Identification and quantitation of multiple variants in RNA virus genomes.

The goal of the study was to identify and characterize RNA virus variants containing mutations spread over genomic distances >5 kb. As proof of concept, high-quality viral RNA of the Dengue 2 component of Takeda's tetravalent dengue vaccine candidate (TDV-2) was used to develop a reverse transcription-polymerase chain reaction protocol to amplify a ∼5.3 kb cDNA segment that contains the three genetic determinants of TDV-2 attenuation. Unique molecular identifiers were incorporated into each viral cDNA molecule for PacBio library preparation to improve the quantitative precision of the observed variants at the attenuation loci. Following assay optimization, PacBio long-read sequencing was validated with multiple clone-derived TDV-2 revertant variants and four complex revertant mixtures containing various compositions of TDV-2 and revertant viruses. PacBio sequencing analysis correctly identified and quantified variant composition in all tested samples, demonstrating that TDV-2 revertants could be identified and characterized and supporting the use of this method in the differentiation and quantification of complex variants of other RNA viruses. Long-read sequencing can identify complex RNA virus variants containing multiple mutations on a single-genome molecule, which is useful for in-depth genetic stability and revertant detection of live-attenuated viral vaccines, as well as research in virus evolution to reveal mechanisms of immune evasion and host cell adaption.

Neutralization fingerprinting technology for characterizing polyclonal antibody responses to dengue vaccines.

Dengue is a major public health threat. There are four dengue virus (DENV) serotypes; therefore, efforts are focused on developing safe and effective tetravalent DENV vaccines. While neutralizing antibodies contribute to protective immunity, there are still important gaps in understanding of immune responses elicited by dengue infection and vaccination. To that end, here, we develop a computational modeling framework based on the concept of antibody-virus neutralization fingerprints in order to characterize samples from clinical studies of TAK-003, a tetravalent vaccine candidate currently in phase 3 trials. Our results suggest a similarity of neutralizing antibody specificities in baseline-seronegative individuals. In contrast, amplification of pre-existing neutralizing antibody specificities is predicted for baseline-seropositive individuals, thus quantifying the role of immunologic imprinting in driving antibody responses to DENV vaccines. The neutralization fingerprinting analysis framework presented here can contribute to understanding dengue immune correlates of protection and help guide further vaccine development and optimization.

Development and validation of an in vitro titrimetric method for determination of classical swine fever viruses in PK-15 cells.

Classical swine fever (CSF) is a highly contagious notifiable disease of pigs caused by CSF virus of Flaviviridae family. Previously, lapinized vaccines were used for the disease control, which has now been replaced with cell culture vaccines. Determination of virus titre is the key factor for development and quality control testing of classical swine fever (CSF) cell culture vaccines. Since CSFV is a non- cytopathic virus, an accurate method for the titration of this virus in cell culture has not yet been reported. Here we present a full proof method of titration of CSF cell culture viruses employing Fluorescent Antibody Technique (FAT) in 24 well plate cover slip culture of PK-15 cells. CSFV monoclonal antibodies (Mab) used in the test bind to the CSF virus particles in the cell cytoplasm of the infected cells and the immune-fluorescence signal is produced by subsequent binding of FITC conjugate with Mab. In this newly developed method, apple green fluorescence is observed in the cytoplasm of the infected cells as the virus multiplies only in the cytoplasm. The nucleus as well as the uninfected cells cytoplasm is stained red without any traces of green fluorescence. Thus, the test clearly differentiates a CSFV infected cell from the uninfected cells in the vicinity, if any, and also from the uninfected controls. The test can also quantify the accurate titres of CSF live viruses in the cell culture vaccines and hence it has wide application in routine virus titration applied for manufacturing of CSF cell culture vaccines, determination of accurate multiplicity of infection (m.o.i.) during infection and quality control of vaccines by the testing laboratories.

Specificity and Breadth of the Neutralizing Antibody Response to a Live-Attenuated Tetravalent Dengue Vaccine.

BACKGROUND: An effective dengue vaccine should ideally induce broadly neutralizing antibody (nAb) responses against all 4 dengue virus (DENV) serotypes. METHODS: We characterized the specificity and breadth of the nAb response to TAK-003, a live-attenuated tetravalent dengue vaccine, in serum samples from phase 2 and 3 clinical trials. RESULTS: Microneutralization tests using postvaccination serum showed comparable neutralization against diverse DENV-1-4 genotypes. Reporter virus particle neutralization assays after depletion of anti-DENV-2 nAbs demonstrated that the nAb response to DENV-1, -3, and -4 comprises both type-specific (TS) and cross-reactive (CR) nAbs. CONCLUSIONS: Therefore, TAK-003 induces broad tetravalent TS and CR nAb responses.

Somatic Hypermutation and Framework Mutations of Variable Region Contribute to Anti-Zika Virus-Specific Monoclonal Antibody Binding and Function.

Zika virus (ZIKV) is a global public health concern due to its ability to cause congenital Zika syndrome and lack of approved vaccine, therapeutic, or other control measures. We discovered eight novel rabbit monoclonal antibodies (MAbs) that bind to distinct ZIKV envelope protein epitopes. The majority of the MAbs were ZIKV specific and targeted the lateral ridge of the envelope (E) protein domain III, while the MAb with the highest neutralizing activity recognized a putative quaternary epitope spanning E protein domains I and III. One of the non-neutralizing MAbs specifically recognized ZIKV precursor membrane protein (prM). Somatic hypermutation of immunoglobulin variable regions increases antibody affinity maturation and triggers antibody class switching. Negative correlations were observed between the somatic hypermutation rate of the immunoglobulin heavy-chain variable region and antibody binding parameters such as equilibrium dissociation constant, dissociation constant, and half-maximal effective concentration value of MAb binding to ZIKV virus-like particles. Complementarity-determining regions recognize the antigen epitopes and are scaffolded by canonical framework regions. Reversion of framework region amino acids to the rabbit germ line sequence decreased anti-ZIKV MAb binding activity of some MAbs. Thus, antibody affinity maturation, including somatic hypermutation and framework region mutations, contributed to the binding and function of these anti-ZIKV MAbs. IMPORTANCE ZIKV is a global health concern against which no vaccine or therapeutics are available. We characterized eight novel rabbit monoclonal antibodies recognizing ZIKV envelope and prM proteins and studied the relationship between somatic hypermutation of complementarity-determining regions, framework regions, mutations, antibody specificity, binding, and neutralizing activity. The results contribute to understanding structural features and somatic mutation pathways by which potent Zika virus-neutralizing antibodies can evolve, including the role of antibody framework regions.

TAK - 021, an inactivated Enterovirus 71 vaccine candidate, provides cross-protection against heterologous sub-genogroups in human scavenger receptor B2 transgenic mice.

BACKGROUND: Enterovirus 71 (EV71) is a major cause of outbreaks of hand, foot and mouth disease, most frequently in children, and is a public health concern in the Asia-Pacific region. Takeda is developing TAK-021, an inactivated EV71 vaccine candidate based on sub-genogroup B2 strain MS87. In a phase I clinical trial, TAK-021 was safe, well tolerated, and immunogenic in healthy adults and elicited cross-neutralizing antibodies against heterologous EV71 sub-genogroup viruses. TAK-021 confers protection from lethal challenge with a mouse-adapted homologous strain in AG129 mice. However, it has not been determined whether TAK-021 can provide cross-protection against heterologous EV71 sub-genogroups. METHODS: We examined the efficacy of TAK-021 against challenge with EV71 sub-genogroups B4, B5, C1, C2, and C4 on day 42 (short-term) and sub-genogroups B5 and C4 on day 120 (long-term) after immunization of human scavenger receptor B2 transgenic (hSCARB2-tg) mice with TAK-021 on days 0 and 28. Antibody titers were monitored over 120 days using plaque reduction neutralization test of the homologous vaccine virus. RESULTS: TAK-021 elicited neutralizing antibody (nAb) in greater than 90% of the mice and nAb persisted through day 120. Challenge of control animals led to weight loss and death, as well as virus detection in various organs and histopathological lesions in the brain. All mice that received two doses of TAK-021 developed nAb and survived a short-term challenge given on day 42, while more than 80% survived a long-term challenge given on day 120. EV71 was detected less frequently and at lower levels in organs of immunized mice compared to non-immunized control mice. CONCLUSIONS: The results show that TAK-021 can confer protection in mice against the EV71 sub-genogroups tested.

An inactivated enterovirus 71 vaccine is safe and immunogenic in healthy adults: A phase I, double blind, randomized, placebo-controlled, study of two dosages.

BACKGROUND: Hand, foot and mouth disease (HFMD), especially that caused by enterovirus 71 (EV71) infection, is a public health concern in the Asia-Pacific region. We report a phase I clinical trial of an EV71 candidate vaccine (INV21) based on a binary ethylenimine inactivated B2 sub-genotype formulated with aluminum hydroxide. METHODS: In this double-blind, placebo-controlled, randomized, dose escalation study adult volunteers received two vaccinations 28 days apart of low or high dose formulations of the candidate vaccine and were then monitored for safety and reactogenicity for four weeks after each dose, and for their immune responses up to 28 weeks. RESULTS: Of 36 adults enrolled, 35 completed the study as planned. Either no or mild adverse events were observed, mainly injection site pain and tiredness. Seroconversion was 100% after two vaccinations. High geometric mean neutralizing antibody titers (GMT) were observed 14 days post first dose, peaking 14 days post second dose (at Day 42) in both high and low dose groups; GMTs on days 14, 28, 42, and 56 were 128, 81, 323, 203 and 144, 100, 451, 351 in low- and high-dose groups, respectively. Titers for both doses declined gradually to Day 196 but remained higher than baseline and the placebo groups, which had low GMTs throughout the duration of the study. Cross-neutralizing antibody activity against heterologous sub-genotypes was demonstrated. CONCLUSION: These data show that the EV71 candidate vaccine is safe and immunogenic in adults and supports further clinical development as a potential pediatric vaccine by initiating a dose-escalation study for determining the dose-dependent safety and immunogenicity of the vaccine in young naïve children.

Selection of antigenically advanced variants of seasonal influenza viruses.

Influenza viruses mutate frequently, necessitating constant updates of vaccine viruses. To establish experimental approaches that may complement the current vaccine strain selection process, we selected antigenic variants from human H1N1 and H3N2 influenza virus libraries possessing random mutations in the globular head of the haemagglutinin protein (which includes the antigenic sites) by incubating them with human and/or ferret convalescent sera to human H1N1 and H3N2 viruses. We also selected antigenic escape variants from human viruses treated with convalescent sera and from mice that had been previously immunized against human influenza viruses. Our pilot studies with past influenza viruses identified escape mutants that were antigenically similar to variants that emerged in nature, establishing the feasibility of our approach. Our studies with contemporary human influenza viruses identified escape mutants before they caused an epidemic in 2014-2015. This approach may aid in the prediction of potential antigenic escape variants and the selection of future vaccine candidates before they become widespread in nature.

Application of Standardized Precipitation Index to assess meteorological drought in Bangladesh.

Bangladesh is one of the vulnerable countries of the world for natural disasters. Drought is one of the common and severe calamities in Bangladesh that causes immense suffering to people in various ways. The present research has been carried out to examine the frequency of meteorological droughts in Bangladesh using the long-term rainfall data of 30 meteorological observatories covering the period of 1948-2011. The study uses the highly effective Standardized Precipitation Index (SPI) for drought assessment in Bangladesh. By assessing the meteorological droughts and the history of meteorological droughts of Bangladesh, the spatial distributions of meteorological drought indices were also analysed. The spatial and temporal changes in meteorological drought and changes in different years based on different SPI month intervals were analysed. The results indicate that droughts were a normal and recurrent feature and it occurred more or less all over the country in virtually all climatic regions of the country. As meteorological drought depends on only rainfall received in an area, anomaly of rainfall is the main cause of drought. Bangladesh experienced drought in the years 1950, 1951, 1953, 1954, 1957, 1958, 1960, 1961, 1962, 1963, 1965, 1966, 1967 and 1971 before independence and after independence Bangladesh has experienced droughts in the years 1972, 1973, 1975, 1979, 1980, 1983, 1985, 1992, 1994, 1995, 2002, 2004, 2006, 2009 and 2011 during the period 1948-2011. The study indicated that Rajshahi and its surroundings, in the northern regions and Jessore and its surroundings areas, the island Bhola and surrounding regions, in the south-west region, were vulnerable. In the Sylhet division, except Srimongal, the areas were not vulnerable but the eastern southern sides of the districts Chittagong, Rangamati, Khagrachhari, Bandarban and Teknaf were vulnerable. In the central regions, the districts of Mymensingh and Faridpur were more vulnerable than other districts.

Efficacy of a Trivalent Hand, Foot, and Mouth Disease Vaccine against Enterovirus 71 and Coxsackieviruses A16 and A6 in Mice.

Hand, foot, and mouth disease (HFMD) has recently emerged as a major public health concern across the Asian-Pacific region. Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are the primary causative agents of HFMD, but other members of the Enterovirus A species, including Coxsackievirus A6 (CVA6), can cause disease. The lack of small animal models for these viruses have hampered the development of a licensed HFMD vaccine or antivirals. We have previously reported on the development of a mouse model for EV71 and demonstrated the protective efficacy of an inactivated EV71 vaccine candidate. Here, mouse-adapted strains of CVA16 and CVA6 were produced by sequential passage of the viruses through mice deficient in interferon (IFN) α/ß (A129) and α/ß and γ (AG129) receptors. Adapted viruses were capable of infecting 3 week-old A129 (CVA6) and 12 week-old AG129 (CVA16) mice. Accordingly, these models were used in active and passive immunization studies to test the efficacy of a trivalent vaccine candidate containing inactivated EV71, CVA16, and CVA6. Full protection from lethal challenge against EV71 and CVA16 was observed in trivalent vaccinated groups. In contrast, monovalent vaccinated groups with non-homologous challenges failed to cross protect. Protection from CVA6 challenge was accomplished through a passive transfer study involving serum raised against the trivalent vaccine. These animal models will be useful for future studies on HFMD related pathogenesis and the efficacy of vaccine candidates.

Investigating the efficacy of monovalent and tetravalent dengue vaccine formulations against DENV-4 challenge in AG129 mice.

Dengue (DEN) is the most important mosquito-borne viral disease, with a major impact on global health and economics, caused by four serologically and distinct viruses termed DENV-1 to DENV-4. Currently, there is no licensed vaccine to prevent DEN. We have developed a live attenuated tetravalent DENV vaccine candidate (TDV) (formally known as DENVax) that has shown promise in preclinical and clinical studies and elicits neutralizing antibody responses to all four DENVs. As these responses are lowest to DENV-4 we have used the AG129 mouse model to investigate the immunogenicity of monovalent TDV-4 or tetravalent TDV vaccines, and their efficacy against lethal DENV-4 challenge. Since the common backbone of TDV is based on an attenuated DENV-2 strain (TDV-2) we also tested the efficacy of TDV-2 against DENV-4 challenge. Single doses of the tetravalent or monovalent vaccines elicited neutralizing antibodies, anti-NS1 antibodies, and cellular responses to both envelope and nonstructural proteins. All vaccinated animals were protected against challenge at 60 days post-immunization, whereas all control animals died. Investigation of DENV-4 viremias post-challenge showed that only the control animals had high viremias on day 3 post-challenge, whereas vaccinated mice had no detectable viremia. Overall, these data highlight the excellent immunogenicity and efficacy profile of our candidate dengue vaccine in AG129 mice.

Modified vaccinia virus Ankara encoding influenza virus hemagglutinin induces heterosubtypic immunity in macaques.

UNLABELLED: Current influenza virus vaccines primarily aim to induce neutralizing antibodies (NAbs). Modified vaccinia virus Ankara (MVA) is a safe and well-characterized vector for inducing both antibody and cellular immunity. We evaluated the immunogenicity and protective efficacy of MVA encoding influenza virus hemagglutinin (HA) and/or nucleoprotein (NP) in cynomolgus macaques. Animals were given 2 doses of MVA-based vaccines 4 weeks apart and were challenged with a 2009 pandemic H1N1 isolate (H1N1pdm) 8 weeks after the last vaccination. MVA-based vaccines encoding HA induced potent serum antibody responses against homologous H1 or H5 HAs but did not stimulate strong T cell responses prior to challenge. However, animals that received MVA encoding influenza virus HA and/or NP had high frequencies of virus-specific CD4(+) and CD8(+) T cell responses within the first 7 days of H1N1pdm infection, while animals vaccinated with MVA encoding irrelevant antigens did not. We detected little or no H1N1pdm replication in animals that received vaccines encoding H1 (homologous) HA, while a vaccine encoding NP from an H5N1 isolate afforded no protection. Surprisingly, H1N1pdm viral shedding was reduced in animals vaccinated with MVA encoding HA and NP from an H5N1 isolate. This reduced shedding was associated with cross-reactive antibodies capable of mediating antibody-dependent cellular cytotoxicity (ADCC) effector functions. Our results suggest that ADCC plays a role in cross-protective immunity against influenza. Vaccines optimized to stimulate cross-reactive antibodies with ADCC function may provide an important measure of protection against emerging influenza viruses when NAbs are ineffective. IMPORTANCE: Current influenza vaccines are designed to elicit neutralizing antibodies (NAbs). Vaccine-induced NAbs typically are effective but highly specific for particular virus strains. Consequently, current vaccines are poorly suited for preventing the spread of newly emerging pandemic viruses. Therefore, we evaluated a vaccine strategy designed to induce both antibody and T cell responses, which may provide more broadly cross-protective immunity against influenza. Here, we show in a translational primate model that vaccination with a modified vaccinia virus Ankara encoding hemagglutinin from a heterosubtypic H5N1 virus was associated with reduced shedding of a pandemic H1N1 virus challenge, while vaccination with MVA encoding nucleoprotein, an internal viral protein, was not. Unexpectedly, this reduced shedding was associated with nonneutralizing antibodies that bound H1 hemagglutinin and activated natural killer cells. Therefore, antibody-dependent cellular cytotoxicity (ADCC) may play a role in cross-protective immunity to influenza virus. Vaccines that stimulate ADCC antibodies may enhance protection against pandemic influenza virus.

Hemozoin as a novel adjuvant for inactivated whole virion influenza vaccine.

Because vaccination is an effective means to protect humans from influenza viruses, extensive efforts have been made to develop not only new vaccines, but also for new adjuvants to enhance the efficacy of existing inactivated vaccines. Here, we examined the adjuvanticity of synthetic hemozoin, a synthetic version of the malarial by-product hemozoin, on the vaccine efficacy of inactivated whole influenza viruses in a mouse model. We found that mice immunized twice with hemozoin-adjuvanted inactivated A/California/04/2009 (H1N1pdm09) or A/Vietnam/1203/2004 (H5N1) virus elicited higher virus-specific antibody responses than did mice immunized with non-adjuvanted counterparts. Furthermore, mice immunized with hemozoin-adjuvanted inactivated viruses were better protected from lethal challenge with influenza viruses than were mice immunized with non-adjuvanted inactivated vaccines. Our results show that hemozoin improves the immunogenicity of inactivated influenza viruses, and is thus a promising adjuvant for inactivated whole virion influenza vaccines.

Deciphering the protective role of adaptive immunity to CHIKV/IRES a novel candidate vaccine against Chikungunya in the A129 mouse model.

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, recently re-emerged in Africa and spread to islands in the Indian Ocean, the Indian subcontinent, and to South East Asia. Viremic travelers have also imported CHIKV to the Western hemisphere highlighting the importance of CHIKV in public health. In addition to the great burden of arthralgic disease, which can persist for months or years, epidemiologic studies have estimated case-fatality rates of ∼0.1%, principally from neurologic disease in older patients. There are no licensed vaccines or effective therapies to prevent or treat human CHIKV infections. We have developed a live CHIKV vaccine (CHIKV/IRES) that is highly attenuated yet immunogenic in mouse models, and is incapable of replicating in mosquito cells. In this study we sought to decipher the role of adaptive immunity elicited by CHIKV/IRES in protection against wild-type CHIKV infection. A single dose of vaccine effectively activated T cells with an expansion peak on day 10 post immunization and elicited memory CD4(+) and CD8(+) T cells that produced IFN-γ, TNF-α and IL-2 upon restimulation with CHIKV/IRES. Adoptive transfer of CHIKV/IRES-immune CD4(+) or CD8(+) T cells did not confer protection against wtCHIKV-LR challenge. By contrast, passive immunization with anti-CHIKV/IRES immune serum provided protection, and a correlate of a minimum protective neutralizing antibody titer was established. Overall, our findings demonstrate the immunogenic potential of the CHIKV/IRES vaccine and highlight the important role that neutralizing antibodies play in protection against an acute CHIKV infection.

Cross-protective immunity against multiple influenza virus subtypes by a novel modified vaccinia Ankara (MVA) vectored vaccine in mice.

Development of an influenza vaccine that provides cross-protective immunity remains a challenge. Candidate vaccines based on a recombinant modified vaccinia Ankara (MVA) viral vector expressing antigens from influenza (MVA/Flu) viruses were constructed. A vaccine candidate, designated MVA/HA1/C13L/NP, that expresses the hemagglutinin from pandemic H1N1 (A/California/04/09) and the nucleoprotein (NP) from highly pathogenic H5N1 (A/Vietnam/1203/04) fused to a secretory signal sequence from vaccinia virus was highly protective. The vaccine elicited strong antibody titers to homologous H1N1 viruses while cross-reactive antibodies to heterologous viruses were not detectable. In mice, this MVA/HA1/C13L/NP vaccine conferred complete protection against lethal challenge with A/Vietnam/1203/04 (H5N1), A/Norway/3487-2/09 (pandemic H1N1) or A/Influenza/Puerto Rico/8/34 (seasonal H1N1) and partial protection (57.1%) against challenge with seasonal H3N2 virus (A/Aichi/68). The protective efficacy of the vaccine was not affected by pre-existing immunity to vaccinia. Our findings highlight MVA as suitable vector to express multiple influenza antigens that could afford broad cross-protective immunity against multiple subtypes of influenza virus.

Nanoemulsion W805EC improves immune responses upon intranasal delivery of an inactivated pandemic H1N1 influenza vaccine.

Currently available influenza vaccines provide suboptimal protection. In order to improve the quality of protective immune responses elicited following vaccination, we developed an oil-in-water nanoemulsion (NE)-based adjuvant for an intranasally-delivered inactivated influenza vaccine. Using a prime-boost vaccination regimen, we show that intranasal vaccines containing the W(80)5EC NE elicited higher titers of serum hemagglutination inhibiting (HAI) antibody and influenza-specific IgG and IgA titers compared to vaccines that did not contain the NE. Similarly, vaccines containing the W(80)5EC NE resulted in higher influenza-specific IgA levels in the bronchoalveolar lavage (BAL) fluid and nasal wash when compared to vaccines formulated without NE. The higher antibody titers in mice immunized with the NE-containing vaccines correlated with reduced viral loads in the lungs and nasal turbinates following a high dose viral challenge. Mice immunized with vaccines containing the W(80)5EC NE also showed a reduction in body weight loss following challenge compared to mice immunized with equivalent vaccines produced without NE. Taken together, our results show that the W(80)5EC NE substantially improves the magnitude of protective influenza-specific antibody responses and is a promising mucosal adjuvant for influenza vaccines and vaccines against other mucosal pathogens.

A single amino acid change resulting in loss of fluorescence of eGFP in a viral fusion protein confers fitness and growth advantage to the recombinant vesicular stomatitis virus.

Using a recombinant vesicular stomatitis virus encoding eGFP fused in-frame with an essential viral replication protein, the phosphoprotein P, we show that during passage in culture, the virus mutates the nucleotide C289 within eGFP of the fusion protein PeGFP to A or T, resulting in R97S/C amino acid substitution and loss of fluorescence. The resultant non-fluorescent virus exhibits increased fitness and growth advantage over its fluorescent counterpart. The growth advantage of the non-fluorescent virus appears to be due to increased transcription and replication activities of the PeGFP protein carrying the R97S/C substitution. Further, our results show that the R97S/C mutation occurs prior to accumulation of mutations that can result in loss of expression of the gene inserted at the G-L gene junction. These results suggest that fitness gain is more important for the recombinant virus than elimination of expression of the heterologous gene.

Morphine modulation of toll-like receptors in microglial cells potentiates neuropathogenesis in a HIV-1 model of coinfection with pneumococcal pneumoniae.

Chronic drug users account for a third of all cases of AIDS in the United States and the progression to AIDS dementia is accelerated in opiate drug abusers. Clinically, microglial activation better correlates with HIV-associated neurocognitive disorders (HAND) than productive HIV-1 infection in the CNS. Moreover, pneumococcal pneumonia is the most common opportunistic infection in individuals with HAND. We show that coinfection with Streptococcus pneumoniae may be a contributing factor in the increased prevalence of HAND in the opioid-dependent population. To date, there have been no studies published implicating the Toll-like receptors (TLR) in the neurocognitive disorders associated with NeuroAIDS in the context of opportunistic infection. Our studies show for the first time, in a morphine-dependent model, synergistic increase and activation of TLR expression in the presence of HIV-1 protein TAT and S. pneumoniae with a significant increase in proinflammatory cytokines (IL-6, TNF-α) levels. Furthermore, concurrent increases in reactive oxygen species and nitric oxide production leading to increased caspase 3 activation are also observed in both murine and human microglial cells. These effects are recapitulated with TLR 2, 4, and 9 cognate ligands (Pam3CSK4, LPS, and CpG) and significantly attenuated in TLR 2 and 4 knock-out mice and TLR2/4 double knock-out mice. Therefor, our findings clearly suggest for the first time that activation of TLRs on microglia cells by morphine and TAT in the context of S. pneumoniae infection may be a potential mechanism for the increased prevalence of HAND in HIV-infected opioid-dependent patients.

Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets.

Highly pathogenic avian H5N1 influenza A viruses occasionally infect humans, but currently do not transmit efficiently among humans. The viral haemagglutinin (HA) protein is a known host-range determinant as it mediates virus binding to host-specific cellular receptors. Here we assess the molecular changes in HA that would allow a virus possessing subtype H5 HA to be transmissible among mammals. We identified a reassortant H5 HA/H1N1 virus-comprising H5 HA (from an H5N1 virus) with four mutations and the remaining seven gene segments from a 2009 pandemic H1N1 virus-that was capable of droplet transmission in a ferret model. The transmissible H5 reassortant virus preferentially recognized human-type receptors, replicated efficiently in ferrets, caused lung lesions and weight loss, but was not highly pathogenic and did not cause mortality. These results indicate that H5 HA can convert to an HA that supports efficient viral transmission in mammals; however, we do not know whether the four mutations in the H5 HA identified here would render a wholly avian H5N1 virus transmissible. The genetic origin of the remaining seven viral gene segments may also critically contribute to transmissibility in mammals. Nevertheless, as H5N1 viruses continue to evolve and infect humans, receptor-binding variants of H5N1 viruses with pandemic potential, including avian-human reassortant viruses as tested here, may emerge. Our findings emphasize the need to prepare for potential pandemics caused by influenza viruses possessing H5 HA, and will help individuals conducting surveillance in regions with circulating H5N1 viruses to recognize key residues that predict the pandemic potential of isolates, which will inform the development, production and distribution of effective countermeasures.

The highly conserved arginine residues at positions 76 through 78 of influenza A virus matrix protein M1 play an important role in viral replication by affecting the intracellular localization of M1.

Influenza A virus matrix protein (M1) plays an important role in virus assembly and budding. Besides a well-characterized basic amino acid-rich nuclear localization signal region at positions 101 to 105, M1 contains another basic amino acid stretch at positions 76-78 that is highly conserved among influenza A and B viruses, suggesting the importance of this stretch. To understand the role of these residues in virus replication, we mutated them to either lysine (K), alanine (A), or aspartic acid (D). We could generate viruses possessing either single or combination substitutions with K or single substitution with A at any of these positions, but not those with double substitutions with A or a single substitution with D. Viruses with the single substitution with A exhibited slower growth and had lower nucleoprotein/M1 quantitative ratio in virions compared to the wild-type virus. In cells infected with a virus possessing the single substitution with A at position 77 or 78 (R77A or R78A, respectively), the mutated M1 localized in patches at the cell periphery where nucleoprotein and hemagglutinin colocalized more often than the wild-type did. Transmission electron microscopy showed that virus possessing M1 R77A or R78A, but not the wild-type virus, was present in vesicular structures, indicating a defect in virus assembly and/or budding. The M1 mutations that did not support virus generation exhibited an aberrant M1 intracellular localization and affected protein incorporation into virus-like particles. These results indicate that the basic amino acid stretch of M1 plays a critical role in influenza virus replication.