Highly Pathogenic Avian Influenza Virus Infection of Mallards with Homo- and Heterosubtypic Immunity Induced by Low Pathogenic Avian Influenza Viruses

The potential role of wild birds as carriers of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 is still a matter of debate. Consecutive or simultaneous infections with different subtypes of influenza viruses of low pathogenicity (LPAIV) are very common in wild duck populations. To better understand the epidemiology and pathogenesis of HPAIV H5N1 infections in natural ecosystems, we investigated the influence of prior infection of mallards with homo- (H5N2) and heterosubtypic (H4N6) LPAIV on exposure to HPAIV H5N1. In mallards with homosubtypic immunity induced by LPAIV infection, clinical disease was absent and shedding of HPAIV from respiratory and intestinal tracts was grossly reduced compared to the heterosubtypic and control groups (mean GEC/100 µl at 3 dpi: 3.0×10² vs. 2.3×10⁴ vs. 8.7×10⁴; p<0.05). Heterosubtypic immunity induced by an H4N6 infection mediated a similar but less pronounced effect. We conclude that the epidemiology of HPAIV H5N1 in mallards and probably other aquatic wild bird species is massively influenced by interfering immunity induced by prior homo- and heterosubtypic LPAIV infections.     

Multi-Dimensional Measurement of Antibody-Mediated Heterosubtypic Immunity to Influenza

The human immune response to influenza vaccination depends in part on preexisting cross-reactive (heterosubtypic) immunity from previous infection by, and/or vaccination with, influenza strains that share antigenic determinants with the vaccine strains. However, current methods for assessing heterosubtypic antibody responses against influenza, including the hemagglutination-inhibition (HAI) assay and ELISA, are time and labor intensive, and require moderate amounts of serum and reagents. To address these issues we have developed a fluorescent multiplex assay, mPlex-Flu, that rapidly and simultaneously measures strain specific IgG, IgA, and IgM antibodies against influenza hemagglutinin (HA) from multiple viral strains. We cloned, expressed and purified HA proteins from 12 influenza strains, and coupled them to multiplex beads. Assay validation showed that minimal sample volumes (<5 μl of serum) were needed, and the assay had a linear response over a four Log₁₀ range. The assay detected nanogram levels of anti-influenza specific antibodies, had high accuracy and reproducibility, with an average percentage coefficient of variation (%CV) of 9.06 for intra-assay and 12.94 for inter-assay variability. Pre- and post-intramuscular trivalent influenza vaccination levels of virus specific Ig were consistent with HAI titer and ELISA measurements. A significant advantage of the mPLEX-Flu assay over the HAI assay is the ability to perform antigenic cartography, determining the antigenic distances between influenza HA’s, without mathematical correction for HAI data issues. For validation we performed antigenic cartography on 14 different post-influenza infection ferret sera assayed against 12 different influenza HA’s. Results were in good agreement with a phylogenetic tree generated from hierarchical clustering of the genomic HA sequences. This is the first report of the use of a multiplex method for antigenic cartography using ferret sera. Overall, the mPlex-Flu assay provides a powerful tool to rapidly assess the influenza antibody repertoire in large populations and to study heterosubtypic immunity induced by influenza vaccination.     

A Human CD4+ T Cell Epitope in the Influenza Hemagglutinin Is Cross-Reactive to Influenza A Virus Subtypes and to Influenza B Virus

The hemagglutinin protein (HA) of the influenza virus family is a major antigen for protective immunity. Thus, it is a relevant target for developing vaccines. Here, we describe a human CD4(+) T cell epitope in the influenza virus HA that lies in the fusion peptide of the HA. This epitope is well conserved in all 16 subtypes of the HA protein of influenza A virus and the HA protein of influenza B virus. By stimulating peripheral blood mononuclear cells (PBMCs) from a healthy adult donor with peptides covering the entire HA protein based on the sequence of A/Japan/305/1957 (H2N2), we generated a T cell line specific to this epitope. This CD4(+) T cell line recognizes target cells infected with influenza A virus seasonal H1N1 and H3N2 strains, a reassortant H2N1 strain, the 2009 pandemic H1N1 strain, and influenza B virus in cytotoxicity assays and intracellular-cytokine-staining assays. It also lysed target cells infected with avian H5N1 virus. We screened healthy adult PBMCs for T cell responses specific to this epitope and found individuals who had ex vivo gamma interferon (IFN-γ) responses to the peptide epitope in enzyme-linked immunospot (ELISPOT) assays. Almost all donors who responded to the epitope had the HLA-DRB1*09 allele, a relatively common HLA allele. Although natural infection or standard vaccination may not induce strong T and B cell responses to this highly conserved epitope in the fusion peptide, it may be possible to develop a vaccination strategy to induce these CD4(+) T cells, which are cross-reactive to both influenza A and B viruses.     

Vaccination against Human Influenza A/H3N2 Virus Prevents the Induction of Heterosubtypic Immunity against Lethal Infection with Avian Influenza A/H5N1 Virus

Annual vaccination against seasonal influenza viruses is recommended for certain individuals that have a high risk for complications resulting from infection with these viruses. Recently it was recommended in a number of countries including the USA to vaccinate all healthy children between 6 and 59 months of age as well. However, vaccination of immunologically naïve subjects against seasonal influenza may prevent the induction of heterosubtypic immunity against potentially pandemic strains of an alternative subtype, otherwise induced by infection with the seasonal strains.Here we show in a mouse model that the induction of protective heterosubtypic immunity by infection with a human A/H3N2 influenza virus is prevented by effective vaccination against the A/H3N2 strain. Consequently, vaccinated mice were no longer protected against a lethal infection with an avian A/H5N1 influenza virus. As a result H3N2-vaccinated mice continued to loose body weight after A/H5N1 infection, had 100-fold higher lung virus titers on day 7 post infection and more severe histopathological changes than mice that were not protected by vaccination against A/H3N2 influenza.The lack of protection correlated with reduced virus-specific CD8+ T cell responses after A/H5N1 virus challenge infection. These findings may have implications for the general recommendation to vaccinate all healthy children against seasonal influenza in the light of the current pandemic threat caused by highly pathogenic avian A/H5N1 influenza viruses.     

Memory T Cells Generated by Prior Exposure to Influenza Cross React with the Novel H7N9 Influenza Virus and Confer Protective Heterosubtypic Immunity

Influenza virus is a source of significant health and economic burden from yearly epidemics and sporadic pandemics. Given the potential for the emerging H7N9 influenza virus to cause severe respiratory infections and the lack of exposure to H7 and N9 influenza viruses in the human population, we aimed to quantify the H7N9 cross-reactive memory T cell reservoir in humans and mice previously exposed to common circulating influenza viruses. We identified significant cross-reactive T cell populations in humans and mice; we also found that cross-reactive memory T cells afforded heterosubtypic protection by reducing morbidity and mortality upon lethal H7N9 challenge. In context with our observation that PR8-primed mice have limited humoral cross-reactivity with H7N9, our data suggest protection from H7N9 challenge is indeed mediated by cross-reactive T cell populations established upon previous priming with another influenza virus. Thus, pre-existing cross-reactive memory T cells may limit disease severity in the event of an H7N9 influenza virus pandemic.     

Comparison of Antiviral Activity between IgA and IgG Specific to Influenza Virus Hemagglutinin: Increased Potential of IgA for Heterosubtypic Immunity

Both IgA and IgG antibodies are known to play important roles in protection against influenza virus infection. While IgG is the major isotype induced systemically, IgA is predominant in mucosal tissues, including the upper respiratory tract. Although IgA antibodies are believed to have unique advantages in mucosal immunity, information on direct comparisons of the in vitro antiviral activities of IgA and IgG antibodies recognizing the same epitope is limited. In this study, we demonstrate differences in antiviral activities between these isotypes using monoclonal IgA and IgG antibodies obtained from hybridomas of the same origin. Polymeric IgA-producing hybridoma cells were successfully subcloned from those originally producing monoclonal antibody S139/1, a hemaggulutinin (HA)-specific IgG that was generated against an influenza A virus strain of the H3 subtype but had cross-neutralizing activities against the H1, H2, H13, and H16 subtypes. These monoclonal S139/1 IgA and IgG antibodies were assumed to recognize the same epitope and thus used to compare their antiviral activities. We found that both S139/1 IgA and IgG antibodies strongly bound to the homologous H3 virus in an enzyme-linked immunosorbent assay, and there were no significant differences in their hemagglutination-inhibiting and neutralizing activities against the H3 virus. In contrast, S139/1 IgA showed remarkably higher cross-binding to and antiviral activities against H1, H2, and H13 viruses than S139/1 IgG. It was also noted that S139/1 IgA, but not IgG, drastically suppressed the extracellular release of the viruses from infected cells. Electron microscopy revealed that S139/1 IgA deposited newly produced viral particles on the cell surface, most likely by tethering the particles. These results suggest that anti-HA IgA has greater potential to prevent influenza A virus infection than IgG antibodies, likely due to increased avidity conferred by its multivalency, and that this advantage may be particularly important for heterosubtypic immunity.     

Cell Mediated Immunity in Virus Infections

The discovery of the molecular nature of T cell-mediated immunity is reviewed in a historical context. Current approaches to understanding virus-induced inflammatory processes are described.     

Individual Variation in Influenza A Virus Infection Histories and Long-Term Immune Responses in Mallards

Wild dabbling ducks (genus Anas) are the main reservoir for influenza A virus (IAV) in the Northern Hemisphere. Current understanding of disease dynamics and epidemiology in this virus-host system has primarily been based on population-level surveillance studies and infection experiments conducted in laboratory settings. Using a combined experimental-natural approach with wild-strain captive mallards (Anas platyrhynchos), we monitored individual IAV infection histories and immunological responses of 10 birds over the course of 15 months. This is the first detailed study to track natural IAV infection histories over several seasons amongst the same individuals growing from juvenile to adults. The general trends in the infection histories of the monitored birds reflected seasonal variation in prevalence at the population level. However, within the study group there were significant differences between individuals in infection frequency as well as in short and long term anti-IAV antibody response. Further observations included individual variation in the number of infecting virus subtypes, and a strong tendency for long-lasting hemagglutinin-related homosubtypic immunity. Specifically, all infections in the second autumn, except one, were of different subtypes compared to the first autumn. The variation among birds concerning these epidemiologically important traits illustrates the necessity for IAV studies to move from the level of populations to examine individuals in order to further our understanding of IAV disease and epidemiology.     

Cooperativity Between CD8+ T Cells,Non-Neutralizing Antibodies,and Alveolar Macrophages Is Important for Heterosubtypic Influenza Virus Immunity

Seasonal epidemics of influenza virus result in ∼36,000 deaths annually in the United States. Current vaccines against influenza virus elicit an antibody response specific for the envelope glycoproteins. However, high mutation rates result in the emergence of new viral serotypes, which elude neutralization by preexisting antibodies. T lymphocytes have been reported to be capable of mediating heterosubtypic protection through recognition of internal, more conserved, influenza virus proteins. Here, we demonstrate using a recombinant influenza virus expressing the LCMV GP33-41 epitope that influenza virus-specific CD8+ T cells and virus-specific non-neutralizing antibodies each are relatively ineffective at conferring heterosubtypic protective immunity alone. However, when combined virus-specific CD8 T cells and non-neutralizing antibodies cooperatively elicit robust protective immunity. This synergistic improvement in protective immunity is dependent, at least in part, on alveolar macrophages and/or other lung phagocytes. Overall, our studies suggest that an influenza vaccine capable of eliciting both CD8+ T cells and antibodies specific for highly conserved influenza proteins may be able to provide heterosubtypic protection in humans, and act as the basis for a potential “universal” vaccine.     

Transfer of Maternal Antibodies against Avian Influenza Virus in Mallards (Anas platyrhynchos)

Maternal antibodies protect chicks from infection with pathogens early in life and may impact pathogen dynamics due to the alteration of the proportion of susceptible individuals in a population. We investigated the transfer of maternal antibodies against avian influenza virus (AIV) in a key AIV host species, the mallard (Anas platyrhynchos). Combining observations in both the field and in mallards kept in captivity, we connected maternal AIV antibody concentrations in eggs to (i) female body condition, (ii) female AIV antibody concentration, (iii) egg laying order, (iv) egg size and (v) embryo sex. We applied maternity analysis to the eggs collected in the field to account for intraspecific nest parasitism, which is reportedly high in Anseriformes, detecting parasitic eggs in one out of eight clutches. AIV antibody prevalence in free-living and captive females was respectively 48% and 56%, with 43% and 24% of the eggs receiving these antibodies maternally. In both field and captive study, maternal AIV antibody concentrations in egg yolk correlated positively with circulating AIV antibody concentrations in females. In the captive study, yolk AIV antibody concentrations correlated positively with egg laying order. Female body mass and egg size from the field and captive study, and embryos sex from the field study were not associated with maternal AIV antibody concentrations in eggs. Our study indicates that maternal AIV antibody transfer may potentially play an important role in shaping AIV infection dynamics in mallards.     

Cloned Defective Interfering Influenza Virus Protects Ferrets from Pandemic 2009 Influenza A Virus and Allows Protective Immunity to Be Established

Influenza A viruses are a major cause of morbidity and mortality in the human population, causing epidemics in the winter, and occasional worldwide pandemics. In addition there are periodic outbreaks in domestic poultry, horses, pigs, dogs, and cats. Infections of domestic birds can be fatal for the birds and their human contacts. Control in man operates through vaccines and antivirals, but both have their limitations. In the search for an alternative treatment we have focussed on defective interfering (DI) influenza A virus. Such a DI virus is superficially indistinguishable from a normal virus but has a large deletion in one of the eight RNAs that make up the viral genome. Antiviral activity resides in the deleted RNA. We have cloned one such highly active DI RNA derived from segment 1 (244 DI virus) and shown earlier that intranasal administration protects mice from lethal disease caused by a number of different influenza A viruses. A more cogent model of human influenza is the ferret. Here we found that intranasal treatment with a single dose of 2 or 0.2 µg 244 RNA delivered as A/PR/8/34 virus particles protected ferrets from disease caused by pandemic virus A/California/04/09 (A/Cal; H1N1). Specifically, 244 DI virus significantly reduced fever, weight loss, respiratory symptoms, and infectious load. 244 DI RNA, the active principle, was amplified in nasal washes following infection with A/Cal, consistent with its amelioration of clinical disease. Animals that were treated with 244 DI RNA cleared infectious and DI viruses without delay. Despite the attenuation of infection and disease by DI virus, ferrets formed high levels of A/Cal-specific serum haemagglutination-inhibiting antibodies and were solidly immune to rechallenge with A/Cal. Together with earlier data from mouse studies, we conclude that 244 DI virus is a highly effective antiviral with activity potentially against all influenza A subtypes.     

A VLP Vaccine Induces Broad-Spectrum Cross-Protective Antibody Immunity against H5N1 and H1N1 Subtypes of Influenza A Virus

The recent threats of influenza epidemics and pandemics have prioritized the development of a universal vaccine that offers protection against a wider variety of influenza infections. Here, we demonstrate a genetically modified virus-like particle (VLP) vaccine, referred to as H5M2eN1-VLP, that increased the antigenic content of NA and induced rapid recall of antibody against HA(2) after viral infection. As a result, H5M2eN1-VLP vaccination elicited a broad humoral immune response against multiple viral proteins and caused significant protection against homologous RG-14 (H5N1) and heterologous A/California/07/2009 H1N1 (CA/07) and A/PR/8/34 H1N1 (PR8) viral lethal challenges. Moreover, the N1-VLP (lacking HA) induced production of a strong NA antibody that also conferred significant cross protection against H5N1 and heterologous CA/07 but not PR8, suggesting the protection against N1-serotyped viruses can be extended from avian-origin to CA/07 strain isolated in humans, but not to evolutionally distant strains of human-derived. By comparative vaccine study of an HA-based VLP (H5N1-VLP) and NA-based VLPs, we found that H5N1-VLP vaccination induced specific and strong protective antibodies against the HA(1) subunit of H5, thus restricting the breadth of cross-protection. In summary, we present a feasible example of direction of VLP vaccine immunity toward NA and HA(2), which resulted in cross protection against both seasonal and pandemic influenza strains, that could form the basis for future design of a better universal vaccine.     

Innate Immunity and the Inter-exposure Interval Determine the Dynamics of Secondary Influenza Virus Infection and Explain Observed Viral Hierarchies

Influenza is an infectious disease that primarily attacks the respiratory system. Innate immunity provides both a very early defense to influenza virus invasion and an effective control of viral growth. Previous modelling studies of virus–innate immune response interactions have focused on infection with a single virus and, while improving our understanding of viral and immune dynamics, have been unable to effectively evaluate the relative feasibility of different hypothesised mechanisms of antiviral immunity. In recent experiments, we have applied consecutive exposures to different virus strains in a ferret model, and demonstrated that viruses differed in their ability to induce a state of temporary immunity or viral interference capable of modifying the infection kinetics of the subsequent exposure. These results imply that virus-induced early immune responses may be responsible for the observed viral hierarchy. Here we introduce and analyse a family of within-host models of re-infection viral kinetics which allow for different viruses to stimulate the innate immune response to different degrees. The proposed models differ in their hypothesised mechanisms of action of the non-specific innate immune response. We compare these alternative models in terms of their abilities to reproduce the re-exposure data. Our results show that 1) a model with viral control mediated solely by a virus-resistant state, as commonly considered in the literature, is not able to reproduce the observed viral hierarchy; 2) the synchronised and desynchronised behaviour of consecutive virus infections is highly dependent upon the interval between primary virus and challenge virus exposures and is consistent with virus-dependent stimulation of the innate immune response. Our study provides the first mechanistic explanation for the recently observed influenza viral hierarchies and demonstrates the importance of understanding the host response to multi-strain viral infections. Re-exposure experiments provide a new paradigm in which to study the immune response to influenza and its role in viral control.     

Murine Norovirus Infection Has No Significant Effect on Adaptive Immunity to Vaccinia Virus or Influenza A Virus

Murine norovirus (MNV) is endemic in many research mouse colonies. Although MNV infections are typically asymptomatic in immunocompetent mice, the effects of MNV infection on subsequent experimental viral infections are poorly documented. Here, we infected C57BL/6 mice with MNV and then with either vaccinia virus or influenza A virus. MNV infection had no effect on CD8⁺ T-cell or antibody responses to secondary viruses or to secondary virus-induced morbidity or mortality. While our findings suggest that MNV has little influence on host immunity in immunocompetent mice, we would urge caution regarding the potential effects of MNV on immune responses to viruses and other pathogens, which must be determined on a system-by-system basis.Human norovirus (NoV) infections cause greater than 90% of nonbacterial gastroenteritis cases (4, 5) and are an important public health concern. Murine noroviruses (MNV) were recently identified (7) as highly pathogenic agents in immunocompromised mice, and serological studies indicate that over 20% of mice in research colonies are exposed to MNV (6). As with NoV, MNV is spread through the fecal-oral route. While NoV rapidly causes gastrointestinal symptoms and fever in healthy individuals, MNV is typically asymptomatic in immunocompetent mice.MNV isolates are both genetically and biologically diverse (13). In wild-type (wt) mice, some strains of MNV are rapidly cleared, while others persist (13). Controlling MNV infections requires elements of both innate and adaptive immunity. Mice with defects in interferon (IFN) signaling pathways demonstrate increased MNV lethality (7, 9). CD4⁺ and CD8⁺ T cells and B cells are all needed for complete MNV clearance (1, 2). Natural exposure of immunocompromised mice to MNV leads to inflammation of the liver, lungs, and peritoneal and pleural cavities (14).It is well established that infection with natural mouse viruses can greatly impact immune responses to infections with other viruses. The prevalence of MNV in research mouse colonies might therefore lead to irreproducible and variable results that significantly impact research efforts. Indeed, MNV was recently reported to alter disease progression in a mouse model of bacterium-induced inflammatory bowel disease (8). Concern over the potential effects of MNV on viral immunology research prompted a dedicated workshop at the 2008 Keystone Viral Immunity meeting (http://www.keystonesymposia.org). In the present study, we examined the effect of MNV infection on adaptive immune responses in wt mice to influenza A virus (IAV) and vaccinia virus (VV).We infected C57BL/6 mice perorally with a high dose (3 × 10⁷ PFU/mouse) of a plaque-purified MNV stock derived from MNV-CR6p2 (13). The capacity of this plaque-purified virus to persist in wt mice has been confirmed by quantitative PCR analysis and a plaque assay (D. Strong, L. Thackray, and H. Virgin, unpublished observation). We confirmed that the mice were infected by measuring anti-MNV antibodies (Abs) by using an enzyme-linked immunosorbent assay (ELISA) (data not shown). For all experiments, mice were infected with MNV at Washington University and shipped 4 to 5 days later to NIAID for further study. To contain MNV, infected mice were housed in microisolator cages in a quarantine room. In some experiments, control mice were housed in the same room as MNV-infected mice. Sera collected from control mice did not contain anti-MNV Abs as determined by ELISA (data not shown), confirming that transmission of MNV between mice housed in microisolator cages can be prevented by proper cage changing and aseptic handling of samples from infected mice.Upon intraperitoneal (i.p.) infection with either VV or IAV, mice mount robust CD8⁺ T-cell responses that peak, respectively, on day 6 or 7. Anti-VV and anti-IAV CD8⁺ T-cell responses in C57BL/6 mice conform to a well-established immunodominance hierarchy (3, 10). To determine to what extent MNV infection alters the magnitude and/or immunodominance hierarchy of CD8⁺ T-cell responses, we infected C57BL/6 mice i.p. with either VV or IAV 19 days following MNV infection. As controls, naïve mice (MNV negative) were infected with either virus. Lymphocytes were isolated from mice 6 days postinfection with VV and 7 days postinfection with IAV. The fraction of antigen-specific CD8⁺ T cells present in spleen and peritoneal exudate cells (PEC) was determined by intracellular IFN-γ staining after stimulation with synthetic peptides. MNV infection had little effect on the magnitude of splenic or PEC CD8⁺ T cells responding to VV (Fig. 1A and B) or IAV (Fig. 1C and D) infection. Regardless of MNV exposure history, splenic and PEC responses were dominated by B8R- and A8R-specific CD8⁺ T cells following VV infection (Fig. 1A and B) and by PA-specific and NP-specific CD8⁺ T cells following IAV infection (Fig. 1C and D).Open in a separate windowFIG. 1.MNV exposure does not alter CD8⁺ T-cell responses to VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.p. with ∼1 × 10⁶ PFU of VV (A and B) or ∼1 × 10⁷ 50% tissue culture infective dose units of IAV (C and D), and specific CD8⁺ T cells were determined by intracellular IFN-γ staining after restimulating lymphocytes with peptides. Lymphocytes isolated from the spleen (A and C) and peritoneal cavity (B and D) were tested. MNV infections were completed 19 days prior to VV or IAV infections. Means and SEM are shown in panels A and C. A two-way analysis of variance and Bonferroni statistical analysis were completed for these experiments. Cells were pooled for peritoneal lavage samples as shown in panels B and D. Four to five mice/group were used for each experiment; data are representative of two independent experiments.To examine the effect of MNV infection on antiviral Ab responses, MNV-infected and control C57BL/6 mice were infected intranasally (i.n.) with a sublethal dose of either VV or IAV. Three weeks later, levels of anti-VV and anti-IAV Abs were determined by ELISA and hemagglutination inhibition assays, respectively. MNV infection did not significantly modify the magnitude of Ab responses to VV (Fig. ​(Fig.2A)2A) or IAV (Fig. ​(Fig.2B).2B). Next, we determined the effect of MNV infection on heavy chain class switching of anti-VV or anti-IAV Ab responses. Anti-VV and anti-IAV Ab responses exhibited similar heavy chain profiles dominated by immunoglobulin G2b (IgG2b) Abs regardless of MNV status (Fig. 2C and D). Thus, the CD8⁺ T-cell and Ab response to both VV and IAV appears to be essentially unaffected by chronic MNV infection. Since IgG anti-VV or anti-IAV Ab responses are entirely dependent on CD4⁺ T-cell help (11, 12), we can also infer that MNV also does not significantly affect CD4⁺ T-cell responses to VV or IAV.Open in a separate windowFIG. 2.MNV exposure does not alter Ab responses to VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with ∼1 × 10³ PFU of VV (A and C) or ∼50 50% tissue culture infective dose units of IAV (B and D), and virus-specific Abs were determined by ELISA (A, C, and D) or hemagglutination inhibition (B). The ELISA results shown in panel A measured the total IgG, while the ELISA results shown in panels C and D measured the individual isotype indicated. MNV infections were completed 19 days prior to VV or IAV infections. Means and standard errors of the means are shown in panels A, C, and D. Means are shown as lines in panel B. A two-way analysis of variance and Bonferroni statistical analysis were completed for experiments shown in panels A, C, and D, and t tests were completed for the experiment shown in panel B. Four to five mice/group were used for each experiment. O.D., optical density; HAI, hemagglutination inhibition.T-cell and Ab responses, together with innate immune mechanisms, collaborate to control viral replication and limit pathogenesis. To examine the effect of chronic MNV infection on VV-induced or IAV-induced pathogenesis, we infected C57BL/6 mice i.n. with a lethal or sublethal dose of VV or IAV and monitored body weight over a 16-day period. MNV-CR6p2 infection had no significant effect on morbidity or mortality from either virus (Fig. ​(Fig.33 and ​and4).4). Since MNV isolates are highly diverse, we decided to examine the effects of a second strain of MNV (MNV-CW3) which is fully cleared in immunocompetent mice. Mice that cleared MNV-CW3 (19 days post-MNV infection) were infected i.n. with VV or IAV. Once again, this strain of MNV had no effect on VV-induced or IAV-induced morbidity or mortality (Fig. ​(Fig.33 and ​and4).4). Future studies should address the extent to which other MNV strains affect the generation of adaptive immune responses to secondary viral infections.Open in a separate windowFIG. 3.MNV does not increase morbidity following subsequent i.n. infection with VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with a sublethal dose of VV (∼1 × 10³ PFU) (A) or IAV (∼50 50% tissue culture infective dose units) (B), and weight loss was recorded for 16 days postinfection. MNV infections were completed 19 days prior to VV or IAV infections. A two-way analysis of variance and Bonferroni statistical analysis were completed. Four to five mice/group were used for each experiment.Open in a separate windowFIG. 4.MNV does not increase mortality following subsequent i.n. infection with VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with VV (∼1 × 10⁴ PFU) (A) or IAV (∼500 50% tissue culture infective dose units) (B), and survival was monitored for 16 days postinfection. MNV infections were completed 19 days prior to VV or IAV infections. Eight to 10 mice/group were used for each experiment.Taken together, these data demonstrate that MNV infection has no significant effects on the measured immune response to VV or IAV. Our results cannot, however, be simply extrapolated to other viruses or microorganisms. Rather, the effect of MNV infection on host immunity in mouse model disease systems needs to be established on a system-by-system basis. Without this knowledge, the possible confounding effects of MNV infection will continue to undermine the confidence in results obtained using mice in colonies in which MNV infections are endemic.