Heterologous prime-boost immunization induces protection against dengue virus infection in cynomolgus macaques.

IMPORTANCE: Currently licensed dengue vaccines do not induce long-term protection in children without previous exposure to dengue viruses in nature. These vaccines are based on selected attenuated strains of the four dengue serotypes and employed in combination for two or three consecutive doses. In our search for a better dengue vaccine candidate, live attenuated strains were followed by non-infectious virus-like particles or the plasmids that generate these particles upon injection into the body. This heterologous prime-boost immunization induced elevated levels of virus-specific antibodies and helped to prevent dengue virus infection in a high proportion of vaccinated macaques. In macaques that remained susceptible to dengue virus, distinct mechanisms were found to account for the immunization failures, providing a better understanding of vaccine actions. Additional studies in humans in the future may help to establish whether this combination approach represents a more effective means of preventing dengue by vaccination.

Identification of inositol monophosphatase as a broad-spectrum antiviral target of ivermectin.

Ivermectin has broad-spectrum antiviral activities. Despite the failure in clinical application of COVID-19, it can serve as a lead compound for the development of more effective broad-spectrum antivirals, for which a better understanding of its antiviral mechanisms is essential. We thus searched for potential novel targets of ivermectin in host cells by label-free thermal proteomic profiling using Huh-7 cells. Inositol monophosphatase (IMPase) was found among the proteins with shifted thermal stability by ivermectin. Ivermectin could inhibit IMPase activity and reduce cellular myo-inositol and phosphatidylinositol-4-phosphate levels. On the other hand, inositol could impair the antiviral activity of ivermectin and lithium, an IMPase inhibitor with known antiviral activity. As phosphatidylinositol phosphate is crucial for the replication of many RNA viruses, inhibition of cellular myo-inositol biosynthesis may be an important antiviral mechanism of ivermectin. Hence, inhibition of IMPase could serve as a potential target for broad-spectrum antiviral development.

Comparative study of the propagation and plaque titration conditions for human coronavirus OC43 as a surrogate for SARS-CoV-2.

The ongoing COVID-19 pandemic is threatening human health globally. The development of effective drugs and vaccines against SARS-CoV-2 is hindered by the limited access to high-biosafety-level facilities. Although human coronavirus (HCoV) OC43, a low-pathogenic endemic human coronavirus, has been used as a surrogate virus for SARS-CoV-2 research, a standard technique for HCoV-OC43 culture and plaque titration has not been established. Our objective was to establish optimized culture and titration protocols for HCoV-OC43. The growth kinetics and permissibility to HCoV-OC43 infection of seven different cell lines were examined concurrently at two different temperatures, 33°C and 37°C. Cell lines exhibiting a cytopathic effect (CPE) were selected for plaque titration. No significant difference in the rate of cell growth was observed at the two temperatures tested. Interestingly, HCoV-OC43 was found not to be a high-temperature-sensitive virus, since it grew well at 37°C. Although RD, LLC-MK2, MRC-5, and HCT-8 cell lines supported virus growth with an obvious cytopathic effect and a high yield of virus after two days of infection, only RD cells were suitable for producing countable plaques. The incubation of the cells with 1.2% low-viscosity Avicel as an overlay medium at 37°C for 4 days appeared to promote clearer and sharper plaque morphology. However, further optimization of the plaque titration protocol is still required due to the continued observation of plaque size variation and hazy zones. We propose a cost-effective protocol for HCoV-OC43 culture and plaque titration that can be implemented at a standard conventional temperature without the need for additional special equipment.

In vitro synergistic antiviral activity of repurposed drugs against enterovirus 71.

Enteroviruses cause viral diseases that are harmful to children. Hand, foot, and mouth disease (HFMD) with neurological complications is mainly caused by enterovirus 71 (EV71). Despite its clinical importance, there is no effective antiviral drug against EV71. However, several repurposed drugs have been shown to have antiviral activity against related viruses. Treatments with single drugs and two-drug combinations were performed in vitro to assess anti-EV71 activity. Three repurposed drug candidates with broad-spectrum antiviral activity were found to demonstrate potent anti-EV71 activity: prochlorperazine, niclosamide, and itraconazole. To improve antiviral activity, combinations of two drugs were tested. Niclosamide and itraconazole showed synergistic antiviral activity in Vero cells, whereas combinations of niclosamide-prochlorperazine and itraconazole-prochlorperazine showed only additive effects. Furthermore, the combination of itraconazole and prochlorperazine showed an additive effect in neuroblastoma cells. Itraconazole and prochlorperazine exert their antiviral activities by inhibiting Akt phosphorylation. Repurposing of drugs can provide a treatment solution for HFMD, and our data suggest that combining these drugs can enhance that efficacy.

Intra-serotypic antigenic diversity of dengue virus serotype 3 in Thailand during 2004-2015.

In addition to the well-known differences among the four dengue serotypes, intra-serotypic antigenic diversity has been proposed to play a role in viral evolution and epidemic fluctuation. A replacement of genotype II by genotype III of dengue virus serotype 3 (DENV3) occurred in Thailand during 2007-2014, raising questions about the role of intra-serotypic antigenic differences in this genotype shift. We characterized the antigenic difference of DENV3 of genotypes II and III in Thailand, utilizing a neutralizing antibody assay with DENV3 vaccine sera and monotypic DENV3 sera. Although there was significant antigenic diversity among the DENV3, it did not clearly associate with the genotype. Our data therefore do not support the role of intra-serotypic antigenic difference in the genotype replacement. Amino acid alignment showed that eight positions are potentially associated with diversity between distinct antigenic subgroups. Most of these amino acids were found in envelope domain II. Some positions (aa81, aa124, and aa172) were located on the surface of virus particles, probably involving the neutralization sensitivity. Notably, the strains of both genotypes II and III showed clear antigenic differences from the vaccine genotype I strain. Whether this differencewill affect vaccine efficacy requires further studies.

Microparticles from human the lower airway show inhibitory activity against respiratory syncytial virus.

Airway microparticles (MPs) have been shown previously to inhibit influenza virus by trapping virions on their surface through their surface viral receptor. It was hypothesized that airway MPs may carry most of the epithelial cell surface molecules, including receptors for respiratory viruses, and may be able to inhibit various respiratory viruses. We show here that MPs from human bronchoalveolar lavage (BAL) can inhibit respiratory syncytial virus (RSV). Those MPs stained positive for the RSV receptor, CX3CR1. Furthermore, incubating the MPs with a monoclonal antibody against CX3CR1 reduced the anti-RSV activity. These data indicate that MPs can contribute to respiratory innate antiviral defense.

Influence of cellular lipid content on influenza A virus replication.

Influenza A virus (IAV) depends on the metabolism of its cellular host to provide energy and essential factors, including lipids, for viral replication. Previous studies have shown that fatty acids (FAs) play an important role in IAV replication and that inhibition of FA biosynthesis can diminish viral replication. However, cellular lipids can either be synthesized intracellularly or be imported from the extracellular environment. Interfering with FA import mechanisms may reduce the cellular lipid content and inhibit IAV replication. To test this hypothesis, MDCK and Detroit 562 cells were infected with IAV followed by exposure to palmitic acid and inhibitors of FA import. Replication of IAV significantly increased when infected cells were supplied with palmitic acid. This enhancement could be reduced by adding an FA import inhibitor. The addition of palmitic acid significantly increased the cellular lipid content, and this increased level was reduced by treatment with an FA import inhibitor. These results show that reducing the cellular lipid level might be an approach for IAV therapy.

The relationship of codon usage to the replication strategy of parvoviruses.

Codon usage is biased in most species, and the pattern of codon usage bias is specific to each species or group of closely related species. Although viruses use the host translational machinery for synthesis of their proteins, their codon usage patterns do not match those of their host. Viral codon usage is determined by a complex interplay of mutational bias, genome composition constraints, translational adaptation to the host, and host cellular innate defense. The codon usage of parvoviruses was previously shown not to be strongly biased and selective pressure was found to be a dominating factor driving codon usage. The family Parvoviridae includes the genus Dependoparvovirus, some of the members of which require a helper virus to complete their replication cycle, whereas the rest of the family can replicate without the need for helper viruses. Here, we show that difference in the replication strategy of these viruses may be an important factor determining viral codon usage. Hierarchical clustering and principal component analysis revealed that the codon usage pattern of adeno-associated viruses (AAVs) of the genus Dependoparvovirus is distinct from that of members of the other genera of vertebrate parvoviruses, and even from that of independent viruses of the genus Dependoparvovirus. Furthermore, the codon usage of human AAVs was found to be similar to that of some human adenoviruses in hierarchical clustering and principal component analysis. This suggests that the codon usage of AAVs is different from that of other parvoviruses because of their distinctive replication strategy and that their codon usage is probably driven by forces similar to those that shaped the codon usage pattern of their helper viruses.

Codon usage of HIV regulatory genes is not determined by nucleotide composition.

Codon usage bias can be a result of either mutational bias or selection for translational efficiency and/or accuracy. Previous data has suggested that nucleotide composition constraint was the main determinant of HIV codon usage, and that nucleotide composition and codon usage were different between the regulatory genes, tat and rev, and other viral genes. It is not clear whether translational selection contributed to the codon usage difference and how nucleotide composition and translational selection interact to determine HIV codon usage. In this study, a model of codon bias due to GC composition with modification for the A-rich third codon position was used to calculate predicted HIV codon frequencies based on its nucleotide composition. The predicted codon usage of each gene was compared with the actual codon frequency. The predicted codon usage based on GC composition matched well with the actual codon frequencies for the structural genes (gag, pol and env). However, the codon usage of the regulatory genes (tat and rev) could not be predicted. Codon usage of the regulatory genes was also relatively unbiased showing the highest effective number of codons (ENC). Moreover, the codon adaptation index (CAI) of the regulatory genes showed better adaptation to human codons when compared to other HIV genes. Therefore, the early expressed genes responsible for regulation of the replication cycle, tat and rev, were more similar to humans in terms of codon usage and GC content than other HIV genes. This may help these genes to be expressed efficiently during the early stages of infection.

Genome polarity of RNA viruses reflects the different evolutionary pressures shaping codon usage.

RNA viruses are classified by their genome polarity and replication strategies. Nucleotide composition and codon usage differ among virus groups, for instance positive-sense RNA (+ssRNA) viruses have higher GC-content than the other RNA virus groups. Codon usage of +ssRNA viruses is closer to humans showing significantly higher codon adaptation index (CAI) than those of negative-sense RNA (-ssRNA), double stranded RNA (dsRNA) and retroviruses. Ambisense viruses have high CAI comparable to that of +ssRNA virus despite their lower GC content, whereas dsRNA viruses have the lowest CAI. This may provide a benefit for +ssRNA viruses as their genomes are used as mRNA. However, analyses for influence of nucleotide composition on codon usage did not show a difference between +ssRNA and -ssRNA viruses. This suggests that genome composition and hence mutational pressure remain the major pressure causing the differences in codon usage among RNA viruses with different genome types.

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.

Expression of importin-α isoforms in human nasal mucosa: implication for adaptation of avian influenza A viruses to human host.

BACKGROUND: Transportation into the host cell nucleus is crucial for replication and transcription of influenza virus. The classical nuclear import is regulated by specific cellular factor, importin-α. Seven isoforms of importin-α have been identified in human. The preference of importin-α3 of avian influenza virus and -α7 isoform of human strains during replication in human cells was previously identified. In addition, both avian and human influenza viruses were shown to use importin-α1 isoform for their replication. FINDING: The mRNA levels of importin-α1, -α3, and -α7 isoforms in human respiratory tract was determined by real-time RT-PCR. The results indicate that mRNA level of importin-α7 was significantly higher than that of importin-α1 (p-value < 0.0001) and importin-α3 (p-value < 0.0001) isoforms in human nasal mucosa while importin-α1 was detected as the highest expression importin-α isoform in lung tissues. CONCLUSIONS: These results may explain the preference of importin-α7 isoforms in seasonal influenza viruses in human upper respiratory tract and may suggest a selective pressure toward importin-α7 in human respiratory tract infection of an avian virus.

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.

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).

ANTI-AMEBIC ACTIVITY OF DIOSGENIN ON NAEGLERIA FOWLERI TROPHOZOITES.

The aim of this study was to investigate the activity of diosgenin against Naegleria fowleri trophozoites at the cellular and molecular levels. Diosgenin (100 µg/ml; 241.2 µM) had a 100% inhibitory effect on N. fowleri trophozoites (5 x 10(5) cell/ml). Scanning electron micrograph revealed diosgenin decreased the number of sucker-like apparatuses and food cup formation among N. fowleri trophozoites at 3 and 6 hours post-exposure, respectively. Diosgenin down-regulated the nf cysteine protease gene expression of N. fowleri trophozoites at 6 and 12 hours post-exposure. The toxicity to mammalian cells caused by diosgenin at therapeutic dose was less than amphotericin B, the current drug used to treat N. fowleri infections. Our findings suggest diosgenin has activity against the surface membrane and the nf cysteine pro tease of N. fowleri trophozoites. However, the other mechanisms of action of diosgenin against N. fowleri trophozoites require further exploration.

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.

Long-term persistence of Chikungunya virus neutralizing antibodies in human populations of North Eastern Thailand.

BACKGROUND: Chikungunya virus (CHIKV) outbreak recurrences in Thailand are unpredictable and separated by unexplained and often long silent epidemiological periods that can last for several years. These silent periods could be explained in part by the fact that infection with one CHIKV strain confers lasting natural immunity, even against other CHIKV strains. In this study we evaluated the persistence of CHIKV-specific neutralizing antibodies in the population of Chumpae District, Khon Kaen Province, nineteen years after a CHIKV outbreak occurred in the same area in 1991. FINDINGS: Overall 39% (44/111) of 111 former patients had neutralizing antibodies reacting against CHIKV ECSA strain. Consistently high titers of neutralizing antibodies were found in 75% (33/44) of all positively-reacting sera, 70% of which (23/33) were collected from individuals amongst the >60 years old age group. Although the prevalence found in Pong Haeng village (70%) was significantly higher than the prevalence detected in the Nong Thum village (14%), control study villages without known previous Chikungunya epidemics had a high Chikungunya neutralizing antibody prevalence (65%). CONCLUSIONS: More than one-third of the pre-exposed population had persisting natural immunity that was more likely boosted by recent and repetitive exposure to the emerging ECSA CHIKV in Thailand. Also, Chikungunya virus appears to largely circulate in the country with a great variability appears between villages or area probably associated with the vector abundance and efficiency. Altogether these results show a potential for a lifelong immunity against CHIKV. Given the rapid spread of the highly pathogenic ECSA strain in Southern Thailand, the development of CHIK vaccine is strongly recommended.