Influenza Virus Subtype H5N1 Research Articles

The effect of garlic (Allium sativum) and red onion (Allium cepa) on the histopathological signs of avian influenza disease in broiler chickens exposed to avian influenza virus subtype H9N2

ویروس آنفلوانزای پرندگان، تحت­تیپ H9N2در ایران شیوع بالائی داشته و از سراسر جهان هم گزارش شده­ است. وقوع بیماری آنفلوانزا باعث خسارت سنگین اقتصادی در صنعت طیور می‌شود. در مطالعه حاضر نقش سیر (Allium sativum) و پیاز قرمز (Allium cepa) در کاهش شدت نشانه­های آسیب‌شناختی بیماری آنفلوانزای پرندگان ناشی از تحت­تیپ H9N2 در جوجه­های گوشتی، بررسی شد. بدین منظور ابتدا سمیت سیر و پیاز مورد بررسی قرار­گرفت. همچنین تعداد 72 قطعه جوجه­ء یک روزه نژاد گوشتی سویه­­ء راس به 9 گروه 8 قطعه­ای تقسیم ­شدند که شامل‌ 4 گروه درمانی، 1 گروه کنترل مثبت و 4 گروه مسمومیت بودند. سپس گروه‌های درمانی در 28 روزگی با ویروس مواجهه داده شدند. پرندگان گروه‌های مواجهه داده شده یک روز بعد از مواجهه به مدت هفت روز، به‌طور جداگانه سیر و پیاز قرمز را همراه غذا دریافت کردند. در ادامه علائم بالینی، نشانه­های آسیب‌شناختی و نیز تیتر آنتی­بادی اختصاصی ضد ویروس آنفلوانزای تحت­تیپ H9N2 در سرم پرندگان مورد آزمایش بررسی شد. سیر و پیاز هیچ اثر سمی بر جوجه‌ها نداشتند. در آسیب‌شناسی روده نیز میزان افزایش سلول‌های جامی‌شکل و قطر پرزها در گروه‌های کنترل منفی و درمانی در مقایسه با گروه کنترل مثبت، بیشتر بود که از نظر آماری نیز اختلاف معنی‌دار داشتند (05/0p<). به نظر می­رسد سیر و پیاز قرمز که موجب کاهش شدت علائم آسیب‌شناختی بیماری آنفلوانزای پرندگان ناشی از تحت­تیپ H9N2 ویروس آنفلوانزا در دئودنوم جوجه­های گوشتی مورد مطالعه شدند، احتمالا با کاهش تعداد ویروس‌ها، در ترمیم مخاط روده نقش دارند.

ANTI-INFLUENZA ACTIVITY OF PLANT EXTRACTS OF THE LAMIACEAE FAMILY

In this work, it was revealed that water and ethanol extracts of the plants Nepeta cataria, Nepeta sibirica, Scutellaria baicalensis, Hyssopus officinalis, Betonica officinalis, and water extracts of Dracocephalum moldavica, Glechoma hederacea, Mentha arvensis, Prunella vulgaris, Melissa officinalis ethanol extracts of Mentha piperita, Mentha crispa, Origanum vulgare, Hyssopus officinalis, Salvia verticillata showed antiviral activity against the influenza virus subtype H5N1. Aqueous extracts of Nepeta cataria and Glechoma hederacea (NI 3.75) showed the highest antiviral effect against the H5N1 subtype.&#x0D; It was revealed the antiviral activity against influenza virus subtype H3N2 of aqueous and ethanol extracts of Scutellaria baicalensis, Mentha piperita, Mentha arvensis, Mentha crispa, aqueous extract of Dracocephalum moldavica and ethanol extracts of Glechoma hederacea, Origanum vulgare, Prunella vulgaris, Hyssopus officinalis, Betonica officinalis, Salvia verticillata. Ethanolic extract of Betonica officinalis (NI 4.25) showed the highest virus neutralizing activity against the H3N2 subtype.&#x0D; It was carried out chemical analysis of the aerial parts of plants of the Lamiaceae family. It was shown that the highest content of flavonols was observed in Dracocephalum nutans (4.47±0.04%), the highest content of tannins was found in Mentha arvensis (17.62 ± 0.78%), and the highest content catechins were found in Nepeta cataria (0.43 ± 0.007%).&#x0D; Thus, plant extracts of the Lamiaceae family are promising sources for further studies to develop new antiviral drugs.

NF-кB pathway genes expression in chicken erythrocytes infected with avian influenza virus subtype H9N2

ABSTRACT 1. Chicken erythrocytes in blood vessels are the most abundant circulating cells, which participate in the host’s immune responses. The transcription factor nuclear factor-kappa B (NF-κB) plays a vital role in the inflammatory response following viral infections. However, the expression of the NF-κB pathway, and other immune-related genes in chicken erythrocytes infected with low pathogenic avian influenza virus (LPAIV H9N2), has not been extensively studied. 2. The following study determined the interaction of LPAIV H9N2 with chicken erythrocytes using indirect immunofluorescence microscopy. This was followed by investigating myeloid differentiation primary response 88 (MyD88), C-C motif chemokine ligand 5 (CCL5), melanoma differentiation-associated protein 5 (MDA5), the inhibitor of nuclear factor-kappa B kinase subunit epsilon (IKBKE), NF-κB inhibitor alpha (NFKBIA), NF-κB inhibitor epsilon (NFKBIE), interferon-alpha (IFN-α), colony-stimulating factor 3 (CSF3) and tumour necrosis factor receptor-associated factor 6 (TRAF6) by mRNA expression using quantitative real-time PCR (qRT-PCR) at four different time intervals (0, 2, 6 and 10 h). 3. There was a significant interaction between erythrocytes and LPAIV H9N2 virus. Furthermore, the mRNA expression of the NF-κB pathway and other immune-related genes were significantly up-regulated at 2 h post-infection in infected chicken erythrocytes, except for TRAF6, which were significantly downregulated. While at 0 h post-infection, IFN-α and CSF3 were significantly upregulated, whereas NFKBIA was significantly downregulated. Further expression of MDA5, CCL5 and NFKBIA was upregulated, while TRAF6 was downregulated at 6 h post-infection. In infected erythrocytes, expression of MyD88, CCL5 and IKBKE was upregulated. However, IFN-α and TRAF6 were downregulated at 10 h post-infection. 4. These results give initial evidence that the NF-κB pathway, and other genes related to immunity, in chicken erythrocytes may contribute to LPAIV subtype H9N2 and induce host immune responses.

Molecular Characterization, Pathogenesis and Immunological Effect of Avian Influenza H9N2 Virus Isolated From Broiler Chickens in Egypt During 2019

Many commercial flocks in Egypt were infected with the low pathogenic avian influenza virus (LPAIV) subtype H9N2, resulting in severe economic losses, particularly when combined with other viral and bacterial diseases. The current study aimed to investigate the genetic evolution of LPAIV (H9N2) in broiler farms in Egypt during 2019 and evaluate its effect on the viral pathogenesis, cytokine response, innate immunity and oxidative state in specific pathogen free (SPF) chicks. Sixty tracheal swabs were collected from broilers and tested by RRT-PCR and the HA gene were sequenced for the genetic evolution. Then this isolated virus was experimentally studied in a total of 20 SPF chicks (21 days old) that were divided into two equal groups. Group-1(G1) was negative control and group-2 (G2) was experimentally infected with the isolated LPAIV (H9N2). The obtained results revealed that all isolated LPAIV (H9N2) in this study were belong to G1-like Eurasian sub-lineages (A/quail/Hong Kong/G1/97-like) and clustered with Israeli strains in group B with specific mutation specific for Egyptian AIV-H9N2 isolated in 2018-2019 forming new subgroup-I. The experimental infection with LPAIV (H9N2) (G2) in SPF chickens, induced mild clinical signs, moderate histopathological changes in lung and spleen, shedding of higher mean LPAIV (H9N2) titre in trachea, up regulation of both the pro-inflammatory (IL-6) and the anti-viral (IFN-γ) cytokines, significant decrease of the serum levels of lysozyme and disturbance of the oxidative state where the serum nitric oxide (NO) and the malondialdehyde (MDA) levels were significantly increased and the antioxidants glutathione (GSH) and superoxide dismutase (SOD) were significantly decreased. In conclusion, the LPAIV (H9N2) was continually circulated in broiler chicken farms and highly evolved. The induced oxidative stress in LPAIV (H9N2) infection could be contributed to the viral pathogenesis, up-regulation of pro-inflammatory and anti-viral cytokines and down-regulation of the innate immune response.

Isolation and molecular characterization of the hemagglutinin gene of H9N2 avian influenza viruses from poultry in Java, Indonesia.

Objective:The avian influenza virus (AIV) subtype H9N2 circulating in Indonesia has raised increasing concern about its impact on poultry and its public health risks. In this study, the H9N2 virus from chicken poultry farms in Java was isolated and characterized molecularly.Materials and Methods:Thirty-three pooled samples of chicken brain, cloacal swab, trachea, and oviduct were taken from multiple chickens infected with AIV in five regions of Java, Indonesia. The samples were isolated from specific pathogenic-free embryonated eggs that were 9 days old. Reverse transcription polymerase chain reaction and sequencing were used to identify H9N2 viruses.Results:This study was successful in detecting and characterizing 13 H9N2 isolates. The sequencing analysis of hemagglutinin genes revealed a 96.9%–98.8% similarity to the H9N2 AIV isolated from Vietnam in 2014 (A/muscovy duck/Vietnam/LBM719/2014). According to the phylogenetic analysis, all recent H9N2 viruses were members of the lineage Y280 and clade h9.4.2.5. Nine of the H9N2 isolates studied showed PSKSSR↓GLF motifs at the cleavage site, while four had PSKSSR↓GLF. Notably, all contemporary viruses have leucine (L) at position 216 in the receptor-binding region, indicating that the virus can interact with a human-like receptor.Conclusion:This study described the features of recent H9N2 viruses spreading in Java’s poultry industry. Additionally, H9N2 infection prevention and management must be implemented to avoid the occurrence of virus mutations in the Indonesian poultry industry.

Evolution of H9N2 avian influenza viruses in Iran, 2017-2019.

Since its first detection in 1998, avian influenza virus (AIV) subtype H9N2 has been enzootic in Iran. To better understand the evolutionary history of H9N2 viruses in Iran, we sequenced 15 currently circulating H9N2 viruses from domestic poultry during 2017-2019 and performed phylogenetic analysis of complete genome sequences. Phylogenetic analyses indicated that the Iranian H9N2 viruses formed multiple well-supported monophyletic groups within the G1-lineage of H9N2 virus. Our analysis of viral population dynamics revealed an increase in genetic diversity until 2007, corresponding to the multiple introductions and diversification of H9N2 viruses into multiple genetic groups (named Iran 1-4 subgroups), followed by a sudden decrease after 2008. Only the Iran 4 subgroup has survived, expanded, and currently circulates in Iran. The H9N2 viruses possessed many molecular markers associated with mammalian adaption in all gene segments, except neuraminidase gene. Considering the presence of mammalian host-specific markers, the public health threat of H9N2 viruses continues. Molecular analysis showed that Iranian H9N2 strains have continued to evolve and recent strains have multiple amino acid changes and addition of potential N-glycosylation on the antigenic sites of haemagglutinin. Continued antigenic and molecular surveillance of H9N2 viruses in poultry and mammals would be required to monitor further increments in viral evolution and their potential threat to public health.

Immunization of turkeys with a DNA vaccine expressing the haemagglutinin gene of low pathogenic avian influenza virus subtype H9N2

Low pathogenic avian influenza H9N2 is still circulating in the Middle East causing respiratory manifestations and severe economic losses in poultry. In the present study, an H9 plasmid-based DNA vaccine targeting the HA gene of H9N2 A/CK/Egypt/SCU8/2014 was developed and evaluated in turkeys. The full length of HA was cloned into vector plasmids under the control of a cytomegalovirus promoter. The in-vitro expression of the recombinant HA was demonstrated in HeLa cells transfected with the plasmids pVAX1-H9 or pCR-H9 using western blot and Immunofluorescent assay (IFA). The efficacy of pVAX-H9 and pCR- H9, naked or saponin-adjuvanted, was evaluated in turkey poults at 3 weeks and challenged with A/CK/Egypt/SCU8/2014 (106 EID50/bird at 3 weeks post-vaccination. The efficacy was assesses based on virus shedding, oropharyngeal and cloacal, as well as seroconversion using haemagglutination inhibition (HI) test. All immunized birds showed high HI antibody titers (7–8 log2) at 3 weeks post-vaccination. None of the birds vaccinated with naked or saponin-adjuvanted pVAX-H9 or pCR-H9 showed any clinical signs. The pVAX-H9 and pCR-H9 alone did not prevent cloacal and oropharyngeal virus shedding, however, saponin-adjuvanted pVAX1-H9 and pCR-H9 prevented cloacal and oropharyngeal virus shedding at 3 and 5 days post challenge, respectively. In conclusion, DNA vaccination with pVAX1-H9 and pCR-H9 could protect turkey from the H9N2 virus, but vaccination regimes need to be improved.

Characterization and Phylogenetic Analysis of the Hemagglutinin Gene in H9 Influenza Viruses from Chickens in Morocco from 2017 to 2019.

Since the early 2000s, the avian influenza virus (AIV) subtype H9N2 has been widely circulating in North African countries, including Libya, Tunisia, and Egypt. For unknown reasons, H9N2 was not detected in Moroccan farms until the end of 2016, and at present, it is endemic in poultry. This study was carried out to evaluate the evolution of H9 AIVs in Morocco from 2017 to 2019. In this study, 16 H9 viruses collected from 2017 to 2019 in Morocco were isolated and sequenced. The genomic signatures and protein sequences of these isolates were analyzed. Moroccan H9 viruses were closely related to viruses isolated from African and Middle Eastern countries, and the hemagglutinin gene resembled those of viruses of G1-like lineage, and multiple mammalian host-associated mutations were detected that favor transmission from avian to mammalian hosts. Other mutations related to virulence were also identified. Our findings suggest that attention be given to the control of H9N2 influenza viruses in chickens and continuous monitoring of the prevalence and pathogenic potential of AIV H9N2 in the poultry population in Morocco.

Immunological and pathological adverse effects of avian influenza virus subtype H9N2 infection in aflatoxicated-broiler chickens

Aflatoxin B1 (AFB1) is a metabolic product of the Aspergillus spp. of molds, which grow on several feedstuffs stored in hot moist conditions. It is one of the immunosuppressive agents that might influence the pathogenesisof avian influenza virus (AIV) subtype H9N2 in broilers, which can exacerbate the disease outcomes. The immunological, biochemical and pathological adverse health effects of an interaction between low levels of dietary aflatoxins (AFs) and H9N2 infection in broiler chickens were investigated. One hundred and eighty of unvaccinated 1-day-old COBB chicks were, therefore, raised for 35 days in the following treatment groups: control, AFs, AFs+H9N2, and H9N2. AFs in the basal diet was added at 200 ppb starting from the first day of age, while H9N2 virus was intra-nasally installed at a dose of 100 μl of 106 EID50/bird of allantois fluid at 23rd day. Humoral and cell-mediated immune responses were evaluated. Evidence of H9N2-AIV viral shedding was also detected. It has been observed that concurrent exposure of AFs and H9N2 virus negatively affected chicken performance traits i.e. lowered feed intake and body weights with exaggerated respiratory and digestive disturbances, and 20% mortality rate. Ten days’ post H9N2 infection, significant (p≤ 0.05) increment in serum transaminases (AST and ALT) and falling in cell-mediated immunity i.e. total leukocyte count, lymphocyte transformation activity and macrophage phagocytic activity were detected. Additionally, AFs+H9N2 significantly (p≤ 0.05) lowered H9N2-HI titers (5.5 Log2) than H9N2 alone (6.3 Log2). Pathologically, aflatoxicated chickens showed hydropic degeneration, hepatocytic vacuolation and necrosis of liver tissues with nephrosis and urates deposition in ureters, as well as bursal and thymic lesions, which were potent in H9N2–inoculated chickens. AFs exposure increased the incidence and titer of H9N2 viral shedding. It could be concluded that dietary contamination with AFs even at very low levels has explanatory effect in H9N2–inoculated broilers, and vice versa.

Commensal gut microbiota can modulate adaptive immune responses in chickens vaccinated with whole inactivated avian influenza virus subtype H9N2

Variations in the composition of commensal gut microbiota have been reported to be major contributors to differences in responses to vaccination among individuals. In chickens, there is limited information on the role of gut microbiota in responses to vaccination. The current study studied the role of gut microbiota in cell- and antibody-mediated immune responses to vaccination with a whole inactivated avian influenza virus, subtype H9N2. A total of 166 one-day-old specific pathogen free layer chickens (SPF) were randomly assigned to treatments, where a combination of antibiotic depletion, and probiotics (a combination of five Lactobacillus species) or fecal microbial transplant (FMT) reconstitution were used to study the dynamics of cell- and antibody-mediated immune responses to primary and secondary vaccinations at days 15 and 29 of age, respectively. Overall, at days 7 and 14 post primary vaccination (p.p.v.), administration of probiotics to non-depleted chickens resulted in significantly higher mean hemagglutination (HI) titre compared to antibiotic treated chickens. Furthermore, at day 21 p.p.v., chickens treated with probiotics or FMT post-antibiotic treatment showed a significantly higher mean HI titre compared to non-depleted chickens treated with probiotics. At day 7 p.p.v., a significantly higher virus specific IgM and IgG titres were observed in non-depleted chickens administered with probiotics compared to antibiotic depleted chickens, and a significantly higher IgG titre was observed in chickens treated with FMT following antibiotic treatment compared to only antibiotic treatment. Analysis of interferon gamma expression in splenocytes to assess cell-mediated immune responses showed a significantly lower expression in antibiotic-treated chickens compared to non-depleted chickens and FMT reconstituted chickens. Taken together, the current study suggests that shifts in the composition of gut microbiota of chickens may result in changes in cell- and antibody-mediated immune responses to vaccination against influenza viruses. Further studies will be needed to highlight the mechanisms involved in this modulation.

Development of a Subtype-Specific Diagnostic System for Influenza Virus H3N2 Using a Novel Virus-Based Systematic Evolution of Ligands by Exponential Enrichment (Viro-SELEX).

Aptamers are oligonucleotide molecules that bind to specific target molecules generated by systematic evolution of ligands by exponential enrichment (SELEX). Aptamers have high future potential for use in diagnostics and therapeutics as molecular probes that recognize target molecules. To develop aptamers against a target protein using a SELEX process, it is necessary to purify the target protein. Purifying a membrane protein, however, is usually a challenging task. Here, we report a novel approach to developing aptamers against membrane proteins. Surrogate viruses containing target proteins on the surface of an enveloped virus (e.g., baculovirus), instead of purified proteins, were used in a new SELEX process. We designated this new SELEX process as "surrogate virus-based SELEX (viro-SELEX)." Using viro-SELEX, we developed a pair of aptamers that specifically interact with the hemagglutinin protein of influenza subtype H3N2. Using the aptamer pair and a lateral flow assay system, we developed a very sensitive point-of-care diagnostic system for specifically detecting influenza virus subtype H3N2.

Isolation and Characterization of Avian Influenza H9N2 Viruses from Different Avian Species in Pakistan 2016-17.

Avian influenza (AI) virus (AIV) subtype H9N2 continues to cause significant outbreaks among commercial and backyard poultry in Pakistan. Despite this, the characterization of H9N2 viruses in avian hosts other than chickens in Pakistan has not been thoroughly investigated. In this study, 12 low pathogenicity avian influenza viruses subtype H9N2 were isolated from peacocks (n = 4), ducks (n = 4), pheasants (n = 2), geese (n = 1), and black swans (n = 1) in Pakistan during 2016 and were characterized on the basis of the hemagglutinin (HA) and neuraminidase genes. All of the viruses possessed an amino acid substitution Q226L in the receptor-binding site of the HA protein, which is known to contribute to increased viral replication and virulence in mammals. In addition, phylogenetic studies showed that these H9N2 AIVs belonged to the Middle East B genetic group of sublineage G1 and were very similar to viruses isolated from an outbreak in chickens in Pakistan in 2017. This demonstrates an epidemiologic link between poultry and other avian species, which is a fact to consider in future H9N2 disease management programs.

Contaminated feed-borne Bacillus cereus aggravates respiratory distress post avian influenza virus H9N2 infection by inducing pneumonia

Avian influenza virus subtype H9N2 is identified in chickens with respiratory disease while Bacillus cereus (B. cereus) has been frequently isolated from chicken feed in China. However, the roles of co-infection with these two pathogens remain unclear. In the present study, SPF chicks were intragastrically administered with 108 CFU/mL of B. cereus for 7 days and then inoculated intranasally with 100 EID50 of H9N2 three days later. Alternatively, chickens were initially inoculated with H9N2 and then with B. cereus for one week. Post administration, typical respiratory distress persisted for 5 days in both co-infection groups. Gizzard erosions developed in the groups B. cereus/H9N2 and B. cereus group on 7th day while in group H9N2/B. cereus on 14th day. More importantly, both air-sac lesions and lung damage increased significantly in the co-infection group. Significant inflammatory changes were observed in the B. cereus group from day 7 to day 21. Moreover, higher loads of H9N2 virus were found in the co-infected groups than in the H9N2 group. Newcastle Disease Virus (NDV) specific antibodies were decreased significantly in the H9N2/B. cereus group compared to the B. cereus and the B. cereus/H9N2 groups. Nonspecific IgA titers were reduced significantly in the B. cereus group and the H9N2/B. cereus group compared to the control group. In addition to this, lower lymphocyte proliferation was found in the con-infection groups and the H9N2 group. Hence, feed-borne B. cereus contamination potentially exacerbates gizzard ulceration and aggravates H9N2-induced respiratory distress by inhibiting antibody-mediated immunity and pathogen clearance. Thus controlling the B. cereus contamination in poultry feed is immediately needed.

Effect of feeding Chinese herb medicine ageratum-liquid on intestinal bacterial translocations induced by H9N2 AIV in mice

BackgroundAs a low pathogenic influenza virus, avian influenza virus subtype H9N2 (H9N2 AIV) often induces high morbidity in association with secondary bacterial infections in chickens or mammals. To explore this phenomenon, the relationship between intestinal microflora changes and bacterial translocations was studied post H9N2 AIV challenge and post AIV infection plus Ageratum-liquid treatment.MethodsIllumina sequencing, histological examination and Neongreen-tagged bacteria were used in this study to research the microbiota composition, intestinal barrier, and bacterial translocation in six weeks of BALB/c mice.ResultsH9N2 AIV infection caused intestinal dysbacteriosis and mucosal barrier damages. Notably, the villus length was significantly reduced (p < 0.01) at 12 dpi and the crypt depth was significantly increased (p < 0.01) at 5 dpi and 12 dpi with infection, resulting in the mucosal regular villus-length/crypt-depth (V/C) was significantly reduced (p < 0.01) at 5 dpi and 12 dpi. Moreover, degeneration and dissolution of the mucosal epithelial cells, loose of the connective tissue and partial glandular atrophy were found in infection group, indicating that intestinal barrier function was weakened. Eventually, intestinal microbiota (Staphylococcus, E. coli, etc.) overrun the intestinal barrier and migrated to liver and lung tissues of the mice at 5 and 12 dpi. Furthermore, the bacteria transferred in mesentery tissue sites from intestine at 36 h through tracking the Neongreen-tagged bacteria. Then the Neongreen-tagged bacteria were isolated from liver at 48 h post intragastrical administration. Simultaneously, Ageratum-liquid could inhibit the intestinal microbiota disorder post H9N2 AIV challenge via the respiratory tract. In addition, this study also illustrated that Ageratum-liquid could effectively prevent intestinal bacterial translocation post H9N2 AIV infection in mice.ConclusionIn this study, we report the discovery that H9N2 AIV infection could damage the ileal mucosal barrier and induce the disturbance of the intestinal flora in BALB/c mice resulting in translocation of intestinal bacteria. In addition, this study indicated that Ageratum-liquid can effectively prevent bacterial translocation following H9N2 infection. These findings are of important theoretical and practical significance in prevention and control of H9N2 AIV infection.

Low Pathogenic Avian Influenza Virus Subtype H9N2 in Poultry in North Africa: Current Status

Low Pathogenic Avian Influenza Virus Subtype H9N2 in Poultry in North Africa: Current Status

Dietary selenium supplementation enhances antiviral immunity in chickens challenged with low pathogenic avian influenza virus subtype H9N2

Selenium supplementation in poultry feeds has been known to have beneficial effects on the bird health and performance; however antiviral effects of selenium have remained largely unknown. In this study, we have evaluated the effects of supplementation of chicken diets with organic (Selenium Enriched Yeast; SEY) and inorganic selenium (Sodium Selenite; SS) on low pathogenicity avian influenza virus (H9N2) shedding in the cloacal and oropharyngeal swab samples as well as examined the expression of immune related genes. Chickens were fed two doses (High- 0.30 mg/kg of feed; Low- 0.15 mg/kg of feed) of selenium supplementation for 2 weeks followed by low pathogenicity avian influenza virus challenge. Our results showed that the cloacal shedding of virus in all the selenium supplemented groups was significantly lower when compared to the non-supplemented control groups. In addition, the oropharyngeal shedding of virus in chickens fed with organic selenium supplementation was significantly lower than that in the chickens that received either inorganic selenium supplemented feed or controls. Furthermore, the expression of interferon stimulated genes (Viperin, OAS: 2′-5′ oligoadenylate synthetase and MDA5: melanoma differentiation-associated gene) in the cecal tonsils was significantly elevated in the selenium treated groups when compared to controls. Additionally, a significantly higher transcription of interferon (IFN)-α, IFN-β and IFN-γ genes in the cecal tonsils and spleens of chickens receiving SEY-L and SS-H supplemented feed was also observed at post virus challenge time points compared to untreated controls. The results of this study demonstrated that supplementation of chicken diets with selenium, can enhance antiviral defense and thus, may have a beneficial effect in controlling viral infections in poultry.

Multifunctional G-quadruplex-based fluorescence probe coupled with DNA-templated AgNCs for simultaneous detection of multiple DNAs and MicroRNAs

A rapid, label-free, and multifunctional fluorescent probe for simultaneous detection of multiple targets was fabricated based on G-quadruplexes and DNA-templated silver nanoclusters (AgNCs). In this work, a probe with two capitated recognized regions was coupled with a locked G-quadruplex at the 5′-terminus and dark AgNC at the 3′-terminus. Upon the addition of the virus subtype H5N1 gene or microRNA-141, only the sequence of the G-quadruplex was released and bound with Thioflavin T (ThT) for a specific fluorescent response. On the contrary, with the presence of the influenza virus subtype H1N1 gene or microRNA-21, the fluorescence intensity was enhanced because of two split AgNCs approaching closely to produce a nanocluster dimer. Subsequently, with multiple target addition, fluorescence signals were produced for both G-quadruplexes and AgNCs. Moreover, this single and duplex detection for virus DNAs and microRNAs, which provides a versatile platform for different targets, was sufficiently sensitive for the expected detection limit, and still possessed unique selectivity with similar oligonucleotides. The simultaneous detection of targets in biological fluids indicated that there is great opportunity for this strategy to be further applied in biomedical research and clinical diagnosis.

Potential Use of Genetically Engineered Variable Lymphocyte Receptor B Specific to Avian Influenza Virus H9N2.

The variable lymphocyte receptor (VLR) B of jawless vertebrates functions as a secreted Ab of jawed vertebrates and has emerged as an alternative Ab with a single polypeptide chain. After observing an upregulated VLRB response in hagfish immunized with avian influenza virus (AIV) subtype H9N2, we screened AIV H9N2-specific VLRB using a mammalian expression system. To improve the binding avidity of the Ag-specific VLRB to the Ag, we enabled multimerization of the VLRB by conjugating it with C-terminal domain of human C4b-binding protein. To dramatically enhance the expression and secretion of the Ag-specific VLRB, we introduced a glycine-serine linker and the murine Ig κ leader sequence. The practical use of the Ag-specific VLRB was also demonstrated through various immunoassays, detected by anti-VLRB Ab (11G5). Finally, we found that the Ag-specific VLRB decreased the infectivity of AIV H9N2. Together, our findings suggest that the generated Ag-specific VLRB could be used for various immunoapplications.

Synthesis of new quinoline scaffolds via a solvent-free fusion method and their anti-microbial properties

Purpose: To develop a robust and simple fusion-based methodology for the synthesis of various 5-(-3- nitrophenyl) pyrimido[5,4-c] quinoline-2,4(1H,3H)-diones (5 – 9). Method: The synthesis involved formation of a Knoevenagel product using barbituric acid and 3- nitrobenzaldehyde which cyclized on fusing with various sulfanilamides in a sealed tube at 170 – 212°C. This resulted in the synthesis of the target quinolines (5 – 9). To evaluate their antibacterial and antiviral properties, the synthesized quinolines were tested against four gram-negative bacterial strains and four poultry viruses. The MIC and IC 50 of each active compound were calculated. Results: Data from NMR, mass spectrometry and elemental analysis confirmed the formation of quinoline scaffolds. Antibacterial screening revealed that all the compounds had antibacterial activities. However, the minimum inhibitory concentration (MIC) of compounds 6 – 8 and 9 against Proteus vulgaris and Klebsiella pneumoniae showed that these compounds were more active than the standard drug ampicillin. Antiviral studies and IC 50 values showed that compounds 5 – 9 were effective against Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV), while compounds 5, 6 and 8 were active against avian influenza virus subtype H9N2 (AIV); compounds 7 and 8 were active against infectious bronchitis virus (IBV). Conclusion: A simple strategy of fusion of Knoevenagel product with aromatic amines can be used to synthesize highly functionalized quinoline scaffolds which are potential drug candidates for development of new antibacterial and antiviral agents. Keywords: Quinoline scaffolds, Barbituric acid, Knoevenagel condensation, Antiviral, Antibacterial

Identification of avian influenza virus subtype H9N2 in chicken farms in Indonesia

Avian influenza virus subtype H9N2 (AIV-H9N2) has become established in domestic poultry in Asia and Africa. AIV-H9N2 has not been reported previously in Indonesia. Here we describe the presence of AIV-H9N2 in chicken farms in Indonesia. Ninety-nine cases were observed in various provinces in Indonesia. Clinical signs, pathologic lesions and egg production were recorded. Confirmation was made using virus isolation, reverse transcriptase PCR (RT-PCR), and sequencing. To construct hemaglutinin (HA) phylogeny, the secondary data of Eurasian lineages were downloaded from GenBank. For neuraminidase, five sequences with the highest similarities with every sequence found in this study were downloaded. Phylogeny was inferred using Neighbor-Joining method in MEGA6 package. Forty-nine AIV-H9N2-positive cases were observed, of which 35 were tested positive for AIV-H9N2 only. The age of the infected chickens was 43.17 ± 16.56 weeks, and their egg production was 35.85 ± 17.80% lower than before outbreak. BLAST search revealed that the nucleotide sequence of the HA-encoding gene identified in this study shared 98% sequence identity with that of A/Muscovy duck/Vietnam/LBM719/2014(H9N2), while its neuraminidase-encoding gene sequences shared 94%, 98%, and 100% identities with three different influenza viruses. The phylogeny shows that the HA of AIV-H9N2 found in this study forms distinct cluster with some Vietnam and China’s sequence data. The NA sequence data form three distinct clusters. We conclude that AIV-H9N2 is widespread in many provinces in Indonesia. To lessen economic losses to the poultry industry, flock biosecurity and vaccination against this virus subtype should be implemented rapidly. Thorough and rigid AIV surveillance is paramount to prevent further veterinary and public health consequences of the circulation of this virus in Indonesia.