M Patrzałek

P Albrecht
email: piotr.albrecht@acn.waw.pl

M Sobczynski

Significant decline in pneumonia admission rate after the introduction of routine 2+1 dose schedule heptavalent pneumococcal conjugate vaccine (PCV7

Significant decline in pneumonia admission

rate after the introduction of routine 2+1 dose schedule heptavalent pneumococcal conjugate vaccine (PCV7) in children under 5 years of age in Kielce, Poland M. Patrzalek, P. Albrecht, M.

Introduction

Acute respiratory infections (ARIs) are among the leading causes of childhood morbidity and mortality, especially in developing countries [START_REF] Williams | Estimates of world-wide distribution of child deaths from acute respiratory infections[END_REF]. The World Health Organization (WHO) data clearly indicate that Streptococcus pneumoniae is the most frequent cause of deaths worldwide and is responsible for >1.6 million deaths annually of children under 5 years of age, with pneumonia being the major cause [START_REF]WHO Global Immunization Vision and Strategy[END_REF]. Before the introduction of heptavalent pneumococcal conjugate vaccine (PCV7), the incidence of pneumonia in children below 5 years of age in Europe and USA was, similar to in Poland, around 34-40/1,000 [START_REF] Foy | Rates of pneumonia during influenza epidemics in Seattle, 1964 to 1975[END_REF][START_REF] Murphy | Pneumonia: an eleven-year study in a pediatric practice[END_REF][START_REF] Mcconnochie | Lower respiratory tract illness in the first two years of life: epidemiologic patterns and costs in a suburban pediatric practice[END_REF]. S. pneumoniae was considered to be responsible for 17-44% of pneumonia-related hospitalizations in children in this age group [START_REF] Michelow | Epidemiology and clinical characteristics of community-acquired pneumonia in hospitalized children[END_REF][START_REF] Korppi | Aetiology of community-acquired pneumonia in children treated in hospital[END_REF][START_REF] Drummond | Community acquired pneumonia-a prospective UK study[END_REF]. PCV7 was included in the vaccination calendar initially in the USA (2000), and then, gradually, in about 45 other countries. Clinical trials showed that PCV7 had originally led to a completely unexpected reduction in the frequency of pneumonia, dating back to children below 5 years of age, depending on the applied analysis protocol, of 23.4% (Northern California Kaiser Permanente; NCKP) [START_REF] Black | Efficacy, safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in children. Northern California Kaiser Permanente Vaccine Study Center Group[END_REF] or 30.3% (NCKP; reanalysis) [START_REF] Black | Effectiveness of heptavalent pneumococcal conjugate vaccine in children younger than five years of age for prevention of pneumonia[END_REF] and intent-to-treat appropriately of 17.7% [START_REF] Black | Efficacy, safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in children. Northern California Kaiser Permanente Vaccine Study Center Group[END_REF] and 25.5% [START_REF] Black | Effectiveness of heptavalent pneumococcal conjugate vaccine in children younger than five years of age for prevention of pneumonia[END_REF]. In the postlicensure surveillance, Grijalva et al. [START_REF] Grijalva | Decline in pneumonia admissions after routine childhood immunisation with pneumococcal conjugate vaccine in the USA: a time-series analysis[END_REF] demonstrated that, for the end of the year 2004 compared with the pre-vaccination era (1997)(1998)(1999), in children up to 2 years of age, a 39% reduction in all-cause pneumonia admission rates and a 65% fall in pneumococcal pneumonia hospitalizations could be observed. Comparing similar periods, Zhou et al. [START_REF] Zhou | Health care utilization for pneumonia in young children after routine pneumococcal conjugate vaccine use in the United States[END_REF] demonstrated in children below 2 years of age a 52% reduction in the hospitalization rate and a 41.1% reduction in the number of clinic visits caused by all-cause pneumonia and 57.6 and 46.9%, respectively, for pneumococcal pneumonia (all differences were statistically significant; p<0.001). Adam and Fehnle [START_REF] Adam | Safety and effectiveness against respiratory tract infections for pneumococcal conjugate vaccine co-administered with routine vaccine combinations[END_REF], in a study in which the 3+1 schedule was being applied, demonstrated a 38.4% reduction in all-cause pneumonia in risk groups, as compared to 10.9% in children without risk factors. Durando et al. [START_REF] Durando | Collaborative Group for Pneumococcal Vaccination in Liguria (2009) Universal childhood immunisation against Streptococcus pneumoniae: the five-year experience of Liguria Region, Italy[END_REF], in an examination carried out on a free-of-charge PCV7 immunization program in Liguria, used a similar scheme to that of the present study (2+1 dose schedule in children aged 3, 5, and 11-12 months) and demonstrated a 15.2% reduction in all-cause pneumonia versus a 70.5% reduction in pneumococcal pneumonia.

In Poland, PCV7 remains the only recommended chargeable vaccination. In October 2008, the vaccinations were implemented at no cost only for children from risk groups. The overall vaccine uptake in Poland in the first year of life exceeded 98% and is constantly increasing for pneumococcal vaccination; it amounted to 41,000, 154,000, and 225,000 doses in 2006, 2007, and 2008, respectively. Starting from the end of 2008 to the end of January 2010, children from risk groups received a total of 50,000 doses of PCV7 (on average, 2,260 doses monthly).

Kielce is, so far, the only city in Poland where PCV7 was implemented as a compulsory and free-of-charge vaccination for all children in the 2+1 dose schedule system. There is only one children's hospital in the city which was allowed to be considered in our quasiexperimental design, in which we attempted to assess the influence of mass pneumococcal vaccination on the radiologically confirmed pneumonia admission rate, comparing their number in children in the age groups 0-1 year and 2-4 years in 2004 and 2005 (before implementing vaccination) and in 2007 and 2008 (two years after implementing city-wide immunization). We introduced the 2+1 dose schedule, which is officially recommended for mass vaccination, is employed in different countries, including Norway, Denmark, Finland, Italy, Ireland, Iceland, Sweden, and the United Kingdom, and also has an indirect effect among the unvaccinated population (herd immunity). The 3+1 dose schedule is recommended in Poland for individual vaccination and in risk groups as giving more reliable protection against invasive pneumococcal disease (IPD). For logistic reasons, we start PCV7 vaccination in the third or fourth month of life, which is somewhat later than that generally recommended. Nonetheless, according to some data [START_REF] Adam | Safety and effectiveness against respiratory tract infections for pneumococcal conjugate vaccine co-administered with routine vaccine combinations[END_REF][START_REF] Käyhty | Immunogenicity and tolerability of a heptavalent pneumococcal conjugate vaccine administered at 3, 5 and 12 months of age[END_REF][START_REF] Esposito | Immunogenicity, safety and tolerability of heptavalent pneumococcal conjugate vaccine administered at 3, 5 and 11 months post-natally to pre-and fullterm infants[END_REF], this delay may render a better immunological response .

We also decided to check whether mass vaccination give rise to the so-called herd immunity effect manifested by a fall of pneumonia incidents in adults.

Materials and methods

In the period 2004-2008, the number of residents of Kielce dropped from approximately 209,000 to approximately 206,000. Despite the consistent drop in population number, the annual birth rate was still growing from 1,675 in 2004 to 1,879 in 2008.

In March 2006, by virtue of decision of the Kielce Municipal Council, city-wide free PCV7 vaccination (Prevenar®) was commenced. It involved children born from 1st January 2006 with permanent place of residence in Kielce. A 2+1 immunization scheme was applied-the first dose at the third or fourth month of life, and the second at the fifth or sixth month of life, combined with routine mandatory vaccinations-DTP, Hib, IPV; the third booster dose was administered in the 12-14th month, after vaccination with MMR vaccine.

According to the Provincial Sanitary and Epidemiological Station in Kielce, the performance rate of the vaccination was approximately 99% in the period analyzed. These data were double-checked. In the same period, the vaccination uptake was similar to other vaccinations: hepatitis B 99.84%; DTP + IPV 99.03%; and MMR 99.21% in 2006; hepatitis B 99.90%; DTP + IPV 99.45%; MMR 99.3%; and Hib 99.34% in 2007. Polyclinic Children's Hospital is the only children's hospital in Kielce, thus, almost all of the children living in the city requiring hospitalization are admitted to there.

To assess the effect of pneumococcal vaccination (PCV7) on the pneumonia-related hospitalization rate, data concerning the number of hospitalizations in 2007 (second year of vaccination) and 2008 (third year) were compared with data concerning pneumonia hospitalizations in 2004 and 2005, i.e., before the vaccination program. In order to ascertain that the reported decline in hospitalization rate was not due to incidental causes, we compared the rates for pneumonia and acute diarrhoea (ICD10 codes: A04, A08, and A09). The hospitalization rate in the analyzed period (2006)(2007)(2008)) demonstrated a rising trend in all of the analyzed codes. Significance of the trends was verified using the log-linear model with quasi-Poisson errors.

Diagnosis of pneumonia was made on the basis of history, physical examination, and routine investigations (blood count, C-reactive protein [CRP], and erythrocyte sedimentation rate [ESR]). To ensure maximum reliability of the analysis, only pneumonia cases confirmed radiologically by two independent radiologists were taken into consideration and all cases of pneumonia were reassessed. Nearly all cases were coded in ICD10 as J18 and only a very small number as J15. None were encrypted as J13.

Data concerning the general number of pneumonia cases in Kielce for all age groups were derived from the Świętokrzyski Provincial Branch of the National Health Fund in Kielce.

The incidence rates were presented as the number of cases per 1,000 population and were calculated on the basis of the data available at the National Centre for Statistics in Poland (GUS). Analysis of covariance (ANCOVA) as a case of the general linear model (GLM) with binomial and negative binomial error structure was used for modeling pneumonia incidence. p-values <0.05 were considered as statistically significant. All data were analyzed using R version 2.2.1 and its "base" and "MASS" packages [START_REF]A language and environment for statistical computing[END_REF].

Results

Table 1 encompasses the number of hospitalizations due to radiologically confirmed pneumonia in children aged 0-1 year and 2-4 years, as well as the size of the population of Kielce in the years 2004, 2005, 2007, and 2008. In 2004 and 2005, i.e., in the period without mass vaccination, the incidence rate in the first year of life was 46.96/1,000 and 35.68/1,000, respectively (mean: 41.32/1,000), and in children aged 2-4 years, it was 6.88/1,000 and 5.32/1,000, respectively (mean: 6.11/1,000). Figure 1 The risk of pneumonia hospitalization in children in their first year of life and between their second and fourth years was modeled by a binomial distribution. Analysis based on the model revealed that the reduction of incidence rate in subsequent years in both age groups, as observed in Fig. 1, varied but was statistically significant (for children in their first year of life p<0.0000001, for children aged 2-4 years p=0.01). As early as in the first year after the implementation of vaccination, the number of hospitalized pneumonia cases in children aged 0-1 year decreased by almost 65%, and in children aged 2-4 years, it decreased by 23%. In 2008, a subsequent, but lower, decrease in hospitalization rate in both groups was observed, as presented in Fig. 1. The decrease was 10% in the group aged 0-1 year and 18% in the group aged 2-4 years. During the same period (2006)(2007)(2008), the frequency of the admission rate for acute diarrhea as the internal control group showed a growing tendency. The log-linear model with quasi-Poisson errors revealed the observed trends to be significant (p=0.0173) for all ICDs and there were no differences between A04, A08, and A09 in the rates of growth because the interaction between year and kind of ICDs was not significant.

Analysis of pneumonia incidence in individuals over 4 years of life revealed a significant diversity. Table 2 Figure 2 presents the incidence rate per 1,000 residents in all of the age groups analyzed. The lowest incidence is observed in individuals aged between 30-49 years.

ANCOVA revealed that patient age is the only factor influencing the risk of disease. The positive slope of the variable indicates that the risk increases with age (p=0.00133). However, no evidence was found to prove that the implementation of mass vaccination resulted in a decrease of the incidence rate in individuals aged over 4 years (p=0.5335). The risk of pneumonia in nonvaccinated people in subsequent years remained unchanged in any age group. Interaction between the year of the study and patient age is not significant (p=0.321). It refers also to the age group 18-39 years, analyzed by Grijalva et al. [START_REF] Grijalva | Decline in pneumonia admissions after routine childhood immunisation with pneumococcal conjugate vaccine in the USA: a time-series analysis[END_REF], with the highest risk of contact with children. In our study, the incidence in this group was 0.71/1,000.

Discussion

We report that, after the introduction of a routine 2+1 dose schedule PCV7 immunization program in Kielce, Poland, admission rates for radiologically confirmed pneumonia decreased significantly for children younger than 5 years old who were the target population of the program. The reduction in the first year of life was 65% and between 2 and 4 years of age, it was 23%. This reduction in radiologically confirmed pneumonia provides an estimate of the vaccine-preventable burden of childhood pneumonia admissions attributable to S. pneumoniae in young children before PCV7 was introduced.

Randomized clinical trials showed that pneumococcal conjugate vaccines prevent pneumonia [START_REF] Black | Efficacy, safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in children. Northern California Kaiser Permanente Vaccine Study Center Group[END_REF][START_REF] Zhou | Health care utilization for pneumonia in young children after routine pneumococcal conjugate vaccine use in the United States[END_REF][START_REF] Adam | Safety and effectiveness against respiratory tract infections for pneumococcal conjugate vaccine co-administered with routine vaccine combinations[END_REF][START_REF] Cutts | Efficacy of nine-valent pneumococcal conjugate vaccine against pneumonia and invasive pneumococcal disease in The Gambia: randomised, double-blind, placebo-controlled trial[END_REF][START_REF] Klugman | Vaccine Trialists Group (2003) A trial of a 9-valent pneumococcal conjugate vaccine in children with and those without HIV infection[END_REF]. Efficacy estimates, however, varied according to the case definition of pneumonia. Vaccine efficacy against clinically diagnosed pneumonia was 6% for PCV7 in the NCKP trial [START_REF] Black | Efficacy, safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in children. Northern California Kaiser Permanente Vaccine Study Center Group[END_REF] and 7% for a nine-valent PCV in the Gambian trial [START_REF] Cutts | Efficacy of nine-valent pneumococcal conjugate vaccine against pneumonia and invasive pneumococcal disease in The Gambia: randomised, double-blind, placebo-controlled trial[END_REF]. Vaccine efficacy estimates were consistently higher when radiological confirmation was included as part of the case definition-18% in the Kaiser Permanente trial [START_REF] Black | Effectiveness of heptavalent pneumococcal conjugate vaccine in children younger than five years of age for prevention of pneumonia[END_REF], 17% in the South African trial [START_REF] Klugman | Vaccine Trialists Group (2003) A trial of a 9-valent pneumococcal conjugate vaccine in children with and those without HIV infection[END_REF], and 37% in the Gambian trial [START_REF] Cutts | Efficacy of nine-valent pneumococcal conjugate vaccine against pneumonia and invasive pneumococcal disease in The Gambia: randomised, double-blind, placebo-controlled trial[END_REF].

When the NCKP data were re-analyzed according to WHO radiological criteria for pneumonia diagnosis, the estimated vaccine efficacy was 26% [START_REF] Black | Effectiveness of heptavalent pneumococcal conjugate vaccine in children younger than five years of age for prevention of pneumonia[END_REF]. The post-marketing study by Grijalva et al. [START_REF] Grijalva | Decline in pneumonia admissions after routine childhood immunisation with pneumococcal conjugate vaccine in the USA: a time-series analysis[END_REF] revealed a 39% decrease in the all-cause pneumonia admission rate among children up to 2 years of age and a 65% decrease in the rate of pneumonia defined as pneumococcal. Zhou et al. [START_REF] Zhou | Health care utilization for pneumonia in young children after routine pneumococcal conjugate vaccine use in the United States[END_REF], comparing similar periods for children under 2 years of age, showed a 52% reduction of hospitalization and a 41.1% reduction of outpatient visits due to all-cause pneumonia and 57.6 and 46.9% for pneumococcal pneumonia, respectively.

Our findings are similar to the results of Grijalva et al. [START_REF] Grijalva | Decline in pneumonia admissions after routine childhood immunisation with pneumococcal conjugate vaccine in the USA: a time-series analysis[END_REF], Zhou et al. [START_REF] Zhou | Health care utilization for pneumonia in young children after routine pneumococcal conjugate vaccine use in the United States[END_REF], and Durando et al. [START_REF] Durando | Collaborative Group for Pneumococcal Vaccination in Liguria (2009) Universal childhood immunisation against Streptococcus pneumoniae: the five-year experience of Liguria Region, Italy[END_REF]. It can be that, in an open population, as in our study, the effect of a PCV7 immunization program is greater than that seen in clinical trials because herd immunity can have an important role in the reduction of disease in both vaccinated and unvaccinated people, through decreases in the nasopharyngeal carriage of vaccine serotypes [START_REF] O'brien | The potential indirect effect of conjugate pneumococcal vaccines[END_REF][START_REF] Levine | Pneumococcal vaccination in developing countries[END_REF]. Decline in the rates of all-cause pneumonia admissions in Kielce, Poland, suggests that S. pneumoniae was a major contributor to the burden of pneumonia admissions in young children in this town [START_REF] Mcintosh | Community-acquired pneumonia in children[END_REF][START_REF]Preventing pneumococcal disease among infants and young children. Recommendations of the Advisory Committee on Immunization Practices (ACIP)[END_REF], similar to in the USA, and provides an estimate of the burden of pneumococcal pneumonia admissions in young children due to PCV7 vaccine serotypes.

Unfortunately, in most cases in Poland, including in the city of Kielce, establishing the etiology of pneumonia is very difficult due to economic, logistic, and technical reasons. The research groups experienced these difficulties while conducting a sponsored epidemiological study [START_REF] Grzesiowski | Polish Pediatric IPD Group (2008) Invasive pneumococcal disease in children up to 5 years of age in Poland[END_REF] concerning the IPD frequency in Poland. In a 2-year period, only 134 samples fit for determining the etiological factor reached the central reference laboratory, of which as many as 108 were culture-negative.

Further, our study suggests a substantial effect on one of the most common reasons for hospital admissions not only in Kielce but throughout Poland and in other countries [START_REF] Levine | Pneumococcal vaccination in developing countries[END_REF][START_REF] Grijalva | Promises and challenges of pneumococcal conjugate vaccines for the developing world[END_REF][START_REF] Wardlaw | Pneumonia: the leading killer of children[END_REF].

Observations similar to those for S. pneumoniae were made for Haemophilus influenzae type B pneumonia in Gambia, where substantial reductions in overall pneumonia were attributable to immunization with conjugate vaccines against this bacterium [START_REF] Mulholland | The Gambian pneumococcal vaccine trialimplications for control of childhood pneumonia[END_REF].

As evidenced in this study, surprisingly favorable results of PCV7 immunization may also be related to the relatively high serotype coverage of PCV7 vaccine in the Polish population, which, according to our findings, amounted to 73-77% in children under 5 years of age [START_REF] Grzesiowski | Polish Pediatric IPD Group (2008) Invasive pneumococcal disease in children up to 5 years of age in Poland[END_REF].

The 3+1 scheme of PCV7 vaccination is generally recognized and applied in many countries. However, the Municipal Council in Kielce decided to apply the 2+1 scheme, as it was not only cheaper, which was a particularly important factor in this case, but also, as evidenced in numerous studies [START_REF] Adam | Safety and effectiveness against respiratory tract infections for pneumococcal conjugate vaccine co-administered with routine vaccine combinations[END_REF][START_REF] Käyhty | Immunogenicity and tolerability of a heptavalent pneumococcal conjugate vaccine administered at 3, 5 and 12 months of age[END_REF][START_REF] Esposito | Immunogenicity, safety and tolerability of heptavalent pneumococcal conjugate vaccine administered at 3, 5 and 11 months post-natally to pre-and fullterm infants[END_REF][START_REF] Van Gils | Effect of reduced-dose schedules with 7-valent pneumococcal conjugate vaccine on nasopharyngeal pneumococcal carriage in children: a randomized controlled trial[END_REF], was as efficient in a mass vaccination pattern as the original 3+1 scheme. The scheme was, thus, approved for population vaccination also in the registration dossier. Also in Poland, the 2+1 scheme proved to be efficient in population vaccination.

Previous findings showed that the group aged between 18 and 39 years had the second highest percentage decline in invasive pneumococcal disease after children younger than 5 years of age, suggesting a vaccine herd effect [START_REF] O'brien | The potential indirect effect of conjugate pneumococcal vaccines[END_REF]. This age group includes parents of young children, so they could have benefited from reduced exposure to pneumococci when their children were immunized with PCV7.

The lack of an apparent decrease in pneumonia incidence in individuals after the fourth year of life, i.e., the so-called population effect, may be related to too a short period of vaccination and our observation, as well as to imperfect coding and classification of pneumonia in adults, in particular, in outpatient settings.

The increase in birth rate, observed despite a systematic decrease in the city population size, may be explained by the willingness of young mothers to take advantage of free vaccination of their children, and, thus, their registration for permanent residence in Kielce.

This exceptionally high vaccination rate (99% vs., for instance, 93.4% reached in the best year in the Liguria Region program [START_REF] Durando | Collaborative Group for Pneumococcal Vaccination in Liguria (2009) Universal childhood immunisation against Streptococcus pneumoniae: the five-year experience of Liguria Region, Italy[END_REF]) resulted not only from the good performance and efficacy of vaccination points, but most likely also from free-of-charge access to the vaccine. The cost of complete PCV7 vaccination, even in the 2+1 scheme, under the conditions of a private healthcare system, amounts to at least 25% of the average monthly salary in Poland.

Our study provides a comprehensive assessment of the changes in pneumonia admission rates after implementation of the PCV7 immunization program in Kielce, Poland. We used a strong quasi-experimental design to assess the effect of PCV7 vaccination. The observed reductions in radiologically confirmed pneumonia admissions suggest that, before the introduction of PCV7, S. pneumoniae was a major contributor to childhood pneumonia in Kielce, Poland, and provide an estimate of the burden of pneumococcal pneumonia admissions in young children due to PCV7 vaccine serotypes. Our results support the constantly growing evidence of the beneficial effects of the pneumococcal conjugate vaccines in children. Further, this substantial effect obtained in this study targeted one of the most common reasons for hospital admissions in Poland.

  presents the number of hospitalized pneumonia cases per 1,000 children residing in Kielce in the form of an average of two years preceding vaccination (2004 and 2005) and two subsequent years (2007 and 2008) after implementation of the vaccination program.

Table 1

 1 presents Size of the child population in selected age groups and the number of pneumonia cases before (2004 and 2005) and after (2007 and 2008) implementation of the vaccination program

	Age (years)	Year
		2004		2005		2007		2008
		N	n	N	n	N	n	N	n
	0-1	3,279	154	3,279	117	3,534	53	3,774	51
	2-4	4,937	34	4,882	26	4,858	23	4,939	19

N-number of living children; n-number of pneumonia cases Fig.

1

Pneumonia hospitalization rates in age groups 0-1 and 2-4 years in Kielce before and after the introduction of pneumococcal vaccinations the population size and number of pneumonia cases within four age groups in particular years. As is clearly visible, the highest incidence involved individuals aged above 65 years. Before the vaccination "era" in 2004, the incidence was 17.40/1,000 in this age group and 19.38/1,000 in 2005 (mean: 18.41/1,000). In 2007, the incidence in this age group was even higher, amounting to 20.48/1,000, but then it decreased to 16.91/1,000 in 2008.

  Incidence rate per 1,000 residents of Kielce in the various age groups analyzed

	Table 2 Size of the population
	of Kielce before (2004 and	Year	Age (years)
	2005) and after (2007 and 2008)
	implementation of the vaccina-		0-29		30-49		50-64		65+
	tion program in particular age groups and number of pneumo-		N	n	N	n	N	n	N	n
	nia cases
		2004	81,479	282	58,787	110	42,247	178	26,942	469
		2005	78,964	222	58,046	77	43,587	191	27,596	535
	N-number of living individu-	2007	74,210	192	57,351	103	45,989	217	28,357	581
	als; n-number of pneumonia	2008	72,114	130	57,079	85	47,056	211	28,845	488
	cases
	Fig. 2


Marieke I Both

Mathilde A Overvest

Mark F Wildhagen

Jean Golding

Hajo I J Wildschut
email: h.wildschut@erasmusmc.nl

Á H I J Wildschut

The association of daily physical activity and birth outcome: a population-based cohort study

Keywords: Birth weight, Gestational duration, Motor activity, Premature birth, Perinatal mortality Abbreviations CI Confidence interval OR Odds ratio ALSPAC Avon Longitudinal Study of Parents and Children antenatal exercise, yoga, squash, badminton, swimming, brisk walking, weight training, cycling

The potential relationship between daily physical activity and pregnancy outcome remains unclear because of the wide variation in study designs and physical activity assessment measures. We sought to prospectively quantify the potential effects of the various domains of physical activity on selected birth outcomes in a large unselected population. The sample consisted of 11,759 singleton pregnancies from the Avon longitudinal study of parents and children, United Kingdom. Information on daily physical activity was collected by postal questionnaire for self-report measures. Main outcome measures were birth weight, gestational age at delivery, preterm birth and survival. After controlling for confounders, a sedentary lifestyle and paid work during the second trimester of pregnancy were found to be associated with a lower birth weight, while 'bending and stooping' and 'working night shifts' were associated with a higher birth weight. There was no association between physical exertion and duration of gestation or survival. Repetitive boring tasks during the first trimester was weakly associated with an increased risk of preterm birth (\37 weeks) (adjusted odds ratio [OR] = 1.25, 95% CI 1.04-1.50). 'Bending and stooping' during the third trimester was associated with a reduced risk of preterm birth (adjusted OR = 0.73, 95% CI 0.63-0.84). Demanding physical activities do not have a harmful effect on the selected birth outcomes while a sedentary lifestyle is associated with a lower birth weight. In the absence of either medical or obstetric complications, pregnant women may safely continue their normal daily physical activities should they wish to do so.

Introduction

Birth weight has a major impact on the general health and survival of infants [START_REF] Stein | Intrauterine growth retardation: epidemiological issues and public health significance[END_REF]. Two important processes govern birth weight, i.e., the duration of gestation and the intrauterine growth rate. Low birth weight is thus caused by either early delivery or intrauterine growth restriction (IUGR), or a combination of both [START_REF] Kaltreider | Epidemiology of preterm delivery[END_REF][START_REF] Tucker | Epidemiology of preterm birth[END_REF][START_REF] Kramer | Determinants of low birth weight: methodological assessment and meta-analysis[END_REF].

There has been limited study on the effect of daily physical activity on birth outcome, and the results are inconsistent. Some researchers report a positive relation between the various domains of physical activity and birth outcome, while others find either a negative relationship or none at all [START_REF] Leiferman | The effect of regular leisure physical activity on birth outcomes[END_REF][START_REF] Sternfeld | Exercise during pregnancy and pregnancy outcome[END_REF][START_REF] Campbell | Recreational exercise and occupational activity during pregnancy and birth weight: a case-control study[END_REF][START_REF] Hatch | Maternal exercise during pregnancy, physical fitness, and fetal growth[END_REF][START_REF] Hall | Effects of aerobic and strength conditioning on pregnancy outcomes[END_REF][START_REF] Hatch | Do standing, lifting, climbing, or long hours of work during pregnancy have an effect on fetal growth?[END_REF][START_REF] Lokey | Effects of physical exercise on pregnancy outcomes: a meta-analytic review[END_REF][START_REF] Klebanoff | The effect of physical activity during pregnancy on preterm delivery and birth weight[END_REF][START_REF] Hatch | Maternal leisure time exercise and timely delivery[END_REF][START_REF] Nieuwenhuijsen | the ALSPAC Study Team. Swimming and birth weight[END_REF][START_REF] Kramer | Aerobic exercise for women during pregnancy[END_REF][START_REF] Marcoux | The effect of leisure time physical activity on the risk of pre-eclampsia and gestational hypertension[END_REF][START_REF] Rudra | A prospective analysis of recreational physical activity and preeclampsia risk[END_REF][START_REF] Magnus | Recreational physical activity and the risk of preeclampsia: a prospective cohort of Norwegian women[END_REF][START_REF] Osterdal | Does leisure time physical activity in early pregnancy protect against pre-eclampsia? Prospective cohort in Danish women[END_REF]. Most prior studies [START_REF] Sternfeld | Exercise during pregnancy and pregnancy outcome[END_REF][START_REF] Hall | Effects of aerobic and strength conditioning on pregnancy outcomes[END_REF][START_REF] Kramer | Aerobic exercise for women during pregnancy[END_REF] focused on the association of vigorous physical activity and birth outcome in selected groups of pregnant women, such as aerobic dancers and elite athletes. Authors of three recent reviews [START_REF] Chasan-Taber | Assessment of recreational physical activity during pregnancy in epidemiologic studies of birthweight and length of gestation: methodologic aspects[END_REF][START_REF] Schlu ¨ssel | Physical activity during pregnancy and maternal-child health outcomes: a systematic literature review[END_REF][START_REF] Lagerros | Physical activity-the more we measure, the more we know how to measure[END_REF] concluded that the majority of epidemiological studies in this field observed a neutral or protective effect of physical activity on selected maternal-child health outcomes. However, given the wide variation in study designs and physical activity assessment measures, it was impossible to provide an overall quantitative estimate of risk.

Physical activity may be defined as 'any bodily movement produced by skeletal muscle that results in energy expenditure' [START_REF] Caspersen | Physical activity, exercise and physical fitness: definitions and distinctions for health-related research[END_REF]. The magnitude of the physiological response to physical activity is determined by age, physical conditions, body weight, body position, concurrent physiological adaptations to pregnancy and psychological factors. Since physical activity has many interrelated dimensions, its quantification is very complex. Several techniques are available for assessing physical activity. Indirect assessment of physical activity by questionnaire is the most practical and widely accepted approach in largescale epidemiological studies [START_REF] Chasan-Taber | Assessment of recreational physical activity during pregnancy in epidemiologic studies of birthweight and length of gestation: methodologic aspects[END_REF][START_REF] Schlu ¨ssel | Physical activity during pregnancy and maternal-child health outcomes: a systematic literature review[END_REF][START_REF] Lagerros | Physical activity-the more we measure, the more we know how to measure[END_REF].

The purpose of our study was to quantify the magnitude of the associations between daily physical activity and birth outcome measures such as birth weight, duration of gestation and perinatal death.

Methods

Data

The research data were derived from a large populationbased cohort consisting of pregnant women who took part in the Avon Longitudinal Study of Parents and Children (ALSPAC) [START_REF] Golding | ALSPAC study team. AL-SPAC-the Avon Longitudinal Study of Parents and Children. I. Study methodology[END_REF]. ALSPAC is a geographically based birth cohort designed to analyze environmental and other factors that might affect the health and development of children. Pregnant women who lived in one of three health districts in Bristol, UK were approached. The core sample consisted of pregnant women who were due to deliver between April 1st 1991 and December 31st 1992. A total of 14,663 pregnant women (i.e., approximately 85% response) were enrolled in the study, completed antenatal questionnaires, and agreed to the collection of detailed birth record information. For the current analyses, women with multiple pregnancies (n = 390), non-white women (n = 2,513) and those who were delivered prior to 16 weeks' gestation (n = 1) were excluded. After these exclusions, the remaining research sample consisted of 11,759 singleton pregnancies, yielding an equivalent number of infants of whom 11,737 were born alive.

Measurement of exposure

Physical activity was assessed by asking questions pertaining to regular physical activities, including housework and occupational activity (Table 1) using a validated postal questionnaire for self-report measures of habitual activity [START_REF] Wildschut | The potential value of a short self-completion questionnaire for the assessment of habitual physical activity in pregnancy[END_REF]. Information concerning daily activities was asked once during the first trimester of pregnancy and once in the second trimester. Employment status was ascertained by the question 'are you currently in paid work?' and was reassessed in the second and third trimester, respectively. Furthermore there were questions enquiring about shift work including night shifts, standing and repetitive boring tasks. The response options were divided in dichotomous categories, i.e., 'yes' and 'no'.

Furthermore, women were asked to report their engagement in leisure time physical activities, including sports such as jogging, cycling, and squash, among others. These variables were categorized into four response options, i.e., 'never', 'less than 1 h per week', 'between 2 and 6 h per week' and 'more than 7 h per week'.

Additional information on the questionnaires can be obtained from the authors.

Measurement of background variables and potential confounders

Variables considered affecting the birth outcome measures of interest were identified from a literature search [START_REF] Kramer | Determinants of low birth weight: methodological assessment and meta-analysis[END_REF][START_REF] Khoury | Congenital malformations and intrauterine growth retardation: a population study[END_REF][START_REF] Herceg | Risk factors and outcomes associated with low birthweight delivery in the Australian Capital Territory 1989-90[END_REF][START_REF] Mercer | The preterm prediction study: a clinical risk assessment system[END_REF][START_REF] Orr | Psychosocial stressors and low birth weight in an urban population[END_REF][START_REF] Zimmer-Gembeck | Low birthweight in a public prenatal care program: behavioral and psychosocial risk factors and psychosocial intervention[END_REF][START_REF] Fairley | Changing patterns of inequality in birthweight and its determinants: a population-based study, Scotland 1980-2000[END_REF][START_REF] Pompeii | Physical exertion at work and the risk of preterm delivery and smallfor-gestational-age birth[END_REF]. These potentially confounding variables were then sought in the ALSPAC data set. Variables with more than 20% missing or inappropriate values were excluded from the analyses since these could unduly distort the effect of other variables in the model. For this reason the variables 'partner's weight', 'partner's height' and 'maternal social economic status based on maternal occupation' were excluded.

Information on background variables was obtained from the pregnant women using self-reported questionnaires. A history of low birth weight was assessed by the question: ''were any of your babies under 5 lb 80z (2,500 g) at birth?''. History of preterm birth by the question; ''were any of your babies born more than 3 weeks early?'' History of hypertension was determined by the response to ''have you ever had hypertension (high blood pressure)?''. Current smoking habits were addressed by the following trimester-specific questions: ''did you smoke regularly during the first 3 months of pregnancy?'' (first trimester); ''did you smoke regularly during the last 2 weeks ?'' (second trimester) and ''how many cigarettes per day are you smoking at the moment''(third trimester). The ALSPAC questionnaires were designed to determine actual smoking habits at each trimester of pregnancy thereby minimizing recall bias and equivocal answers when the mother had decided to quit smoking at some point during each trimester. For each trimester, the responses regarding smoking were regrouped into a dichotomous variable: 'yes-no'. Subjective maternal health (''how would you describe your health?'') was assessed by the following response options: 1 always fit & well; 2 usually fit & well; 3 sometimes unwell; 4 often unwell 5 always unwell. The woman's pre-pregnancy body mass index (BMI) was recoded from a continuous variable into a categorical variable, divided in 6 groups: \20, 20-24.99, 25-29.99, 30-34.99, 35-39.99, C40. The level of maternal stress was measured in the second and third trimester by the derived variable 'Crown Crisp score', adding anxiety, depression and somatic scales into one variable.

Outcome measures

Three birth outcome measures were analyzed, i.e., crude 'birth weight' in grams (continuous variable), 'gestational duration' in weeks as a continuous variable or as 'preterm birth', i.e., before 37 completed weeks of gestation (y/n) as a categorical variable and 'survival' (y/n) as a categorical variable. The variable 'survival' was recoded into two categories, i.e., (1) 'fetal death from 16 weeks' onward or neonatal death occurring the first 7 days after birth' and (2) 'alive after 7 days'.

Statistical analyses

First, to identify an association with the outcomes of interest, univariate analyses of background variables and potential confounders were carried out using chi-square, t-test, ANOVA and correlation coefficients, depending on the continuous or dichotomous nature of the data (SPSS 14.0.1). Next, statistically significant variables (P \ 0.05) emerging from the univariate analyses were entered in the multivariate analyses together with each measurement of exposure. The variable 'birth weight' was adjusted for 'gestational duration'. However, 'gravidity' and 'live births' were excluded due to their similarity to 'parity'. Also, 'maternal height' and 'maternal weight' were excluded due to their inherent similarity to 'maternal BMI'. The multivariate analyses were conducted in three different stages reflecting the trimester specific exposure sequence i.e., (1) variables included were known in the first trimester of pregnancy and emerged as statistically significant from the univariate analyses, (2) statistically significant variables were known in the first and second trimesters were included, and (3) statistically significant variables were included if known in all three trimesters of pregnancy.

Backward linear regression analyses were used for the outcome measures 'birth weight' and 'gestational duration', while binominal logistic regression was used for 'survival' and 'preterm birth'. Regression analyses for all outcome measures were done separately for each physical activity variable. The final results of the three multivariate analyses are reported in the form of regression coefficients ('b') together with the 95% confidence intervals (CI) or odds ratios (OR). In the regression analyses, we used the critical inclusion value for significance at the P = 0.05 level (two tailed probability) and the critical exclusion value at the P = 0.15 level.

Results

Approximately 4.0% (n = 464) of women delivered an infant weighing less than 2,500 grams, while 4.2% (n = 494) delivered prior to 37 weeks' gestation. In total 11,720 out of 11,759 (99.7%) infants survived the antenatal and perinatal period.

Tables 2,3, and 4 demonstrate the univariate associations between the potentially confounding variables with birth weight (Table 2), gestational duration (Table 3) and preterm birth (Table 4). Tables 5 and6 demonstrate the variables of trimester specific exposure to various physical activities showing the adjusted associations with the birth outcome measures following multivariate analyses. A sedentary lifestyle was found to be independently associated with a lower birth weight. The same is true for having paid work in the second trimester. Having night shifts in the second trimester and 'bending and stooping' in the third trimester were significantly associated with a higher birth weight (Table 5). Doing repetitive boring tasks in the first trimester was associated with an increased risk of preterm birth, while 'working night shifts' in the third trimester was associated with a reduction in the risk of preterm birth (Table 6). None of the physical activity variables were found to have an independent, statistically significant association with gestational duration. Physical activity was not associated with fetal and neonatal survival (results not given).

Discussion

After controlling for a large number of potentially confounding variables and other effect modifiers, we find that physically demanding activities, including sporting activities, are not associated with adverse birth outcome in terms of lower birth weight, gestational duration or poor fetal and neonatal survival. In contrast, a sedentary lifestyle is associated with a small but significant negative effect on birth weight (Table 5). The same is true for having paid work in the second trimester. 'Doing repetitive boring tasks' in the first trimester was the only variable that showed an apparent association with an increased likelihood of preterm birth, although the magnitude of this risk was small. The precise underlying mechanism of the latter finding has not been clarified. Previous research has also observed a negative effect of 'doing repetitive, boring tasks' and 'employment status' on birth outcome. Mame `lle et al. [START_REF] Mame `lle | Prematurity and occupational activity during pregnancy[END_REF] characterised these circumstances by the term 'occupational fatigue'. It consisted of five sources, namely posture, work on an industrial machine, physical exertion, mental stress and environment. They demonstrated a statistically significant association between occupational fatigue and preterm birth. Newman et al. [START_REF] Newman | National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network. Occupational fatigue and preterm premature rupture of membranes[END_REF] showed that each source of occupational fatigue was independently associated with a significantly increased risk of preterm premature rupture of membranes among nulliparous women but not among multiparous women.

One unexpected finding was that (working night shifts) was associated with a slightly increased birth weight. This finding could be explained by the so-called 'healthy worker effect'. Women who are employed tend to be in better health at the offset than those who are out of work. This observation could also be explained by the women's The association of daily physical activity and birth outcome characteristics linked to selective implementation of preventive measures (job withdrawal or reassignment) [START_REF] Croteau | Work activity in pregnancy, preventive measures, and the risk of delivering a small-forgestational-age infant[END_REF]. Others [START_REF] Zhu | Shift work, job stress, and late fetal loss: the national birth cohort in Denmark[END_REF][START_REF] Niedhammer | the Lifeways Cross-Generation Cohort Study Steering Group. Occupational predictors of pregnancy outcomes in Irish working women in the Lifeways cohort[END_REF], however, claim that physical demanding work, such as night and shift work, may increase the risk of adverse birth outcome. From a systematic review of the literature on this topic Bonzini et al. [START_REF] Bonzini | Risk of prematurity, low birth weight, and pre-eclampsia in relation to working hours and physical activities: a systematic review[END_REF] concluded that the balance of evidence tends to favour no effect, or an effect that is no more than a moderate. The strength of our study is its size and prospective nature, thereby minimizing recall bias. In fact, the effect of daily physical activity on the selected birth outcomes could be quantified thereby controlling for a large number of potential confounders and effect modifiers. Moreover, pregnant women were asked about their daily physical activities. A relatively small number of studies have provided longitudinal data on the effect of daily physical activity patterns on birth outcome. Using data from the 1988 National Maternal and Infant Health Survey in the United States, Leiferman et al. [START_REF] Leiferman | The effect of regular leisure physical activity on birth outcomes[END_REF] concluded that regular leisure physical activity during pregnancy had no deleterious effect on pregnancy outcome. In contrast, Hegaard et al. [START_REF] Hegaard | Leisure time physical activity is associated with a reduced risk of preterm delivery[END_REF] recently concluded from a population-based longitudinal study among healthy pregnant women in Denmark that moderate-to-heavy leisure time physical activity was associated with a significantly reduced risk of preterm birth. Others [START_REF] Klebanoff | The effect of physical activity during pregnancy on preterm delivery and birth weight[END_REF][START_REF] Croteau | Work activity in pregnancy, preventive measures, and the risk of delivering a small-forgestational-age infant[END_REF][START_REF] Henriksen | Standing at work and preterm delivery[END_REF][START_REF] Mozurkewich | Working conditions and adverse pregnancy outcome: a meta-analysis[END_REF][START_REF] Croteau | Work activity in pregnancy, preventive measures, and the risk of preterm delivery[END_REF] reported that prolonged periods of standing and physically demanding work are associated with a modestly increased risk of preterm delivery. We could not confirm their findings.

This investigation has several limitations. It was based on questionnaires completed by the pregnant woman and her partner. Self-reported data can be criticized because for their subjective nature. Assessment of daily activities during pregnancy is complex because of the various domains of physical activity. The questionnaires used in our survey tried to overcome this problem by adding questions pertaining to household activities, leisure time activities, and employment status, among others. Wildschut et al. [START_REF] Wildschut | The potential value of a short self-completion questionnaire for the assessment of habitual physical activity in pregnancy[END_REF] argued that this strategy facilitates the understanding of the complex relationship between the way of life and pregnancy outcome. Interpretation of the findings, however, could be hampered by the fact that not all questions were properly validated (e.g., leisure time activities).

Most variables were broadly categorised, lacking a measure for the dimension of the dose of activity (i.e., frequency and duration and intensity). Questions were asked with few answer options, for example 'neversometimes-often-always' or had dichotomous options, e.g., with the questions concerning night and shift work. Unfortunately, no data were available on the frequency of night and shift work. Apart from the broad categorisation, some questions were only asked in certain trimesters. For example, information on daily physical activity was only asked in the first and second trimesters of pregnancy. As the length of the questionnaire was restricted, it was decided by the ALPAC research team at the onset of the study not to include questions pertaining to daily physical activity in the third trimester of pregnancy. In addition, this team was anxious to assess whether the activity level of the prospective mother affect her risk of preterm delivery. Consequently, it was deemed necessary to ask these questions prior to the third trimester. The same is true for the questions concerning exercise, such as jogging and swimming, were only asked in the second trimester, not in the first or third trimester. Because of this, it is impossible to be more precise in the conclusions concerning the effects of these activities during specific trimesters. The same is true for occupational activities. The absence of an association between physical activity and fetal and neonatal survival could be explained by a lack of power. Also, because of the multiple testing of the various domains of physical activity, it is possible that the associations that we have found are chance findings. Furthermore, some of the effect sizes showed a major attenuation after adjustment. This could imply that the remaining effect may be a residual confounding effect. Despite the large number of potential effect modifiers tested, the issue of residual confounding by for instance genetic, environmental or lifestyle factors remains unresolved. Finally, the variable 'maternal social economic status' was not included in the regression analyses because of the high percentage of missing values ([20%). Maternal socio-economic status is based on the occupation of the pregnant woman. The relatively high number of missing values (n = 2,170) can be explained mainly by the categories of teenage women and housewives who do not have a formal occupation. Multiple imputation was considered, but due to the non-random nature of the missing values for this variable a sensitivity analyses was disregarded. It is, however, unlikely that the absence of this variable influenced the findings of our study since other measures of maternal socio-economic status were included in the model, such as paternal socio-economic status, maternal education and house ownership.

Despite these methodological limitations, the findings of our large scale study may be used for generating a policy guideline for physical activity of pregnant women attending antenatal care. In the absence of either medical or obstetric complications, pregnant women may be advised to safely continue their normal daily physical activities should they wish to do so. They can be reassured that physically demanding activities, such as exercise and sports, are not associated with the adverse birth outcomes considered in this study. At the same time, however, our findings do not permit firm conclusions about occupational activities in light of the uncertainties associated with paid work and the birth outcomes of interest.

Table 2

 2 General and trimester-

	specific univariate associations
	of potential confounders and
	birth weight (grams; continuous
	variable)

Table 3

 3 General and trimester-

	specific univariate associations
	of potential confounders and
	gestational duration (weeks)

Table 4

 4 General and trimester-specific univariate associations of potential confounders and preterm birth \37 weeks (categorical)

		Effect size	95% CI	P value b
		(OR a )
	General
	Male sex of child	0.76	0.63, 0.91 0.003
	History of LBW	2.36	1.53, 3.62 \0.001
	History of PTB	2.98	2.04, 4.33 \0.001
	History of spontaneous abortion 1.04	0.77, 1.40 NS
	History of hypertension	1.22	1.04, 1.43 0.015
	Hospitalisation during pregnancy 1.59	1.28, 1.97 \0.001
	Parity	0.79	0.69, 0.90 0.001
	Home ownership	0.97	0.88, 1.07 NS
	Marital status (married)	0.92	0.83, 1.02 NS
	Maternal educational level	1.00	0.89, 1.13 NS
	Maternal social economic status 1.04	0.96, 1.14 NS
	Paternal social economic status	0.90	0.82, 0.99 NS
	Maternal BMI (pre-pregnancy)	1.12	0.95, 1.34 NS
	Paternal weight	1.01	1.00, 1.02 NS
	Paternal height	1.00	0.98, 1.02 NS
	Maternal age at LMP	0.78	0.59, 1.04 NS
	Maternal age at delivery	1.27	0.96, 1.69 NS
	Smoking before pregnancy	0.97	0.60, 1.57 NS
	Drug abuse	1.26	0.77, 2.05 NS
	1st trimester
	Smoking	1.02	0.52, 2.00 NS
	Alcohol use	0.89	0.74, 1.08 NS
	2nd trimester
	Smoking	0.85	0.40, 1.80 NS
	Alcohol use	1.10	0.92, 1.31 NS
	Negative mood	0.99	0.97, 1.01 NS
	Poor maternal health	1.13	1.05, 1.23 0.002
	In paid work	1.06	0.81, 1.38 NS
	Shift work	1.14	0.65, 2.01 NS
	Night shifts	1.80	0.77, 4.20 NS
	3rd trimester
	Smoking	0.90	0.45, 1.83 NS
	Alcohol use	1.34	1.23, 1.45 \0.001
	Negative mood	1.02	0.99, 1.05 NS
	Poor maternal health	1.21	1.08, 1.34 0.001
	In paid work	1.15	0.86, 1.53 NS
	Shift work	0.73	0.30, 1.78 NS
	Night shifts	0.48	0.15, 1.60 NS
	a Odd's ratio

b Statistically significant P \ 0.05

Table 5

 5 Trimester-specific associations with birth weight (in grams, continuous variable) (n = 8,879). Statistically significant variables (P \ 0.05) emerging from the univariate analyses (see Table2) were entered in the final model

			Effect size (b a ):	Effect size (b a ):	95% CI	P value
			unadjusted	adjusted
		1st trimester
		Sitting	-38.4	-21.4	-39.3, -3.5	0.019
		Standing	-10.4	-15.6	-33.6, 2.3	0.088
		2nd trimester
		Sitting 2nd and 3rd trim	-37.1	-21.6	-39.6, -3.7	0.018
		In paid work	-43.7	-17.9	-32.0, -3.9	0.009
		Night shifts	52.1	27.6	11.8, 43.5	\0.001
		3rd trimester
	a Beta (regression coefficient) and CI 95% confidence interval	Bend and stoop Night shifts	130.8 71.6	37.8 91.4	20.3, 55.3 -15.0, 197.8	\0.001 0.092

Table 6

 6 Trimester-specific associations with the risk of preterm birth (categorical variable) (n = 11,123). Statistically significant variables (P \ 0.05) emerging from the univariate analyses (see Table4) were entered in the final model

		Effect size	Effect size	95% CI	P value
		(OR a ):	(OR a ):
		unadjusted	adjusted
	1st trimester
	Repetitive boring tasks	1.22	1.25	1.04, 1.50	0.016
	3rd trimester
	Bend and stoop	0.60	0.73	0.63, 0.84	\0.001
	Night shifts	0.48	0.67	0.47, 0.95	0.025

a OR odds ratio, CI 95% confidence interval

Acknowledgments We are very grateful to all the families who took part in this study, the midwives for their help in recruiting them, and the whole ALSPAC team, which includes interviewers, computer and laboratory technicians, clerical workers, research scientists, volunteers, managers, receptionists and nurses. Also, we would like to thank Narada Bouwland, Bastiaan Overvest, Andy Ness and Carl Weiner for their constructive comments on earlier drafts of this paper. The UK Medical Research Council, the Wellcome Trust and the University of Bristol provide core support for ALSPAC. For this study, there were no other sources of funding.

Conflict of interest statements None declared.

Ethical approval

The law and ethics committee for the Avon longitudinal study of parents and children approved this study.

Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.


M G Belkum

B J Netea

A G Kullberg

Vonk

W W J Van De Sande

J R Lo-Ten-Foe

A Van Belkum

M G Netea

B J Kullberg

A G Vonk
email: a.vonk@erasmusmc.nl

Mycoviruses: future therapeutic agents of invasive fungal infections in humans?

Mycoviruses: future therapeutic agents of invasive fungal infections in humans?

Introduction

Fungal infections such as invasive candidiasis and aspergillosis cause a high morbidity and mortality in immunocompromised patients. Although timely administration of antifungal therapy has a significantly positive influence on the outcome of infection in such patients, treatment failure remains a major problem [START_REF] Pfaller | Epidemiology of invasive candidiasis: a persistent public health problem[END_REF][START_REF] Slobbe | Outcome and medical costs of patients with invasive aspergillosis and acute myelogenous leukemia-myelodysplastic syndrome treated with intensive chemotherapy: an observational study[END_REF]. As well as pivotal development of adjunctive immunotherapy to enforce antifungal host immune response, new therapeutic strategies are needed to selectively combat aspergillosis and other invasive opportunistic fungal diseases.

Mycoviruses are viruses that selectively infect fungi. Although the first reports on mycoviruses date from the 1960s [START_REF] Hollings | Viruses associated with a die-back disease of cultivated mushroom[END_REF], our knowledge and understanding of mycoviruses is still in its infancy. Moreover, current mycovirus publications primarily concern economically important fungi such as cultivated mushrooms, yeasts, and fungal pathogens of plants. To our knowledge, the use of mycoviruses as a tool to combat invasive fungal infection in animals and humans has not yet been suggested, let alone explored in any detail.

Mycovirus biology and interaction with the fungal host

The genetic information of mycoviruses is either encoded by double-stranded RNA (dsRNA), single-stranded RNA with or without a reverse transcription step for replication (ss(+)RNA-RT), or double-stranded DNA (dsDNA) [START_REF] Pearson | Mycoviruses of filamentous fungi and their relevance to plant pathology[END_REF]. The 8th report on virus taxonomy of the International Committee for Taxonomy of Viruses (ICTV) lists more than 90 mycoviruses covering 10 viral families [START_REF] Fauquet | Virus taxonomy: eighth report of the International Committee on Taxonomy of Viruses[END_REF]. A complete list of mycoviruses of plant pathogenic fungi can be found in a recent review by Pearson et al. [START_REF] Pearson | Mycoviruses of filamentous fungi and their relevance to plant pathology[END_REF]. Despite the limited data on mycoviruses of human pathogenic fungi, evidence is provided that mycoviruses do exist in various fungal genera that are pathogenic to humans [START_REF] Bozarth | Mycoviruses: a new dimension in microbiology[END_REF][START_REF] Border | Viruses of Penicillium and Aspergillus species[END_REF][START_REF] Van Diepeningen | Dynamics of dsRNA mycoviruses in black Aspergillus populations[END_REF][START_REF] Van Diepeningen | Intra-and interspecies virus transfer in Aspergilli via protoplast fusion[END_REF][START_REF] El-Sherbeini | Viruses in fungi: infection of yeast with the K1 and K2 killer viruses[END_REF][START_REF] Papp | Presence of double-stranded RNA and virus-like particles in Rhizopus isolates[END_REF][START_REF] Vagvolgyi | Detection of doublestranded RNA molecules and virus-like particles in different Mucor species[END_REF][START_REF] Mehta | Virus-like particles and lytic plaque formation in lawns of Candida albicans[END_REF][START_REF] Matte | Isolation and characterization of a RNA-virus like particle from Candida curvata[END_REF][START_REF] Hammond | Aspergillus mycoviruses are targets and suppressors of RNA silencing[END_REF][START_REF] Nesterova | Virus-like particles in Candida tropicalis[END_REF][START_REF] El-Sherbeini | Geneprotein assignments within the yeast Yarrowia lipolytica dsRNA viral genome[END_REF][START_REF] Aoki | A novel mycovirus associated with four double-stranded RNAs affects host fungal growth in Alternaria alternata[END_REF]. The mycoviruses that are associated with fungal genera that can cause human fungal disease and are also formally known by the ICTV are listed in Table 1. In addition, viral Table 1 List of mycoviruses of fungal genera known to be pathogenic to humans formally named and recognised by the ICTV [11-14, 16, 18]. To date, the majority of characterised mycoviruses of pathogenic fungi have dsRNA genomes. These viruses have subsequently been grouped into five families: Partitiviridae, Totiviridae, Chrysoviridae, Reoviridae, and Hypoviridae [START_REF] Fauquet | Virus taxonomy: eighth report of the International Committee on Taxonomy of Viruses[END_REF]. The families mainly differ in the number of genome segments and the way they are packaged. An almost universal trait of the dsRNA mycoviruses is the presence of capsids (except for Hypoviridae), which are selectively porous protein compartments in which the genome and replicase are contained. Nucleotides diffuse into this compartment and nascent positive sense strands are extruded from it. DsRNA mycoviruses are not surrounded by an envelope. A synopsis of the dsRNA viruses and their characteristics relevant to human pathogenic fungi is provided in Fig. 1.

Transmission of mycoviruses

Intracellular transmission

Mycoviruses are mainly spread by vertical transmission through asexual or sexual spore formation, with asexual sporulation being the most efficient means of transmission. Transmission also occurs horizontally via cytoplasmic exchange during cell division and mating with strains that, depending on the genus, do not have to be genetically compatible for virus transfer to occur [START_REF] Van Diepeningen | Intra-and interspecies virus transfer in Aspergilli via protoplast fusion[END_REF][START_REF] Coenen | Factors affecting the spread of double-stranded RNA viruses in Aspergillus nidulans[END_REF][START_REF] Hillman | Viruses of the chestnut blight fungus, Cryphonectria parasitica[END_REF][START_REF] Van Diepeningen | Heterokaryon incompatibility blocks virus transfer among natural isolates of black Aspergilli[END_REF][START_REF] Liu | Evidence for interspecies transmission of viruses in natural populations of filamentous fungi in the genus Cryphonectria[END_REF]. As a rule, however, when two fusing hyphae are not compatible, they recognize each other as nonself, which triggers programmed cell death (PCD). In aspergilli, the genes involved in activation of PCD reactions are currently being characterised and regarded as a promising approach to combat invasive aspergillosis [START_REF] Fedorova | Comparative analysis of programmed cell death pathways in filamentous fungi[END_REF][START_REF] Davies | Potential link between the NIMA mitotic kinase and nuclear membrane fission during mitotic exit in Aspergillus nidulans[END_REF][START_REF] Fedorova | Genomic islands in the pathogenic filamentous fungus Aspergillus fumigatus[END_REF][26]. Genetic incompatibility is mostly followed by death of the hyphal fusion cell, the so-called heterokaryon, and often of surrounding cells. When this occurs, transmission of mycoviruses fails (Fig. 2). Rejection of heterokaryon formation has been observed to be relatively mild or absent when hyphae of less related Aspergillus species fuse [START_REF] Van Diepeningen | Intra-and interspecies virus transfer in Aspergilli via protoplast fusion[END_REF].

Direct transmission of mycoviruses from one fungal species to another occurred following another experimental means, namely, induced fusion of cell wall-free fungi, socalled protoplasts. Using protoplast fusion, mycoviruses may even be transferred between two fungal compartments of strains that are heterokaryon incompatible [START_REF] Van Diepeningen | Intra-and interspecies virus transfer in Aspergilli via protoplast fusion[END_REF].

Extracellular transmission

Bearing in mind that crossing the cellular membrane is a key step in the infectious life cycle of all viruses, it is understandable that the rigid cell wall in fungi serves as a membrane shielding structure constituting a barrier to extracellular virus uptake. For instance, dsRNA mycoviruses with an average size of 40 nm are approximately nine times the pore-size of the cell wall matrix in Candida species [START_REF] Yphantis | Effect of some proteins on the yeast cell membrane[END_REF]. The cell wall thus prevents the virus from reaching the cell membrane to gain entry. Consequently, mycoviruses are unable to initiate fungal infections by extracellular routes. However, whereas the understanding of (enveloped) virus entry via membrane fusion is well advanced for mammalian viruses or viruses that selectively Fig. 1 List of the predominating genome type of the dsRNA mycoviruses. Taxonomic families that are associated with pathogenic fungi are shown with the genome composition, virus structure, associated hypovirulence, as well as the host range of the respective viral families infect bacteria (bacteriophages), the entry mechanisms for non-enveloped viruses, virus-like particles or naked virus-like dsRNA are still in infancy. Therefore, it may well be that the overall routes and mechanisms of extracellular virus transfer still remain to be elucidated. Infection by the extracellular route could thus far only be achieved in special experimental settings using protoplasts. Applying this method, purified viral particles belonging to the dsRNA families of Partitiviridae, Totiviridae, and Reoviridae successfully infected fungal protoplasts [START_REF] El-Sherbeini | Viruses in fungi: infection of yeast with the K1 and K2 killer viruses[END_REF][START_REF] Sasaki | Infection of Rosellinia necatrix with purified viral particles of a member of Partitiviridae (RnPV1-W8)[END_REF][START_REF] Hillman | A reovirus of the fungus Cryphonectria parasitica that is infectious as particles and related to the coltivirus genus of animal pathogens[END_REF].

Experimental introduction of genetic information

Fungi have been transfected using an infectious complementary DNA (cDNA) copy of mycovirus RNA [START_REF] Chen | cDNA-derived hypovirus RNA in transformed chestnut blight fungus is spliced and trimmed of vector nucleotides[END_REF]. Transfected fungi showed cytoplasmically replicating dsRNA, demonstrating that a synthetic transcript corresponding to the coding strand of mycovirus dsRNA can initiate an infection when introduced into fungal protoplasts [START_REF] Chen | cDNA-derived hypovirus RNA in transformed chestnut blight fungus is spliced and trimmed of vector nucleotides[END_REF]. Again, this has putative therapeutic implications.

Mycelial spread of mycoviruses after transmission

In septate fungi, hyphal compartments are interconnected through hyphal pores. Replicating mycoviruses are thus able to spread throughout the colony via plasma streaming [START_REF] Sasaki | Infection of Rosellinia necatrix with purified viral particles of a member of Partitiviridae (RnPV1-W8)[END_REF][START_REF] Chen | cDNA-derived hypovirus RNA in transformed chestnut blight fungus is spliced and trimmed of vector nucleotides[END_REF] (Fig. 2). If the spread of mycoviruses throughout the colony occurs mainly via plasma streaming and not via fusion with neighbouring hyphae, the viral replication rate may be the limiting factor of transmission. Even after successful mycovirus transmission, persistence may not be obtained. This may be the case when compatible hyphae that form a heterokaryon are both infected with a different mycovirus. Co-infections may be generated, but the mycovirus in the acceptor fungus may also be replaced by the mycovirus from the donor. Indeed, viruses are not always detected in the colonies derived from the protoplast/ hyphae fusion products [START_REF] Van Diepeningen | Intra-and interspecies virus transfer in Aspergilli via protoplast fusion[END_REF]. This suggests that mycoviruses can be selectively eliminated upon regeneration and outgrowth of (transfected) protoplasts as a result of intrahost competition between viruses.

Symptoms associated with mycovirus infection

Generally, mycoviruses cause latent and persistent infections [START_REF] Buck | Fungal virology-an overview[END_REF]. Some mycovirus families are connected with variable phenotypic effects such as hypovirulence or killer phenomena in their host. Hypovirulence is, among other characteristics, defined as reduced pigmentation, reduced asexual sporulation, loss of fertility, or reduced growth rate. Hypovirulence-associated mycoviruses have ssRNA or, mainly, dsRNA genomes (Fig. 1) [START_REF] Van Diepeningen | Dynamics of dsRNA mycoviruses in black Aspergillus populations[END_REF][START_REF] Polashock | Movement of a small mitochondrial double-stranded RNA element of Cryphonectria parasitica: ascospore inheritance and implications for mitochondrial recombination[END_REF][START_REF] Park | Characterization of HOG1 homologue, cpMK1, from Cryphonectria parasitica and evidence for hypovirus-mediated perturbation of its phosphorylation in response to hypertonic stress[END_REF][START_REF] Deng | Comparative analysis of alterations in host phenotype and transcript accumulation following hypovirus and mycoreovirus infections of the chestnut blight fungus Cryphonectria parasitica[END_REF][START_REF] Jiang | Molecular characterization of Penicillium chrysogenum virus: reconsideration of the taxonomy of the genus Chrysovirus[END_REF]. The killer phenomena are induced by proteins encoded by satellite dsRNA that are associated with Totiviruses (discussed below) [START_REF] Schmitt | Unique double-stranded RNAs responsible for the anti-Candida activity of the yeast Hanseniaspora uvarum[END_REF].

Isolation of mycoviruses and maintenance

Once fungi are successfully transfected, the observed infection patterns are very stable when subcultured [START_REF] Van Diepeningen | Intra-and interspecies virus transfer in Aspergilli via protoplast fusion[END_REF][START_REF] Sasaki | Infection of Rosellinia necatrix with purified viral particles of a member of Partitiviridae (RnPV1-W8)[END_REF]. Furthermore, viruses present in Aspergillus isolates are highly efficiently transmitted to asexual progeny [START_REF] Van Diepeningen | Heterokaryon incompatibility blocks virus transfer among natural isolates of black Aspergilli[END_REF].

Limited data exist regarding the amount of mycoviruses that can be isolated from infected fungi and their stability after isolation. Most viruses have only been isolated with a relatively low yield. Hillman et al. showed that 3-6 μg of purified reovirus was obtained from each purification reaction, representing 2 l of infected fungi. These isolated Reoviridae were quite stable and retained infectivity upon prolonged storage [START_REF] Hillman | A reovirus of the fungus Cryphonectria parasitica that is infectious as particles and related to the coltivirus genus of animal pathogens[END_REF]. In contrast, Saccharomyces cerevisiae Totivirus preparations stored refrigerated for three weeks became non-infectious [START_REF] El-Sherbeini | Viruses in fungi: infection of yeast with the K1 and K2 killer viruses[END_REF]. Thus, isolation, production, purity and stability of mycoviruses of interest remain to be assessed.

Mycoviruses and toxins

As stated above, some mycovirus infections have a toxinmediated killing capacity. Although a filamentous basidiomycete fungus, Ustilago maydis, is known to secrete killer toxins encoded by cytoplasmic dsRNA mycoviruses that are lethal to mycovirus-free susceptible strains of the same fungus [START_REF] Park | The Ustilago maydis virally encoded KP1 killer toxin[END_REF][START_REF] Gage | KP4 fungal toxin inhibits growth in Ustilago maydis by blocking calcium uptake[END_REF][START_REF] Li | Structure of Ustilago maydis killer toxin KP6 alpha-subunit. A multimeric assembly with a central pore[END_REF], most killer phenotypes and their applications characterized to date are derived from yeasts. Killer yeasts are immune to their own secreted toxins, while remaining susceptible to others. Toxin-coding cytoplasmic dsRNA-mycoviruses have been detected in Saccharomyces cerevisiae and in non-conventional yeasts such as Hanseniaspora uvarum and Zygosaccharomyces bailii [START_REF] El-Sherbeini | Viruses in fungi: infection of yeast with the K1 and K2 killer viruses[END_REF][START_REF] Schmitt | The viral killer system in yeast: from molecular biology to application[END_REF]. In contrast to the S. cerevisiae toxins, killer toxins of non-Saccharomyces yeasts and especially those secreted by virus-infected killer strains of the yeasts H. uvarum and Z. bailii show a broad-spectrum antimycotic potential to human pathogenic strains, such as Candida albicans, Sporothrix schenkii, and Fusarium sp. [START_REF] Schmitt | Unique double-stranded RNAs responsible for the anti-Candida activity of the yeast Hanseniaspora uvarum[END_REF][START_REF] Hodgson | Anti-Candida activity of a novel killer toxin from the yeast Williopsis mrakii[END_REF]. The toxin isolated from Z. bailii, zygocin, effectively kills C. albicans, C. glabrata and C. tropicalis yeast cells and germlings. Moreover, on a molar basis, zygocin is even more effective in killing yeasts than antifungal agents such as clotrimazole or miconazole, suggesting that zygocin is a promising antifungal agent [START_REF] Weiler | Zygocin, a secreted antifungal toxin of the yeast Zygosaccharomyces bailii, and its effect on sensitive fungal cells[END_REF].

The outer cell wall layer of yeasts predominantly consist of mannoproteins, connected to a glucan skeleton consisting of linear and branched beta-1,3-and beta-1,6-D-glucans, and to a much lesser extent of chitin which mainly concentrates in bud scars [START_REF] Cabib | The yeast cell wall, a dynamic structure engaged in growth and morphogenesis[END_REF]. In the search for novel and more selective antifungal agents, fungal cell wall components represent primary targets, as these structures are restricted to yeasts and higher fungi and do not occur in human cells [START_REF] Kurtz | New antifungal drug targets: a vision for the future[END_REF][START_REF] Hector | Compounds active against cell walls of medically important fungi[END_REF]. Each of the mentioned cell wall components has been shown to act as the primary binding site for different yeast killer toxins [START_REF] Schmitt | Mannoprotein of the yeast cell wall as primary receptor for the killer toxin of Saccharomyces cerevisiae strain 28[END_REF][START_REF] Hutchins | Cell wall receptor for yeast killer toxin: involvement of (1 leads to 6)-beta-D-glucan[END_REF][START_REF] Jablonowski | Saccharomyces cerevisiae cell wall chitin, the Kluyveromyces lactis zymocin receptor[END_REF][START_REF] Marquina | Biology of killer yeasts[END_REF]. In addition, for killer toxins secreted by various strains of the yeast Hansenula it was demonstrated that these toxins not only bind to yeast cell wall components, but that they also strongly inhibit de novo L-1,3-D-glucan biosynthesis [START_REF] Yamamoto | Killer toxin from Hansenula mrakii selectively inhibits cell wall synthesis in a sensitive yeast[END_REF][START_REF] Kimura | A novel yeast gene, RHK1, is involved in the synthesis of the cell wall receptor for the HM-1 killer toxin that inhibits beta-1, 3-glucan synthesis[END_REF]. Given the cell wall related activity of mycovirus-encoded killer toxins, antifungal research has indeed focused on the possible use of yeast killer toxins as novel antifungal agents for the treatment of fungal infections [START_REF] Selitrennikoff | Antifungal proteins[END_REF]. Clinical recovery as well as negative mycological test cultures of experimental dermatomycosis in animals proved to be clearly associated with topical killer toxin treatment [START_REF] Polonelli | Potential therapeutic effect of yeast killer toxin[END_REF]. However, although some researchers have shown that topical applications in the treatment of superficial lesions might well be possible, one drawback of most yeast killer proteins is that they exhibit their cytotoxic activity only at temperatures between 20°C and 30°C within a narrow pH range [START_REF] Polonelli | Potential therapeutic effect of yeast killer toxin[END_REF][START_REF] Walker | Interactions between killer yeasts and pathogenic fungi[END_REF]. Therefore, yeast toxins are unsuitable as direct applications for oral and/or intravenous administration [START_REF] Magliani | From yeast killer toxins to antibiobodies and beyond[END_REF]. Killer toxins, however, fuelled the development of new antifungal agents that mimick the antimicrobial activity of killer toxins, so-called killer antibodies and killer antibodyderived peptides. These derivatives have shown activity against various human fungal pathogenic fungi including C. albicans and may be therapeutic when administered parentally. For reviews of killer toxin/antibodies the reader is referred to Marquina et al. [START_REF] Marquina | Biology of killer yeasts[END_REF], Magliani et al. [START_REF] Magliani | From yeast killer toxins to antibiobodies and beyond[END_REF], and Schmitt and Breinig [START_REF] Schmitt | The viral killer system in yeast: from molecular biology to application[END_REF].

Viruses as therapeutic agents for infections in humans

The idea to cure infections in humans with viruses as biological control agents is not new. Most research has been conducted using viruses that specifically and uniquely search and destroy bacteria. These bacteriophages-'eaters of bacteria'-have been known since the early 20th century [START_REF] Sulakvelidze | Bacteriophage therapy[END_REF]. To offer support for the contention that a strategy using mycoviruses in the treatment of medically important fungi can be effective, analogous research using bacteriophages for treating bacterial infections is briefly reviewed.

Bacteriophages are ubiquitous and found in many different environments including soil, human and animal bodies; they do not attack human cells and exist as partners in microbiological eco-systems in mammals. Bacteriophages may be divided into a number of basic types based on morphological and genome analysis. The genome types are dsDNA, ssDNA, dsRNA and ssRNA. The most prevalent bacteriophages are tailed and all tailed bacteriophages have dsDNA. Furthermore, these tail fibres confer host specificity within this group [START_REF] Fauquet | Virus taxonomy: eighth report of the International Committee on Taxonomy of Viruses[END_REF].

Although a large number of publications have suggested that bacteriophages may be effective therapeutic agents in clinical settings, most published studies are uncontrolled or anecdotal and do not meet the criteria of modern evidence [START_REF] Sulakvelidze | Bacteriophage therapy[END_REF]. However, recently, proof of principle has been provided as bacteriophage therapies have been developed for multiresistant bacteria and other difficult to treat infections [START_REF] Stone | Bacteriophage therapy. Stalin's forgotten cure[END_REF][START_REF] Bradbury | Using phages to fight bacteria[END_REF]. For instance, it has been shown that a bacteriophage with lytic activity against vancomycin resistant Enterococcus faecium (VRE) successfully cured mice from VRE bacteraemia [START_REF] Biswas | Bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycinresistant Enterococcus faecium[END_REF]. A review of various experimental infections treated with bacteriophages can be found in the work of O'Flaherty et al. [START_REF] O'flaherty | Bacteriophage and their lysins for elimination of infectious bacteria[END_REF]. In addition, the therapeutic bacteriophage of VRE bacteraemia was well tolerated in a phase I clinical trial [START_REF] Stone | Bacteriophage therapy. Stalin's forgotten cure[END_REF]. Other clinical trials have shown that bacteriophages could be safely administered, even to children, and demonstrated cost effectiveness over antibiotic treatment of infections caused by multidrug-resistant bacteria [START_REF] Bruttin | Human volunteers receiving Escherichia coli phage T4 orally: a safety test of phage therapy[END_REF][START_REF] Fortuna | Bacteriophage therapy in children: facts and prospects[END_REF]. Furthermore, the first results of a randomised, double-blind, placebo-controlled phase I/II clinical trial of a bacteriophage therapeutic targeting long-term ear infections with P. aeruginosa refractory to antibiotic showed both efficacy and safety [START_REF] Wright | A controlled clinical trial of a therapeutic bacteriophage preparation in chronic otitis due to antibiotic-resistant Pseudomonas aeruginosa; a preliminary report of efficacy[END_REF].

In tandem with the resurgent interest in bacteriophages as therapeutic agents and the advent of high throughput sequencing, the number of bacteriophage genomes sequenced to completion has drastically increased to >500 [START_REF] O'flaherty | Bacteriophage and their lysins for elimination of infectious bacteria[END_REF]. Although host species specificity is the rule for most bacteriophages investigated, many bacteriophages have developed efficient methods to change their host range via genetic mechanisms [START_REF] Chibani-Chennoufi | Phage-host interaction: an ecological perspective[END_REF]. The dsDNA Escherichia coli bacteriophage, Phi K1-5, for example, has shown to be capable of infecting E. coli strains that possess either the K1 or the K5 polysaccharide capsule due to possession of two different tail fiber proteins. The arrangement of genes suggests that this bacteriophage can broaden its host range by horizontal gene transfer [START_REF] Scholl | Bacteriophage K1-5 encodes two different tail fiber proteins, allowing it to infect and replicate on both K1 and K5 strains of Escherichia coli[END_REF].

Although bacteriophages have a unique and limited bactericidal spectrum, they are almost as numerous as bacteria and the development of therapeutic bacteriophages is therefore hardly problematic. Furthermore, nowadays, engineering of genes involved in host recognition may be an additional approach for generating an expanded host range.

Host range expansion using genetic engineering

Pyocins

Engineering of genes involved in host recognition results in host range expansion. Evidence is mostly provided by the studies of R-type pyocins. Certain Pseudomonas aeruginosa strains produce R-type pyocins (henceforth called pyocins), protein particles resembling non-flexible and contractile tails of bacteriophages [START_REF] Michel-Briand | The pyocins of Pseudomonas aeruginosa[END_REF]. Although many pyocin genes share common ancestry with bacteriophage genes, pyocins differ from bacteriophages, for they do not contain nucleic acid and cannot replicate. Both bacteriophages and pyocins bind to an accessible receptor on the target bacterium, but pyocins kill the target bacterium by a different mechanism. Pyocins kill bacteria by first binding to a cell surface structure via tail fibers, followed by contraction of the sheath and insertion of their hollow core through the cell envelope resulting in depolarization of the cytoplasmic membrane and cell death [START_REF] Uratani | Pyocin R1 inhibits active transport in Pseudomonas aeruginosa and depolarizes membrane potential[END_REF]. Pyocin has been shown to increase survival and reduce bacterial counts in a lethal murine experimental Pseudomonas peritonitis model [START_REF] Scholl | Antibacterial efficacy of R-type pyocins towards Pseudomonas aeruginosa in a murine peritonitis model[END_REF]. The bactericidal specificity of pyocins is conferred by their tail fibers encoded by prf15. A chaperone, encoded by prf16 immediately downstream of prf15, is required for tail fiber assembly [START_REF] Nakayama | The R-type pyocin of Pseudomonas aeruginosa is related to P2 phage, and the F-type is related to lambda phage[END_REF]. Chimeric tail fusion fibers that bind to presumably different bacterial surface molecules have been engineered. The generated chimeric tail fibers that formed active pyocins resulted from many trials of empirically chosen fusion sites between prf15 and portions of bacteriophage tail/spike fibers. The engineered fusions between N-terminal portions of the pyocin tail fiber and various C-terminal portions of the bacteriophage tail/spike fibers resulted in pyocin host range expansion from Pseudomonas to Yersinia and various E. coli strains [START_REF] Williams | Retargeting R-type pyocins to generate novel bactericidal protein complexes[END_REF][START_REF] Scholl | An engineered Rtype pyocin is a highly specific and sensitive bactericidal agent for the food-borne pathogen Escherichia coli O157:H7[END_REF]. Scholl et al. further envisage that an unlimited amount of chimeric pyocins can be created based on the presence of a vast number and diversity of bacteriophage tail fibres combined with the application of phage display techniques. This may provide even greater diversity of binding specificities than exists naturally on pyocins and could lead to an almost unlimited number of bactericidal agents.

Plant viruses

Similarly, plant viruses have been genetically modified to express heterologous proteins for vaccine development. To this purpose, coat protein genes of several plant viruses have been genetically altered to express foreign antigenic epitopes of human or animal origin as fusions to the viral coat protein. These foreign epitopes are exposed and displayed on the surface of chimeric virus particles [START_REF] Johnson | Presentation of heterologous peptides on plant viruses: genetics, structure, and function[END_REF]. Although the size of the peptide fusions that still permit virus assembly is limited [START_REF] Yusibov | Antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and HIV-1[END_REF], several strategies have been developed to overcome assembly problems for long or difficult peptides. These include "Foot and Mouth Disease Virus 2A peptide coat protein (CP) fusion strategy" and the so-called "leaky stop codon CP fusion strategy", which would cause most of the CP produced to be unfused, while a small portion would be fused to the peptide displayed on the surface [START_REF] De Felipe | Cotranslational, intraribosomal cleavage of polypeptides by the Footand-mouth disease virus 2A peptide[END_REF][START_REF] Borovsky | Expression of Aedes trypsin-modulating oostatic factor on the virion of TMV: a potential larvicide[END_REF][START_REF] Cruz | Assembly and movement of a plant virus carrying a green fluorescent protein overcoat[END_REF].

Fusion proteins have to be compatible with normal viral assembly and fitness, and steric hindrance or interference with virus movement must be avoided to sustain infection. Indeed, recombinant viruses expressing foreign antigenic peptides on their surface can remain infectious and retain the foreign sequences during passage from plant to plant [START_REF] Yusibov | Antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and HIV-1[END_REF][START_REF] Cruz | Assembly and movement of a plant virus carrying a green fluorescent protein overcoat[END_REF]. Many stable mutants have already been prepared that allow specific modification of the capsid surface of plant viruses (for a review, see [START_REF] Lico | Viral vectors for production of recombinant proteins in plants[END_REF]).

Mycoviruses

Likewise, in mycovirus research for vaccine development against aspergillosis, the transposon Ty1 of yeast Saccharomyces cerevisiae has successfully been used for the expression of Aspergillus peptides on its surface. This bifunctional virus-like particle engineered to contain peptides representing dominant T-and B-cell epitopes of Aspergillus proved more potent in stimulating memory immune responses [START_REF] Marchenko | The p1 protein of the yeast transposon Ty1 can be used for the construction of bi-functional virus-like particles[END_REF].

One possible scenario that can be derived from the results of genetic modification of bacteriophages, pyocins, plant viruses, and mycoviruses described above, is the generation of a selective mycovirus that is transmitted extracellular in an efficient way. Genetic modification of structures involved in mycovirus binding to the target fungus may be the first that comes to mind. To date, such a mycovirus is not known to exist. Recently however, the infectious myonecrosis virus (IMNV) was discovered as the cause of infectious myonecrosis in penaeid shrimp. IMNV is tentatively considered as a totivirus due to its RNAdependent RNA-polymerase (RdRp) that matches significantly with the consensus sequence of the RdRp of the Totiviridae family [START_REF] Poulos | Purification and characterization of infectious myonecrosis virus of penaeid shrimp[END_REF]. As outlined above, totiviruses are able to infect fungi and have no known capacity to infect humans, and most totiviruses lack the means to be transmitted extracellularly. IMNV is an exception (Fig. 1) and therefore may be an interesting candidate for genetic engineering to expand its host range [START_REF] Tang | Infectious myonecrosis virus has a totivirus-like, 120-subunit capsid, but with fiber complexes at the fivefold axes[END_REF]. Similar to the surface fibers in other viruses, the IMNV protrusions are almost certainly involved in cell entry, including receptor binding and/or membrane penetration, i.e. confer host specificity. Exploring if genetic modification of IMNV protrusions generates a chimeric virus that is able to selectively infect fungi via an extracellular mode of transmission may be an interesting field of research.

Mycoviruses as gene vectors

Another possible scenario, apart from generating a chimeric mycovirus that is efficiently transmitted extracellularly, may be its use as a gene insertion vector. As such, the virus does not need to be detrimental to the fungus itself, as is the case for most mycoviruses in their natural life cycle, but the mycovirus should be able to function as a "Trojan horse", expressing foreign inserts "upon request" (Table 2). This has already been applied in fighting fungal diseases in plants using killer toxins (discussed above) [START_REF] Donini | Production of an engineered killer peptide in Nicotiana benthamiana by using a Potato virus X expression system[END_REF]. The gene encoding a broad spectrum yeast killer toxin was inserted in Potato virus X and yielded particles displaying an active form of the killer peptide against the plant pathogenic fungi of interest and, additionally, also displayed activity against C. albicans [START_REF] Donini | Production of an engineered killer peptide in Nicotiana benthamiana by using a Potato virus X expression system[END_REF].

Of note, recombinant mycoviruses have to meet various criteria and characteristics. Research into the field of mycoviruses can only be performed in a model system that offers a high virus yield and is safe at the same time with the lowest risk possible of emergence of viruses capable of infecting humans. Characteristics required are summarised in Table 2.

Overcoming the cell wall barrier

As explained before, the fungal cell wall poses a barrier to viral entry. Different types of strategies can be hypothesized to overcome this obstacle. One such strategy may be pretreatment with an antifungal agent to induce holes in the fungal wall thus exposing the cell membrane surface to mycovirus application. Another strategy may be the use of delivery systems. Because dsRNA viruses are approximately 2.5 times smaller than empty Amphotericin B liposomes which measure 100 nm (Gilead Science), liposomes are an interesting delivery system. The delivery system, or the mycovirus itself, may be expressing enzymes that have chitinase of glucanase activity. Again, this idea was strengthened by observations from studying bacteriophages. Bacteria sometimes produce thick polysaccharide capsules for protection in hostile environments which also limits infection with bacteriophages. Some bacteriophages, however, express enzymes with capsule-depolymerizing activities. Bacteriophages expressing these enzymes that are attached via an adapter protein to the virus portal vertex through which the DNA is ejected during infection are able to digest their way through 400-nm thick capsules [START_REF] Scholl | Escherichia coli K1's capsule is a barrier to bacteriophage T7[END_REF][START_REF] Leiman | The structures of bacteriophages K1E and K1-5 explain processive degradation of polysaccharide capsules and evolution of new host specificities[END_REF].

Conclusion

In their natural life cycle, mycoviruses are usually nontransmissible by an extracellular route and cause persistent infections in their host without phenotypic alterations. However, based on results obtained with chimeric plant viruses, bacteriophages and pyocins, genetic engineering of existing mycoviruses is possible. Mycovirus therapy may thus be regarded as an interesting and promising direction for future research and may provide an alternative therapeutic biological agent against invasive fungal infections.

Fig. 2

 2 Fig.2Transfer of mycoviruses between fungal hyphae is dependent on the compatibility of the donor and acceptor hyphae upon contact

  Candida albicans, C. tropicalis, C. lipolytica and C. curvata, but these fungal genera are omitted from Table 1 because they are not listed in the ICTV report

	U01060
	Aspergillus foetidus virus S	Aspergillus niger virus S	M satellites of Saccharomyces	cerevisiae virus L-A	Aspergillus foetidus virus F	Fusarium graminearum virus DK21
			Unassigned Sub-viral RNA encoding killer toxins and requires co-infection	with helper virus for productive multiplication	Unassigned	Unassigned	Confirmed species in italics; tentative and unassigned mycoviruses in regular font.

Data are extracted from Fauquet et al.

[START_REF] Fauquet | Virus taxonomy: eighth report of the International Committee on Taxonomy of Viruses[END_REF]

and from ICTVdB-The Universal Virus Database, version 4. http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/.

particles have also been detected in Alternaria alternata, Rhizopus and Mucor spp.,

Table 2

 2 Ideal characteristics of mycoviruses intended to be used as biocontrol agents of invasive fungal infections in humans

	The mycovirus should	The mycovirus should
		NOT
	have an extracellular mode of	be able to cause infection
	transmission	in the human host
	replicate efficiently in fungi	be able to recombine with
	pathogenic to humans	human pathogenic
		viruses
	cause 100% lysis of the fungal target	be able to integrate into
		the human genome
	have a broad anti-fungal range	induce (serious) side
		effects
	readily be produced in large quantities	induce production of
	with high purity and stability at low	neutralising antibodies
	cost
	have topical and systemic applicability
	have a genome that facilitates genetic
	manipulation
	have modifiable levels of expression of
	foreign inserts when requested and the
	foreign inserts should not interfere
	with normal viral assembly and fitness
	and steric hindrance or interference
	with virus movement must be avoided

Eur J Clin Microbiol Infect Dis (2010) 29:755-763