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.

A novel film spray containing curcumin inhibits SARS-CoV-2 and influenza virus infection and enhances mucosal immunity.

BACKGROUND: Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and influenza virus is still a major worldwide health concern. Plants are a good source of bioactive compounds to be used as preventive measures for both inhibiting the virus binding and enhancing mucosal innate immunity. Curcumin has been shown to possess antiviral activity and modulate innate immunity. Therefore, the purpose of this study was to develop an oro-nasal film spray containing curcumin and determine its antiviral activity against SARS-CoV-2 and influenza virus infection, as well as its effects on mucosal innate immunity and inflammatory cytokines in vitro. METHODS: The antiviral activity of the film spray against SARS-CoV-2, influenza A/H1N1, A/H3N2, and influenza B was assessed in vitro by plaque reduction assay. Cytotoxicity of the film spray to oral keratinocytes and nasal epithelial cells was assessed by MTT assay, and cytotoxicity to Vero and MDCK cells was assessed by an MTS-based cytotoxicity assay. Oral and nasal innate immune markers in response to the film spray were determined by ELISA and by a commercial Milliplex Map Kit, respectively. RESULTS: Our data show that the film spray containing curcumin can inhibit both SARS-CoV-2 and influenza virus infections while maintaining cell viability. Results obtained among 4 viruses revealed that curcumin film spray demonstrated the highest inhibitory activity against SARS-CoV-2 with the lowest EC50 of 3.15 µg/ml and the highest SI value of 4.62, followed by influenza B (EC50 = 6.32 µg/ml, SI = 2.04), influenza A/H1N1 (EC50 = 7.24 µg/ml, SI = 1.78), and influenza A/H3N2 (EC50 > 12.5 µg/ml, SI < 1.03), respectively. Antimicrobial peptides LL-37 and HD-5, IL-6 and TNF-α produced by oral keratinocytes were significantly induced by the film spray, while hBD2 was significantly reduced. CONCLUSION: Film spray containing curcumin possesses multiple actions against SARS-CoV-2 infection by inhibiting ACE-2 binding in target cells and enhancing mucosal innate immunity. The film spray can also inhibit influenza virus infection. Therefore, the curcumin film spray may be effective in preventing the viral infection of both SARS-CoV-2 and influenza.

Molecular characterization and geographical distribution of Zika virus worldwide from 1947 to 2022.

OBJECTIVES: We conducted molecular characterization, demonstrated the geographical distribution of Zika virus (ZIKV) circulating worldwide from 1947 to 2022 and explored the potential genetic recombination site in the Thailand ZIKV genomes. METHODS: We constructed phylogenetic trees based on ZIKV coding sequences (CDS) and determined the geographical distribution of the representative viruses by genetic relationship and timeline. We determined genetic recombination among ZIKV and between ZIKV and other flaviviruses using similarity plot and bootscan analyzes, together with the phylogeny encompassing the CDS and eight subgenomic regions. RESULTS: The phylogenetic trees comprising 717 CDS showed two distinct African and Asian lineages. ZIKV in the African lineage formed two sublineages, and ZIKV in the Asian lineage diversified into the Asian and American sublineages. The 1966 Malaysian isolate was designated the prototype of the Asian sublineage and formed a node of only one member, while the newer viruses formed a distinct node. We detected no genetic recombination in the Thailand ZIKV. CONCLUSION: Five Thailand isolates discovered in 2006 were the second oldest ZIKV after the Malaysian prototype. Our result suggested two independent routes of ZIKV spread from Southeast Asia to Micronesia in 2007 and French Polynesia in 2013 before further spreading to South American countries.

Phenotypic and functional changes of T cell subsets after CoronaVac vaccination.

BACKGROUND: The pandemic coronavirus disease 2019 (COVID-19) is a major global public health concern and several protective vaccines, or preventive/therapeutic approaches have been developed. Sinovac-CoronaVac, an inactivated whole virus vaccine, can protect against severe COVID-19 disease and hospitalization, but less is known whether it elicits long-term T cell responses and provides prolonged protection. METHODS: This is a longitudinal surveillance study of SARS-CoV-2 receptor binding domain (RBD)-specific IgG levels, neutralizing antibody levels (NAb), T cell subsets and activation, and memory B cells of 335 participants who received two doses of CoronaVac. SARS-CoV-2 RBD-specific IgG levels were measured by enzyme-linked immunosorbent assay (ELISA), while NAb were measured against two strains of SARS-CoV-2, the Wuhan and Delta variants. Activated T cells and subsets were identified by flow cytometry. Memory B and T cells were evaluated by enzyme-linked immune absorbent spot (ELISpot). FINDINGS: Two doses of CoronaVac elicited serum anti-RBD antibody response, elevated B cells with NAb capacity and CD4+ T cell-, but not CD8+ T cell-responses. Among the CD4+ T cells, CoronaVac activated mainly Th2 (CD4+ T) cells. Serum antibody levels significantly declined three months after the second dose. INTERPRETATION: CoronaVac mainly activated B cells but T cells, especially Th1 cells, were poorly activated. Activated T cells were mainly Th2 biased, demonstrating development of effector B cells but not long-lasting memory plasma cells. Taken together, these results suggest that protection with CoronaVac is short-lived and that a third booster dose of vaccine may improve protection.

Seroprevalence of anti-SARS-CoV-2 antibodies in Thai adults during the first three epidemic waves.

This study determined the presence of anti-SARS-CoV-2 antibodies in 4964 individuals, comprising 300 coronavirus disease-19 (COVID-19) prepandemic serum samples, 142 COVID-19 patients, 2113 individuals at risk due to their occupations, 1856 individuals at risk due to sharing workplaces or communities with COVID-19 patients, and 553 Thai citizens returning after spending extended periods of time in countries with a high disease prevalence. We recruited participants between May 2020 and May 2021, which spanned the first two epidemic waves and part of the third wave of the COVID-19 outbreaks in Thailand. Their sera were tested in a microneutralization and a chemiluminescence immunoassay for IgG against the N protein. Furthermore, we performed an immunofluorescence assay to resolve discordant results between the two assays. None of the prepandemic sera contained anti-SARS-CoV-2 antibodies, while antibodies developed in 88% (15 of 17) of the COVID-19 patients at 8-14 days and in 94-100% of the patients between 15 and 60 days after disease onset. Neutralizing antibodies persisted for at least 8 months, longer than IgG antibodies. Of the 2113 individuals at risk due to their occupation, none of the health providers, airport officers, or public transport drivers were seropositive, while antibodies were present in 0.44% of entertainment workers. Among the 1856 individuals at risk due to sharing workplaces or communities with COVID-19 patients, seropositivity was present in 1.9, 1.5, and 7.5% of the Bangkok residents during the three epidemic waves, respectively, and in 1.3% of the Chiang Mai people during the first epidemic wave. The antibody prevalence varied between 6.5 and 47.0% in 553 Thai people returning from high-risk countries. This serosurveillance study found a low infection rate of SARS-CoV-2 in Thailand before the emergence of the Delta variant in late May 2021. The findings support the Ministry of Public Health's data, which are based on numbers of patients and contact tracing.

Antiviral effect in association with anti-apoptosis and anti-autophagy of repurposing formoterol fumarate dihydrate on enterovirus A71-infected neuronal cells.

OBJECTIVE: To investigate antiviral activity, anti-apoptosis and anti-autophagy associated with antiviral effect of repurposing formoterol fumarate dihydrate (FFD) against enterovirus A71 (EV-A71) infection in human neuroblastoma cells. METHODS: In vitro antiviral effects of FFD against EV-A71 infection were examined in human neuroblastoma SK-N-SH cells. The impacts on EV-A71 replication were evaluated by progeny virus production, viral RNA synthesis, and viral protein expression. The target of action of FFD against EV-A71 was determined from the effective stage by time-of-addition assay. Moreover, the anti-apoptosis and anti-autophagy activities associated with antiviral effect were observed by detection of apoptosis- and autophagy-related proteins. RESULTS: FFD significantly inhibited EV-A71 replication in neuronal cells through interfering the early stages of replication cycle which might be the steps during uncoating to viral protein synthesis. Additionally, FFD culminated in reducing of EV-A71-induced apoptosis and autophagy with caspase-3-cleaved form and LC3-II expression levels showed markedly decreased while increasing of Bcl-2 and mTOR expression levels. These might indicate the neuroprotective effect of FFD on EV-A71-induced apoptosis and autophagy. CONCLUSIONS: Preliminary mode of action studies showed that repurposing FFD significantly inhibited EV-A71 replication at early stage of viral replication and exhibited anti-apoptosis and anti-autophagy activities in neuronal cells. These findings may provide an opportunity, via drug repurposing of FFD, for a candidate antiviral drug against EV-A71 infection.

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.

Full Genomic Sequences of H5N1 Highly Pathogenic Avian Influenza Virus in Human Autopsy Specimens Reveal Genetic Variability and Adaptive Changes for Growth in MDCK Cell Cultures.

The entire H5N1 highly pathogenic avian influenza viral genomes were identified in the frozen autopsy specimens: the trachea, lung, colon, and intestinal feces from a patient who died of the disease in 2006. Phylogenetic analysis of the viral genomes showed that these viruses belonged to clade 1 and were the reassortants generated from the reassortment of the viruses within the same clade. The sequencing data from the autopsy specimens revealed at least 8 quasispecies of the H5N1 viruses across all 4 specimen types. These sequences were compared to those derived from the virus isolates grown in Madin Darby canine kidney (MDCK) cells. The virus isolates from the trachea, lung, and fecal specimens showed 27 nucleotide substitutions, leading to the changes of 18 amino acid residues. However, there was no change in the amino acid residues that determined the viral virulence. The changes were more commonly observed in the lung, particularly in the HA and NA genes. Our study suggested that the adaptation changes for the viral fitness to survive in a new host species (MDCK cells) might involve many genes, for example, the amino acid substitution 177G or 177W adjacent to the receptor-binding residues in the HA1 globular head and the substitution M315I in PB2. However, a mutation changes near the receptor binding domain may play an important role in determining the cell tropism and is needed to be further explored.

Molecular Characterization of Seasonal Influenza A and B from Hospitalized Patients in Thailand in 2018-2019.

Influenza viruses continue to be a major public health threat due to the possible emergence of more virulent influenza virus strains resulting from dynamic changes in virus adaptability, consequent of functional mutations and antigenic drift in surface proteins, especially hemagglutinin (HA) and neuraminidase (NA). In this study, we describe the genetic and evolutionary characteristics of H1N1, H3N2, and influenza B strains detected in severe cases of seasonal influenza in Thailand from 2018 to 2019. We genetically characterized seven A/H1N1 isolates, seven A/H3N2 isolates, and six influenza B isolates. Five of the seven A/H1N1 viruses were found to belong to clade 6B.1 and were antigenically similar to A/Switzerland/3330/2017 (H1N1), whereas two isolates belonged to clade 6B.1A1 and clustered with A/Brisbane/02/2018 (H1N1). Interestingly, we observed additional mutations at antigenic sites (S91R, S181T, T202I) as well as a unique mutation at a receptor binding site (S200P). Three-dimensional (3D) protein structure analysis of hemagglutinin protein reveals that this unique mutation may lead to the altered binding of the HA protein to a sialic acid receptor. A/H3N2 isolates were found to belong to clade 3C.2a2 and 3C.2a1b, clustering with A/Switzerland/8060/2017 (H3N2) and A/South Australia/34/2019 (H3N2), respectively. Amino acid sequence analysis revealed 10 mutations at antigenic sites including T144A/I, T151K, Q213R, S214P, T176K, D69N, Q277R, N137K, N187K, and E78K/G. All influenza B isolates in this study belong to the Victoria lineage. Five out of six isolates belong to clade 1A3-DEL, which relate closely to B/Washington/02/2009, with one isolate lacking the three amino acid deletion on the HA segment at position K162, N163, and D164. In comparison to the B/Colorado/06/2017, which is the representative of influenza B Victoria lineage vaccine strain, these substitutions include G129D, G133R, K136E, and V180R for HA protein. Importantly, the susceptibility to oseltamivir of influenza B isolates, but not A/H1N1 and A/H3N2 isolates, were reduced as assessed by the phenotypic assay. This study demonstrates the importance of monitoring genetic variation in influenza viruses regarding how acquired mutations could be associated with an improved adaptability for efficient transmission.

T cell mediated immunity against influenza H5N1 nucleoprotein, matrix and hemagglutinin derived epitopes in H5N1 survivors and non-H5N1 subjects.

BACKGROUND: Protection against the influenza virus by a specific antibody is relatively strain specific; meanwhile broader immunity may be conferred by cell-mediated immune response to the conserved epitopes across influenza virus subtypes. A universal broad-spectrum influenza vaccine which confronts not only seasonal influenza virus, but also avian influenza H5N1 virus is promising. METHODS: This study determined the specific and cross-reactive T cell responses against the highly pathogenic avian influenza A (H5N1) virus in four survivors and 33 non-H5N1 subjects including 10 H3N2 patients and 23 healthy individuals. Ex vivo IFN-γ ELISpot assay using overlapping peptides spanning the entire nucleoprotein (NP), matrix (M) and hemagglutinin (HA) derived from A/Thailand/1(KAN-1)/2004 (H5N1) virus was employed in adjunct with flow cytometry for determining T cell functions. Microneutralization (microNT) assay was performed to determine the status of previous H5N1 virus infection. RESULTS: IFN-γ ELISpot assay demonstrated that survivors nos. 1 and 2 had markedly higher T cell responses against H5N1 NP, M and HA epitopes than survivors nos. 3 and 4; and the magnitude of T cell responses against NP were higher than that of M and HA. Durability of the immunoreactivity persisted for as long as four years after disease onset. Upon stimulation by NP in IFN-γ ELISpot assay, 60% of H3N2 patients and 39% of healthy subjects exhibited a cross-reactive T cell response. The higher frequency and magnitude of responses in H3N2 patients may be due to blood collection at the convalescent phase of the patients. In H5N1 survivors, the effector peptide-specific T cells generated from bulk culture PBMCs by in vitro stimulation displayed a polyfunction by simultaneously producing IFN-γ and TNF-α, together with upregulation of CD107a in recognition of the target cells pulsed with peptide or infected with rVac-NP virus as investigated by flow cytometry. CONCLUSIONS: This study provides an insight into the better understanding on the homosubtypic and heterosubtypic T cell-mediated immune responses in H5N1 survivors and non-H5N1 subjects. NP is an immunodominant target of cross-recognition owing to its high conservancy. Therefore, the development of vaccine targeting the conserved NP may be a novel strategy for influenza vaccine design.

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.

Longitudinal study on enterovirus A71 and coxsackievirus A16 genotype/subgenotype replacements in hand, foot and mouth disease patients in Thailand, 2000-2017.

BACKGROUND: Enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16) are the major causative agents of hand, foot and mouth disease (HFMD) worldwide, particularly in the Asia-Pacific region. Several strains have emerged, circulated, and faded out over time in recent decades. This study investigated the EV-A71 and CV-A16 circulating strains and replacement of genotypes/subgenotypes in Thailand during the years 2000-2017. METHODS: The complete VP1 regions of 92 enteroviruses obtained from 90 HFMD patients, one asymptomatic adult contact case, and one encephalitic case were sequenced and investigated for serotypes, genotypes, and subgenotypes using a phylogenetic analysis. RESULTS: The 92 enterovirus isolates were identified as 67 (72.8%) EV-A71 strains comprising subgenotypes B4, B5, C1, C2, C4a, C4b and C5, and 25 (27.2%) CV-A16 strains comprising subgenotypes B1a and B1b. Genotypic/subgenotypic replacements were evidenced during the study period. EV-A71 B5 and C4a have been the major circulating strains in Thailand for more than a decade, and CV-A16 B1a has been circulating for almost two decades. CONCLUSIONS: This study provides chronological data on the molecular epidemiology of EV-A71 and CV-A16 subgenotypes in Thailand. Subgenotypic replacement frequently occurred with EV-A71, but not CV-A16. Monitoring for viral genetic and subgenotypic changes is important for molecular diagnosis, vaccine selection, and vaccine development.

Complete genome analysis demonstrates multiple introductions of enterovirus 71 and coxsackievirus A16 recombinant strains into Thailand during the past decade.

Hand, foot, and mouth disease (HFMD) caused by enteroviruses remains a public health threat, particularly in the Asia-Pacific region during the past two decades. Moreover, the introduction of multiple subgenotypes and the emergence of recombinant viruses is of epidemiological importance. Based on either the full genome or VP1 sequences, 32 enteroviruses (30 from HFMD patients, 1 from an encephalitic patient, and 1 from an asymptomatic contact case) isolated in Thailand between 2006 and 2014 were identified as 25 enterovirus 71 (EV71) isolates (comprising 20 B5, 1 C2, 2 C4a, and 2 C4b subgenotypes) and 7 coxsackievirus A16 (CA16) isolates (comprising 6 B1a and 1 B1b subgenotypes). The EV71 subgenotype C4b was introduced into Thailand for the first time in 2006 and was replaced by subgenotype C4a strains in 2009. Phylogenetic, similarity plot and bootscan analyses of the complete viral genomes identified 12 recombinant viruses among the 32 viral isolates. Only one EV71-B5 isolate out of 20 was a recombinant virus with one region of intratypic or intertypic recombination, while all four EV71-C4 isolates were recombinant viruses having undergone double recombination, and all seven CA16 isolates were recombinant viruses. The recombination breakpoints of these recombinants are located solely within the P2 and P3 regions. Surveillance for circulating strains and subgenotype replacement are important with respect to molecular epidemiology and the selection of the upcoming EV71 vaccine. In addition, the clinical importance of recombinant viruses needs to be further explored.

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.

Influenza Neuraminidase Subtype N1: Immunobiological Properties and Functional Assays for Specific Antibody Response.

Influenza neuraminidase (NA) proteins expressed in TK- cells infected with recombinant vaccinia virus carrying NA gene of highly pathogenic avian influenza H5N1 virus or 2009 pandemic H1N1 (H1N1pdm) virus were characterized for their biological properties, i.e., cell localization, molecular weight (MW), glycosylation and sialidase activity. Immune sera collected from BALB/c mice immunized with these recombinant viruses were assayed for binding and functional activities of anti-NA antibodies. Recombinant NA proteins were found localized in cytoplasm and cytoplasmic membrane of the infected cells. H1N1pdm NA protein had MW at about 75 kDa while it was 55 kDa for H5N1 NA protein. Hyperglycosylation was more pronounced in H1N1pdm NA compared to H5N1 NA according to N-glycosidase F treatment. Three dimensional structures also predicted that H1N1 NA globular head contained 4 and that of H5N1 contained 2 potential glycosylation sites. H5N1 NA protein had higher sialidase activity than H1N1pdm NA protein as measured by both MUNANA-based assay and fetuin-based enzyme-linked lectin assay (ELLA). Plaque reduction assay demonstrated that anti-NA antibody could reduce number of plaques and plaque size through inhibiting virus release, not virus entry. Assay for neuraminidase-inhibition (NI) antibody by ELLA showed specific and cross reactivity between H5N1 NA and H1N1pdm NA protein derived from reverse genetic viruses or wild type viruses. In contrast, replication-inhibition assay in MDCK cells showed that anti-H1N1 NA antibody moderately inhibited viruses with homologous NA gene only, while anti-H5N1 NA antibody modestly inhibited the replication of viruses containing homologous NA gene and NA gene derived from H1N1pdm virus. Anti-H1N1 NA antibody showed higher titers of inhibiting virus replication than anti-H5N1 NA antibody, which are consistent with the results on reduction in plaque numbers and sizes as well as in inhibiting NA enzymatic activity. No assay showed cross reactivity with reassorted PR8 (H1N1) virus and H3N2 wild type viruses.

Homosubtypic and heterosubtypic antibodies against highly pathogenic avian influenza H5N1 recombinant proteins in H5N1 survivors and non-H5N1 subjects.

Six recombinant vaccinia viruses containing HA, NA, NP, M or NS gene insert derived from a highly pathogenic avian influenza H5N1 virus, and the recombinant vaccinia virus harboring plasmid backbone as the virus control were constructed. The recombinant proteins were characterized for their expression and subcellular locations in TK(-) cells. Antibodies to the five recombinant proteins were detected in all 13 sequential serum samples collected from four H5N1 survivors during four years of follow-up; and those directed to rVac-H5 HA and rVac-NA proteins were found in higher titers than those directed to the internal proteins as revealed by indirect immunofluorescence assay. Although all 28 non-H5N1 subjects had no neutralizing antibodies against H5N1 virus, they did have cross-reactive antibodies to those five recombinant proteins. A significant increase in cross-reactive antibody titer to rVac-H5 HA and rVac-NA was found in paired blood samples from patients infected with the 2009 pandemic virus.

Seroprevalence of antibodies to avian influenza virus A (H5N1) among residents of villages with human cases, Thailand, 2005.

In 2005, we assessed the seroprevalence of neutralizing antibodies to avian influenza virus A (H5N1) among 901 residents of 4 villages in Thailand where at least 1 confirmed human case of influenza (H5N1) had occurred during 2004. Although 68.1% of survey participants (median age 40 years) were exposed to backyard poultry and 25.7% were exposed to sick or dead chickens, all participants were seronegative for influenza virus (H5N1).

Surveillance for reassortant virus by multiplex reverse transcription-PCR specific for eight genomic segments of avian influenza A H5N1 viruses.

Avian influenza H5N1 virus is a global threat. An emergence of a reassortant virus with a pandemic potential is a major concern. Here we describe a multiplex reverse transcription-PCR assay that is specific for the eight genomic segments of the currently circulating H5N1 viruses to facilitate surveillance for a virus resulting from reassortment between human influenza virus and the H5N1 virus.