Effect of oil droplet size on the oxidative stability of spray-dried flaxseed oil powders.

The effect of the size of oil droplets on the oxidative stability of flaxseed oil in spray-dried powders was investigated. Maltodextrin with a dextrose equivalent of 25 was used as a wall material, and sodium caseinate and transglutaminase-polymerized sodium caseinate were used as emulsifiers. The oxidative stability of flaxseed oil encapsulated in the spray-dried powders was evaluated using lipid oxidation and conductometric determination tests at 105 °C. The powders containing larger oil droplets exhibited higher surface oil content after spray drying, and higher peroxide value and conductivity after storage at 105 °C. Removal of the surface oil from the powders by washing with hexane significantly decreased the conductivity. The results indicated that the surface oil of the spray-dried flaxseed oil powders affected the oxidation stability.

Protective immunity afforded by inactivated H5N1 (NIBRG-14) vaccine requires antibodies against both hemagglutinin and neuraminidase in mice.

BACKGROUND: Hemagglutination-inhibition (HI) antibody titers correlate with protective immunity to seasonal influenza viruses. However, inactivated H5N1 influenza vaccines from Vietnam 2004 strains afford protection without producing high or even detectable HI antibodies. METHODS: BALB/c mice were immunized twice (at a 3-week interval) with inactivated whole-virus influenza vaccine produced using reverse genetics, with the internal genes of A/PR/8/34 (a high-yield strain) and the hemagglutinin (HA) and neuraminidase (NA) genes of A/Vietnam/1194/04 (H5N1) virus (NIBRG-14) adjuvanted with alum (5 microg of HA). Either HA- or NA-binding antibodies were absorbed from the immune serum. The protective efficacy of these antibodies was determined by injecting the absorbed serum into severe combined immunodeficiency mice, which were then challenged with highly pathogenic H5N1 virus (A/Vietnam/Jp1203/2004; Japanese isolate of A/Vietnam/1203/2004). RESULTS: The NIBRG-14 vaccine elicited levels of anti-HA antibodies similar to levels elicited by the H1N1 vaccines, as well as levels of anti-NA antibodies higher than those elicited by the H1N1 vaccines. The absorption of either anti-HA or anti-NA antibody from immune serum samples obtained from NIBRG-14-vaccinated mice significantly reduced the protective efficacy of the serum. CONCLUSIONS: For NIBRG-14 vaccines to confer protection to vaccinated hosts, both anti-HA and anti-NA antibodies are required. This finding implies that the measurement of both antibody levels may be required for accurate evaluation of vaccine efficacy.

Cross-protection against H5N1 influenza virus infection is afforded by intranasal inoculation with seasonal trivalent inactivated influenza vaccine.

BACKGROUND: Avian H5N1 influenza A virus is an emerging pathogen with the potential to cause substantial human morbidity and mortality. We evaluated the ability of currently licensed seasonal influenza vaccine to confer cross-protection against highly pathogenic H5N1 influenza virus in mice. METHODS: BALB/c mice were inoculated 3 times, either intranasally or subcutaneously, with the trivalent inactivated influenza vaccine licensed in Japan for the 2005-2006 season. The vaccine included A/NewCaledonia/20/99 (H1N1), A/NewYork/55/2004 (H3N2), and B/Shanghai/361/2002 viral strains and was administered together with poly(I):poly(C(12)U) (Ampligen) as an adjuvant. At 14 days after the final inoculation, the inoculated mice were challenged with either the A/HongKong/483/97, the A/Vietnam/1194/04, or the A/Indonesia/6/05 strain of H5N1 influenza virus. RESULTS: Compared with noninoculated mice, those inoculated intranasally manifested cross-reactivity of mucosal IgA and serum IgG with H5N1 virus, as well as both a reduced H5N1 virus titer in nasal-wash samples and increased survival, after challenge with H5N1 virus. Subcutaneous inoculation did not induce a cross-reactive IgA response and did not afford protection against H5N1 viral infection. CONCLUSIONS: Intranasal inoculation with annual influenza vaccine plus the Toll-like receptor-3 agonist, poly(I):poly(C(12)U), may overcome the problem of a limited supply of H5N1 virus vaccine by providing cross-protective mucosal immunity against H5N1 viruses with pandemic potential.

Intranasal immunization with H5N1 vaccine plus Poly I:Poly C12U, a Toll-like receptor agonist, protects mice against homologous and heterologous virus challenge.

The avian H5N1 influenza virus has the potential to cause a new pandemic. Since it is difficult to predict which strain of influenza will cause a pandemic, it is advantageous to produce vaccines that confer cross-protective immunity. Mucosal vaccine administration was reported to induce cross-protective immunity by inducing secretion of IgA at the mucosal surface. Adjuvants can also enhance the development of fully protective mucosal immunity. Here we show that a new mucosal adjuvant, poly I:poly C12U (Ampligen), a Toll-like receptor 3 agonist proven to be safe in a Phase III human trial, is an effective adjuvant for H5N1 influenza vaccination. Intranasal administration of a candidate influenza vaccine with Ampligen resulted in secretion of IgA, and protected mice that were subsequently challenged with homologous A/Vietnam/1194/2004 and heterologous A/HK/483/97 and A/Indonesia/6/2005 virus.

Prophylactic effects of chitin microparticles on highly pathogenic H5N1 influenza virus.

Highly pathogenic avian influenza virus (H5N1) is an emerging pathogen with the potential to cause great harm to humans, and there is concern about the potential for a new influenza pandemic. This virus is resistant to the antiviral effects of interferons and tumor necrosis factor-alpha. However, the mechanism of interferon-independent protective innate immunity is not well understood. The prophylactic effects of chitin microparticles as a stimulator of innate mucosal immunity against a recently obtained strain of H5N1 influenza virus infection were examined in mice. Clinical parameters and the survival rate of mice treated by intranasal application of chitin microparticles were significantly improved compared to non-treated mice after a lethal influenza virus challenge. Flow cytometric analysis revealed that the number of natural killer cells that expressed tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and that had migrated into the cervical lymph node was markedly increased (26-fold) after intranasal treatment with chitin microparticles. In addition, the level of IL-6 and interferon-gamma-inducible protein-10 (IP-10) in the nasal mucosa after H5N1 influenza virus challenge was decreased by prophylactic treatment with chitin microparticles. These results suggest that prophylactic intranasal administration of chitin microparticles enhanced the local accumulation of natural killer cells and suppressed hyper-induction of cytokines, resulting in an innate immune response to prevent pathogenesis of H5N1 influenza virus.

Inactivated influenza H5N1 whole-virus vaccine with aluminum adjuvant induces homologous and heterologous protective immunities against lethal challenge with highly pathogenic H5N1 avian influenza viruses in a mouse model.

In response to recent outbreaks of H5N1 highly pathogenic avian influenza virus (HPAIV), the development of an effective H5N1 influenza vaccine is urgently important. We assessed the efficacy of two inactivated H5N1 whole-virus vaccines, rgHK213/03 and rgVNJP1203/04, generated by reverse genetics in a mouse model in the presence or absence of aluminum hydroxide (alum) adjuvant. Mice immunized with rgHK213/03 vaccine produced sufficient levels of serum antibodies that were cross-reactive to recent heterologous HPAIV-H5N1 virus, A/Turkey/12/06. The vaccinated mice also elicited protective immunity against challenge with both homologous and heterologous HPAIV-H5N1 viruses. These immune responses were enhanced by addition of alum adjuvant, resulting in antigen sparing of vaccine. On the other hand, mice immunized with rgVNJP1203/04 vaccine had low levels of serum antibodies and less protective immunity than that elicited with rgHK213/03 vaccine regardless of addition of alum adjuvant. Our study suggests that rgHK213/03 vaccine is still useful as a backup vaccine for recent H5N1 viruses and that if rgVNJP1203/04 vaccine is employed, more vaccine antigen would be necessary to induce sufficient immunity.

Rapid diagnosis of H5N1 avian influenza virus infection by newly developed influenza H5 hemagglutinin gene-specific loop-mediated isothermal amplification method.

Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) is a unique gene amplification method that can be completed within 35 min at 62.5 degrees C. In the present study, RT-LAMP was used to develop a rapid and sensitive laboratory diagnostic system for the H5N1 highly pathogenic avian influenza (HPAI). The sensitivity of the system was 0.1-0.01 plaque-forming units per reaction for HPAI-H5N1 viruses belonging to the genetically and antigenically distinct clade 1, represented by A/Vietnam/JP1203/2004, and clade 2, represented by A/Indonesia/JP283/2006. This RT-LAMP sensitivity is 10-fold higher than the sensitivity of standard one-step RT-PCR. By using viral RNAs extracted from avian influenza viruses of H1-H15 hemagglutinin (HA) subtypes and human pathogenic respiratory viruses, it was confirmed that the RT-LAMP system amplifies specifically RNA of the H5 subtype virus. The system detected H5-HA genes in throat swabs collected from humans as well as from wild birds. These results suggest that the present RT-LAMP system is a useful diagnostic tool for surveillance of recent outbreaks of the HPAI-H5N1 virus.

Development of H5-RT-LAMP (loop-mediated isothermal amplification) system for rapid diagnosis of H5 avian influenza virus infection.

We developed a rapid and sensitive diagnosis system for H5N1 highly pathogenic avian influenza (HPAI) virus infection using an unique gene amplification method, reverse transcriptase loop-mediated isothermal amplification (RT-LAMP). The sensitivity of the system was found to be 100-fold higher than that of ordinary one-step RT-PCR. Moreover, by using viral RNAs extracted from influenza viruses of all 15 HA subtypes, the RT-LAMP system was confirmed to amplify only the RNA of H5 subtype virus. In the surveillance of H5N1 virus infection of wild birds, we detected two positive cases from dead crows found near the affected area with H5N1-HPAI by using RT-LAMP system, although one of two positive cases was missed by RT-PCR. These results suggested that our newly developed RT-LAMP system specific for H5 virus would be a beneficial diagnostic tool for surveillance of recent outbreaks caused by H5N1-HPAI viruses.

Antibodies to human-related H3 influenza A virus in Baikal seals (Phoca sibirica) and ringed seals (Phoca hispida) in Russia.

Antibodies to influenza A virus were detected using enzyme-linked immunosorbent assay (ELISA) in the sera from two of seven Baikal seals (Phoca sibrica) and from five of six ringed seals (Phoca hispida) in Russia. In a hemagglutination-inhibition test using H1-H15 reference influenza A viruses, ELISA-positive sera from one Baikal seal and four ringed seals reacted to A/Aichi/2/68 (H3N2) and A/Bangkok/1/79 (H3N2) strains. One ringed seal serum sample reacted to A/seal/Massachusetts/1/80 (H7N7). The present results suggested that human-related H3 viruses were prevalent in Baikal seals and ringed seals inhabiting the central Russian Arctic.

Influenza B virus BM2 protein is a crucial component for incorporation of viral ribonucleoprotein complex into virions during virus assembly.

Influenza B virus contains four integral membrane proteins in its envelope. Of these, BM2 has recently been found to have ion channel activity and is considered to be a functional counterpart to influenza A virus M2, but the role of BM2 in the life cycle of influenza B virus remains unclear. In an effort to explore its function, a number of BM2 mutant viruses were generated by using a reverse genetics technique. The BM2DeltaATG mutant virus synthesized BM2 at markedly lower levels but exhibited similar growth to wild-type (wt) virus. In contrast, the BM2 knockout virus, which did not produce BM2, did not grow substantially but was able to grow normally when BM2 was supplemented in trans by host cells expressing BM2. These results indicate that BM2 is a required component for the production of infectious viruses. In the one-step growth cycle, the BM2 knockout virus produced progeny viruses lacking viral ribonucleoprotein complex (vRNP). The inhibited incorporation of vRNP was regained by trans-supplementation of BM2. An immunofluorescence study of virus-infected cells revealed that distribution of hemagglutinin, nucleoprotein, and matrix (M1) protein of the BM2 knockout virus at the apical membrane did not differ from that of wt virus, whereas the sucrose gradient flotation assay revealed that the membrane association of M1 was greatly affected in the absence of BM2, resulting in a decrease of vRNP in membrane fractions. These results strongly suggest that BM2 functions to capture the M1-vRNP complex at the virion budding site during virus assembly.

Neurotropism of the 1997 Hong Kong H5N1 influenza virus in mice.

The direct transmission of H5N1 influenza A viruses from chickens to humans in Hong Kong in 1997 emphasized the need to have information on the pathogenesis of avian influenza virus infection in mammals. H5N1 influenza viruses isolated from patients during the incident killed experimentally infected mice. The principal lesions of the mice were broncho-interstitial pneumonia and nonsuppurative encephalitis. Infectious viruses and/or viral antigens were detected in the brain as well as in the trigeminal and vagal ganglia but not in the blood of the mice. These findings suggest that the virus reached the brain through the vagus and/or trigeminal nerves following replication in the respiratory mucosa. The results imply that neurotropism of the H5N1 virus in mice is a novel characteristic in the pathogenesis of infection by human influenza virus isolates.

Intranasal immunization with formalin-inactivated virus vaccine induces a broad spectrum of heterosubtypic immunity against influenza A virus infection in mice.

It has been known that influenza A virus infection induces a cross-protective immunity against infection by viruses with different subtypes of viral envelope proteins, hemagglutinin (HA) and neuraminidase (NA). This heterosubtypic immunity is generally mediated by cytotoxic T lymphocytes (CTL) reactive to specific epitopes in the viral internal proteins, such as nucleoprotein and matrix protein. By contrast, immunization with inactivated virus antigens has been thought to be unable to generate heterosubtypic immunity, since inactivated antigens do not usually induce CTL responses. However, we show that intranasal immunization with formalin-inactivated intact virus, but not ether-split vaccines, induced a broad spectrum of heterosubtypic protective immunity in mice. The protection may be mediated by the mucosal immune response, most likely secretory IgA antibodies to the viral proteins. This approach may overcome limitations in the efficacy of inactivated influenza vaccines and confer potent immunity to humans against viruses with new pandemic potential.

Serological evidence of transmission of human influenza A and B viruses to Caspian seals (Phoca caspica).

Seroepidemiological surveillance of influenza in Caspian seals (Phoca caspica) was conducted. Antibodies to influenza A virus were detected in 54% (7/13), 57% (4/7), 40% (6/15) and 26% (11/42) of the serum samples collected in 1993, 1997, 1998 and 2000 by enzyme-linked immunosorbent assay (ELISA). In an hemagglutination-inhibition (HI) test using H1-H15 reference influenza A viruses as antigens, more than half of the examined ELISA-positive sera reacted with an H3N2 prototype strain A/Aichi/2/68. These sera were then examined by HI test with a series of naturally occurring antigenic variants of human H3N2 virus, and H3 viruses of swine, duck, and equine origin. The sera reacted strongly with the A/Bangkok/1/79 (H3N2) strain, which was prevalent in humans in 1979-1981. The present results indicate that human A/Bangkok/1/79-like virus was transmitted to Caspian seals probably in the early 1980s, and was circulated in the population. Antibodies to influenza B virus were detected by ELISA in 14% (1/7) and 10% (4/42) serum samples collected from Caspian seals in 1997 and 2000, respectively. Our findings indicate that seal might be a reservoir of both influenza A and B viruses originated from humans.

Intranasal administration of a synthetic peptide vaccine encapsulated in liposome together with an anti-CD40 antibody induces protective immunity against influenza A virus in mice.

Mucosal immunity is critical for protection from viral infections. We attempted to activate mucosal cytotoxic T lymphocytes (CTLs) specific for influenza A virus nucleoprotein (NP) which play an important role in protective immunity. It has been shown that dendritic cells (DCs) activated by signaling via CD40-CD40 ligand (CD40L) interaction are required for the differentiation of naive CD8(+) T cells into antigen-specific CTLs in a non-mucosal environment. We herein inoculated mice intranasally with an anti-CD40 monoclonal antibody (anti-CD40 mAb) and NP366-374 peptide, corresponding to a CTL epitope on NP, encapsulated in liposome (liposomal NP366-374) to induce protective CTL responses against influenza A virus. Intranasal but not subcutaneous immunization with liposomal NP366-374 effectively induced mucosal immunity to reduce virus replication in the lung, suggesting that anti-CD40 mAb also functioned as a mucosal adjuvant. Interestingly, neither MHC class I- nor class II-deficient mice immunized intranasally with these materials were resistant to the infection. Since anti-CD40 mAb was considered to help replace CD4(+) T cells, another help of CD4(+) T cells are presumably required for the induction of CTL activity in the lung. This approach may prove promising for developing vaccines to induce mucosal CTL responses, and seems to highlight differences between mucosal and non-mucosal immunity.

Seroepidemiological evidence of avian H4, H5, and H9 influenza A virus transmission to pigs in southeastern China.

Pig serum samples collected in southeastern China were examined for antibodies to influenza A viruses. Since the hemagglutination inhibition (HI) test does not accurately detect antibodies to the hemagglutinins (HAs) of "avian" influenza viruses, we utilized the neutralization (NT) test to detect subtype-specific antibodies to the HA of avian viruses in pig sera. Neutralizing antibodies to H1, H3, H4, and H5 influenza viruses were detected in the serum samples collected in 1977-1982 and 1998, suggesting that pigs in China have been sporadically infected with avian H4 and H5 viruses in addition to swine and human H1 and H3 viruses. Antibodies to H9 virus, on the other hand, were found only in the sera collected in 1998, not in those collected in 1977-1982, correlating with the recent spread in poultry and subsequent isolation of H9N2 viruses from pigs and humans in 1998. The present results indicate that avian influenza viruses have been transmitted to pig populations in southeastern China.

Polymorphisms and the differential antiviral activity of the chicken Mx gene.

The nucleotide sequence of chicken Mx cDNA was reported earlier using the White Leghorn breed in Germany, but it showed no enhanced resistance to viruses. In this study, the nucleotide sequences of chicken Mx cDNA were determined in many breeds. A total of 25 nucleotide substitutions, of which 14 were deduced to cause amino acid exchanges, were detected, suggesting that the chicken Mx gene is very polymorphic. Transfected cell clones expressing chicken Mx mRNA were established after the Mx cDNA was constructed with an expression vector and introduced into mouse 3T3 cells, and the Mx genes from some breeds were demonstrated to confer positive antiviral responses to influenza virus and vesicular stomatitis virus. On the basis of the comparison among the antiviral activities associated with many Mx variations, a specific amino acid substitution at position 631 (Ser to Asn) was considered to determine the antivirally positive or negative Mx gene. Thus, a single amino acid substitution influences the antiviral activity of Mx in domesticated chickens.