Vaccination of pregnant macaques protects newborns against mucosal simian immunodeficiency virus infection

Simian immunodeficiency virus (SIV) infection of newborn rhesus macaques is a rapid, sensitive animal model of human pediatric AIDS. Newborn macaques were readily infected by uncloned SIVmac following oral-conjunctival exposure and had persistently high viremia and rapid development of AIDS. In contrast, when 3 pregnant macaques were vaccinated against SIV, 2 of the newborns that had transplacentally acquired antiviral antibodies were protected against mucosal SIV infection at birth. These results suggest that intervention strategies such as active immunization of human immunodeficiency virus (HIV)-infected pregnant women and anti-HIV immunoglobulin administration may decrease the rate of perinatal HIV infection.     

Antibody-secreting cells specific for simian immunodeficiency virus antigens in lymphoid and mucosal tissues of immunized macaques

OBJECTIVES: To examine whether the route of immunization affects the induction of antibody-secreting cells (ASC) in the circulation of macaques. The distribution of ASC in the rectal mucosa and lymphoid tissues following challenge with simian immunodeficiency virus (SIV) was investigated. DESIGN: Macaques were immunized with recombinant SIV gp120 and p27 antigens by the targeted iliac lymph node (TILN) route of immunization or the nasal and rectal route, augmented by intramuscular immunization [naso-rectal intramuscular (NRI)]. The macaques were challenged with live SIV by the rectal route and ASC were assayed in the circulation before and after SIV challenge, and in the tissues removed at post-mortem. METHODS: ASC were examined in the circulation by Elispot assay. Mononuclear cells were prepared from peripheral blood, iliac and axillary lymph nodes and spleen. Rectal tissue was treated by enzyme digestion to elute mononuclear cells. RESULTS: TILN and NRI immunization induced circulating IgA and IgG ASC to both gp120 and p27. Following rectal challenge with SIV, TILN macaques were protected from infection whereas NRI route-immunized and unimmunized controls became infected. IgA ASC to p27 were increased significantly in the iliac lymph nodes of the TILN immunized macaques compared with unimmunized controls (P < 0.05). Only IgA ASC were found in the rectal mucosa of the immunized protected macaques but both IgA and IgG ASC were detected in the unimmunized infected macaques. Overall the number of IgG ASC specific for p27 was significantly higher in the infected NRI and control macaques than in the protected macaques (P < 0.02). A progressive increase in IgG but not IgA ASC was detected in the peripheral blood mononuclear cells of the unimmunized infected macaques. CONCLUSIONS: The results suggest that cells secreting IgA antibodies to p27 in the iliac lymph nodes of the TILN immunized macaques correlate significantly with protection from infection. The unimmunized infected macaques showed a progressive increase in IgG ASC in the peripheral blood after SIV challenge; this was found in the iliac and axillary lymph nodes and also in the spleen, suggesting that it is an immune response to the SIV infection.     

Selective amplification of simian immunodeficiency virus genotypes after intrarectal inoculation of rhesus monkeys

Animal models for sexual transmission of human immunodeficiency virus can define the influences of virus type, dose, and route of inoculation on infection and clinical outcome. We used an uncloned simian immunodeficiency virus stock (SIVmac) to inoculate cells in vitro and to inoculate rhesus monkeys by intravenous and intrarectal routes. The distribution of virus genotypes present in each of these infection examples was characterized by DNA sequence analysis of viral long terminal repeats (LTRs). Our analysis of LTR sequences from in vitro and in vivo infections revealed three main genotypes: one genotype was observed only for in vitro infection, and two other genotypes were recovered only from infected animals. By comparing animals inoculated with high intrarectal doses of SIVmac and those inoculated with low doses, we demonstrated that unique subsets of the stock were selected after intrarectal infection. Our findings indicate that minor genotypes present in the stock cross the rectal mucosa and are amplified selectively to become prominent in peripheral blood mononuclear cells from acutely infected animals. Studies with a molecular recombinant of SIV and human immunodeficiency virus type 1 sequences, SHIV, showed that viral LTR sequences do not undergo especially rapid sequence variation or rearrangement after intrarectal inoculation. The mucosal barrier exerts a significant influence on infection and disease progression by reducing the efficiency of SIVmac infection and by permitting distinct, pathogenic genotypes to become established in the host.     

Passive immunization of newborn rhesus macaques prevents oral simian immunodeficiency virus infection

To determine if passively acquired antiviral antibodies modulate virus transmission and disease progression in human pediatric AIDS, the potential of pre- and postexposure passive immunization with hyperimmune serum to prevent oral simian immunodeficiency virus (SIV) infection or disease progression in newborn rhesus macaques was tested. Untreated neonates became infected after oral SIV inoculation and had high viremia, and most animals developed fatal AIDS within 3 months. In contrast, SIV hyperimmune serum given subcutaneously prior to oral SIV inoculation protected 6 newborns against infection. When this SIV hyperimmune serum was given to 3 newborns 3 weeks after oral SIV inoculation, viremia was not reduced, and all 3 infants died within 3 months of age due to AIDS and immune-complex disease. These results suggest that passively acquired antihuman immunodeficiency virus (HIV) IgG may decrease perinatal HIV transmission. However, anti-HIV IgG may not impart therapeutic benefit to infants with established HIV infection.     

Live attenuated simian immunodeficiency virus (SIV)mac in macaques can induce protection against mucosal infection with SIVsm

OBJECTIVE: To investigate whether vaccination of macaques with attenuated simian immunodeficiency virus (SIV)macC8 could induce long-term protective immunity against rectal exposure to SIVsm and intravenous exposure to the more divergent HIV-2. DESIGN AND METHODS: Eight months after vaccination with live attenuated SIVmacC8, four cynomolgus monkeys were challenged with SIVsm intrarectally and another four vaccinated monkeys were challenged with HIV-2 intravenously. Sixteen months after SIVmacC8 vaccination, another two monkeys were challenged with SIVsm across the rectal mucosa. Two vaccinees shown to be protected against SIVsm were rechallenged 8 months after the first challenge. Ten naive animals were used as controls. Serum antigenaemia, virus isolation, antibody responses, cell-mediated immunity and CD4+ and CD8+ T-cell subpopulations were monitored. PCR-based assays were used to distinguish between virus populations. RESULTS: At the time of challenge, eight out of 10 vaccinees were PCR-positive for SIVmacC8 DNA but no virus could be isolated from peripheral blood mononuclear cells. After SIVsm challenge, three out of six vaccinees were repeatedly SIVsm PCR-negative. In one of the three infected monkeys, the challenge virus was initially suppressed but the monkey ultimately developed AIDS after increased replication of the pathogenic virus. Rechallenged monkeys remained protected. All HIV-2-challenged vaccinees became superinfected. All controls became infected with either SIVsm or HIV-2. At the time of challenge the vaccinees had neutralizing antibodies to SIVmac but no demonstrable cross-neutralizing antibodies to SIVsm or HIV-2. Titres of antigen-binding or neutralizing antibodies did not correlate with protection. Cytotoxic T-cell responses to SIV Gag/Pol and virus-specific T-cell proliferative responses were low. CONCLUSION: The live attenuated SIVmacC8 vaccine was able to induce long-term protection against heterologous intrarectal SIVsm challenge in a proportion of macaques but not against the more divergent HIV-2, which was given intravenously.     

Rapid clearance of simian immunodeficiency virus particles from plasma of rhesus macaques

Perturbation of the equilibrium between human immunodeficiency virus type 1 (HIV-1) and the infected host by administering antiretroviral agents has revealed the rapid turnover of both viral particles and productively infected cells. In this study, we used the infusion of simian immunodeficiency virus (SIV) particles into rhesus macaques to obtain a more accurate estimate of viral clearance in vivo. Consistently, exogenously infused virions were cleared from plasma with an extremely short half-life, on the order of minutes (a mean of 3.3 min). This new estimate is approximately 100-fold lower than the upper bound of 6 h previously reported for HIV-1 in infected humans. In select animals, multiple tissues were collected at the completion of each experiment to track the potential sites of virion clearance. Detectable levels of SIV RNA were found in lymph nodes, spleen, lungs, and liver, but not in other tissues examined. However, only approximately 1 to 10% or less of the infused virions were accounted for by the thorough tissue sampling, indicating that the vast majority of the infused particles must have been degraded over a short period of time. Should the rapid clearance of virions described here be applicable to infected patients, then HIV-1 production and thus the number of productively infected CD4(+) T lymphocytes or the viral burst size must be proportionally higher than previous minimal estimates.     

Early physiological abnormalities after simian immunodeficiency virus infection

Central nervous system (CNS) damage and dysfunction are devastating consequences of HIV infection. Although the CNS is one of the initial targets for HIV infection, little is known about early viral-induced abnormalities that can affect CNS function. Here we report the detection of early physiological abnormalities in simian immunodeficiency virus-infected monkeys. The acute infection caused a disruption of the circadian rhythm manifested by rises in body temperature, observed in all five individuals between 1 and 2 weeks postinoculation (p.i.), accompanied by a reduction in daily motor activity to 50% of control levels. Animals remained hyperthermic at 1 and 2 months p.i. and returned to preinoculation temperatures at 3 months after viral inoculation. Although motor activity recovered to baseline values at 1 month p.i., activity levels then decreased to approximately 50% of preinoculation values over the next 2 months. Analysis of sensory-evoked responses 1 month p.i. revealed distinct infection-induced changes in auditory-evoked potential peak latencies that persisted at 3 months after viral inoculation. These early physiological abnormalities may precede the development of observable cognitive or motor deficiencies and can provide an assay to evaluate agents to prevent or alleviate neuronal dysfunction.     

Coinfection of macaques with simian immunodeficiency virus and simian T cell leukemia virus type I: effects on virus burdens and disease progression

To test the hypothesis that coinfection with human immunodeficiency virus (HIV) and human T cell leukemia/lymphoma virus types I or II (HTLV-I or -II) accelerates progression to AIDS, pig-tailed macaques were inoculated with the simian counterparts, SIV and STLV-I. During 2 years of follow-up of singly and dually infected macaques, no differences in SIV burdens, onset of disease, or survival were detected. However, in the first coinfected macaque that died of AIDS (1 year after infection), >50% of CD4+ and CD8+ lymphocytes expressed CD25. On the basis of the low incidence of HTLV-I- and STLV-I-associated disease during natural infections, this early evidence of neoplastic disease was unexpected. While these results demonstrate that coinfection with SIV and STLV-I has no influence on the development of immunodeficiency disease, they do establish a reliable macaque model of persistent STLV-I infection.     

Fas antigen expression and apoptosis of lymphocytes in macaques infected with simian immunodeficiency virus strain mac

The surface of early Earth was exposed to both UVC radiation (< 280 nm) and higher doses of UVB (280-315 nm) compared with the surface of present day Earth. The degree to which this radiation environment acted as a selection pressure on organisms and biological systems has rarely been theoretically examined with respect to the biologically effective irradiances that ancient organisms would receive. Here action spectra for DNA inactivation and isolated chloroplast inhibition are used to estimate biologically effective irradiances on archean Earth. Comparisons are made with present day Earth. The theoretical estimations on the UV radiation screening required to protect DNA on archean Earth compare well with field and laboratory observations on protection strategies found in present day microbial communities. They suggest that many physical and biological methods may have been effective and would have allowed for the radiation of life even under the high UV radiation regimes of archean Earth. Such strategies would also have provided effective reduction of photoinhibition by UV radiation. The data also suggest that the UV regime on the surface of Mars is not a life limiting factor per se, although other environmental factors such as desiccation and low temperatures may contribute towards the apparent lack of a surface biota.     

Human immune responses to influenza virus vaccines administered by systemic or mucosal routes

Healthy adult volunteers were immunized by parenteral or oral routes with trivalent inactivated influenza vaccine (A/Chile/1/83 (H1N1), A/Mississippi/1/85 (H3N2), and B/Ann Arbor/1/86), or intranasally with live attenuated, cold-adapted influenza type A/Texas/1/85 (H1N1) reassortant virus. In all volunteers, cells spontaneously secreting IgA, IgG or IgM antibodies specific to influenza virus were detected in peripheral blood on days 6-13 after immunization, and specific IgA, IgG and IgM antibodies to influenza vaccine were measured in sera and external secretions (saliva and nasal lavage). Following systemic immunization, a raise in specific antibodies of all isotypes was observed in sera beginning on day 13. Although small variations in IgA and IgM antibodies in saliva and nasal lavages were detected, antigen-specific IgG significantly increased between days 13 and 27. Intranasal administration of attenuated virus induced IgA and IgG antibodies in serum as well as in secretions. Serum antibodies were not substantially influenced by oral immunization, only a small increase in all isotypes was observed in volunteers' sera 21 days after ingestion of vaccine. However, in secretions, antigen-specific IgA and IgG responses were detected one week after immunization and reached a peak response on day 20. These studies show that different routes of immunization can be effective for the induction of specific antibodies, and support the concept of the common mucosal immune system in humans by demonstrating that the oral or intranasal administration of antigen-induced specific antibodies of IgA isotype in external secretions, preceded by the transient appearance in peripheral blood of specific antibody-producing cells.     

Prophylaxis after occupational exposure to human immunodeficiency virus (HIV)

Reasons for seeking consultation among health care workers due to potential or supposed risk of HIV infection were analyzed. From August 1990 till July 1996 41 health care providers were consulted including: 22 nurses, 1 student of nursing college, 3 midwives, 4 laboratory workers and 7 physicians (surgeons and gynaecologist). Type of exposure to HIV and applying of safety precautions were evaluated in each case. In 10 cases the offer of postexposure prophylaxis with zidovudine was accepted (6 nurses, 1 student of nursing college, 3 surgeons). Exposure to HIV was described as: needlestick immediately after it was used in a HIV/AIDS patient, injury with a surgical needle while operating on an HIV infected blood. In the remaining cases the fear of HIV infection was due to work without protective gloves (nurses, laboratory workers), performing surgery on HIV (+) patient, (surgeons, nurses) or short-time contact of HIV infected blood with undamaged skin (nurses). Following conclusions can be drawn from our study: 1. Health care workers undertake safety precautions only when they are informed about HIV seropositivity of the patient. 2. Patients whose HIV serologic status is not known are considered not to create health risk for medical staff. 3. The level of knowledge of health care workers about risk of acquiring HIV infection, lack of risk and ways of diminishing the risk is poor. 4. None of followed health care workers was HIV-seropositive after occupational exposure to HIV.     

Pathogenesis of simian immunodeficiency virus pneumonia: an immunopathological response to virus

We investigated the effects of dietary factors on the pH and the ammonia emission from slurry of growing-finishing pigs. Sixteen male hybrid pigs (80 to 90 kg BW) were allotted to one of four diets based on barley-wheat, tapioca, barley-tapioca, and sugar beet pulp. Diets were formulated to have similar NE and CP contents and a similar lysine:NE ratio. Diets differed in nonstarch polysaccharide content (NSP) and dietary electrolyte balance (dEB). Urine and feces were daily collected quantitatively in metabolism cages and mixed as a slurry at the end of the collection period. After mixing, the pH and the ammonia emission from the slurry were measured daily in a laboratory setup for 7 d at 20 degrees C. The type of diet affected the pH of the slurry and the ammonia emission (P < .001). The pH of the slurry from pigs fed the sugar beet pulp-based diet was .8 unit lower and ammonia emission was 52 to 53% lower than that of the other three diets. The low dEB and high NPS sugar beet pulp-based diet increased the VFA concentration and reduced the pH and ammonia emission from the slurry. We conclude that dietary NSP and dEB influence the pH and ammonia emission from slurry of growing-finishing pigs.     

Slower evolution of human immunodeficiency virus type 1 quasispecies during progression to AIDS

The evolution of human immunodeficiency virus type 1 (HIV-1) quasispecies at the envelope gene was studied from the time of infection in 11 men who experienced different rates of CD4+ cell count decline and 6 men with unknown dates of infection by using DNA heteroduplex mobility assays. Quasispecies were genetically homogeneous near the time of seroconversion. Subsequently, slower proviral genetic diversification and higher plasma viremia correlated with rapid CD4+ cell count decline. Except for the fastest progressors to AIDS, highly diverse quasispecies developed in all subjects within 3 to 4 years. High quasispecies diversity was then maintained for years until again becoming more homogeneous in a subset of late-stage AIDS patients. Individuals who maintained high CD4+ cell counts showed continuous genetic turnover of their complex proviral quasispecies, while more closely related sets of variants were found in longitudinal samples of severely immunocompromised patients. The limited number of variants that grew out in short-term PBMC cocultures were rare in the uncultured proviral quasispecies of healthy, long-term infected individuals but more common in vivo in patients with low CD4+ cell counts. The slower evolution of HIV-1 observed during rapid progression to AIDS and in advanced patients may reflect ineffective host-mediated selection pressures on replicating quasispecies.     

Efficacy of 9-(2-phosphonylmethoxyethyl)adenine treatment against chronic simian immunodeficiency virus infection in macaques

Long-tailed macaques chronically infected with simian immunodeficiency virus (SIV) were treated for 4 or 8 weeks with daily subcutaneous doses of the antiretroviral compound 9-(2-phosphonylmethoxyethyl)adenine (PMEA). The efficacy of PMEA was evaluated by monitoring cell-free virus in plasma, virus titer and viral DNA in peripheral blood mononuclear cells, and absolute numbers of lymphocyte subsets. In mock-treated control macaques, virus titers changed minimally. However, in treated macaques, PMEA exhibited impressive effects, leading to the disappearance of virus in the blood within the first week of treatment and lasting through the fourth week of treatment. The results indicate that PMEA can effectively reduce SIV in chronically infected macaques and offer an optimistic perspective for therapeutic intervention against human immunodeficiency virus infection.     

Common themes of antibody maturation to simian immunodeficiency virus, simian-human immunodeficiency virus, and human immunodeficiency virus type 1 infections

Characterization of virus-specific immune responses to human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) is important to understanding the early virus-host interactions that may determine the course of virus infection and disease. Using a comprehensive panel of serological assays, we have previously demonstrated a complex and lengthy maturation of virus-specific antibody responses elicited by attenuated strains of SIV that was closely associated with the development of protective immunity. In the present study, we expand these analyses to address several questions regarding the nature of the virus-specific antibody responses to pathogenic SIV, SIV/HIV-1 (SHIV), and HIV-1 infections. The results demonstrate for the first time a common theme of antibody maturation to SIV, SHIV, and HIV-1 infections that is characterized by ongoing changes in antibody titer, conformational dependence, and antibody avidity during the first 6 to 10 months following virus infection. We demonstrate that this gradual evolution of virus-specific antibody responses is independent of the levels of virus replication and the pathogenicity of the infection viral strain. While the serological assays used in these studies were useful in discriminating between protective and nonprotective antibody responses during evaluation of vaccine efficacy with attenuated SIV, these same assays do not distinguish the clinical outcome of infection in pathogenic SIV, SHIV, or HIV-1 infections. These results likely reflect differences in the immune mechanisms involved in mediating protection from virus challenge compared to those that control an established viral infection, and they suggest that additional characteristics of both humoral and cellular responses evolve during this early immune maturation.     

Mycobacterium avium complex in macaques with AIDS is associated with a specific strain of simian immunodeficiency virus and prolonged survival after primary infection

Mycobacterium avium complex (MAC) in simian immunodeficiency virus (SIV)-infected macaques is a frequent opportunistic infection that shares many features with the condition in human AIDS patients. A retrospective analysis of necropsies on 135 macaques with SIV-induced simian AIDS that received neither antiretroviral nor antimicrobial therapy revealed that 17% (23/135) were infected with MAC. MAC developed in 31.3% (21/67) of the animals inoculated with uncloned SIVmac251 versus 1.9% (1/53) and 6.7% (1/15) of the animals inoculated with the molecular clones SIVmac239 and SIVmac239/316EM, respectively (P = .001). This is the first example in which the risk of infection with a specific opportunistic organism was affected by the infecting strain of immunodeficiency virus. In addition, animals with MAC had a longer mean survival after primary infection and lower CD4 cell counts at death than animals that did not develop this opportunistic infection. The SIV-inoculated macaque is a valuable model in which to study the pathogenesis of MAC in the immunocompromised host.     

Administration of interleukin 13 to simian immunodeficiency virus-infected macaques: induction of intestinal epithelial atrophy

Increase Th2 cytokine production may contribute to some clinical manifestations of HIV infection, and studies have suggested that IL-13 rather than IL-4 is involved in these conditions. We directly tested this hypothesis by administrating IL-13 to SIV-infected macaques. SIV-infected rhesus macaques received a daily subcutaneous injection for 21 days of either IL-13 (10 microg/kg/day) or a placebo. The four macaques treated with IL-13 experienced body weight loss (9.95 +/- 0.71%) related to intestinal tract damage: they all suffered from a complete atrophy of duodenal villi. This was presumably due to premature epithelial cell death: proliferating Ki67+ cells in glandular crypts were as numerous as in control animals, but many epithelial cells developed apoptosis. The duodenal mucosa was infiltrated with cells expressing CD56 and PEN5, two markers of NK cells, and there was a deregulation of local cytokine and chemokine production characterized by a decrease in IL-10 gene expression (25% of controls) and an increase in gene expression for IFN-gamma (4-fold control), MIP-1alpha (8-fold control), and MIP-1beta (13-fold control). Thus, IL-13 can induce digestive epithelial cell injury in vivo in primates infected with a retrovirus. Therefore, its role should be considered in digestive manifestations of HIV infection as well as in other disorders associated with intestinal epithelial atrophy.     

Diverse host responses and outcomes following simian immunodeficiency virus SIVmac239 infection in sooty mangabeys and rhesus macaques

Sooty mangabeys naturally infected with simian immunodeficiency virus (SIV) do not develop immunodeficiency despite the presence of viral loads of 10(5) to 10(7) RNA copies/ml. To investigate the basis of apathogenic SIV infection in sooty mangabeys, three sooty mangabeys and three rhesus macaques were inoculated intravenously with SIVmac239 and evaluated longitudinally for 1 year. SIVmac239 infection of sooty mangabeys resulted in 2- to 4-log-lower viral loads than in macaques and did not reproduce the high viral loads observed in natural SIVsmm infection. During acute SIV infection, polyclonal cytotoxic T-lymphocyte (CTL) activity coincident with decline in peak plasma viremia was observed in both macaques and mangabeys; 8 to 20 weeks later, CTL activity declined in the macaques but was sustained and broadly directed in the mangabeys. Neutralizing antibodies to SIVmac239 were detected in the macaques but not the mangabeys. Differences in expression of CD38 on CD8(+) T lymphocytes or in the percentage of naive phenotype T cells expressing CD45RA and CD62L-selection did not correlate with development of AIDS in rhesus macaques. In macaques, the proportion of CD4(+) T lymphocytes expressing CD25 declined during SIV infection, while in mangabeys, CD25-expressing CD4(+) T lymphocytes increased. Longitudinal evaluation of cytokine secretion by flow cytometric analysis of unstimulated lymphocytes revealed elevation of interleukin-2 and gamma interferon in a macaque and only interleukin-10 in a concurrently infected mangabey during acute SIV infection. Differences in host responses following experimental SIVmac239 infection may be associated with the divergent outcome in sooty mangabeys and rhesus macaques.     

Neutralization sensitivity of cell culture-passaged simian immunodeficiency virus

CEMx174- and C8166-45-based cell lines which contain a secreted alkaline phosphatase (SEAP) reporter gene under the control of a tat-responsive promoter derived from either SIVmac239 or HIV-1(NL4-3) were constructed. Basal levels of SEAP activity from these cell lines were low but were greatly stimulated upon transfection of tat expression plasmids. Infection of these cell lines with simian immunodeficiency virus (SIV) or human immunodeficiency virus type 1 (HIV-1) resulted in a dramatic increase in SEAP production within 48 to 72 h that directly correlated with the amount of infecting virus. When combined with chemiluminescent measurement of SEAP activity in the cell-free supernatant, these cells formed the basis of a rapid, sensitive, and quantitative assay for SIV and HIV infectivity and neutralization. Eight of eight primary isolates of HIV-1 that were tested induced readily measurable SEAP activity in this system. While serum neutralization of cloned SIVmac239 was difficult to detect with other assays, neutralization of SIVmac239 was readily detected at low titers with this new assay system. The neutralization sensitivities of two stocks of SIVmac251 with different cell culture passage histories were tested by using sera from SIV-infected monkeys. The primary stock of SIVmac251 had been passaged only twice through primary cultures of rhesus monkey peripheral blood mononuclear cells, while the laboratory-adapted stock had been extensively passaged through the MT4 immortalized T-cell line. The primary stock of SIVmac251 was much more resistant to neutralization by a battery of polyclonal sera from SIV-infected monkeys than was the laboratory-adapted virus. Thus, SIVmac appears to be similar to HIV-1 in that extensive laboratory passage through T-cell lines resulted in a virus that is much more sensitive to serum neutralization.