Inactivated SARS-CoV-2 vaccine candidate immunization on non-human primate animal model: B-cell and T-cell responses immune evaluation.

Background: SARS-CoV-2 vaccine was proven to be an effective and efficient measure for mitigating pandemic. COVID-19 infection and mortality subsided along with the increaseing COVID-19 vaccination coverage. Vaccine and health resource equity are predominant factors in COVID-19 pandemic management. Vaccine development for Indonesia, aims to ensure a sustainable pandemic control and steady national stability restoration. A decent vaccine must induce immunity against COVID-19 with minimum adverse reaction. Immunogenicity and ability to induce neutralizing antibody evaluation needs to be performed as part of the SARS-CoV-2 inactivated vaccine development from East Java, Indonesia isolate (Vaksin Merah Putih-INAVAC). Objective: This research demonstrated INAVAC performance in inducing the production neutralizing antibody along with its effects on CD4+ and CD8+ cells response in Macaca fascicularis (non-human primate). Methods: Two dosages of 3 µg and 5 µg were tested, compared to sham (NaCl 0.9%) in 10 Macaca fascicularis (2 injection intramuscular with 14 days interval). All animals were monitored daily for clinical signs. Nasopharyngeal samples were analyzed using qRT-PCR while the serum were tested using ELISA and neutralization assay, whereas PBMCs were flowcytrometrically analyzed to measure CD4+ and CD8+ population. Results: It is observed that both vaccine doses could stimulate relatively similar immune response and neutralizing antibody (end GMT post challenge = 905,1), whereas higher CD8+ cells response were reported in the 5 µg group after the 3rd day post-challenge. The dose of vaccine that produce adequate immune cell stimulation with neutralizing antibody induction can be adopted to clinical study, as favorable result of these parameters could predict minimum adverse reaction from inflammation response with balanced immune response. Conclusions: Therefore, it is concluded that Vaksin Merah Putih-INAVAC with 3 µg dose showed a favorable potential to be developed and tested as human vaccine.

Adipose Mesenchymal Stem Cell Metabolites Oral Gel Enhance Pro-Angiogenic Factors Expression, Angiogenesis, and Clinical Outcome of Oral Ulcer Rat Model.

OBJECTIVE: Enhancing wound healing capacity is one of the main principles in oral ulcer management. Efficient oral ulcer management will accelerate clinical symptom amelioration and prevent complications. Adipose mesenchymal stem cell metabolites (AdMSCM), a novel biological product, contains a plethora of bioactive mediators that can induce a series of processes in wound healing. This study will analyze the clinical outcome, angiogenesis, and expression of FGF-2 and VEGFA in the oral ulcer rat model after AdMSCM oral gel application. MATERIALS AND METHODS: Twenty healthy male Wistar rats (Rattus novergicus) were used to create oral ulcer animal models. AdMSCM oral gel treatment was performed three times daily for 3 and 7 days. Clinical outcome was assessed by measuring the major diameter of the ulcer; the angiogenesis was evaluated through histological assessment; the expression of VEGFA and FGF-2 was assessed using the immunohistochemistry method. STATISTICAL ANALYSIS: This study uses parametric comparative analysis using one-way analysis of variance (ANOVA) and post-hoc Tukey's HSD test RESULTS: The application of AdMSCM oral gel in an oral ulcer rat model significantly enhanced the clinical outcome (p < 0.05). In addition, similar results were shown in the histologic assessment of angiogenesis and supported by the significant increase of VEGFA and FGF-2 expression. CONCLUSIONS: AdMSCM oral gel accelerates oral ulcer healing processes, proven by the enhancement of angiogenesis, pro-angiogenic factors expression, and clinical outcomes.

Whole-genome sequence and genesis of an avian influenza virus H5N1 isolated from a healthy chicken in a live bird market in Indonesia: accumulation of mammalian adaptation markers in avian hosts.

Background: Influenza A viruses are a major pathogen that causes significant clinical and economic harm to many animals. In Indonesia, the highly pathogenic avian influenza (HPAI) H5N1 virus has been endemic in poultry since 2003 and has caused sporadic deadly infections in humans. The genetic bases that determine host range have not yet been fully elucidated. We analyzed the whole-genome sequence of a recent H5 isolate to reveal the evolution toward its mammalian adaptation. Methods: We determined the whole-genome sequence of A/chicken/East Java/Av1955/2022 (hereafter, "Av1955") from a healthy chicken in April 2022 and conducted phylogenetic and mutational analysis. Results: Phylogenetic analysis revealed that Av1955 belonged to the H5N1 clade 2.3.2.1c (Eurasian lineage). The six gene segments (PB1, PB2, HA, NP, NA, and NS) out of the eight segments derived from viruses of H5N1 Eurasian lineage, one (PB2) from the H3N6 subtype and the remaining one (M) from the H5N1 clade 2.1.3.2b (Indonesian lineage). The donor of the PB2 segment was a reassortant among three viruses of H5N1 Eurasian and Indonesian lineages and the H3N6 subtype. The HA amino acid sequence contained multiple basic amino acids at the cleavage site. Mutation analysis revealed that Av1955 possessed the maximal number of mammalian adaptation marker mutations. Conclusions: Av1955 was a virus of H5N1 Eurasian lineage. The HA protein contains an HPAI H5N1-type cleavage site sequence, while the virus was isolated from a healthy chicken suggesting its low pathogenicity nature. The virus has increased mammalian adaptation markers by mutation and intra- and inter-subtype reassortment, gathering gene segments possessing the most abundant maker mutations among previously circulating viruses. The increasing mammalian adaptation mutation in avian hosts suggests that they might be adaptive to infection in mammalian and avian hosts. It highlights the importance of genomic surveillance and adequate control measures for H5N1 infection in live poultry markets.

Knowledge, attitude, and practices associated with avian influenza among undergraduate university students of East Java Indonesia: A cross-sectional survey.

Background: Several public health strategic actions are required for effective avian influenza (AI) prevention and control, as well as the development of a communication plan to keep undergraduate students sufficiently informed on how to avoid or reduce exposure. The aim of the survey was to measure the level of knowledge, attitudes and practices (KAPs) toward AI among undergraduate university students in East Java, Indonesia, and observe the correlation between KAPs and the factors associated with the control and prevention of AI.  Methods: A cross-sectional survey was conducted among undergraduate students to collect information about AI-related KAPs. Students were selected from three faculties of Universitas Airlangga Surabaya Indonesia (Faculty of Veterinary Medicine, Faculty of Fisheries and Marine, and Faculty of Science and Technology). Students voluntarily responded to a pre-designed questionnaire. Results: A total of 425 students (222 female; and 203 male), of ages ranging from 18 years (n=240) to 20-30 years (n=185), responded to the survey. This cohort consisted of 157 students from the Faculty of Fisheries and Marine, 149 from the Faculty of Veterinary Medicine, and 119 from the Faculty of Science and Technology. The results indicated that appropriate knowledge was obtained by 76.94% of students; significantly higher levels were seen in Faculty of Veterinary Medicine students as compared to the other two faculties (p<0.05). 72.89% of students documented positive attitudes; veterinary medicine students had significantly more positive attitudes than other faculties (p<0.05). Proactive behaviors were observed in 56.90% of students. The aggregate scores for KAPs were 6.93 ± 0.77 (range: 0-9) for knowledge, 7.6 ± 1.25 (range: 0-10) for attitude, and 9.1 ± 1.5 (range: 0-12) for practice.

Phylogenetic relationship and genotype variation of six Newcastle disease viruses isolated from duck in Indonesia.

BACKGROUND AND AIM: Newcastle disease viruses (NDVs) are frequently acquired from all ages and types of bird species. In general, ducks are considered as potential reservoirs for different genotypes of NDV and are resistant even to velogenic NDV strains. This research was conducted to genotypically and phylogenetically characterize NDV isolates collected from unvaccinated ducks from Indonesia. MATERIALS AND METHODS: A total of 200 samples were collected through cloacal swabs and were inoculated in the allantoic sacs of 8-day-old specific pathogen-free eggs. Hemagglutination (HA) activity was analyzed through a HA test, and isolated viruses were characterized by reverse transcription-polymerase chain reaction targeting the complete fusion (F)-gene of NDV using three primer sets. One primer set was specific for the F protein cleavage site sequences of velogenic, mesogenic, and lentogenic NDV strains. RESULTS: The results demonstrated that three isolates (NDV/Duck/B104/19, NDV/Duck/B125/19, and NDV/Duck/BK43/19) belonged to genotype VII and one (NDV/Duck/TD19/19) to genotype VI. Other isolates (NDV/Duck/A74/19 and NDV/Duck/M147/19) belonged to genotype II Class II. Based on the F protein cleavage site and the pathogenicity tests, two isolates (NDV/Duck/B104/19 and NDV/Duck/B125/19) were categorized as velogenic viruses and four (NDV/Duck/BK43/19, NDV/Duck/TD19/19, NDV/Duck/A74/19, and NDV/Duck/M147/19) as lentogenic viruses. CONCLUSION: The results indicate that NDVs from unvaccinated ducks from Indonesia carry various genotypes and pathotypes of NDVs; therefore, these viruses are still circulating in the environment and might pose a risk of Newcastle disease outbreak.

Nucleotide mutation analyses of isolated lentogenic newcastle disease virus in live bird market.

Newcastle Disease (ND) is a major viral disease in Indonesia. It is an RNA virus belongs to Paramyxovirinae. It is well known that RNA virus is easily to mutate. In some cases, this mutation could generate virulence alteration. It is noted that mutation of NDV which has avirulent amino acid sequence on the cleavage site, could mutate to be virulent Newcastle Disease Virus (NDV). It is needed to analyze the nucleotide and amino acid mutations and the effect of those to its virulence. The aim of this study was to analyze nucleotide and amino acid mutations of original isolated Lentogenic Newcastle Disease Virus (NDV). Samples were collected from cloacal swab of native chicken (Gallus gallus domesticus) suspected to be infected by Lentogenic NDV from live bird markets. They were inoculated into embryonated eggs, to isolate the virus. HA and HI assays were conducted to confirm that they were NDV. Positive samples were processed into serial passages in embryonated egg to observe their death time. Samples caused mortality of the embryonated eggs more than 90 hours post infection were suspected as Lentogenic NDV. They were processed to RT-PCR then sequenced. Lentogenic NDV confirmation was done by comparing amino acid at Fusion protein cleavage site of the samples to Lasota/JF950510. Nucleotide and amino acid mutations were analyzed. The result showed that some nucleotide mutations were capable to change sequences of amino acid but the virulence of the samples remained the same to the reference sequence.

Antigenic site of nucleoprotein gene from Indonesian rabies virus isolates.

BACKGROUND AND AIM: Several molecular studies on rabies virus (RABV) have been conducted in Indonesia, but it does not give clear information about molecular characteristics of previous RABV isolate in Indonesia. This study was conducted to know the characteristic of circulating RABV to determine a suitable method to control the spreading of RABV in Indonesia. MATERIALS AND METHODS: Samples of infected RABV from dog brain were collected from Sumatera, Kalimantan, Sulawesi, and Bali Islands. All samples were examined based on nucleoprotein encoding gene to determine the molecular characteristics based on homology and phylogenetic tree compared to Pasteur Virus and RABV that came from another country within Asia (Indonesia, China, Thailand, India, and Korea). The collected samples were processed by one-step reverse transcriptase-polymerase chain reaction using nucleoprotein encoding gene followed by sequencing. The amino acid of its antigenic site of isolated RABV was also analyzed. RESULTS: The results showed that isolated RABV has 84-85% similarity compared to Pasteur. According to phylogenetic construction, isolated samples do not share the same lineage toward Pasteur. The homology scores of isolated samples compared to RABV within Asia such as Indonesia, China, Thailand, India, and Korea were 98-99%, 92-93%, 88-89%, 86-88%, and 85-88%, respectively. According to antigenic site analysis compared to Pasteur, it was found that there were amino acid mutations within antigenic Site IV of nucleoprotein. Amino acid mutation from isoleucine to valine occurred in amino acid number 240 of 6 Kalimantan, 7 Kalimantan, and 8 Kalimantan. Amino acid mutation from alanine to aspartate and asparagine to threonine occurred within the same antigenic site in amino acid number 246 and 273 of C4 isolate from Sulawesi. CONCLUSION: According to homology and phylogenetic tree analyses, isolated RABV remained different compared to RABV within Asia and Pasteur. The amino acid mutation occurred in antigenic site of nucleoprotein encoding gene.