ISOLATION AND CHARACTERIZATION OF AVIAN INFLUENZA VIRUS H5N1 SUBTYPE FIELD ISOLATES FROM BALI FOR VACCINE CANDIDATES

A research on the isolation and characterization of the Avian Influenza H5N1 subtype field isolate has been carried out at the BSL-3 Laboratory of PT Sanbio Laboratories, Bogor. The aim of the study was to prepare a candidate for the H5N1 subtype Avian Influenza virus vaccine. Virus isolates were taken from field isolates from Bali. A total of seven field H5N1 AI subtypes from Bali were characterized in Bogor. The isolates were: isolate 3A, isolate 4A, isolate 9C, isolate 10 A, isolate 10 C, isolate P65, isolate P67. The passage of isolates was carried out on 9-day-old embryonic Specific Pathogenic (SPF) chicken eggs by injecting 0.1 mL of SPF isolates/eggs through the allantoic cavity. Each isolate was placed in five SPF eggs and then incubated in an incubator at 37 C and candled every day. Since day 2-4 post inoculation, embryo death has occurred. The eggs are harvested by their allantoic fluid and tested for haemagglutination test(HA/HI). The HI test results were confirmed by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using the front primer FPHA232_13 (ATTGGTTAYCATGCAAAYAACTCG) and the back primer BPHA232_597 (GGAAAYATAGGTRGTTGGRTTYTGATAG) The results were five of the seven isolates were positive AI subtype B5 585 - 581 The five isolates of AI subtype H5N1 were subsequently sequenced, the results were all positive for AI virus subtype H5N1 clade 2.3.2. Each field isolate was given the name A / Chicken / Bali3A / GAY / 2019; A / Chicken / Bali9C / GAY / 2019; A / Chicken / BaliA4 / GAY / 2019; A / Chicken / Bali10A / GAY / 2016 and A / Chicken / Bali10C / GAY / 2019. One A / Chicken / Bali 9C / GAY / 2016 isolate was subsequently repeated 7 times until a stable H5N1 subtype AI virus titer was obtained. The results of matching with bioinformatics turned out that A / Chicken / Bali 9C / GAY / 2016 isolates had a kinship of 98.62% with AI subtype H5N1 Banyuwangi, amounting to 98.45% with AI subtype H5N1 Lamongan, amounting to 98.10% with AI-H5N1 Lumajang, 97.58% with AI-H5N1 Kediri, 97.07% with AIH5N1 Blitar, 96.72% with AI-H5N1 Denpasar, 96.72% with AI-H5N1 Buleleng and 96.72% with AI-H5N1 Sukoharjo. The conclusion is one of isolate namely A / Chicken / Bali 9C / GAY / 2019 including AI subtype H5N1 clade 2.3.2, is’t stable at passage on SPF eggs, has a kinship of 96.72% with A / duck / Sukoharjo / BBVW-1428- 9/2012, the virus content is 106.9 ELD50 so it is potential for vaccine candidates.

Study on the Antiviral Activities and Hemagglutinin-Based Molecular Mechanism of Novel Chlorogenin 3-O-β-Chacotrioside Derivatives against H5N1 Subtype Viruses

The objective of this study was to investigate the inhibitory effect of chlorogenin 3-O-β-chacotrioside derivatives against H5N1 subtype of the highly pathogenic avian influenza (HPAI) viruses and its molecular mechanism. A series of novel small molecule pentacyclic triterpene derivatives were designed and synthesized and their antiviral activities on HPAI H5N1 viruses were detected. The results displayed that the derivatives UA-Nu-ph-5, XC-27-1 and XC-27-2 strongly inhibited wild-type A/Duck/Guangdong/212/2004 H5N1 viruses with the IC50 values of 15.59 ± 2.4 μM, 16.83 ± 1.45 μM, and 12.45 ± 2.27 μM, respectively, and had the selectivity index (SI) > 3, which was consistent with the efficacy against A/Thailand/kan353/2004 pseudo-typed viruses. Four dealt patterns were compared via PRNT. The prevention dealt pattern showed the strongest inhibitory effects than other patterns, suggesting that these derivatives act on the entry process at the early stages of H5N1 viral infection, providing protection for cells against infection. Further studies through hemagglutinin inhibition (HI) and neuraminidase inhibitory (NAI) assay confirmed that these derivatives inhibited H5N1 virus replication by interfering with the viral hemagglutinin function. The derivatives could recognize specifically HA protein with binding affinity constant KD values of 2.57 × 10−4 M and 3.67 × 10−4 M. In addition, through site-directed mutagenesis combined with a pseudovirion system, we identified that the high-affinity docking sites underlying interaction were closely associated with amino acid residues I391 and T395 of HA. However, the potential binding sites of the derivatives with HA did not locate at HA1 sialic acids receptor binding domain (RBD). Taken together, these study data manifested that chlorogenin 3-O-β-chacotrioside derivatives generated antiviral effect against HPAI H5N1 viruses by targeting the hemagglutinin fusion machinery.

Antiviral activity of Siberian wild and cultivated plants

The article presents data on the antiviral activity of ethanol and aqueous extracts isolated from the herb plants Dracocephalum nutans, Glechoma hederacea, Melissa officinalis, Berteroa incana, Aegopodium podagraria, and Veronica longifolia against the A/Aichi/2/68 (H3N2) and A/chicken/ Kurgan/05/2005 (H5N1) subtypes of influenza A virus (IAV) in MDCK cell culture. It was found that the ethanol extracts of Glechoma hederacea, Berteroa incana, and Aegopodium podagraria have the antiviral activity in vitro in MDCK cell culture against H3N2 subtype of IAV. The aqueous extracts of Glechoma hederacea, Melissa officinalis, Aegopodium podagraria, and Veronica longifolia and ethanol extracts of Berteroa incana and Veronica longifolia are active against H5N1 subtype of IAV. None of the extracts of Dracocephalum nutans showed significant activity against the studied subtypes. The greatest efficiency was shown by the ethanol extract of Aegopodium podagraria against the H5N1 subtype and aqueous extracts from Glechoma hederacea and Melissa officinalis.against the H3N2 subtype. The ethanol extract of Berteroa incana has a high activity against both IAV subtypes, which makes this sample the most promising for creating new drugs for the prevention and treatment of influenza virus.

Deteksi, Isolasi, dan Identifikasi Avian influenza Subtipe H5N1 pada Unggas di Pulau Jawa, Indonesia Tahun 2016 (DETECTION, ISOLATION AND IDENTIFICATION OF H5N1 SUBTYPE AVIAN INFLUENZA IN POULTRY IN JAVA ISLAND, INDONESIA, 2016)

H5N1 subtype Avian influenza has harmed for poultry in Indonesia currently. Since clade 2.3.2 Avian influenza has been found in waterfowl from 2012, mortality of waterfowl has been occured until now. Change of extreme weather condition with high rainfall and flood, small poultry that does not adjust stricted biosecurity and without vaccination program, and chain sale of poultry on traders can increase case and transmission of Avian influenza in poultry. The objective of study was to isolate and identify Avian influenza on poultry in Central Java, West Java, East Java and Banten in 2016. The study was conducted by using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) method to identify H5N1 subtype Avian influenza on poultry. The study was also conducted by isolation of H5N1 subtype Avian influenza from poultry into 9-11 days of embrionated chicken eggs. The study result represented that a number of 24 pool samples were positive reacted on RT-PCR with matrix primer, a total of 15 pool samples was positive reacted on RT-PCR with H5 primer and as many as 11 pool samples were positive reacted on RT-PCR with N1 primer. Two isolates of H5N1 subtype Avian influenza were isolated samples collected from the Serang City and six isolates of H5N1 subtype Avian influenza from Avian influenza case on waterfowl in Lamongan District. EID50 titer isolates of Avian influenza virus subtype H5N1 were > 108. The conclusion of this study was H5N1 subtype Avian influenza detected on samples collected from cloacal, trachea swab and organs of poultry from Pekalongan District, Brebes District, Serang City, and Lamongan District in 2016. H5N1 subtype Avian influenza can be grown and isolated from samples collected of Serang City and Lamongan District in 2016. H5N1 subtype Avian influenza can be detected, isolated, and indentified in poultry in Java Island, Indonesia in 2016.