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Since their discovery in the United States in 1963, outbreaks of infection with equine influenza virus (H3N8) have been associated with serious respiratory disease in horses worldwide. Genomic analysis suggests that equine H3 viruses are of an avian lineage, likely originating in wild birds. Equine-like internal genes have been identified in avian influenza viruses isolated from wild birds in the Southern Cone of South America. However, an equine-like H3 hemagglutinin has not been identified. We isolated 6 distinct H3 viruses from wild birds in Chile that have hemagglutinin, nucleoprotein, nonstructural protein 1, and polymerase acidic genes with high nucleotide homology to the 1963 H3N8 equine influenza virus lineage. Despite the nucleotide similarity, viruses from Chile were antigenically more closely related to avian viruses and transmitted effectively in chickens, suggesting adaptation to the avian host. These studies provide the initial demonstration that equine-like H3 hemagglutinin continues to circulate in a wild bird reservoir.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and has since caused a global pandemic resulting in millions of cases and deaths. Diagnostic tools and serological assays are critical for controlling the outbreak, especially assays designed to quantitate neutralizing antibody levels, considered the best correlate of protection. As vaccines become increasingly available, it is important to identify reliable methods for measuring neutralizing antibody responses that correlate with authentic virus neutralization but can be performed outside biosafety level 3 (BSL3) laboratories. While many neutralizing assays using pseudotyped virus have been developed, there have been few studies comparing the different assays to each other as surrogates for authentic virus neutralization. Here, we characterized three enzyme-linked immunosorbent assays (ELISAs) and three pseudotyped vesicular stomatitis virus (VSV) neutralization assays and assessed their concordance with authentic virus neutralization. The most accurate assays for predicting authentic virus neutralization were luciferase- and secreted embryonic alkaline phosphatase (SEAP)-expressing pseudotyped virus neutralizations, followed by green fluorescent protein (GFP)-expressing pseudotyped virus neutralization, and then the ELISAs. IMPORTANCE The ongoing COVID-19 pandemic is caused by infection with severe acute respiratory syndrome virus 2 (SARS-CoV-2). Prior infection or vaccination can be detected by the presence of antibodies in the blood. Antibodies in the blood are also considered to be protective against future infections from the same virus. The “gold standard” assay for detecting protective antibodies against SARS-CoV-2 is neutralization of authentic SARS-CoV-2 virus. However, this assay can only be performed under highly restrictive biocontainment conditions. We therefore characterized six antibody-detecting assays for their correlation with authentic virus neutralization. The significance of our research is in outlining the advantages and disadvantages of the different assays and identifying the optimal surrogate assay for authentic virus neutralization. This will allow for more accurate assessments of protective immunity against SARS-CoV-2 following infection and vaccination.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and has since caused a global pandemic resulting in millions of cases and deaths1-4. Diagnostic tools and serological assays are critical for controlling the outbreak5-7, especially assays designed to quantitate neutralizing antibody levels, considered the best correlate of protection8-11. As vaccines become increasingly available12, it is important to identify reliable methods for measuring neutralizing antibody responses that correlate with authentic virus neutralization but can be performed outside of biosafety level 3 (BSL3) laboratories. While many neutralizing assays using pseudotyped virus have been developed, there have been few studies comparing the different assays to each other as surrogates for authentic virus neutralization9,10,13,14. Here we characterized three enzyme-linked immunosorbent assays (ELISAs) and three pseudotyped VSV virus neutralization assays and assessed their concordance with authentic virus neutralization. The most accurate assays for predicting authentic virus neutralization were luciferase and secreted embryonic alkaline phosphatase (SEAP) expressing pseudotyped virus neutralizations, followed by GFP expressing pseudotyped virus neutralization, and then the ELISAs.

As companion animals, dogs and cats live in close contact with humans, generating the possibility of interspecies pathogen transmission events. Equine origin H3N8 and avian origin H5N1 influenza virus have been reported in dogs and cats respectively since 2004 with outbreaks associated with different strains recorded for both species in Asia and North America. To date, there have been no reports of influenza viruses from companion animals in South America. To fill this gap in knowledge, we performed active epidemiological surveillance in shelters that received abandoned animals, backyard production systems and veterinary clinics between May 2017 and January 2019 to estimate the burden of influenza infection in cats and dogs in the central region of Chile. Blood samples, oropharyngeal swabs or both were collected for influenza A virus detection by RT-qPCR, NP-ELISA, and hemagglutination inhibition assay. Logistic regression models were performed to assess the association between NP-ELISA-positivity and variables including sex and animal origin. The percentage of ELISA-positive samples was 43.5% (95% CI: 37.0 to 50.1) and 23.3% (95% CI: 10.6 to 42.7) for dogs and cats, respectively. No association was found between NP-ELISA results and sex or animal origin for either dogs or cats. Two ELISA positive samples showed hemagglutination inhibition titers against pandemic H1N1 influenza. One dog sample tested positive by RT-qPCR, indicating an overall RT-qPCR positivity in dogs of 1.1% (95% CI: 0.05 to 6.7). None of the tested cat samples were positive by this assay.

SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4+ T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4+ T (TFH) cell responses contribute to this outstanding immunogenicity. Using fine needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we evaluated the T cell receptor sequences and phenotype of lymph node TFH. Mining of the responding TFH T cell receptor repertoire revealed a strikingly immunodominant HLA-DPB1∗04-restricted response to S167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific TFH cells peak one week after the second immunization, S-specific TFH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust TFH cell responses play in establishing long-term immunity by this efficacious human vaccine., Analysis of draining lymph nodes of individuals vaccinated BNT162b2 mRNA vaccine against SARS-CoV-2 identifies viral spike-specific follicular helper CD4+ T cells that persist for months and contribute to long-term immunity.

The continual emergence of novel influenza A strains from non-human hosts requires constant vigilance and the need for ongoing research to identify strains that may pose a human public health risk. Since 1999, canine H3 influenza A viruses (CIVs) have caused many thousands or millions of respiratory infections in dogs in the United States. While no human infections with CIVs have been reported to date, these viruses could pose a zoonotic risk. In these studies, the National Institutes of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) network collaboratively demonstrated that CIVs replicated in some primary human cells and transmitted effectively in mammalian models. While people born after 1970 had little or no pre-existing humoral immunity against CIVs, the viruses were sensitive to existing antivirals and we identified a panel of H3 cross-reactive human monoclonal antibodies (hmAbs) that could have prophylactic and/or therapeutic value. Our data predict these CIVs posed a low risk to humans. Importantly, we showed that the CEIRS network could work together to provide basic research information important for characterizing emerging influenza viruses, although there were valuable lessons learned., Author summary The 2009 influenza pandemic was a stark reminder that ongoing vigilance is critical to protect the public from an influenza pandemic. The continual evolution of influenza viruses and emergence from animal reservoirs, leads to the need to quickly identify strains that pose a public health risk. In these studies, members of the National Institutes of Allergy and Infectious Diseases (NIAID) Centers for Excellence in Influenza Research and Surveillance (CEIRS) network worked together to demonstrate that the emerging canine H3 influenza viruses posed a low risk to public health and identified several therapeutic options in the event of an emergence. In addition to providing important new basic research, many lessons were learned that may be important in dealing with any emerging disease outbreak.

SARS-CoV-2 infection causes diverse outcomes ranging from asymptomatic infection to respiratory distress and death. A major unresolved question is whether prior immunity to endemic, human common cold coronaviruses (hCCCoV) impacts susceptibility to SARS-CoV-2 infection or immunity following infection and vaccination. Therefore, we analyzed samples from the same individuals before and after SARS-CoV-2 infection or vaccination. We found hCCCoV antibody levels increase after SARS-CoV-2 exposure, demonstrating cross-reactivity. However, a case-control study indicates baseline hCCCoV antibody levels are not associated with protection against SARS-CoV-2 infection. Rather, higher magnitudes of pre-existing betacoronavirus antibodies correlate with more SARS-CoV-2 antibodies following infection, an indicator of greater disease severity. Additionally, immunization with hCCCoV spike proteins before SARS-CoV-2 immunization impedes generation of SARS-CoV-2 neutralizing antibodies in mice. Together, these data suggest pre-existing hCCCoV antibodies hinder SARS-CoV-2 antibody-based immunity following infection and provide insight on how pre-existing coronavirus immunity impacts SARS-CoV-2 infection, which is critical considering emerging variants., Graphical Abstract, A major unresolved question is whether prior immunity to endemic, human common cold coronaviruses (hCCCoV) impacts susceptibility to SARS-CoV-2 infection. Lin et al. analyze hCCCoV antibodies in the same individuals before and after SARS-CoV-2 infection, finding pre-existing betacoronavirus antibodies may hinder SARS-CoV-2 effective immunity following infection.

While the circulation of avian influenza viruses (IAV) in wild birds in the northern hemisphere has been well documented, data from South America remain sparse. To address this gap in knowledge, we undertook IAV surveillance in wild birds in parts of Central and Northern Chile between 2012 and 2015. A wide diversity of hemagglutinin (HA) and neuraminidase (NA) subtypes were identified and 16 viruses were isolated including low pathogenic H5 and H7 strains, making this the largest and most diverse collection of Chilean avian IAVs to date. Unlike IAVs isolated from wild birds in other South American countries where the genes were most like viruses isolated from wild birds in either North America or South America, the Chilean viruses were reassortants containing genes like viruses isolated from both continents. In summary, our studies demonstrate that genetically diverse avian IAVs are circulating in wild birds in Chile highlighting the need for further investigation in this understudied area of the world.

Human astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, classic, MLB-type and VA-type have been reported in humans. One limitation towards a better understanding of these viruses has been the lack of a physiologically relevant cell culture model that supports growth of all clades of HAstV. Herein, we demonstrate infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. A detailed investigation of infection of VA1, a member of the non-canonical HAstV-VA/HMO clade, showed robust replication in HIE derived from different patients and from different intestinal regions independent of the cellular differentiation status. Flow cytometry and immunofluorescence analysis revealed that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes, in HIE cultures. RNA profiling of VA1-infected HIE uncovered that the host response to infection is dominated by interferon (IFN)-mediated innate immune responses. A comparison of the antiviral host response in non-transformed HIE and transformed human colon carcinoma Caco-2 cells highlighted significant differences between these cells, including an increased magnitude of the response in HIE. Additional studies confirmed the sensitivity of VA1 to exogenous IFNs, and indicated that the endogenous IFN response of HIE to curtail the growth of strains from all three clades. Genotypic variation in the permissiveness of different HIE lines to HAstV could be overcome by pharmacologic inhibition of JAK/STAT signaling. Collectively, our data identify HIE as a universal infection model for HAstV and an improved model of the intestinal epithelium to investigate enteric virus-host interactions., Author summary Human astroviruses (HAstV) are understudied positive-strand RNA viruses that typically cause gastroenteritis mostly in children and the elderly, but more recent studies also implicate them in neurological disease in immunocompromised patients. To better understand these viruses, a physiologically relevant cell culture model that supports growth of all clades of HAstV would be highly beneficial. Herein, we demonstrated robust infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts from different patients and intestinal regions, making HIE a valuable model to study HAstV biology. Using this system, we identify for the first time that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes. Analysis of the antiviral host response to infection demonstrated that HIE respond to infection with a type I and III interferon response. This response reduced HAstV replication and when blocked resulted in increased infection. Establishment of the HIE system for HAstV research lays the foundation for future basic and translational discoveries.

The influence of living in small remote villages on the diversity of viruses in the nasal mucosa was investigated in three Colombian villages with very different levels of geographic isolation. Metagenomic analysis was used to characterize viral nucleic acids in nasal swabs from 63 apparently healthy young children. Sequences from human virus members of the families Anelloviridae, Papillomaviridae, Picornaviridae, Herpesviridae, Polyomaviridae, Adenoviridae, and Paramyxoviridae were detected in decreasing proportions of children. The number of papillomavirus infections detected was greater among Hispanic children most exposed to outside contacts, while anellovirus infections were more common in the isolated indigenous villages. The diversity of the other human viruses detected did not differ among the villages. Closely related variants of rhinovirus A or B were identified in 2 to 4 children from each village, reflecting ongoing transmission clusters. Genomes of viruses not currently known to infect humans, including members of the families Parvoviridae, Partitiviridae, Dicistroviridae, and Iflaviridae and circular Rep-encoding single-stranded DNA (CRESS-DNA) virus, were also detected in nasal swabs, possibly reflecting environmental contamination from insect, fungal, or unknown sources. Despite the high levels of geographic and cultural isolation, the overall diversity of human viruses in the nasal passages of children was not reduced in highly isolated indigenous villages, indicating ongoing exposure to globally circulating viruses. IMPORTANCE Extreme geographic and cultural isolation can still be found in some indigenous South American villages. Such isolation may be expected to limit the introduction of otherwise common and widely distributed viruses. Very small population sizes may also result in rapid local viral extinction due to a lack of seronegative subjects to maintain transmission chains for rapidly cleared viruses. We compared the viruses in the nasal passages of young children in three villages with increasing levels of geographic isolation. We found that isolation did not reduce the overall diversity of viral infections. Multiple infections with nearly identical rhinoviruses could be detected within each village, likely reflecting recent viral introductions and transmission clusters among epidemiologically linked members of these very small communities. We conclude that, despite their geographic isolation, remote indigenous villages show evidence of ongoing exposure to globally circulating viruses.

Human astroviruses are a major cause of pediatric gastroenteritis, especially in immunocompromised children. We conducted a retrospective study to demonstrate that diverse astrovirus genotypes can co-circulate in pediatric oncology patients. A subset of cases is associated with long-term virus shedding (range 17-183 days).

Noroviruses are a leading cause of gastroenteritis worldwide. Although infections in healthy individuals are self-resolving, immunocompromised individuals are at risk for chronic disease and severe complications. Chronic norovirus infections in immunocompromised hosts are often characterized by long-term virus shedding, but it is unclear whether this shed virus remains infectious. We investigated the prevalence, genetic heterogeneity, and temporal aspects of norovirus infections in 1140 patients treated during a 6-year period at a pediatric research hospital. Additionally, we identified 20 patients with chronic infections lasting 37 to &gt, 418 days. Using a new human norovirus in vitro assay, we confirmed the continuous shedding of infectious virus for the first time. Shedding lasted longer in male patients and those with diarrheal symptoms. Prolonged shedding of infectious norovirus in immunocompromised hosts can potentially increase the likelihood of transmission, highlighting the importance of isolation precautions to prevent nosocomial infections.

Human noroviruses (HuNoVs) are a leading cause of foodborne disease and severe childhood diarrhea, and they cause a majority of the gastroenteritis outbreaks worldwide. However, the development of effective and long-lasting HuNoV vaccines and therapeutics has been greatly hindered by their uncultivability. We recently demonstrated that a HuNoV replicates in human B cells, and that commensal bacteria serve as a cofactor for this infection. In this protocol, we provide detailed methods for culturing the GII.4-Sydney HuNoV strain directly in human B cells, and in a coculture system in which the virus must cross a confluent epithelial barrier to access underlying B cells. We also describe methods for bacterial stimulation of HuNoV B cell infection and for measuring viral attachment to the surface of B cells. Finally, we highlight variables that contribute to the efficiency of viral replication in this system. Infection assays require 3 d and attachment assays require 3 h. Analysis of infection or attachment samples, including RNA extraction and RTRT-qPCR, requires similar to 6 h.

Human parainfluenza virus type 1 (hPIV-1) is the most common cause of laryngotracheobronchitis (croup), resulting in tens of thousands of hospitalizations each year in the United States alone. No licensed vaccine is yet available. We have developed murine PIV-1 (Sendai virus [SeV]) as a live Jennerian vaccine for hPIV-1. Here, we describe vaccine testing in healthy 3- to 6-year-old hPIV-1-seropositive children in a dose escalation study. One dose of the vaccine (5 × 105, 5 × 106, or 5 × 10750% egg infectious doses) was delivered by the intranasal route to each study participant. The vaccine was well tolerated by all the study participants. There was no sign of vaccine virus replication in the airway in any participant. Most children exhibited an increase in antibody binding and neutralizing responses toward hPIV-1 within 4 weeks from the time of vaccination. In several children, antibody responses remained above incoming levels for at least 6 months after vaccination. Data suggest that SeV may provide a benefit to 3- to 6-year-old children, even when vaccine recipients have preexisting cross-reactive antibodies due to previous exposures to hPIV-1. Results encourage the testing of SeV administration in young seronegative children to protect against the serious respiratory tract diseases caused by hPIV-1 infections.

Phylogenetic analysis of the influenza hemagglutinin gene (HA) has suggested that commercial pigs in Chile harbor unique human seasonal H1-like influenza viruses, but further information, including characterization of these viruses, was unavailable. We isolated influenza virus (H1N2) from a swine in a backyard production farm in Central Chile and demonstrated that the HA gene was identical to that in a previous report. Its HA and neuraminidase genes were most similar to human H1 and N2 viruses from the early 1990s and internal segments were similar to influenza A(H1N1)pdm09 virus. The virus replicated efficiently in vitro and in vivo and transmitted in ferrets by respiratory droplet. Antigenically, it was distinct from other swine viruses. Hemagglutination inhibition analysis suggested that antibody titers to the swine Chilean H1N2 virus were decreased in persons born after 1990. Further studies are needed to characterize the potential risk to humans, as well as the ecology of influenza in swine in South America.

The disease mechanisms associated with the onset of astrovirus diarrhea are unknown. Unlike other enteric virus infections, astrovirus infection is not associated with an inflammatory response or cellular damage. In vitro studies in differentiated Caco-2 cells demonstrated that human astrovirus serotype 1 (HAstV-1) capsid protein alone disrupts the actin cytoskeleton and tight junction complex, leading to increased epithelial barrier permeability. In this study, we show that oral administration of purified recombinant turkey astrovirus 2 (TAstV-2) capsid protein results in acute diarrhea in a dose- and time-dependent manner in turkey poults. Similarly to that induced by infectious virus, TAstV-2 capsid-induced diarrhea was independent of inflammation or histological changes but was associated with increased intestinal barrier permeability, as well as redistribution of sodium hydrogen exchanger 3 (NHE3) from the membrane to the cytoplasm of the intestinal epithelium. Unlike other viral enterotoxins that have been identified, astrovirus capsid induces diarrhea after oral administration, reproducing the natural route of infection and demonstrating that ingestion of intact noninfectious capsid protein may be sufficient to provoke acute diarrhea. Based on these data, we hypothesize that the astrovirus capsid acts like an enterotoxin and induces intestinal epithelial barrier dysfunction., IMPORTANCE Acute gastroenteritis, with its sequela diarrhea, is one of the most important causes of childhood morbidity and mortality worldwide. A variety of infectious agents cause gastroenteritis, and in many cases, an enterotoxin produced by the agent is involved in disease manifestations. Although we commonly think of bacteria as a source of toxins, at least one enteric virus, rotavirus, produces a protein with enterotoxigenic activity during viral replication. In these studies, we demonstrate that oral administration of the turkey astrovirus 2 (TAstV-2) structural (capsid) protein induces acute diarrhea, increases barrier permeability, and causes relocalization of NHE3 in the small intestine, suggesting that rotavirus may not be alone in possessing enterotoxigenic activity.

Astroviruses are small, nonenveloped, single-stranded RNA viruses that cause diarrhea in a wide variety of mammals and birds. On the surface of the viral capsid are globular spikes that are thought to be involved in attachment to host cells. To understand the basis of species specificity, we investigated the structure of an avian astrovirus capsid spike and compared it to a previously reported human astrovirus capsid spike structure. Here we report the crystal structure of the turkey astrovirus 2 (TAstV-2) capsid surface spike domain, determined to 1.5-Å resolution, and identify three conserved patches on the surface of the spike that are candidate avian receptor-binding sites. Surprisingly, the overall TAstV-2 capsid spike structure is unique, with only distant structural similarities to the human astrovirus capsid spike and other viral capsid spikes. There is an absence of conserved putative receptor-binding sites between the human and avian spikes. However, there is evidence for carbohydrate-binding sites in both human and avian spikes, and studies with human astrovirus 1 (HAstV-1) suggest a minor role in infection for chondroitin sulfate but not heparin. Overall, our structural and functional studies provide new insights into astrovirus host cell entry, species specificity, and evolution.

While much is known about the prevalence of influenza viruses in North America and Eurasia, their prevalence in birds and mammals in South America is largely unknown. To fill this knowledge gap and provide a baseline for future ecology and epidemiology studies, we conducted 2 years of influenza surveillance in the eastern plains (Los Llanos) region of Colombia. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) identified influenza viruses in wild birds, domestic poultry, swine and horses. Prevalence ranged from 2.6% to 13.4% across species. Swine showed the highest prevalence and were infected primarily with 2009 pandemic H1N1 (pH1N1) viruses genetically related to those in humans. In addition, we isolated H5N2 viruses from two resident species of whistling ducks (genus Dendrocygna) that differed completely from previous South American isolates, instead genetically resembling North American wild bird viruses. Both strains caused low pathogenicity in chickens and mammals. The prevalence and subtype diversity of influenza viruses isolated from diverse species within a small area of Colombia highlights the need for enhanced surveillance throughout South America, including monitoring of the potential transmissibility of low-pathogenic H5N2 viruses from wild birds to domestic poultry and the emergence of reassortant viruses in domestic swine.

SummaryObjectiveNo serological studies have been performed in Mexico to assess the seroprevalence of influenza A/H1N1/2009 in groups of people according to the potential risk of transmission. The aim of this study was to determine the seroprevalence of antibodies against influenza A/H1N1/2009 in subjects in Mexico grouped by risk of transmission.MethodsTwo thousand two hundred and twenty-two subjects were categorized into one of five occupation groups according to the potential risk of transmission: (1) students, (2) teachers, (3) healthcare workers, (4) institutional home residents aged >60 years, and (5) general population. Seroprevalence by potential transmission group and by age grouped into decades was determined by a virus-free ELISA method based on the recombinant receptor-binding domain of the hemagglutinin of influenza A/H1N1/2009 virus as antigen (85% sensitivity; 95% specificity). The Wilson score, Chi-square test, and logistic regression models were used for the statistical analyses.ResultsSeroprevalence for students was 47.3%, for teachers was 33.9%, for older adults was 36.5%, and for the general population was 33.0%, however it was only 24.6% for healthcare workers (p=0.011). Of the students, 56.6% of those at middle school, 56.4% of those at high school, 52.7% of those at elementary school, and 31.1% of college students showed positive antibodies (p

The hemagglutinin-neuraminidase (HN) glycoprotein is utilized by human parainfluenza viruses for binding to the host cell. By the use of glycan array assays, we demonstrate that, in addition to the first catalytic-binding site, the HN of human parainfluenza virus type 1 has a second site for binding covered by N-linked glycan. Our data suggest that attachment of the first site to sialic acid (SA)-linked receptors triggers exposure of the second site. We found that both sites bind to α2-3-linked SAs with a preference for a sialyl-Lewis(x) motif. Binding to α2-3-linked SAs with a sulfated sialyl-Lewis motif as well as to α2-8-linked SAs was unique for the second binding site. Neither site recognizes α2-6-linked oligosaccharides.

The human immune system has evolved to recognize antigenic diversity, a strength that has been harnessed by vaccine developers to combat numerous pathogens (e.g., pneumococcus, influenza virus, rotavirus). In each case, vaccine cocktails were formulated to include antigenic variants of the target. To combat HIV-1 diversity, we assembled a cocktail vaccine comprising dozens of envelopes, delivered as recombinant DNA, vaccinia virus, and protein for testing in a clinical trial. One vaccinee has now completed vaccinations with no serious adverse events. Preliminary analyses demonstrate early proof-of-principle that a multi-envelope vaccine can elicit neutralizing antibody responses toward heterologous HIV-1 in humans.

Little is known about intrinsic epithelial cell responses against astrovirus infection. Here we show that human astrovirus type 1 (HAstV-1) infection induces type I interferon (beta interferon [IFN-β]) production in differentiated Caco2 cells, which not only inhibits viral replication by blocking positive-strand viral RNA and capsid protein synthesis but also protects against HAstV-1-increased barrier permeability. Excitingly, we found similar results in vivo using a murine astrovirus (MuAstV) model, providing new evidence that virus-induced type I IFNs may protect against astrovirus replication and pathogenesis in vivo . IMPORTANCE Human astroviruses are a major cause of pediatric diarrhea, yet little is known about the immune response. Here we show that type I interferon limits astrovirus infection and preserves barrier permeability both in vitro and in vivo . Importantly, we characterized a new mouse model for studying astrovirus replication and pathogenesis.

A central obstacle to the design of a global HIV vaccine is viral diversity. Antigenic differences in envelope proteins result in distinct HIV serotypes, operationally defined such that antibodies raised against envelope molecules from one serotype will not bind envelope molecules from a different serotype. The existence of serotypes has presented a similar challenge to vaccine development against other pathogens. In such cases, antigenic diversity has been addressed by vaccine design. For example, the poliovirus vaccine includes three serotypes of poliovirus, and Pneumovax(ρ) presents a cocktail of 23 pneumococcal variants to the immune system. It is likely that a successful vaccine for HIV must also comprise a cocktail of antigens. Here, data relevant to the development of cocktail vaccines, designed to harness diverse, envelope-specific Bcell and T-cell responses, are reviewed.

Since emerging in 2013, the avian-origin H7N9 influenza viruses have resulted in over 400 human infections, leading to 115 deaths to date. Although the epidemiology differs from human highly pathogenic avian H5N1 influenza virus infections, there is a similar rapid progression to acute respiratory distress syndrome. The aim of these studies was to compare the pathological and immunological characteristics of a panel of human H7N9 and H5N1 viruses in vitro and in vivo . Although there were similarities between particular H5N1 and H7N9 viruses, including association between lethal disease and spread to the alveolar spaces and kidney, there were also strain-specific differences. Both H5N1 and H7N9 viruses are capable of causing lethal infections, with mortality correlating most strongly with wider distribution of viral antigen in the lungs, rather than with traditional measures of virus titer and host responses. Strain-specific differences included hypercytokinemia in H5N1 infections that was not seen with the H7N9 infections regardless of lethality. Conversely, H7N9 viruses showed a greater tropism for respiratory epithelium covering nasal passages and nasopharynx-associated lymphoid tissue than H5N1 viruses, which may explain the enhanced transmission in ferret models. Overall, these studies highlight some distinctive properties of H5N1 and H7N9 viruses in different in vitro and in vivo models. IMPORTANCE The novel avian-origin H7N9 pandemic represents a serious threat to public health. The ability of H7N9 to cause serious lung pathology, leading in some cases to the development of acute respiratory distress syndrome, is of particular concern. Initial reports of H7N9 infection compared them to infections caused by highly pathogenic avian (HPAI) H5N1 viruses. Thus, it is of critical importance to understand the pathology and immunological response to infection with H7N9 compared to HPAI H5N1 viruses. We compared these responses in both in vitro and in vivo models, and found that H5N1 and H7N9 infections exhibit distinct pathological, immunological, and tissue tropism differences that could explain differences in clinical disease and viral transmission.

To determine the seroprevalence of astrovirus MLB1 (MLB1), an indirect enzyme-linked immunosorbent assay (ELISA) was established. MLB1 seropositivity was high in children

Optimal targets for anti-human immunodeficiency virus (HIV) moieties are those regions of the viral genome that are greatly conserved. The primer binding site (PBS) of HIV is an 18-nucleotide sequence complementary to the 3' end of tRNA(Lys3) that serves as the primer for HIV-1 reverse transcription. All HIV-1 isolates analyzed to date contain a PBS complementary to tRNA(Lys3) illustrating the conservation of this sequence. We investigated the activity of a hammerhead ribozyme targeting the PBS of HIV-1. CEMss cells transduced with retroviral vectors containing either the PBS hammerhead ribozyme or its complementary sequence (as a control) in the R region of the vector long terminal repeat (LTR) were challenged with HIV-1NL4-3. Surprisingly80% inhibition of HIV-1 production was observed with the vector containing the (control) sequence complementary to the PBS ribozyme. We propose that the LTR-driven vector transcript containing 18 nucleotides identical to the HIV-1 PBS may act like an RNA decoy to titrate viral proteins such as reverse transcriptase and nucleocapsid away from genuine viral transcripts, thus compromising virus replication.

We conducted a clinical trial to determine the feasibility of growth factor mobilization of CD34+ progenitor cells in human immunodeficiency virus type 1 (HIV-1)-infected individuals. Eight asymptomatic, HIV-1-infected adults (median CD4+ T-cell count, 415 cells/microL), received 480 micrograms/d of granulocyte colony-stimulating factor (G-CSF) for 6 days without evidence of viral activation. Despite concerns that HIV-1 might inhibit hematopoiesis, CD34+ cells were successfully mobilized to the periphery of all donors, independent of the baseline CD4+ T-cell count, and the status of antiretroviral therapy. Leukapheresis was performed on day 6, and yielded a median of 194 x 10(6) CD34+ cells per leukapheresis (n = 7). CD34-enriched cells from the leukapheresis were predominantly myeloid-committed, but between 0.2% and 1.7% were primitive CD34+/CD38- progenitors. A median of 21.7% of the mobilized CD34+ cells were dimly positive for CD4. Consequently, CD34(+)-enriched cells were purified on the cell sorter (mean purity, 97.7% +/- 2.4%; n = 7), and examined for HIV-1 DNA. Purified CD34+ cells from two of seven donors were polymerase chain reaction (PCR)-positive for HIV-1, but only from one of three samples from each donor. We conclude that G-CSF can safely mobilize CD34+ progenitor cells in HIV-1-infected subjects, and that these cells are suitable for consideration in gene-transfer strategies.

A novel H1N1 influenza virus emerged in 2009 (pH1N1) to become the first influenza pandemic of the 21st century. This virus is now cocirculating with highly pathogenic H5N1 avian influenza viruses in many parts of the world, raising concerns that a reassortment event may lead to highly pathogenic influenza strains with the capacity to infect humans more readily and cause severe disease. To investigate the virulence of pH1N1-H5N1 reassortant viruses, we created pH1N1 (A/California/04/2009) viruses expressing individual genes from an avian H5N1 influenza strain (A/Hong Kong/483/1997). Using several in vitro models of virus replication, we observed increased replication for a reassortant CA/09 virus expressing the hemagglutinin (HA) gene of HK/483 (CA/09-483HA) relative to that of either parental CA/09 virus or reassortant CA/09 expressing other HK/483 genes. This increased replication correlated with enhanced pathogenicity in infected mice similar to that of the parental HK/483 strain. The serial passage of the CA/09 parental virus and the CA/09-483HA virus through primary human lung epithelial cells resulted in increased pathogenicity, suggesting that these viruses easily adapt to humans and become more virulent. In contrast, serial passage attenuated the parental HK/483 virus in vitro and resulted in slightly reduced morbidity in vivo , suggesting that sustained replication in humans attenuates H5N1 avian influenza viruses. Taken together, these data suggest that reassortment between cocirculating human pH1N1 and avian H5N1 influenza strains will result in a virus with the potential for increased pathogenicity in mammals.

To combat HIV-1 diversity we are developing a multienvelope vaccine (comprising DNA vaccinia virus and protein vectors). Toward this goal we conducted a phase I clinical trial of EnvPro a gp140 protein formulated in alum. The vaccine was well tolerated and elicited an immune response in every trial participant. (authors)

Human parainfluenza viruses are responsible for tens of thousands of hospitalizations and hundreds of thousands of emergency room visits each year in the United States. Included in this group of viruses is human parainfluenza virus-type 1 (hPIV-1), the main causative agent of laryngotracheobronchitis (croup) in young children, for which there is currently no licensed vaccine. In response to this need, we have developed a Jennerian vaccine that employs the use of murine PIV-1 (Sendai virus, SeV) to produce cross-reactive antibodies towards hPIV-1. The vaccine was administered as a single intranasal dose to healthy, hPIV-1 seropositive, 3-6 year old children in a dose escalation study (5 x 105, 5 x 106 or 5 x 107 EID50). Importantly, the vaccine was well tolerated by all participants. No replication of the viral vaccine was detected in the airway of any participant. Within four weeks of vaccination, most participants showed an increase in binding and neutralizing antibodies towards hPIV-1. For several participants, antibody responses remained elevated at six months post-vaccination. The results of this study suggest that hPIV-1 vaccination may be beneficial to boost immunity in seropositive children, and encourage testing of the vaccine in young, seronegative children who are most at risk for respiratory virus disease.

Despite decades of work, an effective HIV vaccine remains elusive. In an effort to elicit protective immunity, investigators have sought to define vaccines able to elicit durable HIV-specific B-cell and T-cell activities. Additionally, vaccines are sought which can induce antibodies of a variety of isotypes, as each isotype possesses unique attributes in terms of opsonization, Fc receptor binding capacity, complement fixation and location. One prominent new vaccine strategy, applied to numerous distinct antigenic systems is the prime boost-regimen, with DNA, vaccinia virus (VV), and/or purified recombinant protein. To examine the durability, location and isotype distribution of responses induced by prime-boost regimens, we tested successive immunizations with DNA, VV and protein (D-V-P), comparing three forms of protein inoculations: (i) purified protein administered intramuscularly with complete Freunds adjuvant, (ii) purified protein administered intranasally, and (iii) purified protein conjugated to oxidized mannan, administered intranasally. We found that all three protocols elicited serum antibodies of multiple isotypes, with serum IgA being most prominent among mice immunized with mannan-conjugated protein. All D-V-P protocols, regardless of protein form or route, also elicited antibody responses at mucosal surfaces. In bronchoalveolar lavage, a tendency toward IgA production was again most prominent in mice boosted with the protein-mannan conjugate. Both B-cell and T-cell responses were sustained for more than 1 year post-immunization following each form of vaccination. Contemporaneous with long-lasting serum and mucosal antibodies were antibody forming cells in the bone marrow of primed animals. Results highlight the D-V-P vaccination strategy as a promising approach for attaining durable, multi-isotype B-cell and T-cell activities toward HIV.

A critical goal of HIV vaccine development is the identification of safe and immunogenic vectors. Recombinant vaccinia virus is a highly effective vaccine vector, with demonstrated capacity to protect animals from various viral pathogens, including rabies. Unlike many other candidate vaccine vectors, vast human experience exists with the parenteral smallpox vaccine. However, consideration of recombinant vaccinia virus as a modern vaccine is complicated by the relatively high prevalence of immunocompromised persons compared to such prevalence 4 or more decades ago (when smallpox vaccination was still routine). Administering vaccine by the subcutaneous (SQ) route, rather than the traditional scarification route, could address these concerns. SQ administration could prevent transmission of vaccinia virus to potentially vulnerable persons; it could also avoid the most common adverse events, which are cutaneous in nature. However, previous studies suggest that elicitation of immune response against passenger gene products following SQ administration requires development of a superficial pox lesion, defeating the intention of SQ administration. This is the first report to demonstrate that SQ administration of recombinant vaccinia virus does elicit immune response to the passenger protein in the absence of a cutaneous pox lesion. Results further show that a multi-envelope HIV vaccine can elicit antibody responses toward heterologous HIV-1 not represented by primary sequence in the vaccine. These findings have global implications because they support the consideration of recombinant vaccinia virus as a valuable HIV vaccine vector system.

Members of the relatively new Astroviridae family are non-enveloped, single-stranded RNA viruses that cause diarrheal disease in a wide range species. Human astroviruses, of which there are eight canonical strains, are a leading cause of gastroenteritis in children

Primary central nervous system lymphoma (PCNSL), observed among immunocompromised AIDS patients, has not been reported during chemotherapy for acute lymphoblastic leukaemia (ALL). We report a case of PCNSL occurring in a child receiving intensive multiagent chemotherapy for B-cell ALL. In situ hybridization studies demonstrated Epstein-Barr virus genome in both tumours, suggesting a possible link between the two diseases. The clinical response of the PCNSL to conservative therapy highlights the importance of accurately diagnosing such EBV-related disorders, especially in patients where immune compromise can be reversed.