Cryo-EM structure of Influenza A virus NS1 and antiviral protein kinase PKR complex.

Influenza A virus is the cause of a widespread human disease with high morbidity and mortality rates. The influenza virus encodes non-structural protein 1 (NS1), an exceedingly multifunctional virulence component. NS1 plays essential roles in viral replication and evasion of the cellular innate immune system. Protein kinase RNA-activated also known as protein kinase R (PKR) phosphorylates translation initiation factor eIF-2α on serine 51 to inhibit protein synthesis in virus-infected mammalian cells. Consequently, PKR activation inhibits mRNA translation, which results in the assert of both viral protein synthesis and cellular and possibly apoptosis in response to virus infection. Host signaling pathways are important in the replication of influenza virus, but the mechanisms involved remain to be characterized. Herein, the structure of NS1 and PKR complex was determined using Cryo-EM. We found the N91, E94, and G95 residues of PKR bind directly with N188, D125, and K126, respectively, of NS1. Furthermore, the study shows that PKR peptide offers a potential treatment for Influenza A virus infections.

Bioactive prenylated c6-c3 derivatives from the roots of Illicium brevistylum.

Three new prenylated C6-C3 compounds (1-3), together with two known prenylated C6-C3 compounds (4-5) and one known C6-C3 derivative (6), were isolated from the roots of Illicium brevistylum A. C. Smith. The structures of 1-3 were elucidated by spectroscopic methods including 1D and 2D NMR, HRESIMS, CD experiments and ECD calculations. The structure of illibrefunone A (1) was confirmed by single-crystal X-ray diffraction analysis. All compounds were evaluated in terms of their anti-inflammatory potential on nitric oxide (NO) generation in lipopolysaccharide-stimulated murine RAW264.7 macrophages and murine BV2 microglial cells, antiviral activity against Coxsackievirus B3 (CVB3) and influenza virus A/Hanfang/359/95 (H3N2). Compounds 3 and 4 exhibited potent inhibitory effects on the production of NO in RAW 264.7 cells with IC50 values of 20.57 and 12.87 µM respectively, which were greater than those of dexamethasone (positive control). Compounds 1 and 4-6 exhibited weak activity against Coxsackievirus B3, with IC50 values ranging from 25.87 to 33.33 µM.

A multiplex real-time RT-PCR system to simultaneously diagnose 16 pathogens associated with swine respiratory disease.

AIMS: Swine respiratory disease (SRD) is a major disease complex in pigs that causes severe economic losses. SRD is associated with several intrinsic and extrinsic factors such as host health status, viruses, bacteria, and environmental factors. Particularly, it is known that many pathogens are associated with SRD to date, but most of the test to detect those pathogens can be normally investigated only one pathogen while taking time and labor. Therefore, it is desirable to develop rapidly and efficiently detectable methods those pathogens to minimize the damage caused by SRD. METHODS AND RESULTS: We designed a multiplex real-time RT-PCR (RT-qPCR) system to diagnose simultaneously 16 pathogens, including nine viruses and seven bacteria associated with SRD, on the basis of single qPCR and RT-qPCR assays reported in previous studies. Multiplex RT-qPCR system we designed had the same ability to single RT-qPCR without significant differences in detection sensitivity for all target pathogens at minimum to maximum genomic levels. Moreover, the primers and probes used in this system had highly specificity because the sets had not been detected pathogens other than the target and its taxonomically related pathogens. Furthermore, our data demonstrated that this system would be useful to detect a causative pathogen in the diagnosis using oral fluid from healthy pigs and lung tissue from pigs with respiratory disorders collected in the field. CONCLUSIONS: The rapid detection of infected animals from the herd using our system will contribute to infection control and prompt treatment in the field.

Novel 1,2,4-Triazole- and Tetrazole-Containing 4H-Thiopyrano[2,3-b]quinolines: Synthesis Based on the Thio-Michael/aza-Morita-Baylis-Hillman Tandem Reaction and Investigation of Antiviral Activity.

A novel method for synthesizing 1,2,4-triazole- and tetrazole-containing 4H-thiopyrano[2,3-b]quinolines using a new combination of the thio-Michael and aza-Morita-Baylis-Hillman reactions was developed. Target compounds were evaluated for their cytotoxicities and antiviral activities against influenza A/Puerto Rico/8/34 virus in MDCK cells. The compounds showed low toxicity and some exhibited moderate antiviral activity. Molecular docking identified the M2 channel and polymerase basic protein 2 as potential targets. We observed that the antiviral activity of thiopyrano[2,3-b]quinolines is notably affected by both the nature and position of the substituent within the tetrazole ring, as well as the substituent within the benzene moiety of quinoline. These findings contribute to the further search for new antiviral agents against influenza A viruses among derivatives of thiopyrano[2,3-b]quinoline.

Alloferon and Zanamivir Show Effective Antiviral Activity against Influenza A Virus (H1N1) Infection In Vitro and In Vivo.

The use of vaccines is the most effective and reliable method for the prevention of viral infections. However, research on evaluation of effective therapeutic agents for use in treatment after infection is necessary. Zanamivir was administered through inhalation for treatment of pandemic influenza A/H1N1 in 2009. However, the emergence of drug-resistant strains can occur rapidly. Alloferon, an immunomodulatory drug developed as an NK cell activator, exerts antiviral effects against various viruses, particularly influenza viruses. Therefore, alloferon and zanamivir were administered in combination in an effort to improve the antiviral effect of zanamivir by reducing H1N1 resistance. First, we confirmed that administration of combined treatment would result in effective inhibition of viral proliferation in MDCK and A549 cells infected with H1N1. Production of IL-6 and MIP-1α in these cells and the activity of p38 MAPK and c-Jun that are increased by H1N1 were inhibited by combined treatment. Mice were then infected intranasally with H1N1, and examination of the antiviral efficacy of the alloferon/zanamivir combination was performed. The results showed that combined treatment after infection with H1N1 prevented weight loss, increased the survival rate, and improved lung fibrosis. Combined treatment also resulted in reduced infiltration of neutrophils and macrophages into the lungs. Combined treatment effectively inhibited the activity of p38 MAPK and c-Jun in lung tissue, which was increased by infection with H1N1. Therefore, the combination of alloferon/zanamivir effectively prevents the development of H1N1-mediated inflammation in the lungs by inhibiting the production of inflammatory mediators and migration of inflammatory cells into lung tissue.

Polyhydroxy p-Terphenyls from a Mangrove Endophytic Fungus Aspergillus candidus LDJ-5.

Six undescribed polyhydroxy p-terphenyls, namely asperterphenyllins A-F, were isolated from an endophytic fungus Aspergillus candidus LDJ-5. Their structures were determined by NMR and MS data. Differing from the previously reported p-terphenyls, asperterphenyllin A represents the first p-terphenyl dimer connected by a C-C bond. Asperterphenyllin A displayed anti-influenza virus A (H1N1) activity and protein tyrosine phosphatase 1B (PTP1B) inhibitory activity with IC50 values of 53 µM and 21 µM, respectively. The anti-influenza virus A (H1N1) activity and protein tyrosine phosphatase 1B (PTP1B) inhibitory activity of p-terphenyls are reported for the first time. Asperterphenyllin G exhibited cytotoxicity against nine cell lines with IC50 values ranging from 0.4 to 1.7 µM. Asperterphenyllin C showed antimicrobial activity against Proteus species with a MIC value of 19 µg/mL.

Structure and Activities of the NS1 Influenza Protein and Progress in the Development of Small-Molecule Drugs.

The influenza virus causes human disease on a global scale and significant morbidity and mortality. The existing vaccination regime remains vulnerable to antigenic drift, and more seriously, a small number of viral mutations could lead to drug resistance. Therefore, the development of a new additional therapeutic small molecule-based anti-influenza virus is urgently required. The NS1 influenza gene plays a pivotal role in the suppression of host antiviral responses, especially by inhibiting interferon (IFN) production and the activities of antiviral proteins, such as dsRNA-dependent serine/threonine-protein kinase R (PKR) and 2'-5'-oligoadenylate synthetase (OAS)/RNase L. NS1 also modulates important aspects of viral RNA replication, viral protein synthesis, and virus replication cycle. Taken together, small molecules that target NS1 are believed to offer a means of developing new anti-influenza drugs.

The Molecular Basis for Antigenic Drift of Human A/H2N2 Influenza Viruses.

Influenza A/H2N2 viruses caused a pandemic in 1957 and continued to circulate in humans until 1968. The antigenic evolution of A/H2N2 viruses over time and the amino acid substitutions responsible for this antigenic evolution are not known. Here, the antigenic diversity of a representative set of human A/H2N2 viruses isolated between 1957 and 1968 was characterized. The antigenic change of influenza A/H2N2 viruses during the 12 years that this virus circulated was modest. Two amino acid substitutions, T128D and N139K, located in the head domain of the H2 hemagglutinin (HA) molecule, were identified as important determinants of antigenic change during A/H2N2 virus evolution. The rate of A/H2N2 virus antigenic evolution during the 12-year period after introduction in humans was half that of A/H3N2 viruses, despite similar rates of genetic change.IMPORTANCE While influenza A viruses of subtype H2N2 were at the origin of the Asian influenza pandemic, little is known about the antigenic changes that occurred during the twelve years of circulation in humans, the role of preexisting immunity, and the evolutionary rates of the virus. In this study, the antigenic map derived from hemagglutination inhibition (HI) titers of cell-cultured virus isolates and ferret postinfection sera displayed a directional evolution of viruses away from earlier isolates. Furthermore, individual mutations in close proximity to the receptor-binding site of the HA molecule determined the antigenic reactivity, confirming that individual amino acid substitutions in A/H2N2 viruses can confer major antigenic changes. This study adds to our understanding of virus evolution with respect to antigenic variability, rates of virus evolution, and potential escape mutants of A/H2N2.

Evaluation of a multiplex PCR assay for detection of respiratory viruses and Mycoplasma pneumoniae in oropharyngeal swab samples from outpatients.

BACKGROUND: Respiratory viruses, such as influenza viruses, initially infect the upper airways but can manifest as severe lower respiratory tract infections in high-risk patients with significant morbidity and mortality. For syndromic diagnosis, several multiplex nucleic acid amplification tests have been developed for clinics, of which SureX 13 Respiratory Pathogen Multiplex Kit (ResP) can simultaneously detect 13 pathogens directly from airway secretion specimens. The organisms identified are influenza virus A, influenza virus A pdmH1N1 (2009), influenza virus A H3N2, influenza virus B, adenovirus, boca virus, rhinovirus, parainfluenza virus, coronavirus, respiratory syncytial virus, human metapneumovirus, Mycoplasma pneumoniae, and Chlamydia. METHODS: This study provides performance evaluation data of this assay by comparing with pathogen-specific PCRs from oropharyngeal swab samples. RESULTS: Ten pathogens were detected in this assay, of which rhinovirus, adenovirus, and influenza virus A pdmH1N1 (2009) were the most common. The overall agreement between the ResP and the comparator tests was 93.8%. The ResP demonstrated 86.5% agreement for positive results and 97.8% agreement for negative results. CONCLUSION: The ResP assay demonstrated a highly concordant performance comparing with pathogen-specific PCRs for detection of respiratory pathogens in oropharyngeal swabs from outpatients and could aid in the diagnosis of respiratory infections in a variety of clinical scenarios.

[Increasing the Uniformity of Genome Fragment Coverage for High-Throughput Sequencing of Influenza A Virus].

The high variability of the influenza A virus poses a significant threat to public health, therefore monitoring viral strains and studying their genetic properties are important tasks. One part of this monitoring includes sequencing of influenza A viruses of any subtype and analysis of their whole genomes, which is especially important in cases of interspecies adaptation and reassortment. High-throughput sequencing technologies have significantly extended the capabilities of influenza virus epidemiological surveillance. The preparation stages for next generation sequencing (NGS) of influenza A virus include whole genome amplification using one-step RT-PCR, the results of which vary greatly depending on the sample type and quality, that, in turn, affects the coverage of virus fragments and the sequencing results in general. In this work, we propose to supplement the aforementioned technique of whole genome amplification of influenza A virus with sequential suppression PCRs to obtain an even coverage of viral segments of different lengths, which allows sequencing of samples with lower read coverage without decreasing the sequencing quality.

Influenza A virus nucleoprotein is acetylated by histone acetyltransferases PCAF and GCN5.

Histone acetylation plays crucial roles in transcriptional regulation and chromatin organization. Viral RNA of the influenza virus interacts with its nucleoprotein (NP), whose function corresponds to that of eukaryotic histones. NP regulates viral replication and has been shown to undergo acetylation by the cAMP-response element (CRE)-binding protein (CBP) from the host. However, whether NP is the target of other host acetyltransferases is unknown. Here, we show that influenza virus NP undergoes acetylation by the two host acetyltransferases GCN5 and P300/CBP-associated factor (PCAF) and that this modification affects viral polymerase activities. Western blot analysis with anti-acetyl-lysine antibody on cultured A549 human lung adenocarcinoma epithelial cells infected with different influenza virus strains indicated acetylation of the viral NP. A series of biochemical analyses disclosed that the host lysine acetyltransferases GCN5 and PCAF acetylate NP in vitro MS experiments identified three lysine residues as acetylation targets in the host cells and suggested that Lys-31 and Lys-90 are acetylated by PCAF and GCN5, respectively. RNAi-mediated silencing of GCN5 and PCAF did not change acetylation levels of NP. However, interestingly, viral polymerase activities were increased by the PCAF silencing and were decreased by the GCN5 silencing, suggesting that acetylation of the Lys-31 and Lys-90 residues has opposing effects on viral replication. Our findings suggest that epigenetic control of NP via acetylation by host acetyltransferases contributes to regulation of polymerase activity in the influenza A virus.

Emergence and Containment of Canine Influenza Virus A(H3N2), Ontario, Canada, 2017-2018.

Canine influenza virus (CIV) A(H3N2) was identified in 104 dogs in Ontario, Canada, during December 28, 2017-October 30, 2018, in distinct epidemiologic clusters. High morbidity rates occurred within groups of dogs, and kennels and a veterinary clinic were identified as foci of infection. Death attributable to CIV infection occurred in 2 (2%) of 104 diagnosed cases. A combination of testing of suspected cases, contact tracing and testing, and 28-day isolation of infected dogs was used, and CIV transmission was contained in each outbreak. Dogs recently imported from Asia were implicated as the source of infection. CIV H3N2 spread rapidly within groups in this immunologically naive population; however, containment measures were apparently effective, demonstrating the potential value of prompt diagnosis and implementation of CIV control measures.

Identification of Etiologic Agents of the Pertussis-like Syndrome in Children by Real-time PCR Method.

The aim of this study was to recognize the identity and frequency of etiologic agents of the pertussis-like syndrome in children < 2 years of age. A cross-sectional hospital-based study conducted from August 2014 to August 2015. All children < 2 years of age (n=100) who were suspected as pertussis infected were enrolled in this study and tested for Bordetella pertussis, adenovirus (Adv), respiratory syncytial virus (RSV), human metapneumovirus (hMPV), and influenza virus A (INF-A) by real-time PCR technique. RSV was the most detected pathogen (20%), followed by B. pertussis (18%), Adv (16%), INF-A (11%), and hMPV (10%). Co-infection was observed in 8 patients (11%) and the combinations of RSV/INF-A (n=3, 4%), and AdV/B. pertussis (n=3, 4%) were more frequent. RSV, B. pertussis, and hMPV were more frequent pathogens among infants < 4 months of age. However, Adv and INF-A were more frequent pathogens among children > 6 months of age. In this study, RSV was the most frequent identified pathogen (n=20, 20%), followed by B. pertussis (n=18, 18%) and AdV (n=16, 16%). Pertussis was more frequent in spring (8%) and summer (6%). In addition, clinical symptoms of pertussis were the same as some viral pathogens, which can lead to misdiagnosis of infection. Therefore, diagnosis of pertussis should be established on the bases of both the clinical symptoms and the laboratory methods.

Multi-antigen avian influenza a (H7N9) virus-like particles: particulate characterizations and immunogenicity evaluation in murine and avian models.

BACKGROUND: Human infection with avian influenza A virus (H7N9) was first reported in China in March 2013. Since then, hundreds of cases have been confirmed showing severe symptoms with a high mortality rate. The virus was transmitted from avian species to humans and has spread to many neighboring areas, raising serious concerns over its pandemic potential. Towards containing the disease, the goal of this study is to prepare a virus-like particle (VLP) that consists of hemagglutinin (HA), neuraminidase (NA) and matrix protein 1 (M1) derived from the human isolate A/Taiwan/S02076/2013(H7N9) for potential vaccine development. RESULTS: Full length HA, NA, and M1 protein genes were cloned and expressed using a baculoviral expression system, and the VLPs were generated by co-infecting insect cells with three respective recombinant baculoviruses. Nanoparticle tracking analysis and transmission electron microscopy were applied to verify the VLPs' structure and antigenicity, and the multiplicity of infection of the recombinant baculoviruses was adjusted to achieve the highest hemagglutination activity. In animal experiments, BALB/c mice and specific-pathogen-free chickens receiving the VLP immunization showed elevated hemagglutination inhibition serum titer and antibodies against NA and M1 proteins. In addition, examination of cellular immunity showed the VLP-immunized mice and chickens exhibited an increased splenic antigen-specific cytokines production. CONCLUSIONS: The H7N9 VLPs possess desirable immunogenicity in vivo and may serve as a candidate for vaccine development against avian influenza A (H7N9) infection.

Community- and hospital-acquired infections with oseltamivir- and peramivir-resistant influenza A(H1N1)pdm09 viruses during the 2015-2016 season in Japan.

We report five cases of community- and hospital-acquired infections with oseltamivir- and peramivir-resistant A(H1N1)pdm09 viruses possessing the neuraminidase (NA) H275Y mutation during January-February 2016 in Japan. One case was hospitalized and was receiving oseltamivir for prophylaxis. The remaining four cases were not taking antiviral drugs at the time of sampling. These cases were geographically distant and epidemiologically unrelated. The five viruses showed ~300-fold rise in IC50 values against oseltamivir and peramivir, defined as highly reduced inhibition according to the WHO definition. Overall, the prevalence of the H275Y A(H1N1)pdm09 viruses was 1.8 % (5/282). The resistant viruses possessed the V241I, N369 K, and N386 K substitutions in the NA that have been previously reported among A(H1N1)pdm09 to alter transmission fitness. Analysis of Michaelis constant (Km) revealed that two of the isolates had reduced NA affinity to MUNANA, while the other three isolates displayed a slightly decreased affinity compared to the sensitive viruses. Further studies are needed to monitor the community spread of resistant viruses and to assess their transmissibility.

Nanodiamond enhances immune responses in mice against recombinant HA/H7N9 protein.

BACKGROUND: The continuing spread of the newly emerged H7N9 virus among poultry in China, as well as the possibility of human-to-human transmission, has attracted numerous efforts to develop an effective vaccine against H7N9. The use of nanoparticles in vaccinology is inspired by the fact that most pathogens have a dimension within the nano-size range and therefore can be processed efficiently by the immune system, which leads to a potent immune response. Herein, we report a facile approach to increase antigen size to achieve not only fast but also effective responses against the recombinant HA/H7N9 protein via a simple conjugation of the protein onto the surface of nanodiamond particles. RESULTS: In this study, trimeric Haemagglutinin (H7) that is transiently expressed in N. benthamiana was purified using affinity chromatography, and its trimeric state was revealed successfully by the cross-linking reaction. The trimeric H7 solution was subsequently mixed with a nanodiamond suspension in different ratios. The successful conjugation of the trimeric H7 onto the surface of nanodiamond particles was demonstrated by the changes in size and Zeta-potential of the particles before and after protein coating, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and Western-blot analysis. Next, biofunction of the protein-nanodiamond conjugates was screened using a haemagglutination assay. A mixture containing 5 µg of trimeric H7 and 60 µg of nanodiamond corresponds to a ratio of 1:12 (w/w) of agglutinated chicken red blood cells at HA titer of 1024, which is 512-fold higher than the HA titer of free trimeric H7. After the 2nd and 3rd immunization in mice, ELISA and Western blot analyses demonstrated that the physical mixture of trimeric H7 protein and nanodiamond (1:12, w/w) elicited statistically significant stronger H7-specific-IgG response demonstrated by higher amounts of H7N9-specific IgG (over 15.4-fold with P < 0.05 after the second immunization). CONCLUSIONS: These results indicated a potential effect inherent to nanodiamond towards modulating immune systems, which should be further evaluated and broadly applied in nanovaccine development.

Excess all-cause and influenza-attributable mortality in Europe, December 2016 to February 2017.

Since December 2016, excess all-cause mortality was observed in many European countries, especially among people aged ≥ 65 years. We estimated all-cause and influenza-attributable mortality in 19 European countries/regions. Excess mortality was primarily explained by circulation of influenza virus A(H3N2). Cold weather snaps contributed in some countries. The pattern was similar to the last major influenza A(H3N2) season in 2014/15 in Europe, although starting earlier in line with the early influenza season start.

[Action mechanism of Mahuang Xixin Fuzi decoction for mice with influenza based on metabolomics information].

This study aimed to analyze the endogenous metabolite changes in the serum of mice infected with H1N1 virus after intervention by Mahuang-Xixin-Fuzi decoction (MXF) based on metabolomics method, investigate potential biomarkers and related metabolic pathways, and explore the therapeutic mechanism of MXF through metabolomics technology. Thirty-six Kunming (KM) mice were randomly divided into three groups: normal group, model group and MXF group. Influenza virus H1N1 was used by nasal drip to establish influenza mice model. The mice in MXF group were orally administrated with MXF for 6 consecutive days after inoculation, and the other two groups were given with equal volume of saline solution in the same way. Body weight, rectal temperature, morbidity and mortality were recorded daily. Serum samples were collected 24 hours after the last administration for HPLC-TOF-MS analysis. The results showed that as compared with the normal group, the body weight and rectal temperature were decreased in model group, and their lung index and mortality rate were significantly increased (P<0.05); MXF had good therapeutic effects on the abnormity of body weight, rectal temperature, lung index and high mortality rate of mice infected with H1N1 virus. The original data collected from the serum samples were analyzed with R language, MPP, SIMCA-P and other software, and significant changes were found in 14 kinds of endogenous substances from mice serum (P<0.05). As compared with model group, the potential metabolic markers in MXF group recovered to normal levels to a certain degree after being intervened by MXF. Further analysis with MetPA data platform showed that, the pathways involved in 14 metabolites included glucose metabolism, arachidonic acid metabolism, glycerophospholipids and sphingolipids metabolism etc. The metabolomics study and pharmacological experiment showed that MXF might play a role of efficacy by improving glucose metabolism, regulating arachidonic acid metabolism, glycerophospholipid and sphingolipid metabolic pathways.

Austalides S-U, New Meroterpenoids from the Sponge-Derived Fungus Aspergillus aureolatus HDN14-107.

Three new meroterpenoids, named austalides S-U (1-3), were isolated from the culture of a sponge-derived fungus Aspergillus aureolatus HDN14-107, together with eleven known austalides derivates (4-14). Their structures, including absolute configurations, were assigned on the basis of NMR, MS data, and TDDFT ECD calculations. Compound 1 is the first case of austalides with the terpene ring fused to the chroman ring in trans configuration. Compounds 3 and 5 exhibited activities against influenza virus A (H1N1), with IC50 values of 90 and 99 µM, respectively.

Immunization with live nonpathogenic H5N3 duck influenza virus protects chickens against highly pathogenic H5N1 virus.

Development of an effective, broadly-active and safe vaccine for protection of poultry from H5N1 highly pathogenic avian influenza viruses (HPAIVs) remains an important practical goal. In this study we used a low pathogenic wild aquatic bird virus isolate А/duck/Moscow/4182/2010 (H5N3) (dk/4182) as a live candidate vaccine. We compared this virus with four live 1:7 reassortant anti-H5N1 candidate vaccine viruses with modified hemagglutinin from either A/Vietnam/1203/04 (H5N1) or A/Kurgan/3/05 (H5N1) and the rest of the genes from either H2N2 cold-adapted master strain A/Leningrad/134/17/57 (rVN-Len and rKu-Len) or H6N2 virus A/gull/Moscow/3100/2006 (rVN-gull and rKu-gull). The viruses were tested in parallel for pathogenicity, immunogenicity and protective effectiveness in chickens using aerosol, intranasal and oral routes of immunization. All five viruses showed zero pathogenicity indexes in chickens. Viruses rVN-gull and rKu-gull were immunogenic and protective, but they were insufficiently attenuated and caused significant mortality of 1-day-old chickens. The viruses with cold-adapted backbones (rVN-Len and rKu-Len) were completely nonpathogenic, but they were significantly less immunogenic and provided lower protection against lethal challenge with HPAIV A/Chicken/Kurgan/3/05 (H5N1) as compared with three other vaccine candidates. Unlike other four viruses, dk/4182 was both safe and highly immunogenic in chickens of any age regardless of inoculation route. Single administration of 106 TCID50 of dk/4182 virus via drinking water provided complete protection of 30-days-old chickens from 100 LD50 of the challenge virus. Our results suggest that low pathogenic viruses of wild aquatic birds can be used as safe and effective live poultry vaccines against highly pathogenic avian viruses.