Evaluation of different platforms for the detection of anti-SARS coronavirus-2 antibodies, Thailand.

BACKGROUND: Antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) help determine previous infection in individuals, regardless of whether they are asymptomatic or symptomatic. The detection of antibodies serves several purposes, including supporting other assays for disease diagnosis, conducting seroepidemiological studies, and evaluating vaccines. Many platforms of immunological methods for anti-SARS-CoV-2 antibody detection and their performance require validation. METHODS: This study evaluated the test performance of three autoanalyzer-based assays (Architect IgG, Vitros IgG, and Vitros total Ig) and one manual ELISA (Wantai total Ig) against a microneutralization (microNT) assay on the detection of SARS-CoV-2 antibodies. Furthermore, an indirect immunofluorescence assay verified the discordant results between the microNT and commercial assays. The test sensitivity, specificity, positive predictive value, and negative predictive value were determined based on four groups of 1005 serum samples: 102 COVID-19 prepandemic sera, 45 anti-SARS-CoV-2 positive sera, 366 sera of people at risk, and 492 sera of citizens returning from countries with a high prevalence of infection. RESULTS: The analyses as a whole showed that the performance of these commercial assays was comparable. Each group was also analysed separately to gain further insight into test performance. The Architect did not detect two positive sera of people at risk (prevalence of infection 0.55%). The other methods correctly identified these two positive sera but yielded varying false-positive results. The group of returning travellers with an infection rate of 28.3% (139 of 492) better differentiated the test performance of individual assays. CONCLUSIONS: High-throughput Architect and Vitros autoanalyzers appear appropriate for working on large sample sizes in countries that can afford the cost. The Wantai ELISA, while requiring more individual time and technical skill, may provide reliable results at a lower cost. The selection of assays will depend on the laboratory facilities and feasibility.

Replication and cytokine profiles of different subgenotypes of enterovirus 71 isolated from Thai patients in peripheral blood mononuclear cells.

Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are common causative agents of mild and self-limiting symptoms of childhood hand, foot, and mouth disease (HFMD). However, some EV71-infected HFMD patients can develop severe neurological and/or fatal cardiopulmonary complications. In Thailand, HFMD associated with the EV71 subgenotypes C4a and B5 were reported to be associated with diverse outcomes. However, variations in enterovirus subgenotypes and virulence factors have not been fully elucidated; this study elucidated these variations in peripheral blood mononuclear cells (PBMCs) exposed to different subgenotypes of isolated enteroviruses for 24 and 48 h. Following infection, viral titers were determined by plaque assay. Infected cells and intracellular cytokines were quantified using flow cytometry, and multiplex assay was used to examine cytokine release. All isolated subgenotypes showed replication capability in PBMCs; specifically, the replication titer of EV71 C4a tended to be higher than titers of EV71 B5 and CA16. Additionally, the infectivity of EV71 B5 was higher in monocytes than in lymphocytes. Compared with EV71 B5, EV71 C4a and CA16 had greater ability to induce intra- and extracellular cytokine responses. These findings provide new insights into variations in cellular immune responses to different EV71 subgenotypes isolated from Thai patients, which should be considered for the development of vaccines and therapeutic agents.

Elevation of Cleaved p18 Bax Levels Associated with the Kinetics of Neuronal Cell Death during Japanese Encephalitis Virus Infection.

Japanese encephalitis virus (JEV) infection induces uncontrolled neuronal apoptosis, leading to irreversible brain damage. However, the mechanism of JEV-induced neuronal apoptosis has not been clearly elucidated. This study aimed to investigate both virus replication and neuronal cell apoptosis during JEV infection in human neuroblastoma SH-SY5Y cells. As a result, the kinetic productions of new viral progeny were time- and dose-dependent. The stimulation of SH-SY5Y cell apoptosis was dependent on the multiplicity of infections (MOIs) and infection periods, particularly during the late period of infection. Interestingly, we observed that of full-length Bax (p21 Bax) level started to decrease, which corresponded to the increased level of its cleaved form (p18 Bax). The formation of p18 Bax resulting in cytochrome c release into the cytosol appeared to correlate with JEV-induced apoptotic cell death together with the activation of caspase-3/7 activity, especially during the late stage of a robust viral infection. Therefore, our results suggest another possible mechanism of JEV-induced apoptotic cell death via the induction of the proteolysis of endogenous p21 Bax to generate p18 Bax. This finding could be a new avenue to facilitate novel drug discovery for the further development of therapeutic treatments that could relieve neuronal damage from JEV infection.

Seroprevalence of antibodies to enterovirus 71 and coxsackievirus A16 among people of various age groups in a northeast province of Thailand.

BACKGROUND: Hand, foot and mouth disease (HFMD) is endemic among population of young children in Thailand. The disease is mostly caused by enterovirus 71 (EV71) and coxsackievirus A16 (CA16). METHODS: This study conducted serosurveillance for neutralizing (NT) antibodies to EV71 subgenotypes B5 and C4a, and to CA16 subgenotypes B1a and B1b, in 579 subjects of various ages using a microneutralization assay in human rhabdomyosarcoma (RD) cells. These test viruses were the major circulating subgenotypes associated with HFMD in Thailand during the study period. RESULTS: We found that the levels of seropositivity against all 4 study viruses were lowest in the age group of 6-11 months, i.e., 5.5% had antibody to both EV71 subgenotypes, while 14.5% and 16.4% had antibody to CA16 subgenotypes B1a and B1b, respectively. The percentages of subjects with antibodies to these 4 viruses gradually increased with age, but were still less than 50% in children younger than 3 years. These laboratory data were consistent with the epidemiological data collected by the Ministry of Public Health which showed repeatedly that the highest number of HFMD cases was in children aged 1 year. Analyses of amino acid sequences of the test viruses showed 97% identity between the two subgenotypes of EV71, and 99% between the two subgenotypes of CA16. Nevertheless, the levels of seropositivity and antibody titer against the two subgenotypes of EV71 and of CA16 were not significantly different. CONCLUSIONS: This study clearly demonstrated NT antibody activity across EV71-B5 and EV71-C4a subgenotypes, and also across CA16-B1a and CA16-B1b subgenotypes. Moreover, there were no significant differences by gender in the seropositive rates and antibody levels to any of the 4 virus subgenotypes.

Satellite telemetry tracks flyways of Asian Openbill storks in relation to H5N1 avian influenza spread and ecological change.

BACKGROUND: Asian Openbills, Anastomus oscitans, have long been known to migrate from South to Southeast Asia for breeding and nesting. In Thailand, the first outbreak of H5N1 highly pathogenic avian influenza (HPAI) infection in the Openbills coincided with the outbreak in the poultry. Therefore, the flyways of Asian Openbills was determined to study their role in the spread of H5N1 HPAI virus to poultry and wild birds, and also within their flocks. RESULTS: Flyways of 5 Openbills from 3 colonies were monitored using Argos satellite transmitters with positioning by Google Earth Programme between 2007 and 2013. None of the Openbills tagged with satellite telemeters moved outside of Thailand. Their home ranges or movement areas varied from 1.6 to 23,608 km2 per month (95% utility distribution). There was no positive result of the viral infection from oral and cloacal swabs of the Openbills and wild birds living in the vicinity by viral isolation and genome detection during 2007 to 2010 whereas the specific antibody was not detected on both Openbills and wild birds by using microneutralization assay after 2008. The movement of these Openbills did not correlate with H5N1 HPAI outbreaks in domestic poultry but correlated with rice crop rotation and populations of the apple snails which are their preferred food. Viral spread within the flocks of Openbills was not detected. CONCLUSIONS: This study showed that Openbills played no role in the spread of H5N1 HPAI virus, which was probably due to the very low prevalence of the virus during the monitoring period. This study revealed the ecological factors that control the life cycle of Asian Openbills.

H5N1 NS genomic segment distinctly governs the influenza virus infectivity and cytokine induction in monocytic cells.

BACKGROUND: The level of virulence of H5N1 highly pathogenic avian influenza (HPAI) virus was higher than those of the other virus subtypes. It has been suggested that the nonstructural (NS) gene might be a factor contributing to H5N1 HPAI virulence. OBJECTIVES: To determine the efficiency of the NS genomic segment of H5N1 HPAI virus on governing viral infectivity and cytokine induction in monocytic cells compared to other virus strain/subtypes. METHODS: By reverse genetics, five reassortant influenza viruses carrying the NS genomic segment derived from seasonal influenza A(H1N1), 2009 pandemic A(H1N1), A(H3N2) or H5N1 HPAI virus in the backbone of A/Puerto Rico/8/34 H1N1 (PR8) virus were constructed together with the reassorted PR8 virus control, i.e., rgH1N1sea-NS, rgH1N1pdm-NS, rgH3N2-NS, rgH5N1-NS and rgPR8 viruses, respectively. These reverse genetics-derived viruses (rg-viruses) were used to infect monocytic cells for 24 hours prior to determining intracellular influenza nucleoprotein (NP) levels and cytokine induction by flow cytometry. RESULTS: U937 cells were significantly more susceptible to rgPR8 control virus than THP-1 cells; thus, U937 cells were chosen for further study. The number of U937-infected cells (NP+ cells) and the numbers of infected cells that expressed IFN-α (NP+IFN-α+ cell) obtained with rgH5N1-NS virus infection were significantly higher than the others, except for cells infected with the rgH1N1pdm-NS virus. Nevertheless, the numbers of U937 cells that expressed NP+IL-1ß+ were comparable upon infection with any of the rg-viruses; almost none expressed TNF-α. CONCLUSIONS: The H5N1 NS genomic segment distinctly up-regulated the viral infectivity and induction of IFN-α compared to the rgPR8, rgH1N1sea-NS and rgH3N2-NS viruses.

A LONG-TERM SEROSURVEY OF AVIAN INFLUENZA H5 AMONG WILD BIRDS IN NAKHON SAWAN PROVINCE, THAILAND.

An outbreak of HPAIV H5N1 in Nakhon Sawan province, Thailand, in 2004 caused sporadic deaths of Asian openbill storks ( Anastomus oscitans). An investigation was undertaken to determine if this virus occurs and circulates in wild birds in Nakhon Sawan province. Following the outbreak, a widespread serosurvey was conducted using the hemagglutination inhibition assay and microneutralization assay to detect antibodies against AIV H5. From 2007 to 2014, blood was collected from a total of 753 wild birds, representing 10 orders and 44 species. The results reveal that 10 serum samples were positive for AIV H5 antibodies. These seropositive results, found in the orders Ciconiiformes and Anseriformes, demonstrate that waterfowl serve as a reservoir host of AIV. Moreover, the seroprevalences in streak-eared bulbul showed habitat sharing with waterfowl or duck.

Neuraminidase Activity and Resistance of 2009 Pandemic H1N1 Influenza Virus to Antiviral Activity in Bronchoalveolar Fluid.

UNLABELLED: Human bronchoalveolar fluid is known to have anti-influenza activity. It is believed to be a frontline innate defense against the virus. Several antiviral factors, including surfactant protein D, are believed to contribute to the activity. The 2009 pandemic H1N1 influenza virus was previously shown to be less sensitive to surfactant protein D. Nevertheless, whether different influenza virus strains have different sensitivities to the overall anti-influenza activity of human bronchoalveolar fluid was not known. We compared the sensitivities of 2009 pandemic H1N1, seasonal H1N1, and seasonal H3N2 influenza virus strains to inhibition by human bronchoalveolar lavage (BAL) fluid. The pandemic and seasonal H1N1 strains showed lower sensitivity to human BAL fluid than the H3N2 strains. The BAL fluid anti-influenza activity could be enhanced by oseltamivir, indicating that the viral neuraminidase (NA) activity could provide resistance to the antiviral defense. In accordance with this finding, the BAL fluid anti-influenza activity was found to be sensitive to sialidase. The oseltamivir resistance mutation H275Y rendered the pandemic H1N1 virus but not the seasonal H1N1 virus more sensitive to BAL fluid. Since only the seasonal H1N1 but not the pandemic H1N1 had compensatory mutations that allowed oseltamivir-resistant strains to maintain NA enzymatic activity and transmission fitness, the resistance to BAL fluid of the drug-resistant seasonal H1N1 virus might play a role in viral fitness. IMPORTANCE: Human airway secretion contains anti-influenza activity. Different influenza strains may vary in their susceptibilities to this antiviral activity. Here we show that the 2009 pandemic and seasonal H1N1 influenza viruses were less sensitive to human bronchoalveolar lavage (BAL) fluid than H3N2 seasonal influenza virus. The resistance to the pulmonary innate antiviral activity of the pandemic virus was determined by its neuraminidase (NA) gene, and it was shown that the NA inhibitor resistance mutation H275Y abolished this resistance of the pandemic H1N1 but not the seasonal H1N1 virus, which had compensatory mutations that maintained the fitness of drug-resistant strains. Therefore, the innate respiratory tract defense may be a barrier against NA inhibitor-resistant mutants, and evasion of this defense may play a role in the emergence and spread of drug-resistant strains.

Exposure to cold impairs interferon-induced antiviral defense.

It is commonly believed that exposure to low temperature increases susceptibility to viral infection in the human respiratory tract, but a molecular mechanism supporting this belief has yet to be discovered. In this study, we investigated the effect of low temperature on viral infection and innate defense in cell lines from the human respiratory tract and found that interferon-induced antiviral responses were impaired at low temperatures. Cells maintained at 25°C and 33°C expressed lower levels of myxovirus resistance protein 1 (MxA) and 2'5'-oligoadenylate synthetase 1 (OAS1) mRNAs when compared to cells maintained at 37°C after infection by seasonal influenza viruses. Exogenous ß-interferon treatment reduced the viral replication at 37°C, but not at 25°C. Our results suggest that the impairment of interferon-induced antiviral responses by low temperature is one of several mechanisms that could explain an increase in host susceptibility to respiratory viruses after exposure to cold temperature.

Cross-reactive antibodies against H7N9 and H5N1 avian influenza viruses in Thai population.

BACKGROUD: Avian influenza H5N1 and H7N9 viruses have jumped across species from avian to humans and become a threat to public health. Not much is known about pre-existing cross-reactive antibodies against these avian viruses in human population. OBJECTIVE: To determine the prevalence of cross-reactive anti-HA and anti-NA antibodies to avian influenza H5N1 and H7N9 viruses in Thai population. METHOD: Archival serum samples from 100 blood donors and 21 patients infected with 2009 pandemic influenza A (H1N1) (pdmH1N1) virus were investigated by hemagglutination-inhibition (HAI) and neuraminidase-inhibition (NAI) assays for anti-HA and anti-NA antibodies, respectively. The test antigens comprised 2 human viruses (pdmH1N1 and H3N2 viruses), and 6 reassortant viruses carrying HA and NA genes of avian H5N1 or H7N9 virus generated by reverse genetics. RESULTS: HAI antibody titers ≥ 10 were found in 58, 89, 0 and 15% of blood donors as tested against pdmH1N1, H3N2, H5N1 and H7N9 viruses, respectively. On the other hand, NAI antibodies were detected in 98, 94, 73 and 53% of blood donors when reverse genetic-derived viruses harboring NA gene from pdmH1N1, H3N2, H5N1 or H7N9 virus were used as the test antigens. Moreover, 66.7% of pdmH1N1 patients who had > 4 fold increase in HAI antibody titers in paired sera developed > 4 fold increase in NAI antibody titers. CONCLUSIONS: Anti-NA antibody has broader reactivity than anti-HA antibody, therefore, it can be a supplement to anti-HA antibody in the prevention against novel influenza viruses.

The significance of naturally occurring neuraminidase quasispecies of H5N1 avian influenza virus on resistance to oseltamivir: a point of concern.

Viral adaptability and survival arise due to the presence of quasispecies populations that are able to escape the immune response or produce drug-resistant variants. However, the presence of H5N1 virus with natural mutations acquired without any drug selection pressure poses a great threat. Cloacal samples collected from the 2004-2005 epidemics in Thailand from Asian open-billed storks revealed one major and several minor quasispecies populations with mutations on the oseltamivir (OTV)-binding site of the neuraminidase gene (NA) without prior exposure to a drug. Therefore, this study investigated the binding between the NA-containing novel mutations and OTV drug using molecular dynamic simulations and plaque inhibition assay. The results revealed that the mutant populations, S236F mutant, S236F/C278Y mutant, A250V/V266A/P271H/G285S mutant and C278Y mutant, had a lower binding affinity with OTV as compared with the WT virus due to rearrangement of amino acid residues and increased flexibility in the 150-loop. This result was further emphasized through the IC50 values obtained for the major population and WT virus, 104.74 nM and 18.30 nM, respectively. Taken together, these data suggest that H5N1 viruses isolated from wild birds have already acquired OTV-resistant point mutations without any exposure to a drug.

Sialic acid content in human saliva and anti-influenza activity against human and avian influenza viruses.

It was shown previously that human saliva has higher antiviral activity against human influenza viruses than against H5N1 highly pathogenic avian influenza viruses, and that the major anti-influenza activity was associated with sialic-acid-containing molecules. To further characterize the differential susceptibility to saliva among influenza viruses, seasonal influenza A and B virus, pandemic H1N1 virus, and 15 subtypes of avian influenza virus were tested for their susceptibility to human and chicken saliva. Human saliva showed higher hemagglutination inhibition (HI) and neutralization (NT) titers against seasonal influenza A virus and the pandemic H1N1 viruses than against influenza B virus and most avian influenza viruses, except for H9N2 and H12N9 avian influenza viruses, which showed high HI and NT titers. To understand the nature of sialic-acid-containing anti-influenza factors in human saliva, α2,3- and α2,6-linked sialic acid was measured in human saliva samples using a lectin binding and dot blot assay. α2,6-linked sialic acid was found to be more abundant than α2,3-linked sialic acid, and a seasonal H1N1 influenza virus bound more efficiently to human saliva than an H5N1 virus in a dot blot analysis. These data indicated that human saliva contains the sialic acid type corresponding to the binding preference of seasonal influenza viruses.

Immunobiological properties of influenza A (H7N9) hemagglutinin and neuraminidase proteins.

Recombinant vaccinia viruses harboring the complete hemagglutinin (HA) or neuraminidase (NA) genes from the influenza A/Anhui/1/2013 (H7N9) virus were constructed (rVac-H7 HA and rVac-N9 NA viruses). The HA and NA proteins were expressed in the cytoplasm and on the plasma membrane of thymidine-kinase-negative (TK(-)) cells infected with these recombinant viruses. Only one form of the HA protein was expressed in infected TK(-) cells, with a molecular weight (MW) of 75 kDa, but three forms were found when the culture medium was supplemented with trypsin (MWs of 75, 50 and 27 kDa), which was similar to what was found in Madin-Darby canine kidney (MDCK) cells infected with reverse genetic (rg) influenza viruses carrying HA genes of H7N9 virus origin. One form of hyperglycosylated NA protein with a MW of 75 kDa was produced in rVac-N9-NA-virus-infected TK(-) or MDCK cells. The MW decreased to 55 kDa after deglycosylation. The hyperglycosylated recombinant NA protein demonstrated sialidase activity in a fetuin-based neuraminidase assay. The rVac-H7 HA and rVac-N9 NA viruses elicited significantly higher anti-HA and anti-NA antibody titers in BALB/c mice that were immunized once than in ICR mice. The anti-HA and anti-NA antibodies showed activity against homosubtypic HA or NA, but not against heterosubtypic HA or NA, as determined by hemagglutination-inhibition and microneutralization assays for anti-HA antibodies and neuraminidase-inhibition and replication-inhibition assays for anti-NA antibodies. Taken together, our data demonstrated immunobiological properties of recombinant HA and NA proteins that might be useful for vaccine development.

The N-linked glycosylation site at position 158 on the head of hemagglutinin and the virulence of H5N1 avian influenza virus in mice.

N-linked glycosylation of the influenza virus hemagglutinin (HA) protein plays crucial roles in HA structure and function, evasion of neutralizing antibodies, and susceptibility to innate soluble antiviral factors. The N-linked glycosylation site at position 158 of highly pathogenic H5N1 virus was previously shown to affect viral receptor-binding preference. H5N1 viruses show heterogeneity with respect to the presence of this glycosylation site. Clade 1 viruses that caused outbreaks in Southeast Asia in 2004 contained this glycosylation site, while the site is absent in the more recent clade 2 viruses. Here, we show that elimination of this glycosylation site increases viral virulence in mice. The mutant lacking the glycosylation site at position 158 showed unaltered growth kinetics in vitro and a comparable level of sensitivity to a major antiviral protein found in respiratory secretions, surfactant protein D (SP-D).

Susceptibility of human and avian influenza viruses to human and chicken saliva.

Oral cavity can be an entry site of influenza virus and saliva is known to contain innate soluble anti-influenza factors. Influenza strains were shown to vary in their susceptibility to those antiviral factors. Whether the susceptibility to the saliva antiviral factors plays any role in the host species specificity of influenza viruses is not known. In this study, the antiviral activity of human and chicken saliva against human and the H5N1 avian influenza viruses were investigated by hemagglutination inhibition (HI) and neutralization (NT) assays. In comparison to human influenza viruses, H5N1 isolates showed reduced susceptibility to human saliva as measured by HI and NT assays. Interestingly, an H5N1 isolate that bind to both α2,3- and α2,6-linked sialic acid showed much higher HI titers with human saliva, suggesting that the susceptibility profile was linked to the receptor-binding preference and the presence of α2,6-linked sialic in human saliva. On the other hand, the H5N1 isolates showed increased HI titers but reduced NT titers to chicken saliva as compared to human influenza isolates. The human salivary antiviral components were characterized by testing the sensitivity to heat, receptor destroying enzyme (RDE), CaCl2/EDTA dependence, and inhibition by mannan, and shown to be α- and γ-inhibitors. These data suggest that the H5N1 HPAI influenza virus had distinctive susceptibility patterns to human and chicken saliva, which may play some roles in its infectivity and transmissibility in these hosts.

Simultaneous discrimination and detection of influenza A(H1N1)pdm09 and seasonal influenza A viruses using a rapid immunogold biosensor.

A rapid immunogold biosensor for the simultaneous discrimination of influenza A(H1N1)pdm09 and seasonal influenza A viruses was developed successfully. Monoclonal antibodies (mAbs) that were specific for the hemagglutinin protein of the A(H1N1)pdm09 virus were produced, and the best mAb pairs were selected. Using an mAb that was specific for the influenza A nucleoprotein, a rapid immunogold biosensor for the discrimination and detection of A(H1N1)pdm09/seasonal influenza viruses was developed. When tested with 72 virus isolates, the system achieved 100 % detection of the A(H1N1)pdm09 virus without cross-reactivity against seasonal influenza A (H1, H3 subtypes) and B viruses, parainfluenza viruses, respiratory syncytial viruses, and adenoviruses. The detection limits for A(H1N1)pdm09 and seasonal strains were 5 × 10(2)-7.5 × 10(3) and 1 × 10(3)-7.5 × 10(5) TCID50/mL, respectively. When tested with 49 clinical specimens, the specificity was high (100 %). The sensitivity for the detection of A(H1N1)pdm09 and seasonal strains was 90 % and 100 %, respectively, which correlated with the results of real-time reverse transcription polymerase chain reaction as a reference method. The ability of the system to detect and discriminate the A(H1N1)pdm09 strain from the seasonal strains suggests that this method may be beneficial for investigation of outbreaks and diagnostic applications. Furthermore, this method might be a useful platform for developing a rapid diagnostic system for the simultaneous discrimination of other influenza virus subtypes during future outbreaks.

Higher correlation between neutralizing antibodies and surrogate neutralizing or binding antibodies in COVID-19 patients than vaccine recipients.

This study determined the seropositive rates and levels of antibodies to severe acute respiratory syndrome coronavirus-2 in 50 patients and 108 vaccinees using microneutralization test (MNT), surrogate virus neutralization test (sVNT), chemiluminescent microparticle immunoassay (CMIA), and electrochemiluminescence immunoassay (ECLIA). MNT, as the reference method, employed living clade S and Delta viruses to measure neutralizing (NT) antibodies, while sVNT employed wild type strain and Delta receptor-binding domains (RBD) as the test antigens to measure sVNT antibodies. CMIA and ECLIA employed only one version of RBD to measure the binding antibodies. Our study performed S gene sequencing of the test virus to exclude undesired mutants that might lead to changes in antibody levels in MNT assay. We showed that spike protein amino acid sequences of our Delta virus contained 13 amino acid changes, with 3 related to the reduced neutralization. The MNT assay showed a significant reduction in seropositive rates and antibody levels in the patients' sera when the Delta variant replaced clade S as the test virus. In contrast, the seropositive rates determined by sVNT assay using wild type strain RBD and Delta RBD were non-significantly different, suggesting that sVNT assay could not identify the difference between the antigenicity of wild type RBD and Delta RBD. Furthermore, the correlation between the levels of NT and sVNT antibodies was moderate with the patients' sera but modest with the post-vaccination sera. The seropositive rates in the patients, as determined by CMIA or ECLIA, were not different from the MNT assay using clade S, but not Delta, as the test virus. In all analyses, the correlations between the antibody levels measured by MNT and the other 3 assays were modest to moderate, with the r-values of 0.3500-0.7882.

Sero-epidemiological investigation and cross-neutralization activity against SARS-CoV-2 variants in cats and dogs, Thailand.

Epidemiological data on SARS-CoV-2 infection in companion animals have been thoroughly investigated in many countries. However, information on the neutralizing cross-reactivity against SARS-CoV-2 variants in companion animals is still limited. Here, we explored the neutralizing antibodies against SARS-CoV-2 in cats and dogs between May 2020 and December 2021 during the first wave (a Wuhan-Hu-1-dominant period) and the fourth wave (a Delta-dominant period) of the Thailand COVID-19 outbreak. Archival plasma samples of 1,304 cats and 1,795 dogs (total = 3,099) submitted for diagnosis and health checks were collected at the Prasu-Arthorn Veterinary Teaching Hospital, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom. A microneutralization test was used to detect neutralizing antibodies against the ancestral Wuhan-Hu-1 and the Delta variants. A plasma sample with neutralizing titers ≥10 was considered positive. Our results showed relatively low seroprevalence with seropositive samples detected in 8 out of 3,099 individuals (0.26, 95% CI 0.11-0.51%). Among these cases, SARS-CoV-2 neutralizing antibodies from both the ancestral Wuhan-Hu-1 and the Delta variants were found in three out of eight cases in two cats (n = 2) and one dog (n = 1). Furthermore, neutralizing antibodies specific to only the ancestral Wuhan-Hu-1 variant were exclusively found in one cat (n = 1), while antibodies against only the Delta variant were detected in four dogs (n = 4). Additionally, the neutralizing cross-activities against SARS-CoV-2 variants (Alpha, Beta, and Omicron BA.2) were observed in the seropositive cats with limited capacity to neutralize the Omicron BA.2 variant. In summary, the seropositivity among cats and dogs in households with an unknown COVID-19 status was relatively low in Thailand. Moreover, the neutralizing antibodies against SARS-CoV-2 found in the seropositive cats and dogs had limited or no ability to neutralize the Omicron BA.2 variant. Thus, monitoring SARS-CoV-2 infection and sero-surveillance, particularly in cats, is imperative for tracking virus susceptibility to the emergence of new SARS-CoV-2 variants.

Safety and immunogenicity of locally produced trivalent inactivated influenza vaccine (Tri Fluvac) in healthy Thai adults aged 18-64 years in Nakhon Phanom: A Phase III double blinded, three-arm, randomized, controlled trial.

BACKGROUND: Domestic influenza vaccine production facilitates a sustainable supply for mitigating seasonal influenza and improves national health security by providing infrastructure and experience for pandemic vaccine production, if needed. METHODS: A Phase III, double blind, randomized controlled trial was conducted from Sep 2019-Oct 2020 in healthy adults 18-64 years in Nakhon Phanom, Thailand. Randomization (3:3:1) compared study vaccine (Tri Fluvac), saline placebo, and an active comparator (licensed vaccine). Primary outcomes were superior efficacy compared to placebo based on RT-PCR-confirmed influenza virus infection within 12 months and non-inferiority compared to active comparator based on immunogenicity (HAI assay) at 28 days. Safety was also assessed. RESULTS: The trial enrolled 4,284 participants (Tri Fluvac = 1,836; placebo = 1,836; active comparator = 612). There were 29 RT-PCR positive influenza infections (10 Tri Fluvac, 5.5/1,000 PY; 19 placebo, 10.4/1,000PY; 0 comparator) for an absolute protective efficacy of 46.4 (95 % CI = -22.0-76.5) compared with placebo, but the power was 43.7 %. Seroconversion difference rates between Tri Fluvac and comparator at Day 28 were 1.74 (95 % CI: -2.77, 6.25), 2.22 (-2.40, 6.84), and -0.57 (-5.41, 4.27) for A(H1N1), A(H3N2), and B strains, respectively. Adverse and severe adverse events occurred in 175 (9.5 %) Tri Fluvac, 177 (10.8 %) placebo, and 66 (10.8 %) comparator arms (p-value = 0.437, Tri Fluvac vs. comparator) CONCLUSIONS: Tri Fluvac was well tolerated, and immunogenicity was non-inferior to the active comparator, meeting U.S. Food and Drug Administration (FDA) criteria for adult vaccine licensure. Few acute respiratory infections were reported during intense COVID-19 pandemic restrictions, resulting in insufficient power to evaluate clinical efficacy.

A serine-to-asparagine mutation at position 314 of H5N1 avian influenza virus NP is a temperature-sensitive mutation that interferes with nuclear localization of NP.

We have generated a temperature-sensitive (ts) mutant from a human isolate of the H5N1 avian influenza virus by classical adaptation in cell culture. After 20 passages at low temperature, the virus showed a ts phenotype. The ts mutant also showed an attenuated phenotype after nasal inoculation in mice. Using reverse genetics, we generated reassortants carrying individual genomic segments of the wild-type and mutant viruses in an A/Puerto Rico/8/34 background, and found that the nucleoprotein (NP) gene could confer the ts phenotype. This mutant NP contains a serine-to-asparagine mutation at position 314 (S314N). The mutant NP protein showed a defect in nuclear localization at high temperature in mammalian cells.