Maria R. Castrucci

Early Alterations of the Receptor-Binding Properties of H1, H2, and H3 Avian Influenza Virus Hemagglutinins after Their Introduction into Mammals

ABSTRACT Interspecies transmission of influenza A viruses circulating in wild aquatic birds occasionally results in influenza outbreaks in mammals, including humans. To identify early changes in the receptor binding properties of the avian virus hemagglutinin (HA) after interspecies transmission and to determine the amino acid substitutions responsible for these alterations, we studied the HAs of the initial isolates from the human pandemics of 1957 (H2N2) and 1968 (H3N2), the European swine epizootic of 1979 (H1N1), and the seal epizootic of 1992 (H3N3), all of which were caused by the introduction of avian virus HAs into these species. The viruses were assayed for their ability to bind the synthetic sialylglycopolymers 3′SL-PAA and 6′SLN-PAA, which contained, respectively, 3′-sialyllactose (the receptor determinant preferentially recognized by avian influenza viruses) and 6′-sialyl(N-acetyllactosamine) (the receptor determinant for human viruses). Avian and seal viruses bound 6′SLN-PAA very weakly, whereas the earliest available human and swine epidemic viruses bound this polymer with a higher affinity. For the H2 and H3 strains, a single mutation, 226Q→L, increased binding to 6′SLN-PAA, while among H1 swine viruses, the 190E→D and 225G→E mutations in the HA appeared important for the increased affinity of the viruses for 6′SLN-PAA. Amino acid substitutions at positions 190 and 225 with respect to the avian virus consensus sequence are also present in H1 human viruses, including those that circulated in 1918, suggesting that substitutions at these positions are important for the generation of H1 human pandemic strains. These results show that the receptor-binding specificity of the HA is altered early after the transmission of an avian virus to humans and pigs and, therefore, may be a prerequisite for the highly effective replication and spread which characterize epidemic strains.

Balanced Hemagglutinin and Neuraminidase Activities Are Critical for Efficient Replication of Influenza A Virus

ABSTRACT The SD0 mutant of influenza virus A/WSN/33 (WSN), characterized by a 24-amino-acid deletion in the neuraminidase (NA) stalk, does not grow in embryonated chicken eggs because of defective NA function. Continuous passage of SD0 in eggs yielded 10 independent clones that replicated efficiently. Characterization of these egg-adapted viruses showed that five of the viruses contained insertions in the NA gene from the PB1, PB2, or NP gene, in the region linking the transmembrane and catalytic head domains, demonstrating that recombination of influenza viral RNA segments occurs relatively frequently. The other five viruses did not contain insertions in this region but displayed decreased binding affinity toward sialylglycoconjugates, compared with the binding properties of the parental virus. Sequence analysis of one of the latter viruses revealed mutations in the hemagglutinin (HA) gene, at sites in close proximity to the sialic acid receptor-binding pocket. These mutations appear to compensate for reduced NA function due to stalk deletions. Thus, balanced HA-NA functions are necessary for efficient influenza virus replication.

The Role of Antigen in the Localization of Naive, Acutely Activated, and Memory CD8+ T Cells to the Lung During Influenza Pneumonia

The role of Ag in the recruitment and localization of naive, acutely activated, and memory CD8+ T cells to the lung during influenza infection was explored using TCR-transgenic (Tg) mice. Naive, Thy1.2+CD8+ OT-I TCR-Tg cells were primed and recruited to the lung after transfer into congenic Thy1.1+ recipients challenged with a genetically engineered influenza virus (influenza A/WSN/33 (WSN)-OVAI) containing the Kb restricted OVA257–264 epitope (siinfekl) in the viral neuraminidase stalk. However, if the transferred animals were infected with a similar influenza virus that expressed an irrelevant Kb epitope (WSN-PEPII), no TCR-Tg T cells were detectable in the lung, although they were easily visible in the lymphoid organs. Conversely, there were substantial numbers of OT-I cells found in the lungs of WSN-PEPII-infected mice when the animals had been previously, or were concurrently, infected with a recombinant vaccinia virus expressing OVA. Similar results were obtained with nontransgenic populations of memory CD8+ T cells reactive to a murine γ-herpesvirus-68 Ag. Interestingly, the primary host response to the immunodominant influenza nucleoprotein epitope was not affected by the presence of memory or recently activated OT-I T cells. Thus, although Ag is required to activate the T cells, the subsequent localization of T cells to the lung during a virus infection is a property of recently activated and memory T cells and is not necessarily driven by Ag in the lung.

Influence of host species on the evolution of the nonstructural (NS) gene of influenza A viruses.

The matrix (M) and nonstructural (NS) genes of influenza A viruses each encode two overlapping proteins. In the M gene, evolution of one protein affects that of the other. To determine whether or not this evolutionary influence operating between the two M proteins also occurs in the NS gene, we sequenced the NS genes of 36 influenza A viruses isolated from a broad spectrum of animal species (wild and domestic birds, horses, pigs, humans, and sea mammals) and analyzed them phylogenetically, together with other previously published sequences. These analyses enabled us to conclude the following host species-related points that are not found in the other influenza A virus genes and their gene products. (1) The evolution of the two overlapping proteins encoded by the NS gene are lineage-dependent, unlike the M gene where evolutionary constraints on the Ml protein affect the evolution of the M2 protein (Ito et al.. J. Virol. 65 (1991) 5491 5498). (2) The gull-specific lineage contained nonH13 gull viruses and the non-gull avian lineage contained H13 gull viruses, indicating that the gull-specific lineage does not link to the H13 HA subtype in the NS gene unlike findings with other genes. (3) The branching topology of the recent equine lineage (H7N7 viruses isolated after 1973 and H3N8) indicates recent introduction of the NS, M, and PB2 genes into horses from avian sources by genetic reassortment.

Influenza – A Model of an Emerging Virus Disease

Influenza A viruses continue to emerge from the aquatic avian reservoir and cause pandemics. Phylogenetic analysis of the nucleotide sequence of all eight influenza A virus RNA segments indicate that all of the influenza viruses in mammalian hosts originate from the avian gene pool. In contrast to the rapid progressive changes in both the nucleotide and amino acid sequences of mammalian virus gene lineages, avian virus genes show far less variation and, in most cases, appear to be in evolutionary stasis. There are periodic exchanges of influenza virus genes or whole viruses between species giving rise to pandemics of diseases in humans, lower animals and birds. The periodic emergence of influenza viruses in mammalian species has been illustrated by the appearance of a new influenza virus in horses in northern China in 1989. Phylogenetic analysis of classical H1N1, avian-like H1N1 and human H3N2 viruses circulating in Italian pigs reveals that genetic reassortment is taking place between avian- and human-like viruses in the European pig population. These studies provide evidence supporting the possibility that pigs serve as a mixing vessel for reassortment between influenza viruses in mammalian and avian hosts and raise the question of whether the next pandemic of influenza will emerge in Europe!

Immunogenicity of influenza vaccine (1993–1994 winter season) in HIV-seropositive and -seronegative ex-intravenous drug users

The humoral response (haemagglutination inhibiting antibodies) to trivalent split influenza vaccine for the 1993-94 winter season (A/Beijing/32/92 (H3N2), A/Singapore/6/86 (H1N1) and B/Panama/45/90) was evaluated in a group of young HIV-seropositive ex-intravenous heroin users and compared with responses measured in HIV-seronegative individuals with a similar history. HIV-negative volunteers showed an overall positive response suggesting that previous heroin use did not influence their humoral response to influenza vaccine. Comparable results were obtained in HIV-positive subjects with CD4+ lymphocyte counts > 500 microliters-1, whereas impaired reactivity was found in HIV-positive volunteers with CD4+ counts < 500 microliters-1. Booster vaccination did not increase antibody levels in any of the groups studied, although the data did not exclude a positive influence of a second vaccine dose on persistence of antibody at 120 days after the first dose. No significant changes were observed in p24 antigenemia levels in HIV-positive individuals after vaccination.

Investigation of an imported case of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection in Florence, Italy, May to June 2013

On 31 May 2013, the first case of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection in Italy was laboratory confirmed in a previously healthy adult man, who developed pneumonia with moderate respiratory distress after returning from a holiday in Jordan. Two secondary cases were identified through contact tracing, among family members and colleagues who had not previously travelled abroad. Both secondary cases developed mild illness. All three patients recovered fully.

Primary CD8+ T-Cell Response to Soluble Ovalbumin Is Improved by Chloroquine Treatment In Vivo

ABSTRACT The efficiency of cross-presentation of exogenous antigens by dendritic cells (DCs) would seem to be related to the level of antigen escape from massive degradation mediated by lysosomal proteases in an acidic environment. Here, we demonstrate that a short course of treatment with chloroquine in mice during primary immunization with soluble antigens improved the cross-priming of naïve CD8+ T lymphocytes in vivo. More specifically, priming of chloroquine-treated mice with soluble ovalbumin (OVA), OVA associated with alum, or OVA pulsed on DCs was more effective in inducing OVA-specific CD8+ T lymphocytes than was priming of untreated mice. We conclude that chloroquine treatment improves the cross-presentation capacity of DCs and thus the size of effector and memory CD8+ T cells during vaccination.

The Early Expression of Glycoprotein B from Herpes Simplex Virus Can Be Detected by Antigen-Specific CD8+ T Cells

ABSTRACT The immune response to cutaneous herpes simplex virus type 1 (HSV-1) infection begins with remarkable rapidity. Activation of specific cytotoxic T lymphocytes (CTL) begins within hours of infection, even though the response within the draining lymph nodes peaks nearly 5 days later. HSV gene products are classified into three main groups, α, β, and γ, based on their kinetics and requirements for expression. In C57BL/6 mice, the immunodominant epitope from HSV is derived from glycoprotein B (gB498-505). While gB is considered a γ or “late” gene product, previous reports have indicated that some level of gene expression may occur soon after infection. Using brefeldin A as a specific inhibitor of viral antigen presentation to major histocompatibility complex class I-restricted CTL, we have formally addressed the timing of gB peptide expression in an immunologically relevant manner following infection. Presentation of gB peptide detected by T-cell activation was first observed within 2 h of infection. Comparison with another viral epitope expressed early during infection, HSV-1 ribonucleotide reductase, demonstrated that gB is presented with the same kinetics as this classical early-gene product. Moreover, this rapidity of gB expression was further illustrated via rapid priming of naïve transgenic CD8+ T cells in vivo after HSV-1 infection of mice. These results establish that gB is expressed rapidly following HSV-1 infection, at levels capable of effectively stimulating CD8+ T cells.

Human infection with highly pathogenic A(H7N7) avian influenza virus, Italy, 2013.

During an influenza A(H7N7) virus outbreak among poultry in Italy during August–September 2013, infection with a highly pathogenic A(H7N7) avian influenza virus was diagnosed for 3 poultry workers with conjunctivitis. Genetic analyses revealed that the viruses from the humans were closely related to those from chickens on affected farms.