Although several studies have used single recombinant viral proteins as ELISA antigens for measurement of antibody response to hRSV and hMPV [14, 15], this is the first time that the same set of sera comprising a wide range of ages is tested to compare the specific antibody response against the F protein of the two viruses. To do so, the fusion protein of each virus was partially purified and used in an ELISA to detect the presence of specific antibodies in a large cohort of individuals with ages ranging from 0 to 89 years. Since the F protein of each virus was expressed in different systems and bearing in mind that post-transductional modifications do not occur in bacteria and the ones in insect cells differ from the ones in mammalian cells, the antigenicity of both proteins was first tested by Western Blot analysis with a positive pool of human sera. A band corresponding with the expected molecular size of each recombinant fusion protein was confirmed (Figure 1A and 1B).
By the age of 2 to 5 years most children have been infected at least once with both hRSV and hMPV [1, 4]. In the present report, the youngest group of individuals with ages between 0 and 2 years showed the lowest antibody response to either virus. A broad range of ages are included in this group with a high percentage of children younger than 1 year old. The immaturity of the immune system of these infants could explain the low antibody level. Furthermore, the level of antibodies in this group could be even lower, since some of these sera samples may contain maternally derived antibodies, which may not represent true seropositivity due to infection. A rise in the antibody response against both viruses in the groups of individuals with ages between 2–69 years old was detected, probably as result of re-infections. Finally, the oldest group of patients from 70 to 89 years old, showed a significant antibody response towards both viruses, however they still comprise one of the groups at high risk of infection with elevated rates of morbidity . Previous studies have shown that the percentage of neutralizing antibodies against hRSV in the frail people (> 80 years old) is very low, despite having a considerable level of specific antibodies against the virus. This poor neutralizing antibody response is probably one of the causes for the susceptibility of this group of individuals to develop severe disease after infection with hRSV . A recent study has analyzed the neutralizing ability to inhibit hMPV replication using the same set of sera used in the present work . No correlation between neutralizing ability of a given serum and antibody levels was found. Most strikingly there was an already high neutralizing capacity of the sera of the youngest group of patients although this group had the highest rate of seronegativity. For the 70–79 age group the seropositivity was 57.9 % versus 90 % neutralization. The same was true for the other age groups in which the presence of antibodies does not necessarily mean that there is also a high level of neutralizing antibodies. Although neutralizing ability is commonly associated with the presence of neutralizing antibodies in a serum sample, in the Lusebrink et al. study the authors claim that is not solely the neutralizing antibodies which inhibit hMPV replication but also other neutralizing factors, such us interferon. Taking this into consideration it is not surprising that both studies do not correlate, although the set of sera used for both studies was identical. In concert with the study by Lüsebrink et al. it appears likely that neutralizing antibodies play a minor role in the control of hMPV and hRSV infections in humans, a hypothesis that is supported by the fact that virtually all approaches to develop a potent and long lasting vaccine against both viruses have failed so far.
In the present study the F protein of NL/1/99 strain belonging to the B1 sublineage of hMPV and the F protein of the Long strain of subgroup A of hRSV have been used. Although, as mentioned in the introduction, the variability of the F protein belonging to the different subgroups and sublineages of hRSV and hMPV is low, in the developed ELISAs, some anti-F antibodies could be disregarded and therefore false negative results could be potentially obtained in both cases. However, it is well known that anti-hRSV antibodies directed against the F protein are cross-reactive for strains of both subtypes (A and B) , and studies on hMPV using the F protein as a subunit vaccine have indicated similar antibody reactivity patterns against homologous and heterologous hMPV infections [20, 21].
Finally, as shown in Table 1, most age groups showed that the prevalence of hRSV was higher than that of hMPV, however children with ages from 0 to 9 years old had higher rates of seropositivity to hMPV than to hRSV. Several groups have studied the seroprevalence of hMPV and hRSV in young children, with different results. For instance, in The Netherlands all children have been infected with hMPV by the age of 5 . In Canada, the majority of patients over 16 years old were seropositive for hMPV . In Japan, Ebihara et al found that seropositive rates of hMPV were lower than those of hRSV in all children between 1 month and 5 years and a recent study in China showed that seropositive rates of hMPV in children 6 months to 6 years of age were significatively lower than seropositive rates of hRSV [22, 23]. This variety of results may reflect different infection patterns between the two viruses, but also different seroprevalence patterns between geographical locations.
In summary, the present study shows that the distribution of hRSV and hMPV in the general healthy population in the area of Bonn (Germany) is very similar, with hRSV infections more frequent that those of hMPV in most age groups. This kind of analysis appears to be an appropriate instrument for seroepidemiological studies, although it should be used in combination with neutralization test for a more comprehensive analysis.