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Indian Journal of Medical Microbiology, Vol. 29, No. 4, October-December, 2011, pp. 363-367 Original Article Seroepidemiological study of human metapneumovirus in New Delhi, India S Banerjee1, WM Sullender2, RK Ahuja3, S Broor4 1 Department of Microbiology, All India Institute of Medical Sciences, New Delhi- 110029, India Date of Submission: 29-Apr-2011 Code Number: mb11089 PMID: 22120795 Abstract
Purpose: There are a few seroepidemiological studies reported on human metapneumovirus (hMPV) as hMPV was only discovered in the year 2001. This respiratory virus has been reported to be ubiquitous and associated with respiratory tract infections in all age groups. The present study aimed at determining the prevalence of antibodies to hMPV in children and adults of 1 month to 55 years of age. Keywords: Enzyme linked immunosorbant assay, human metapneumovirus, seroprevalence Introduction A new paramyxovirus isolated in the Netherlands from children with respiratory tract infections in 2001 was identified as a member of the genus Metapneumovirus, of subfamily Pneumovirinae, of the family Paramyxoviridae. Being the only member of the genus that infects human, it was designated as human metapneumovirus (hMPV). It has been detected worldwide from children, adults, elderly and immunocompromised individuals. [1],[2],[3],[4],[5],[6] However, hMPV was only recently detected in India in 2004. [7] There are no data from developing countries that provide serologic information of the age-related prevalence of infections with hMPV. Titres of antibodies to hMPV were determined to define the age-related prevalence of hMPV infections in a small study group from Delhi without any concurrent respiratory infection. Materials and Methods Study samples Serum samples of children and adults of either sex visiting hospital for illness other than respiratory diseases were included in the study. Twenty sera were randomly selected in the age groups of 0-5 years, >5-15 years, >15-25 years, >25-35 years, >35-55 years. The Ethics Committee of the institute approved the use of these remnant samples from the clinical virology laboratory. Antigen preparation LLCMK2 (rhesus monkey kidney) cell monolayers were infected with 1 ml of 1:100 dilution of the standard strain of hMPV, CAN97-83 (kindly provided by Dr. Guy Boivin, Quebec, Canada) in the presence of 5 μg/ml of trypsin (Sigma Aldrich Corp., St. Louis, MO, USA). The cells were observed daily for 21 days. The infected/uninfected monolayer was scraped into the medium when 75-100% cytopathic effect was observed. The cells, centrifuged and the pelleted were re-suspended in 500 μl lysis buffer (25mM Tris, 0.15M NaCl, 1% Triton X-100, 1% Deoxycholic acid). Cell lysate was incubated on ice for 15 min and clarified by centrifugation at 4°C. The clarified cell lysate was used as antigen for ELISA. Both mock antigen (antigen prepared from uninfected LLCMK2 cells) and viral lysate antigen (antigen prepared from LLCMK2 cells infected with hMPV) were used in ELISA. The antigens were aliquoted and stored in -20°C. Determination of optimal dilution of antigens and anti-human IgG horse radish peroxidase conjugate for ELISA To determine the optimal dilution of anti-human IgG horse radish peroxidase (HRP) conjugate (Bangalore Genei Ltd, Bangalore, India) to be used in the ELISA, different dilutions of conjugate were tested with human serum by checker board titration. Similarly, to determine the optimal dilution of antigens (viral lysate antigen and mock antigen) to be used in the ELISA, different dilutions of antigens were tested with a known hMPV-positive serum (obtained from Dr. Wayne Sullender at UAB) by the checker board titration method. Human metapneumovirus lysate ELISA Wells of high binding polystyrene micro titre plates (Corning Inc., MA, USA) were coated with 50 μl of optimal dilution of hMPV lysate/mock antigen and the plates were dried overnight at 37°C. The non-specific sites of the plates were then blocked with 5% blotto in PBST (5% dry milk in PBS containing 0.5% Tween 20) and the plates were incubated at 37°C for 1 h. Fifty microlitres of serum samples in double dilution (1:20 to 1:2560; diluent: 5% blotto in PBST) were added in duplicate to wells coated with viral lysate/mock antigen. The plates were incubated at 37°C for 1 h. This was followed by 10 washes with PBST. Fifty microlitres of optimal dilution of antihuman IgG HRP conjugate (Bangalore Genei Ltd, Bangalore, India) (diluent: 1X PBS supplemented with 5% goat serum) was added to each well of the plates. The plates were incubated at 37°C for 1 h and washed 10 times with PBST. Fifty microlitres of tetramethylbenzidine solution (TMB) (Bangalore Genei Ltd, Bangalore, India) was added to the wells and the plates were incubated in dark for 20 min at room temperature. The chromogenic reaction was then stopped by the addition of 50 μl of 2M H 2 SO 4 and OD was measured at 450 nm. A known serum positive for hMPV antibody was included in each run as a positive control, to ensure the reproducibility of the results. The cut-off value was calculated in the following two ways. In one method, cut off value was calculated by doubling the mean OD value of triplicate wells containing known positive serum with mock antigen. [8],[9] The OD obtained due to binding of anti-hMPV antibodies in sera to mock antigen reflects the non-specific binding of the anti-hMPV antibodies as well that of the secondary conjugate. In the second method, the cut off was calculated by tripling the mean OD of three wells having PBS instead of sera to bind to viral lysate antigen. The OD obtained by PBS and viral lysate interaction would reflect only the non-specific binding of the secondary conjugate. The mean OD of the negative controls were doubled in the first method and tripled in the second method to get a stringent cut-off value. For both these methods, the cut off was calculated to be ~ 0.2. The sample-specific OD value was calculated as the value at 450 nm for mock antigen subtracted from the mean OD value for the hMPV antigen. A sample was considered positive when the specific OD was higher than the cut off value of 0.2. To calculate the titre of antibody to hMPV in each of the serum samples the highest dilution of serum that was positive for hMPV antibody was determined. This is the end point dilution of the serum sample. The titre of antibody to hMPV for that sample was the reciprocal of the end point dilution. Statistical analysis The chi square test was done to evaluate significant differences in the seropositivity between different age groups (0-5 years, >5 to 15 years, >15 to 25 years, >25 to 35 years and >35 to 55 years) in the study. The Kruskal-Wallis test (NPar test) was used to evaluate differences in the (median) titre of antibodies to hMPV between the different age groups. Results Cut-Off OD value for ELISA The mean OD at 450 nm of a known positive serum reacted with mock antigen (negative control) was 0.098 and the mean OD at 450 nm of viral lysate antigen reacted with PBS (negative control) was found to be 0.062. The cut off OD value used in this study was set at 0.200 i.e., approximately twice the OD 450 nm value of mock antigen reacted with hMPV positive serum or, three times the OD 450 nm value of negative control with no test sera. Optimal concentration of anti-human horse radish peroxidase conjugate for ELISA The dilution of the conjugate that gave a specific OD of ≥ 0.200 with the maximum dilution (1:1280) of human sera was 1:60,000 (1 unit). Four units of conjugate was used in the assay i.e., ~ 1:15,000. Optimal concentration of antigens for ELISA The highest dilution of antigen which gave a specific OD of ≥ 0.200 with the known hMPV positive serum at dilution 1:160 was 1:150. This was considered as one unit of antigen. The optimal concentration of antigen used in the ELISA was 1 unit of antigen (1:150). Seroprevalence of Human Metapneumovirus Antibody to hMPV was found in 79 of the 100 serum samples tested. [Table - 1] shows hMPV seroprevalence by age groups. The maximum seroprevalence was seen in adults of >35 to 55 years of age (90%), and the minimum seroprevalence was found in children of 1 month to 5 years of age (60%) [Figure - 1]. There were no statistically significant differences in the percent seropositivity between different age groups [χ2 =6.27, P=0.18, (NS)]. Geometric Mean Titre of Human Metapneumovirus Antibody in Different Age Groups Geometric mean titre of hMPV antibody in each age group is also shown in [Table - 1] and [Figure - 1]. The lowest GMT was seen in the broad age group 0-5 years (180); however, within the sub-group 0-6 months, antibody titres were higher (508). The antibody titres subsequently declined until 2 years of age and then began to rise. High antibody titres were subsequently maintained throughout life and peaked in the age group 35-55 years. No statistically significant differences were found in the median titre in different age groups [χ2 =5.08, P=0.28, (NS)]. Discussion In this study, an in-house ELISA was developed using hMPV-infected LLCMK-2 cell lysate as antigen to screen sera quantitatively for hMPV IgG antibodies. Serological studies have been carried out with viral lysate antigen, [4] as well as recombinant viral proteins as antigen [10],[11],[12] and the latter was found to be more sensitive. [13] One issue in our in-house serological assay is that whether the antigen (prepared from hMPV strain of one serotype) used in the assay will be able to detect antibodies against both serotypes. Several studies have shown 100% concordance in detecting antibodies to both the lineages of hMPV using recombinant proteins (especially N, M and F proteins) as well as infected LLCMK-2 cells. [10],[11],[12],[14],[15] Thus, our in-house ELISA also most probably detected antibodies to both serotypes of hMPV. A high seroprevalence of hMPV antibody was detected in the present study. By 5 years of age, 85% of children had been exposed to hMPV infection. This is similar to the previous findings that have shown that hMPV infection is common in childhood. [14],[15],[16],[17] In children below 5 years of age, seropositivity was found to be 50% in the Netherlands [8] and 45%-50% in Japan. [14],[15] However, a Canadian study reported very low seroprevalence (13.5%) of hMPV antibody in children below 5 years of age [12] which may be due to variation in the prevalence of hMPV in different geographical locations. In the present study, the seroprevalence to hMPV was 50% in infants below 1 year of age. The seropositivity increased to 67% in children >1 to 2 years of age and further to 71% in children >2 to 5 years of age. Consistent with our results are results from the Netherlands, that reported 25% of children of 6 months to 1 year age, 55% of children of 1 to 2 years and 70% of children of 2 to 5 years age had serological evidence of hMPV infection. [16] In previous studies from Japan and China [14],[15],[18] a high seropositivity (60-74.5%) in infants below 6 months of age have been reported, which declined till 1 year of age and then again increased in children with increasing age and by 5-6 years of age 76-90% of seropositivity was observed. In a study by Leung et al., very high seropositivity was observed in infants within 5 months of age, which was found to decrease till 2 years of age after which the prevalence to hMPV antibody increased with age. [11] Among hospital referred South African children, a decline in seropositive rate (22%) was observed up to 10 months. From 10 months onward, the seropositive rate increased to 92% in children aged 2 to 3 years. [19] In all these studies, including the present study, the trend of acquisition of antibodies to hMPV was similar. The high seroprevalence (50%) in children within 6 months of age may be due to maternally derived antibodies, which further signifies the high seroprevalence in adult population that might lead to high seropositivity in these infants. Among children >6 months to 1 year of age, the seroprevalence to hMPV was low, may be due to waning maternally acquired antibody. In children >1 year of age, the seroprevalence to hMPV rose, as is observed in the Dutch, Canadian, Japanese, Chinese and the present study. In the study by Leung et al., seropositivity increased in children above 2 years of age. [11] All these reports suggested that primary infections with hMPV occur in children > 1 or 2 years of age. The seroprevalence to hMPV rose to 80% in children >5 years from 60% in children < 5 years of age. This high seroprevalence was observed throughout adulthood and the highest seraprevalence of 90% was seen in adults above >35 years. In the Netherlands and Japan, 100% seoprevalence was observed in children >5 years and 10 years of age respectively. [14],[16] In contrast to the results of the present study and that of the Netherlands, Japan and China, in the Canadian study, the seroprevalence to hMPV was found to be very low in children below 15 years (32.3%) of age and 99% seroprevalence was reported in individuals above 16 years of age. [12] This further suggests that primary infection in the Netherlands, [16] Japan, [14] China [18] and in India may have occurred much earlier than in Canada. [12] Consistent with the results of this study that showed a high seroprevalence to hMPV in adults are reports elsewhere [4],[12],[14] that showed 95-100% seropositivity in adults. In this study, the titre of antibody to hMPV in children within 5 years of age was high (180) with the highest titres (508) in first 6 months of life which may be as a result of active placental IgG transport of maternal antibody. However, due to waning maternally acquired immunity, the titre was found to decrease in children 6 months to 2 years of age. The increase in antibody titre was seen in children above 2 years of age which may be due to primary infection with hMPV. This is consistent with other reports that suggest a primary infection with hMPV in children > 2 years of age. [14],[16] The titre of antibody to hMPV kept on increasing with age with the highest titre in the age group >35 to 55 years i.e., 419. In studies in the Netherlands and Japan, a high titre of hMPV antibody was maintained throughout adulthood. [14],[16] The results of the present study only show the trend of acquisition of hMPV antibody in different age groups. The differences in seropositivity and antibody titre in different age groups in this study were found to be statistically non-significant because of the small sample size and wide range of titre of antibodies in each age group. In conclusion, our results indicate that infection with hMPV is common in this population. Most individuals are likely to be infected during childhood. The continued increase in antibody titres into adulthood provides evidence that re-infection with hMPV is common. This study is the first to assess the seroprevalence of hMPV in India. Acknowledgement We thank Council for Scientific and Industrial research (CSIR) for financial support to Sagarika Banerjee and National Institute of Health (NIH) for providing financial assistance for the project. We would also like to thank Mr. Yashpal Yadav and Ms. Prema Nair for technical assistance. References
Copyright 2011 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb11089t1.jpg] [mb11089f1.jpg] |
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