Generation, characterization, and protective ability of mouse monoclonal antibodies against the HA of A (H1N1) influenza virus.

Influenza virus infections pose a continuous threat to human health. Although vaccines function as a preventive and protective tool, they may not be effective due to antigen drift or an inaccurate prediction of epidemic strains. Monoclonal antibodies (mAbs) have attracted wide attention as a promising therapeutic method for influenza virus infections. In this study, three hemagglutinin (HA)-specific mAbs, named 2A1, 2H4, and 2G2, respectively, were derived from mice immunized with the HA protein from A/Michigan/45/2015(H1N1). The isolated mAbs all displayed hemagglutination inhibition activity and the 2G2 mAb exhibited the strongest neutralization effect. Two amino acid mutations (A198E and G173E), recognized in the process of selection of mAb-resistant mutants, were located in antigenic site Sb and Ca1, respectively. In prophylactic experiments, all three mAbs could achieve 100% protection in mice infected with a lethal dose of A/Michigan/45/2015(H1N1). A dose of 1 mg/kg for 2H4 and 2G2 was sufficient to achieve a full protective effect. Therapeutic experiments showed that all three mAbs could protect mice from death if they received the mAb administration at 6 h postinfection, and 2G2 was still protective after 24 h. Our findings indicate that these three mAbs may have potential prevention and treatment value in an H1N1 epidemic, as well as in the study of antigen epitope recognition.

Rapid emergence of a PB2 D701N substitution during adaptation of an H9N2 avian influenza virus in mice.

H9N2 avian influenza viruses (AIVs) have been isolated frequently from multiple avian species and, occasionally, from humans. To explore the potential molecular basis of cross-species transmission of H9N2 AIVs, an H9N2 AIV (A/chicken/Zhejiang/221/2016) was serially passaged in mouse lung. The results showed that the mouse-adapted H9N2 virus exhibited higher virulence and replicated more efficiently in mouse lung and liver. Whole-genome sequencing showed an amino acid substitution, D701N, in the PB2 protein, which is likely associated with the increased replicative ability of H9N2 virus in mice. The rapid emergence of adaptive substitutions indicates the necessity of continuous monitoring of H9N2 virus in poultry.

Increased virulence of a novel reassortant H1N3 avian influenza virus in mice as a result of adaptive amino acid substitutions.

In this study, a novel multiple-gene reassortant H1N3 subtype avian influenza virus (AIV) (A/chicken/Zhejiang/81213/2017, CK81213) was isolated in Eastern China, whose genes were derived from H1 (H1N3), H7 (H7N3 and H7N9), and H10 (H10N3 and H10N8) AIVs. This AIV belongs to the avian Eurasian-lineage and exhibits low pathogenicity. Serial lung-to-lung passages of CK81213 in mice was performed to study the amino acid substitutions potentially related to the adaptation of H1 AIVs in mammals. And the mouse-adapted H1N3 virus showed greater virulence than wild-type H1N3 AIV in mice and the genomic analysis revealed a total of two amino acid substitutions in the PB2 (E627K) and HA (L67V) proteins. Additionally, the results of the animal study indicate that CK81213 could infect mice without prior adaption and become highly pathogenic to mice after continuous passage. Our findings show that routine surveillance of H1 AIVs is important for the prediction of influenza epidemics.

Development of a monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay for detection of H7N9 subtype avian influenza virus.

To establish a rapid detection method for H7N9 avian influenza virus (AIV), monoclonal antibodies (mAbs) against hemagglutinin (HA) of H7N9 were developed to establish an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA). AC-ELISA achieved high specificity and sensitivity, with a detection limit of 3.9 ng/mL for H7N9 HA protein (A/Zhejiang/DTID-ZJU01/2013), and 2-2 HA unit/100 µL for live H7N9 AIV. The inter- and intra-assay coefficient of variation was less than 10%. Compared with conventional virus isolation detection, the sensitivity and specificity were 94.96% and 88.24%, respectively. AC-ELISA proved to be a rapid and practical technique for the detection of H7N9 AIV.

Antigen-capture ELISA and immunochromatographic test strip to detect the H9N2 subtype avian influenza virus rapidly based on monoclonal antibodies.

BACKGROUND: The H9N2 subtype of avian influenza virus (AIV) has become the most widespread subtype of AIV among birds in Asia, which threatens the poultry industry and human health. Therefore, it is important to establish methods for the rapid diagnosis and continuous surveillance of H9N2 subtype AIV. METHODS: In this study, an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and a colloidal gold immunochromatographic test (ICT) strip using monoclonal antibodies (MAbs) 3G4 and 2G7 were established to detect H9N2 subtype AIV. RESULTS: The AC-ELISA method and ICT strip can detect H9N2 subtype AIV quickly, and do not cross-react with other subtype AIVs or other viruses. The detection limit of AC-ELISA was a hemagglutinin (HA) titer of 4 for H9N2 subtype AIV per 100 µl sample, and the limit of detection of the HA protein of AIV H9N2 was 31.5 ng/ml. The ICT strip detection limit was an HA titer of 4 for H9N2 subtype AIV per 100 µl sample. Moreover, both detection methods exhibited good reproducibility and repeatability, with coefficients of variation < 5%. For detection in 200 actual poultry samples, the sensitivities and specificities of AC-ELISA were determined as 93.2% and 98.1%, respectively. The sensitivities and specificities of the ICT strips were determined as 90.9% and 97.4%, respectively. CONCLUSIONS: The developed AC-ELISA and ICT strips displayed high specificity, sensitivity, and stability, making them suitable for rapid diagnosis and field investigation of H9N2 subtype AIV.

Isolation and characterization of two novel reassortant H5N6 avian influenza viruses from waterfowl in eastern China.

Waterfowl are considered to be the natural hosts of avian influenza virus. In 2017, two reassortant highly pathogenic H5N6 avian influenza viruses of clade 2.3.4.4, subclade II, were identified in wild birds in eastern China. Genome sequencing and phylogenetic and antigenicity analysis showed that the viruses originated from multiple reassortments. To evaluate their pathogenicity in mammals, 15 BALB/c mice were infected with these viruses, and survival and weight loss were monitored for 14 days. Infection was associated with moderate pathogenicity in the mice, and the viruses could replicate in the lungs without prior adaptation. Thus, the existence of these viruses poses a continuous threat to both birds and humans.

Methylene blue photochemical treatment as a reliable SARS-CoV-2 plasma virus inactivation method for blood safety and convalescent plasma therapy for COVID-19.

BACKGROUND: In 2020, a new coronavirus, SARS-CoV-2, quickly spread worldwide within a few months. Although coronaviruses typically infect the upper or lower respiratory tract, the virus RNA can be detected in plasma. The risk of transmitting coronavirus via transfusion of blood products remains. As more asymptomatic infections are identified in COVID-19 cases, blood safety has become particularly important. Methylene blue (MB) photochemical technology has been proven to inactivate lipid-enveloped viruses with high efficiency and safety. The present study aimed to investigate the SARS-CoV-2 inactivation effects of MB in plasma. METHODS: The SARS-CoV-2 virus strain was isolated from Zhejiang University. The live virus was harvested from cultured VERO-E6 cells, and mixed with MB in plasma. The MB final concentrations were 0, 1, 2, and 4 µM. The "BX-1 AIDS treatment instrument" was used at room temperature, the illumination adjusted to 55,000 ± 0.5 million Lux, and the plasma was irradiated for 0, 2, 5, 10, 20, and 40 mins using light at a single wavelength of 630 nm. Virus load changes were measured using quantitative reverse transcription- PCR. RESULTS: BX-1 could effectively eliminate SARS-CoV-2 within 2 mins in plasma, and the virus titer declined to 4.5 log10 TCID50 (median tissue culture infectious dose)/mL. CONCLUSION: BX-1 is based on MB photochemical technology, which was designed to inactivate HIV-1 virus in plasma. It was proven to be safe and reliable in clinical trials of HIV treatment. In this study, we showed that BX-1 could also be applied to inactivate SARS-CoV-2. During the current outbreak, this technique it has great potential for ensuring the safety of blood transfusions, for plasma transfusion therapy in recovering patients, and for preparing inactivated vaccines.

Development of an antigen-capture enzyme-linked immunosorbent assay and immunochromatographic strip based on monoclonal antibodies for detection of H6 avian influenza viruses.

Continuous surveillance has shown that H6 subtype avian influenza viruses (AIVs) are prevalent in poultry and occasionally break the species barrier to infect humans. It is therefore necessary to establish a specific, rapid and sensitive method to screen H6 AIVs. In this study, a panel of monoclonal antibodies (mAbs) against the hemagglutinin (HA) of an H6 AIV isolate was produced. The purified mAbs have high affinity and specificity for H6 AIVs. An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and immunochromatographic strip were developed based on two mAbs (1D7 and 1F12). The AC-ELISA results showed high sensitivity with a limit of detection (LOD) of 3.9 ng/ml for H6 HA protein and 0.5 HAU (HA units)/100 µl for live H6 subtype AIVs. The average recovery of the AC-ELISA with allantoic fluid, respiratory specimens, and cloacal swabs was 91.907 ± 1.559%, 82.977 ± 1.497% and 73.791 ± 2.588%, respectively. The intra- and inter-assay coefficient of variation was less than 10%. The LOD of immunochromatographic strip was 1 HAU when evaluated by the naked eye, and the detection time was less than 10 min without any equipment. Storage at room temperature or 4 °C for 30 days or 60 days had no effect on sensitivity and specificity of the strip. Thus, the AC-ELISA and immunochromatographic strips described here could be a secondary method to diagnose H6 AIV infections with high specificity, sensitivity, and stability.

Development of a colloidal gold-based immunochromatographic strip test using two monoclonal antibodies to detect H7N9 avian influenza virus.

H7N9 low pathogenic avian influenza viruses (AIVs) emerged in China in 2013 and mutated into highly pathogenic strains in 2017, causing disease in infected birds and humans. Thus, the development of rapid, specific, and sensitive detection methods is urgently required. Herein, two specific monoclonal antibodies against H7N9 AIV were produced to develop a colloidal gold-based immunochromatographic test strip to detect H7N9 AIV. High specificity, repeatability, and sensitivity were achieved, with a detection limit of two hemagglutinin units or 102.55 50% tissue culture infective dose. This assay may represent a powerful tool to rapidly detect H7N9 influenza viruses in the future.

Development and evaluation of a TaqMan MGB RT-PCR assay for detection of H5 and N8 subtype influenza virus.

BACKGROUND: Highly pathogenic influenza A (H5N8) viruses have caused several worldwide outbreaks in birds and are of potential risk to humans. Thus, a specific, rapid and sensitive method for detection is urgently needed. METHODS: In the present study, TaqMan minor groove binder probes and multiplex real-time RT-PCR primers were designed to target the H5 hemagglutinin and N8 neuraminidase genes. A total of 38 strains of avian influenza viruses and other viruses were selected to test the performance of the assay. RESULTS: The results showed that only H5 and N8 avian influenza viruses yielded a positive signal, while all other subtypes avian influenza viruses and other viruses were negative. High specificity, repeatability, and sensitivity were achieved, with a detection limit of 10 copies per reaction. CONCLUSIONS: The developed assay could be a powerful tool for rapid detection of H5N8 influenza viruses in the future.

Molecular characterization of H10 subtype avian influenza viruses isolated from poultry in Eastern China.

In recent years, avian-origin H10 influenza viruses have proved capable of infecting human beings, and they pose a potential public health threat. Seven H10 avian influenza viruses (AIVs), H10N3 (n = 2), H10N7 (n = 1), and H10N8 (n = 4), were isolated from chickens in Zhejiang Province, Eastern China, during surveillance of AIVs in live poultry markets in 2016 and 2017. Phylogenetic analysis indicated that Zhejiang H10 strains received gene segments from H10, H3, and H7 viruses from birds in East Asia. Animal inoculation tests showed that these isolates have low pathogenicity in mice and can replicate in this species. Our findings suggest these H10 AIVs have the ability to adapt to chicken or other poultry, and highlight the need of long-term surveillance.

On channel estimation schemes for APD-based DDM-OFDM-PONs under sub-Nyquist sampling.

Implementing preprocessing in a delay-division multiplexing (DDM) orthogonal frequency-division multiplexing (OFDM) passive optical network (PON) requires a priori knowledge of channel responses, which need to be estimated under the constraint of sub-Nyquist analog-to-digital sampling. The localized approach allocates subcarriers in different frequency zones to training symbols in different time slots for channel estimation without spectral overlap. Unfortunately, the localized scheme is susceptible to inaccurate estimation when using an avalanche photodiode (APD), due to variations in APD saturation associated with different training symbols. Instead of localizing all subcarriers of a training symbol in a single frequency zone, we propose distributing training subcarriers through various frequency zones. This distributed scheme would prevent spectral overlap and also reduce the degree of variation in APD saturation, thereby improving the accuracy of channel estimation. Alternatively, we propose an orthogonal scheme in which each training symbol uses all of the subcarriers simultaneously. The orthogonality specified among consecutive training symbols should make it possible to estimate the channel response with low computational complexity. We conducted experiments to compare various schemes used for channel estimation in a 25-Gbps APD-based OFDM-PON. Our results revealed that the orthogonal scheme achieved the best results, and the localized scheme provided the worst channel estimates. We demonstrate the application of the orthogonal scheme in a penalty-free DDM system at 1/32 of the Nyquist rate, which provided a loss budget of 28 dB after fiber transmission over a distance of 25 km.

Isolation and characterization of novel reassortant H6N1 avian influenza viruses from chickens in Eastern China.

BACKGROUND: The H6N1 subtype of avian influenza viruses (AIVs) can infect people with an influenza-like illness; the H6N1 viruses possess the ability for zoonotic transmission from avians into mammals, and possibly pose a threat to human health. METHODS: In 2017, live poultry markets (LPMs) in Zhejiang Province were surveyed for AIVs. To better understand the genetic relationships between these strains from Eastern China and other AIVs, all gene segments of these strains were sequenced and compared with sequences available in GenBank. In this study, we analyzed the receptor-binding specificity, antigenic characteristics, and pathogenicity of these two H6N1 viruses. RESULTS: In 2017, two H6N1 AIVs were isolated from chickens during surveillance for AIVs in LPMs in Eastern China. Phylogenetic analysis showed that these strains shared genetic characteristics from H6, H10, H1, and H4 AIVs found in ducks and wild birds in East Asia. These AIV strains were able to replicate in mice without prior adaptation. CONCLUSIONS: In this study, we report the discovery of new strains of H6N1 viruses from chickens with novel gene reassortments. Our results suggest that these chickens play an important role generating novel reassortments in AIVs, and emphasize the need for continued surveillance of AIV strains circulating in poultry.

Establishment of a multiplex real-time RT-PCR assay for rapid identification of H6 subtype avian influenza viruses.

The H6 subtype avian influenza viruses (AIVs) possess the capacity for zoonotic transmission from avian species to humans. Establishment of a specific, rapid and sensitive method to screen H6 AIVs is necessary. Based on the conserved domain of the matrix and H6 AIV hemagglutinin genes, two TaqMan minor-groove-binder probes and multiplex real-time RT-PCR primers were designed in this study. The multiplex real-time RT-PCR assay developed in this study had high specificity and repeatability and a detection limit of 30 copies per reaction. This rapid diagnostic method will be useful for clinical detection and surveillance of H6 AIVs in China.

Isolation and molecular characterization of an H5N1 swine influenza virus in China in 2015.

In 2015, an H5N1 influenza virus was isolated from a pig in Zhejiang Province, Eastern China. This strain was characterized by whole-genome sequencing with subsequent phylogenetic analysis. Phylogenetic analysis showed that all segments from this strain belonged to clade 2.3.2 and that it had received its genes from poultry influenza viruses in China. A Glu627Lys mutation associated with pathogenicity was observed in the PB2 protein. This strain was moderately pathogenic in mice and was able to replicate without prior adaptation. These results suggest that active surveillance of swine influenza should be used as an early warning system for influenza outbreaks in mammals.

Development of a TaqMan MGB RT-PCR assay for the detection of type A and subtype H10 avian influenza viruses.

H10 subtype avian influenza viruses have caused several epidemics in poultry and mammals, and specific, rapid and sensitive methods for detection are urgently needed. Herein, TaqMan minor groove binder (MGB) probes and multiplex real-time RT-PCR primers were designed based on gene regions encoding conserved domains of the nucleoprotein and H10 hemagglutinin. The developed multiplex real-time RT-PCR assay displayed high specificity, repeatability, and a detection limit of 10 copies per reaction. This diagnostic method could prove valuable for the rapid detection of H10 subtype AIVs in China.

Characterization of reassortant H1-subtype avian influenza viruses isolated from poultry in Zhejiang Province in China from 2013 to 2015.

From 2013 to 2015, 32 H1-subtype avian influenza viruses (AIVs), H1N2 (n = 12), H1N3 (n = 14), H1N4 (n = 4) and H1N9 (n = 2), were isolated from poultry in Zhejiang Province in eastern China. These strains were characterized by whole-genome sequencing with subsequent phylogenetic analysis and genetic comparison. Phylogenetic analysis of all eight viral genes showed that these strains clustered in the AIV Eurasian lineage. These strains were found to be minimally pathogenic in mice and were able to replicate in mice without prior adaptation. Continued surveillance is needed, considering the important role of poultry in AIV reassortment.

High-throughput sequencing identifies HIV-1-replication- and latency-related miRNAs in CD4+ T cell lines.

MicroRNAs are potent gene expression regulators involved in regulating various biological processes, including host-pathogen interactions. In this study, we used high-throughput sequencing to investigate cellular miRNA signatures related to HIV-1 replication and latent infection in CD4+ T cell lines, which included HIV-1-replicating H9/HTLV-IIIB, HIV-1-latently-infected CEM-Bru cells, and their parental uninfected H9 and CEM-SS cells. Relatively few miRNAs were found to be modulated by HIV-1 replication or latent infection, while the cell-lineage-specific miRNA difference was more pronounced, irrespective of HIV-1 infection. In silico analysis showed that some of our HIV-1 infection-regulated miRNA profiles echoed previous studies, while others were novel. In addition, some of the miRNAs that were differentially expressed between the productively and latently infected cells seemed to participate in shaping the differential infection state. Thus, the newly identified miRNA profiles related to HIV-1 replication and latency provide information about the interplay between HIV-1 and its host.

Amino acid substitutions occurring during adaptation of an emergent H5N6 avian influenza virus to mammals.

Avian influenza viruses (AIVs) are known to cross species barriers, and emergent highly pathogenic H5N6 AIVs pose a serious threat to human health and the poultry industry. Here, we serially passaged an H5N6 virus 10 times in BALB/c mice. The pathogenicity of the wild-type 6D2 (WT-6D2) and mammal-adapted 6D2 strain (MA-6D2) were compared. The viral titer in multiple organs and the death rate for MA-6D2 were significantly higher than for WT-6D2. We provide evidence that the mutations HA A150V, NA R143K and G147E, PB2 E627K, and PA A343T may be important for adaptation of H5N6 AIVs to mammals.