to detect AIV and that the detection limit can reach 6.7 copies/µL and 12 copies/µL for the M and NP gene, respectively. In addition, this assay showed no cross-reactivity with other avian-derived RNA viruses such as NDV, IBDV, and IBV. Moreover, the detection system presented 97.5% consistency and agreement with rRT-PCR and virus isolation for detecting samples from poultry. This portable and accurate method has great potential for AIV detection in the field. CONCLUSION: An RT-RPA/CRISPR method was developed for rapid, sensitive detection of AIV. The new system presents a good potential as an accurate, user-friendly, and inexpensive platform for point-of-care testing applications.

Analysis of avian influenza A (H3N8) viruses in poultry and their zoonotic potential, China, September 2021 to May 2022.

BackgroundTwo human cases of avian influenza A (H3N8) virus infection were reported in China in 2022.AimTo characterise H3N8 viruses circulating in China in September 2021-May 2022.MethodsWe sampled poultry and poultry-related environments in 25 Chinese provinces. After isolating H3N8 viruses, whole genome sequences were obtained for molecular and phylogenetic analyses. The specificity of H3N8 viruses towards human or avian receptors was assessed in vitro. Their ability to replicate in chicken and mice, and to transmit between guinea pigs was also investigated.ResultsIn total, 98 H3N8 avian influenza virus isolates were retrieved from 38,639 samples; genetic analysis of 31 representative isolates revealed 17 genotypes. Viruses belonging to 10 of these genotypes had six internal genes originating from influenza A (H9N2) viruses. These reassorted viruses could be found in live poultry markets and comprised the strains responsible for the two human infections. A subset of nine H3N8 viruses (including six reassorted) that replicated efficiently in mice bound to both avian-type and human-type receptors in vitro. Three reassorted viruses were shed by chickens for up to 9 days, replicating efficiently in their upper respiratory tract. Five reassorted viruses tested on guinea pigs were transmissible among these by respiratory droplets.ConclusionAvian H3N8 viruses with H9N2 virus internal genes, causing two human infections, occurred in live poultry markets in China. The low pathogenicity of H3N8 viruses in poultry allows their continuous circulation with potential for reassortment. Careful monitoring of spill-over infections in humans is important to strengthen early-warning systems and maintain influenza pandemic preparedness.

Correction: Genetics, Receptor Binding Property, and Transmissibility in Mammals of Naturally Isolated H9N2 Avian Influenza Viruses.

[This corrects the article DOI: 10.1371/journal.ppat.1004508.].

Amino Acid Mutations A286V and T437M in the Nucleoprotein Attenuate H7N9 Viruses in Mice.

The low-pathogenic H7N9 influenza viruses that emerged in 2013 acquired an insertion of four amino acids in their hemagglutinin cleavage site and thereby became highly pathogenic to chickens in 2017. Previous studies indicated that these highly pathogenic H7N9 viruses are virulent in chickens but have distinct pathotypes in mice. A/chicken/Guangdong/SD098/2017 (CK/SD098) is avirulent, with a 50% mouse lethal dose (MLD50) of >7.5 log10 50% egg infectious dose (EID50), whereas A/chicken/Hunan/S1220/2017 (CK/S1220) is virulent in mice, with an MLD50 of 3.2 log10 EID50 In this study, we explored the genetic determinants that contribute to the difference in virulence between these two H7N9 viruses by generating a series of reassortants and mutants in the CK/S1220 virus background and testing their virulence in mice. We found that the reassortant CK/1220-SD098-NP, carrying the nucleoprotein (NP) of CK/SD098, was avirulent in mice, with an MLD50 of >107.5 EID50 The NPs of these two viruses differ by two amino acids, at positions 286 and 437. We further demonstrated that the amino acid mutations A286V and T437M of NP independently slowed the process of NP import to and export from the nucleus and thus jointly impaired the viral life cycle and attenuated the virulence of these H7N9 viruses in mice. Our study identified new virulence determinants in NP and provided novel targets for the development of live attenuated vaccines and antiviral drugs against influenza viruses.IMPORTANCE The H7N9 influenza viruses that emerged in China in 2013 have caused over 1,500 human infections, with a mortality rate of nearly 40%. The viruses were initially low pathogenic but became highly pathogenic in chickens at the beginning of 2017 and caused severe disease outbreaks in poultry. Several studies suggested that the highly pathogenic H7N9 viruses have increased virulence in mammals; however, the genetic basis of the virulence of H7N9 viruses in mammals is not fully understood. Here, we found that two amino acids, 286A and 437T, in NP are prerequisites for the virulence of H7N9 viruses in mice and the mutations A286V and T437M collectively eliminate the virulence of H7N9 viruses in mice. Our study further demonstrated that the virulence of influenza viruses is a polygenic trait, and the newly identified virulence-related residues in NP may provide new targets for attenuated influenza vaccine and antiviral drug development.

The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry.

Influenza A virus (IAV) coopts numerous host factors to complete its replication cycle. Here, we identify free fatty acid receptor 2 (FFAR2) as a cofactor for IAV entry into host cells. We found that downregulation of FFAR2 or Ffar2 expression significantly reduced the replication of IAV in A549 or RAW 264.7 cells. The treatment of A549 cells with small interfering RNA (siRNA) targeting FFAR2 or the FFAR2 pathway agonists 2-(4-chlorophenyl)-3-methyl-N-(thiazol-2-yl)butanamide (4-CMTB) and compound 58 (Cmp58) [(S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide] dramatically inhibited the nuclear accumulation of viral nucleoprotein (NP) at early time points postinfection, indicating that FFAR2 functions in the early stage of the IAV replication cycle. FFAR2 downregulation had no effect on the expression of sialic acid (SA) receptors on the cell membrane, the attachment of IAV to the SA receptors, or the activity of the viral ribonucleoprotein (vRNP) complex. Rather, the amount of internalized IAVs was significantly reduced in FFAR2-knocked-down or 4-CMTB- or Cmp58-treated A549 cells. Further studies showed that FFAR2 associated with ß-arrestin1 and that ß-arrestin1 interacted with the ß2-subunit of the AP-2 complex (AP2B1), the essential adaptor of the clathrin-mediated endocytosis pathway. Notably, siRNA knockdown of either ß-arrestin1 or AP2B1 dramatically impaired IAV replication, and AP2B1 knockdown or treatment with Barbadin, an inhibitor targeting the ß-arrestin1/AP2B1 complex, remarkably decreased the amount of internalized IAVs. Moreover, we found that FFAR2 interacted with three G protein-coupled receptor (GPCR) kinases (i.e., GRK2, GRK5, and GRK6) whose downregulation inhibited IAV replication. Together, our findings demonstrate that the FFAR2 signaling cascade is important for the efficient endocytosis of IAV into host cells.IMPORTANCE To complete its replication cycle, IAV hijacks the host endocytosis machinery to invade cells. However, the underlying mechanisms of how IAV is internalized into host cells remain poorly understood, emphasizing the need to elucidate the role of host factors in IAV entry into cells. In this study, we identified FFAR2 as an important host factor for the efficient replication of both low-pathogenic and highly pathogenic IAV. We revealed that FFAR2 facilitates the internalization of IAV into target cells during the early stage of infection. Upon further characterization of the role of FFAR2-associated proteins in virus replication, we found that the FFAR2-ß-arrestin1-AP2B1 signaling cascade is important for the efficient endocytosis of IAV. Our findings thus further our understanding of the biological details of IAV entry into host cells and establish FFAR2 as a potential target for antiviral drug development.

Automatic Classification of Anterior Chamber Angle Based on Ultrasound Biomicroscopy Images.

INTRODUCTION: Evaluating the anterior chamber angle (ACA) is important for the early diagnosis and treatment of primary angle-closure glaucoma. The assessment of ultrasound biomicroscopy (UBM) images usually requires well-trained ophthalmologists and screening for patients with narrow ACA is usually time- and labor-intensive. Therefore, the automatic assessment of UBM could be cost-effective and valuable in daily practice. OBJECTIVE: The objective of this study is to develop an automatic method for localizing and classifying ACA based on UBM images. METHODS: UBM images were collected and a coarse-to-fine method was used to localize the apex of the angle recess. By analyzing the grayscale features around the angle recess, closed angles were identified, and the rest were then classified as open or narrow angles, based on the degree of ACA. Using manual classification as the reference standard, the overall accuracy (OAcc), sensitivity (Sen), specificity (Spe), and balanced accuracy of the automatic classification method were evaluated. RESULTS: A total of 540 UBM images from 290 participants were analyzed. Using these UBM images and the proposed method, the ACA was classified as open, narrow, or closed. During processing, the method localized the angle recess with 95% accuracy. The OAcc of the ACA classification was 77.8%, and the Spe and Sen of our method were 85.8 and 81.7% for angle closure; 88.9 and 75.6% for open angles; 91.9 and 76.1% for narrow angles, respectively. CONCLUSIONS: Our method of automatic angle localization and classification based on UBM images is feasible and reliable. The automatic classification of ACA provides a basis and reference for future studies.

Identification of Key Amino Acids in the PB2 and M1 Proteins of H7N9 Influenza Virus That Affect Its Transmission in Guinea Pigs.

Efficient human-to-human transmission is a prerequisite for a novel influenza virus to cause an influenza pandemic; however, the genetic determinants of influenza virus transmission are still not fully understood. In this study, we compared the respiratory droplet transmissibilities of four H7N9 viruses that are genetic closely related and found that these viruses have dissimilar transmissibilities in guinea pigs: A/Anhui/1/2013 (AH/1) transmitted efficiently, whereas the other three viruses did not transmit. The three nontransmissible viruses have one to eight amino acid differences compared with the AH/1 virus. To investigate which of these amino acids is important for transmission, we used reverse genetics to generate a series of reassortants and mutants in the AH/1 background and tested their transmissibility in guinea pigs. We found that the neuraminidase (NA) of the nontransmissible virus A/chicken/Shanghai/S1053/2013 had low enzymatic activity that impaired the transmission of AH/1 virus, and three amino acid mutations-V292I and K627E in PB2 and D156E in M1-independently abolished the transmission of the AH/1 virus. We further found that an NA reassortant and three single-amino-acid mutants replicated less efficiently than the AH/1 virus in A549 cells and that the amino acid at position 156 of M1 affected the morphology of H7N9 viruses. Our study identifies key amino acids in PB2 and M1 that play important roles in H7N9 influenza virus transmission and provides new insights into the transmissibility of influenza virus.IMPORTANCE Efficient transmission is a prerequisite for a novel influenza virus to cause an influenza pandemic; however, the genetic determinants of influenza virus transmission remain poorly understood. H7N9 influenza viruses, which emerged in 2013 in China, have caused over 1,560 human infection cases, showing clear pandemic potential. Previous studies have shown that the H7N9 viruses differ in their transmissibility in animal models. In this study, we found two amino acids in PB2 (292V and 627K) and one in M1 (156D) that are extremely important for H7N9 virus transmission. Of note, PB2 292V and M1 156D appear in most H7N9 viruses, and the PB2 627K mutation could easily occur when the H7N9 virus replicates in humans. Our study thus identifies new amino acids that are important for influenza virus transmission and suggests that just a few key amino acid changes can render the H7N9 virus transmissible in mammals.

Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication.

Transcription and replication of the influenza A virus (IAV) genome occur in the nucleus of infected cells and are carried out by the viral ribonucleoprotein complex (vRNP). As a major component of the vRNP complex, the viral nucleoprotein (NP) mediates the nuclear import of the vRNP complex via its nuclear localization signals (NLSs). Clearly, an effective way for the host to antagonize IAV infection would be by targeting vRNP nuclear import. Here, we identified phospholipid scramblase 1 (PLSCR1) as a binding partner of NP by using a yeast two-hybrid (Y2H) screen. The interaction between NP and PLSCR1 in mammalian cells was demonstrated by using co-immunoprecipitation and pull-down assays. We found that the stable overexpression of PLSCR1 suppressed the nuclear import of NP, hindered the virus life cycle, and significantly inhibited the replication of various influenza subtypes. In contrast, siRNA knockdown or CRISPR/Cas9 knockout of PLSCR1 increased virus propagation. Further analysis indicated that the inhibitory effect of PLSCR1 on the nuclear import of NP was not caused by affecting the phosphorylation status of NP or by stimulating the interferon (IFN) pathways. Instead, PLSCR1 was found to form a trimeric complex with NP and members of the importin α family, which inhibited the incorporation of importin ß, a key mediator of the classical nuclear import pathway, into the complex, thus impairing the nuclear import of NP and suppressing virus replication. Our results demonstrate that PLSCR1 negatively regulates virus replication by interacting with NP in the cytoplasm and preventing its nuclear import.

Development of a duplex TaqMan real-time RT-PCR assay for simultaneous detection of newly emerged H5N6 influenza viruses.

BACKGROUND: In 2017-2018, a new highly pathogenic H5N6 avian influenza virus (AIV) variant appeared in poultry and wild birds in Asian and European countries and caused multiple outbreaks. These variant strains are different from the H5N6 virus associated with human infection in previous years, and their genetic taxonomic status and antigenicity have changed. Therefore, revision of the primers and probes of fluorescent RT-PCR is important to detect the new H5N6 subtype AIV in poultry and reduce the risk of an epidemic in birds or humans. METHODS: In this study, the primers and probes including three groups of HA and four groups of NA for H5N6 influenza virus were evaluated. Then a set of ideal primer and probes were selected to further optimize the reaction system and established a method of double rRT-PCR assay. The specificity of this method was determined by using H1~H16 subtype AIV. RESULTS: The results showed that fluorescence signals were obtained for H5 virus in FAM channel and N6 virus in VIC channel, and no fluorescent signal was observed in other subtypes of avian influenza viruses. The detection limit of this assay was 69 copies for H5 and 83 copies for N6 gene. And, the variability tests of intra- and inter-assay showed excellent reproducibility. Moreover, this assay showed 100% agreement with virus isolation method in detecting samples from poultry. CONCLUSION: The duplex rRT-PCR assay presented here has high specificity, sensitivity and reproducibility, and can be used for laboratory surveillance and rapid diagnosis of newly emerged H5N6 subtype avian influenza viruses.

MicroRNA-203 Increases Cell Radiosensitivity via Directly Targeting Bmi-1 in Hepatocellular Carcinoma.

BACKGROUND: B-cell-specific moloney leukemia virus insertion site 1 (Bmi-1) plays important roles in various cancers, but its regulation through microRNAs (miRNAs) and its functions in hepatocellular carcinoma (HCC) remains unclear. METHODS: We evaluated the expression and prognostic significance of Bmi-1 in HCC by using tissue samples and The Cancer Genome Atlas (TCGA) data sets. The relationship between miRNAs and Bmi-1 was verified by bioinformatics prediction and immunofluorescence. Colony formation and apoptosis assays were used to reveal the effect of miR-203 on radiosensitivity. RESULTS: The Bmi-1 mRNA and protein were upregulated in HCC tissues. Cox regression multivariate analyses showed that Bmi-1 overexpression was an independent prognostic parameter for HCC patients. The expression level of Bmi-1 was negatively associated with miR-203 levels in HCC tissues. Dual-luciferase reporter assays showed that miR-203 could target the 3' untranslated region (3'-UTR) of Bmi-1 directly. Overexpression of miR-203 in HepG2 and Smmc-7721 cells increases their sensitivity to ionizing radiation in vitro and in vivo. Moreover, the improved cell radiosensitivity induced by miR-203 could be rescued by restoration of Bmi-1 expression. CONCLUSIONS: Bmi-1 could improve the predictive accuracy for HCC patients' survival. Moreover, miR-203 enhance cell radiosensitivity in vitro and in vivo by targeting Bmi-1 in HCC.

Area of the cone interdigitation zone in healthy Chinese adults and its correlation with macular volume.

BACKGROUND: Numerous studies have suggested that the integrity of the cone interdigitation zone (IZ) could be considered to be a marker of photoreceptor damage and its recovery. However, little is known about the IZ in healthy eyes. Our present study was to measure the cone IZ area by optical coherence tomography (OCT), and determine its distribution in healthy adults. METHODS: This was a cross-sectional non-interventional study. We involved a group of 158 emmetropic or low myopic (from -3D to + 0.5D) eyes in 97 healthy adult volunteers. All subjects underwent thorough ophthalmologic examinations and the posterior pole was scanned by OCT. The cone IZ area in healthy adults and its correlation with macular volume and other factors was analyzed. RESULTS: The cone IZ was visible and clear in all 158 eyes, and the IZ area was successfully measured by 6 radical scans centered on the fovea. The mean IZ area was 30.22 ± 12.70 mm2, and ranged from 5.91 to 57.47 mm2. The IZ area exhibited a normal distribution (P = 0.635) with 95% confidence interval of 28.06-32.29 mm2. The IZ area was significantly correlated with the retinal and outer nuclear layer (ONL) volumes within the macula. CONCLUSIONS: The cone IZ area could be measured using a commercially available OCT system. The IZ area showed high variability among healthy adults, and this might be related to the variability in the photoreceptor distribution in healthy adults.

Glycine at Position 622 in PB1 Contributes to the Virulence of H5N1 Avian Influenza Virus in Mice.

UNLABELLED: We isolated two H5N1 viruses, A/duck/Hunan/S4020/2008 (DK/08) and A/chicken/Guangxi/S2039/2009 (CK/09), from live-bird markets during routine surveillance and found that these two viruses are genetically similar but differ in their replication and virulence in mice. The CK/09 virus is lethal for mice with a 50% mouse lethal dose (MLD50) of 1.6 log10 50% egg infectious doses (EID50), whereas the DK/08 virus is nonpathogenic for mice with an MLD50 value of 6.2 log10 EID50. We explored the genetic basis of the virulence difference of these two viruses by generating a series of reassortant viruses and mutants in the lethal virus CK/09 background and evaluating their virulence in mice. We found that the PB1 gene of the DK/08 virus dramatically attenuated the virulence of the CK/09 virus and that the amino acid at position 622 in PB1 made an important contribution. We further demonstrated that the mutation of glycine (G) to aspartic acid (D) at position 622 in PB1 partially impaired the binding of PB1 to viral RNA, thereby dramatically decreasing the polymerase activity and attenuating H5N1 virus virulence in mice. Our results identify a novel virulence-related marker of H5N1 influenza viruses and provide a new target for live attenuated vaccine development. IMPORTANCE: H5N1 avian influenza viruses have caused the deaths of nearly 60% of the humans that they have infected since 1997 and clearly represent a threat to public health. A thorough understanding of the genetic basis of virulence determinants will provide important insights for antiviral drug and live attenuated vaccine development. Several virulence-related markers in the PB2, PA, M1, and NS1 proteins of H5N1 viruses have been identified. In this study, we isolated two H5N1 avian influenza viruses that are genetically similar but differ in their virulence in mice, and we identified a new virulence-related marker in the PB1 gene. We found that the mutation of glycine (G) to aspartic acid (D) at position 622 in PB1 partially impairs the binding of PB1 to viral RNA, thereby attenuating H5N1 virus virulence in mice. This newly identified virulence-related marker could be applied to the development of live attenuated vaccines against H5N1 influenza.

Characterization of Clade 7.2 H5 Avian Influenza Viruses That Continue To Circulate in Chickens in China.

The H5N1 avian influenza viruses emerged in Southeast Asia in the late 20th century and have evolved into multiple phylogenetic clades based on their hemagglutinin (HA)-encoding genes. The clade 7.2 viruses were first detected in chickens in northern China in 2006, and vaccines specifically targeted to the clade were developed and have been used in poultry in China since 2006. During routine surveillance and disease diagnosis, we isolated seven H5 viruses between 2011 and 2014 that bear the clade 7.2 HA genes. Here, we performed extensive studies to understand how the clade 7.2 H5 viruses have evolved in chickens in China. Full genome sequence analysis revealed that the seven viruses formed two subtypes (four H5N1 viruses and three H5N2 viruses) and four genotypes by deriving genes from other influenza viruses. All of the viruses had antigenically drifted from the clade 7.2 viruses that were isolated in 2006. Pathogenicity studies of four viruses, one from each genotype, revealed that all of the viruses are highly pathogenic in chickens, but none of them could replicate in ducks. The four viruses exclusively bound to avian-type receptors and replicated only in the turbinates and/or lungs of mice; none of them were lethal to mice at a dosage of 106 50% egg infective doses (EID50). Our study indicates that although the clade 7.2 viruses have not been eradicated from poultry through vaccination, they have not become more dangerous to other animals (e.g., ducks and mice) and humans. IMPORTANCE: Animal influenza viruses can acquire the ability to infect and kill humans. The H5N1 viruses have been a concern in recent decades because of their clear pandemic potential. We sorted H5N1 influenza viruses into different phylogenetic clades based on their HA genes. The clade 7.2 viruses were detected in chickens in several provinces of northern China in 2006. Vaccines for these viruses were subsequently developed and have been used ever since to control infection of poultry. Here, we analyzed the genetic and biologic properties of seven clade 7.2 viruses that were isolated from chickens between 2011 and 2014. We found that after nearly 9 years of circulation in chickens, the clade 7.2 viruses still exclusively bind to avian-type receptors and are of low pathogenicity to mice, suggesting that these H5 viruses pose a low risk to human public health.

Genetics, Receptor Binding, Replication, and Mammalian Transmission of H4 Avian Influenza Viruses Isolated from Live Poultry Markets in China.

UNLABELLED: H4 avian influenza virus (AIV) is one of the most prevalent influenza virus subtypes in the world. However, whether H4 AIVs pose a threat to public health remains largely unclear. Here, we analyzed the phylogenetic relationships, receptor binding properties, replication, and transmissibility in mammals of H4 AIVs isolated from live poultry markets in China between 2009 and 2012. Genomic sequence analysis of 36 representative H4 viruses revealed 32 different genotypes, indicating that these viruses are undergoing complex and frequent reassortment events. All 32 viruses tested could replicate in the respiratory organs of infected mice without prior adaptation. Receptor binding analysis demonstrated that the H4 AIVs bound to α-2,6-linked glycans, although they retained the binding preference for α-2,3-linked glycans. When we tested the direct-contact transmission of 10 H4 viruses in guinea pigs, we found that three viruses did not transmit to any of the contact animals, one virus transmitted to one of three contact animals, and six viruses transmitted to all three contact animals. When we further tested the respiratory droplet transmissibility of four of the viruses that transmitted efficiently via direct contact, we found that three of them could transmit to one or two of the five exposed animals. Our study demonstrates that the current circulating H4 AIVs can infect, replicate in, and transmit to mammalian hosts, thereby posing a potential threat to human health. These findings emphasize the continual need for enhanced surveillance of H4 AIVs. IMPORTANCE: Numerous surveillance studies have documented the wide distribution of H4 AIVs throughout the world, yet the biological properties of H4 viruses have not been well studied. In this study, we found that multiple genotypes of H4 viruses are cocirculating in the live poultry markets of China and that H4 viruses can replicate in mice, possess human-type receptor binding specificity, and transmit between guinea pigs via direct contact. Strikingly, some H4 strains also can transmit via respiratory droplet, albeit with limited efficiency. These results clearly show the potential threat posed by H4 viruses to public health.