Brendan ; Crowley
email: bcrowley@stjames.ie

Aisling Salter

Bairbre Ni Laoi

Identified in Adults with Haematological Malignancy Attending an Irish Hospital Between 2004 -2009

Keywords: haematological malignancy, HRSV subgroups, G-protein gene, phylogenetics, genotypes

Figure 2 A maximum likelihood tree based on nucleotide sequences from the second variable region of the G protein gene for Irish HRSV subgroup A isolates from St. James's Hospital between 2004/2005 and 2008/2009. The sequences of the Irish strain are indicated by solid squares. HRSV A control sequence, A Strain Long (GenBank M17212) as used as an outgroup. Bootstrap resampling was carried out using 1,000 replicates of the dataset and the values are written as a percentile beside the appropriate branch. Bootstrap values <70% are not shown. Scale bars indicate 0.1 nucleotide substitutions per site. 164x212mm (96 x 96 DPI)

Introduction

Human respiratory syncytial virus (HRSV) is recognised as a significant pathogen among the elderly, in particular those individuals with cardiopulmonary disease, and also immunocompromised hosts [START_REF] Falsey | Respiratory syncytial virus infection in adults[END_REF]. The main routes of transmission are by contact and droplet spread [START_REF] Hall | Respiratory syncytial virus and parainfluenza virus[END_REF]. It is associated with significant morbidity and mortality in haematopoetic stem cell transplant recipients [START_REF] Avetisyan | Respiratory syncytial virus infection in recipients of allogeneic stem-cell transplantation: a retrospective study of the incidence, clinical features, and outcome[END_REF]. In a study involving post-bone marrow transplant patients, it was found that nosocomial infection with HRSV occurred in almost half (48%) of them [START_REF] Whimbey | Community 30 respiratory virus infections among hospitalized adult bone marrow transplant recipients[END_REF]. Furthermore, progression of HRSV infection from upper respiratory tract infection to lower respiratory tract infection is associated with increased risk of death [START_REF] Avetisyan | Respiratory syncytial virus infection in recipients of allogeneic stem-cell transplantation: a retrospective study of the incidence, clinical features, and outcome[END_REF].

HRSV strains have been classified on the basis of reactivity of the virus with monoclonal antibodies directed against the attachment glycoprotein, a type II integral membrane protein, which is highly glycosylated with both N-linked and O-linked sugars [START_REF] Wertz | Structure and cell surface maturation of the attachment glycoprotein of human respiratory syncytial virus in a cell line deficient in O glycosylation[END_REF]] and is the main target for neutralising and protective humoral immune responses [START_REF] Galiano | Genetic and antigenic variability of human respiratory syncytial virus (groups a and b) isolated over seven consecutive seasons in Argentina (1995 to 2001)[END_REF].

Therefore, antigenic variability as a consequence of mutations in the two hypervariable regions of the G protein contributes to the ability of the virus to cause HRSV reinfections throughout life. These two hypervariable regions are separated by a central conserved region of 13 amino acids [Johnson et al., 1987]. The second hypervariable region of the C-terminal of the G protein has only 44% amino acid homology between the two groups of HRSV while the transmembrane and cytoplasmic domains of the G protein have 83% identity on comparison of amino acid [Johnson et al., 1987]. Furthermore, it has been shown that there is approximately 20% sequence variation in this region of the G protein among RSV isolates belonging to the same antigenic group [Melero et al., 1997]. Not surprisingly, sequencing of the variable regions of the G protein gene has been used widely in subdividing further the two groups into genotypes and therefore facilitating differentiation between RSV isolates.

Eight HRSV subgroup A genotypes have been described so far, namely GA1 to GA7 and South Africa A1 (SAA1) [START_REF] Peret | Circulation patterns of group A and B human respiratory syncytial virus genotypes in 5 communities in North America[END_REF][START_REF] Peret | Circulation patterns of genetically distinct group A and B strains of human respiratory syncytial virus in a community[END_REF][START_REF] Venter | Genetic diversity and molecular epidemiology of respiratory syncytial virus over four consecutive seasons in South Africa: identification of new subgroup A and B genotypes[END_REF]. HRSV B viruses have been divided into genotypes GB1-4 [START_REF] Peret | Circulation patterns of group A and B human respiratory syncytial virus genotypes in 5 communities in North America[END_REF][START_REF] Peret | Circulation patterns of genetically distinct group A and B strains of human respiratory syncytial virus in a community[END_REF]), SAB1-3 [Venter et al., 2002[START_REF] Venter | Genetic diversity and molecular epidemiology of respiratory syncytial virus over four consecutive seasons in South Africa: identification of new subgroup A and B genotypes[END_REF], and BA [START_REF] Trento | Major changes in the G protein of human respiratory syncytial virus isolates introduced by a duplication of 60 nucleotides[END_REF]. Of these HRSV B genotypes, the BA genotype is relatively new and is characterised by a 60 nucleotide duplication in the G glycoprotein gene of HRSV group B [START_REF] Trento | Major changes in the G protein of human respiratory syncytial virus isolates introduced by a duplication of 60 nucleotides[END_REF]]. An increasing incidence of strains containing this duplication has been reported from Buenos Aires [START_REF] Trento | Natural history of human respiratory syncytial virus inferred from phylogenetic Page 33 of 35 John Wiley & Sons Journal of Medical Virology 29 analysis of the attachment (G) glycoprotein with a 60-nucleotide duplication[END_REF]. This variant of HRSV probably arose when the viral polymerase duplicated sequences within the G protein gene. It originated from an ancestor between 1998 and 1999, just before the first isolation of these variants in 1999, and can be used as a natural tag to study the evolution and dissemination of HRSV worldwide [START_REF] Trento | Natural history of human respiratory syncytial virus inferred from phylogenetic Page 33 of 35 John Wiley & Sons Journal of Medical Virology 29 analysis of the attachment (G) glycoprotein with a 60-nucleotide duplication[END_REF]. Phylogenetic analysis of this strain of HRSV B determined that there was sequence divergence within the BA genotype and further subdivided it into subgroups BA-I to BA-VI [START_REF] Trento | Natural history of human respiratory syncytial virus inferred from phylogenetic Page 33 of 35 John Wiley & Sons Journal of Medical Virology 29 analysis of the attachment (G) glycoprotein with a 60-nucleotide duplication[END_REF]. There have been reports of the BA genotype circulating in Argentina, Australia, Belgium, Canada, Kenya, New Zealand, Japan and Uruguay [START_REF] Matheson | Distinct patterns of evolution between respiratory syncytial virus subgroups A and B from New Zealand isolates collected over thirty-seven years[END_REF][START_REF] Tozer | Identification of Australian human respiratory syncytial virus strains containing a 60-nucleotide duplication within the G glycoprotein gene[END_REF][START_REF] Trento | Major changes in the G protein of human respiratory syncytial virus isolates introduced by a duplication of 60 nucleotides[END_REF][START_REF] Trento | Natural history of human respiratory syncytial virus inferred from phylogenetic Page 33 of 35 John Wiley & Sons Journal of Medical Virology 29 analysis of the attachment (G) glycoprotein with a 60-nucleotide duplication[END_REF].

Materials and Methods

Sample selection and Storage

A total of 60 samples collected from patients attending St. James' Hospital (SJH) over the period from November 2004 to March 2009 were selected for this study. Samples included nasopharyngeal aspirates and bronchoalveolar lavages (BALs). These samples were confirmed positive for human respiratory syncytial virus (HRSV) by direct immunofluorescence (Imagen TM RSV A & B, Oxoid, UK). An aliquot of NPA or BAL (approx 1.5ml) from each sample was frozen at -80°C in an RNase and DNase free tubes until required.

Human respiratory syncytial virus (HRSV) RNA extraction

Template RNA was extracted from 200µl of each sample using the QIAamp MinElute Virus Spin Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions.

Reverse Transcription Polymerase Chain Reaction (RT-PCR)

The template RNA was converted to complementary DNA (cDNA) using the High Capacity Archive kit (Applied Biosystem Warrington, UK) following the manufacturer's instructions.

Amplification the C-terminal of G protein gene

To amplify the C-terminal region of the G protein gene, hemi-nested primers (Gr5, FV and F1)

were used [START_REF] Da Silva | Genetic variability in the G protein gene of human respiratory syncytial virus isolated from the Campinas metropolitan region, Brazil[END_REF] for 3 minutes, followed by 45 cycles of 94°C for 30 seconds (denaturation), 57°C for 30 seconds (primer annealing) and 72°C 1 minute (extension) and a final elongation step of 72°C for 10 minutes. The PCR product was purified using the Qiaquick® PCR Purification kit (Qiagen GmbH, Hilden, Germany) following the manufacturer's instructions.

DNA Sequencing and Phylogenetic analysis

Automated DNA sequencing was performed an all samples using the ABI Prism 3130xl

Analyser (Applied Biosystems, Warrington, UK). Sequence traces were analysed and a consensus sequence for all samples were obtained using the Lasergene® SeqMan sequence analysis software (DNASTAR, Madison, Wisconsin). All nucleotide and amino acid sequences were aligned using Clustal W [Thompson et al., 1997] and sequence polymorphisms within the amplified region were identified by comparison with a variety of sequences collected from GenBank which represented circulating subtypes of HRSV A and B during the defined season.

Aligned sequences were then translated into protein using the protein translation function within the Lasergene® MegAlign software (DNASTAR, Madison, Wisconsin). Amino acid sequences were compared to the GenBank sequences at known mutation sites and any changes in amino acids were noted. The sequences analysed correspond to amino acid residues 220 to 298 of the prototype A2 strain (GenBank M11486) [START_REF] Wertz | Nucleotide sequence of the G protein gene of human respiratory syncytial virus reveals an unusual type of viral membrane protein[END_REF] and amino acid residues 219 to 315 of the BA4128/99B strain (GenBank AY333364) [START_REF] Trento | Major changes in the G protein of human respiratory syncytial virus isolates introduced by a duplication of 60 nucleotides[END_REF]. Known substitution sites and any changes in amino acids were noted.

Phylogenetic reconstructions, all employing maximum likelihood, were carried out using PAUP*4.0b10 (Swofford, 2002). The 'best fit' models of evolution for the aligned datasets were determined using the Modeltest version 3.06 [Posada and Crandall, 1998]) by comparing the likelihood of the different parameters for all models of substitution. Model parameters were then estimated using a heuristic search strategy and selecting those with the highest likelihood.

Bootstrap resampling was carried out using PAUP* with a 1000 replicates performed to examine the robustness of the trees produced. In total, two phylogenetic trees were constructed.

Identification of potential glycosylation sites in the G protein of HRSV A and HRSV B isolates

Potential N-glycosylation sites were identified in both HRSV A and HRSV B isolates from the amino acid sequences, where the amino acid motif, asparagines X threonine (NXT), was seen, where X is not proline. To estimate the numbers of potentially O-glycosylated serine and threonine residues, the NetOglyc software (version 3.1) [START_REF] Hansen | Prediction of Oglycosylation of mammalian proteins: specificity patterns of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase[END_REF] was used.

Analysis of Synonymous vs Non-Synonymous mutations

Synonymous and non-synonymous mutations were analysed by the method of Nei and Gojobori [START_REF] Nei | Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions[END_REF]

Results

Study population and clinical details

A total of 980 samples, including nasopharyngeal aspirates (98%) and bronchial washings (2%)

were collected for laboratory diagnosis of respiratory viruses at St. James's Hospital Dublin between December 2004 and March 2009. All samples were collected from adult patients, aged between 19 and 77 years, who had haematological malignancies and were on immunosuppressive therapy. Most patients (85%) had undergone haematopoietic stem cell transplantation. Eighty-three samples were confirmed positive for HRSV infection by direct immunofluorescence during this time period but only 60 samples from 58 patients were available for analysis in this study. Interestingly, all HRSV-positive samples were consistent with community-aquired HRSV infection and collected from patients attending the Outpatient Clinic.

However, almost 50% (n=31) of these patients were hospitalised for treatment with aerosolised ribavirin 2g every 8 hours because of risk factors for severe complications of HRSV infection.

The latter included patients who were within 2 months of haematopoetic stem cell transplant, on immunosuppression for acute or chronic GVHD, lymphopenic (< 0.3/mm 3 ) or received alemtuzumab or anti-thymocyte globulin (ATG) as part of conditioning regimen. The underlying haematological diseases in these patients, 70% of whom were male, included chronic myeloid leukaemia (n=10), chronic lymphocytic leukaemia (n=10), acute lymphoblastic leukaemia (n=10), acute myeloid leukaemia (n=9), non-Hodgkins lymphoma (n=8), multiple myeloma (n=7), mantle cell lymphoma (n=2) and aplastic anaemia (n=2). The main clinical presentations involved the upper respiratory tract, and included cough (100%), rhinorrhoea (100%) and fever (50%). However, three patients (2%) who were diagnosed with pneumonia and respiratory

Phylogenetic analysis and genotype distribution of HRSV subgroup A and B sequences.

The G protein gene in 59 of the 60 subgroup A and B HRSV isolates were successfully amplified and sequenced. One isolate, RSV 18, failed to amplify successfully but was shown to belong to subgroup B based on molecular analysis of the SH gene (data not shown). A maximum likelihood phylogenetic tree was constructed of the G protein gene of the 31 subgroup A HRSV isolates collected from St. James's Hospital (SJH) over that past five winter seasons, including a selection of strains that represented the different genotypes of HRSV A isolates collected from the GenBank database (Figure 2). The reference strain Long (GenBank M17212) was used as an outgroup in the phylogenetic tree. In this study the HRSV A isolates belonged to two genotypes, 14 (45%) were genotype GA2, and 17 (55%) belonged to genotype GA5. The rates of divergence between prototype A2 and the Irish strains were 12% to 18% at nucleotide level, and 13% to 18% at the amino acid level. Differences of 13% were observed at both the nucleotide isolates from this study that belonged to genotype GA5 showed close similarity to isolates from Uruguay (AY571777), Argentina (AY910841), Kenya (AY660678), Brazil (FJ427335 and EU625728), South Africa (AY146437) and Belgium (AY343560) (Figure 2). Among the GA2 isolates a high similarity was seen to isolates from Kenya (AY524663 and AY524651) and Singapore (AY114149) (Figure 2).

Maximum likelihood phylogenetic trees were constructed for the G protein gene of all the HRSV B isolates in this study along with a variety of strains collected from the GenBank database that This genotype is defined by a 60 nucleotide (nt) duplication at position 781-840 in the second variable region of the G protein gene. All of the HRSV B isolates from this study belonged to subgroup BA-III within the BA genotype, the first one being detected in December 2004. There was 5% to 9% divergence at both nucleotide and amino acid level, respectively, between the Irish HRSV B strains and the prototype BA strain. Among the Irish HRSV B strains, up to 4%

divergence was observed at both nucleotide level and amino acid level. The majority of the isolates had a high similarity with two isolates from a study in Spain, DQ227395 and DQ227396 [START_REF] Trento | Natural history of human respiratory syncytial virus inferred from phylogenetic Page 33 of 35 John Wiley & Sons Journal of Medical Virology 29 analysis of the attachment (G) glycoprotein with a 60-nucleotide duplication[END_REF], and an isolate (AY751086) from a Belgian study in 2001 [START_REF] Zlateva | Genetic variability and molecular evolution of the human respiratory syncytial virus subgroup B attachment G protein[END_REF] (Figure 3). However, one isolate, RSV 9, clustered with strains of HRSV subgroup B including DQ227381, DQ227392 and DQ227387, all isolated in Spain [START_REF] Trento | Natural history of human respiratory syncytial virus inferred from phylogenetic Page 33 of 35 John Wiley & Sons Journal of Medical Virology 29 analysis of the attachment (G) glycoprotein with a 60-nucleotide duplication[END_REF] and another isolate (AB175819) from a study in Japan [START_REF] Sato | Molecular epidemiology of respiratory syncytial virus infections among children with acute respiratory symptoms in a community over three seasons[END_REF] (Figure 3). It was observed that all of the HRSV B isolates in this study were quite conserved from season to season with very little variability among the amino acid sequences observed (Table 1). However, all of the 2006/2007 isolates (RSV 21, RSV 23, RSV 25, RSV 26 and RSV 29, RSV 33, RSV 34 and RSV 36) formed a distinct cluster with the strain DQ227395 (Figure 3) each had amino acid substitution S297F, which occurred with the potential N-glycosylation site at position 296 (Table 1). Also in this cluster of isolates was isolate HRSV 53 (Figure 3), which had a different amino acid substitution, S297L (Table 1).

Sequence analysis of the G glycoprotein

Predicted amino acid sequences of the C-terminal end of the G protein gene in all the HRSV A isolates in this study were compared with prototype A2 strain. Seventeen of the HRSV A isolates were predicted to encode G proteins of 298 amino acids in length and 14 of them encoded a G protein of 297 amino acids (Table 2). Interestingly, the length of the G protein was genotype-specific in this study as G proteins of amino acid length of 297 belonged to the GA2 genotype while G proteins of 298 amino acids in length belonged to genotype GA5.

Furthermore, genotype-specific amino acid substitutions were observed at several positions among the HRSV subgroup A isolates. For instance, genotype GA2 was characterised by S269T

(serine to threonine) and P289S (proline to serine). Similarly, GA5 was distinguished by amino acid substitutions V225A (valine to alanine), S250N (serine to asparagine), N251S (asparagine to serine), P274T (proline to threonine) and V279I (valine to isoleucine).

In this study, the partial G protein gene sequences of the BA genotypes were predicted to encode proteins of three different lengths, 312, 315 and 319 amino acids (Table 1). The majority of the isolates (23/28) had predicted amino acid lengths of 312 residues, one isolate encoded a protein of 315 amino acids and four isolates had 319 amino acids (Table 1). A serine (S) to proline (P) amino acid change occurred within the 20-amino acid duplicated region at position 247 in all of the Irish BA strains compared to the sequence of the prototype BA strain. A second conserved amino acid substitution was also observed at position 270 where a threonine (T) was substituted for an isoleucine (I) when compared with the prototype BA strain. The majority of isolates also had amino acid substitutions at positions 271 (V271A) and 287 (H287Y) (Table 1).

Glycosylation sites

Three putative N-glycosylation sites (NXT, where X is not proline) were identified on the second variable region of the G protein among the majority (19/31) of the HRSV A strains (Table 2).

Two potential sites were identified in 10 of the 31 isolates, one potential site in one isolate (RSV 59) and four potential sites in one isolate (RSV 13) (Table 2). The potential N-glycosylation site, at position 294 was conserved among all of the isolates while that at residue 237 was also observed among the majority of the isolates (20/31) (Table 2). Among the GA2 genotypes, 12 of the isolates had a conserved N-glycosylation pattern at residue 251 consistent with the site being genotype-specific. The remaining two GA2 isolates (RSV 37 and RSV 59) lost this site due to an amino acid substitution at position 251 (N251S and N251Y, respectively) (Table 2).

Furthermore, three isolates (RSV 13, RSV 37 and RSV 39) were observed to have gained an Nglycosylation site at position 244, where isoleucine was replaced by asparagine (Table 2).

Two N-glycosylation sites were identified among the group B strains at the C-terminal end of the G protein gene. One of these, at amino acid 296, was conserved among all isolates examined while the second site, amino acid 310, was found in the majority (26/28) of HRSV subgroup B isolates (Table 1). Two of the isolates analysed, RSV 9 and RSV 55, acquired N-glycosylation sites as a result of amino acid substitutions at K278N and D253N, respectively.

One of the features of the G protein is the large number of serine and threonine residues that are potential O-linked sugar acceptors [START_REF] Wertz | Nucleotide sequence of the G protein gene of human respiratory syncytial virus reveals an unusual type of viral membrane protein[END_REF]. The analysis of the deduced 78 to 79 amino acid sequences of the group A strains by the program NetOglyc predicted 25 to 34 potential serine and threonine residues to be O-glycosylated with G scores of between 0.5 and In addition to serine and threonine residues, two repeats of the KPX---TTKX were present among subgroup A strains and may be associated with extensive O-glycosylation of the G protein (Table 2) [Cane et al., 1991[START_REF] Roca | Genetic variability among group A and B respiratory syncytial viruses in Mozambique: identification of a new cluster of group B isolates[END_REF]. This motif was not identified among the subgroup B isolates.

Synonymous vs Non-Synonymous mutations

To study the evolutionary divergence of the HRSV A and B sequences the number of synonymous (silent) and non-synonymous (amino acid altering) nucleotide substitutions was estimated using SNAP (www.hiv.lanl.gov) [START_REF] Korber | HIV Signature and Sequence Variation Analysis[END_REF]. On average, the synonymous mutation/non-synonymous mutation (dS/dN) ratio for all the HRSV groups A samples (i.e. GA5

and GA2) in this study was 1.59. The average synonymous mutation/non-synonymous mutation (dS/dN) ratios for genotypes GA2 and GA5 were 1.64 and 3.13, respectively. Ratios greater than one demonstrated a high abundance of synonymous mutations. This suggested neutral selection pressure on the variable region for all of the HRSV A isolates. Similarly, the mean dS/dN ratio for all of the HRSV B samples in the study was 2.04 which again suggested neutral selection. subgroup A viruses were detected more frequently as found in this study. Studies in Germany, Sweden, Belgium, Argentina, Japan, Kenya and India [START_REF] Ostlund | Molecular epidemiology and genetic variability of respiratory syncytial virus (RSV) in Stockholm, 2002-2003[END_REF][START_REF] Parveen | Genetic variability in the G protein gene of group A and B respiratory syncytial viruses from India[END_REF][START_REF] Reiche | Genetic variability of group A human respiratory syncytial virus strains circulating in Germany from 1998 to 2007[END_REF][START_REF] Sato | Molecular epidemiology of respiratory syncytial virus infections among children with acute respiratory symptoms in a community over three seasons[END_REF][START_REF] Scott | Molecular epidemiology of respiratory syncytial virus in Kilifi district, Kenya[END_REF][START_REF] Viegas | Molecular epidemiology of human respiratory syncytial virus subgroup A over a six-year period (1999-2004) in Argentina[END_REF][START_REF] Zlateva | Subgroup prevalence and genotype circulation patterns of human respiratory syncytial virus in Belgium during ten successive epidemic seasons[END_REF] have reported a predominance of circulating HRSV A between 2004 and 2009.

Two of the eight known genotypes of HRSV A, namely GA2 and GA5, were shown to be been cocirculating since 1998 [START_REF] Frabasile | Antigenic and genetic variability of human respiratory syncytial viruses (group A) isolated in Uruguay and Argentina: 1993-2001[END_REF][START_REF] Ostlund | Molecular epidemiology and genetic variability of respiratory syncytial virus (RSV) in Stockholm, 2002-2003[END_REF][START_REF] Parveen | Genetic variability in the G protein gene of group A and B respiratory syncytial viruses from India[END_REF][START_REF] Sato | Molecular epidemiology of respiratory syncytial virus infections among children with acute respiratory symptoms in a community over three seasons[END_REF][START_REF] Zlateva | Molecular evolution and circulation patterns of human respiratory syncytial virus subgroup a: positively selected sites in the attachment G glycoprotein[END_REF]. This continuous and predominant circulation of the genotypes GA2 and GA5 demonstrate that these strains are stable and have become epidemic in many countries.

In this study, HRSV subgroup A amino acid sequences were predicted to be 297 to 298 residues in length. This was in agreement with previous studies in Uruguay, Argentina and Germany where these changes in protein length were also observed and they correlated with the different genotypes [Frabasile et al., 2003, Reiche and[START_REF] Reiche | Genetic variability of group A human respiratory syncytial virus strains circulating in Germany from 1998 to 2007[END_REF]. It was found that G proteins of 297 amino acids grouped into genotypes GA1 and GA2, whereas G proteins of 298 amino acids in length represented the GA3 and GA5 genotypes [START_REF] Frabasile | Antigenic and genetic variability of human respiratory syncytial viruses (group A) isolated in Uruguay and Argentina: 1993-2001[END_REF]. The results in the current study supported this observation, however, since several genotypes can have the same number of amino acid residues, sequence analysis is an absolute requirement for confirmation of HRSV A genotype.

Genotype-specific amino acid substitutions facilitated differentiation between genotypes GA2 and GA5 in this study. Two were identified in the GA2 genotypes (S269T and P289S) while seven were observed in the GA5 isolates (V225A, S250N, N251S, P274T, V279I, T295I and R297D). All of these substitutions were identified previously in the corresponding genotypes in a study of German isolates examined from the 1998 season to the 2007 season [START_REF] Reiche | Genetic variability of group A human respiratory syncytial virus strains circulating in Germany from 1998 to 2007[END_REF]. Interestingly, some of these substitutions were also observed in strains from Argentina, South Africa and New York [START_REF] Galiano | Genetic and antigenic variability of human respiratory syncytial virus (groups a and b) isolated over seven consecutive seasons in Argentina (1995 to 2001)[END_REF], Peret et al., 1998, Venter et The relatively new genotype of HRSV B genotype, BA, was the only one identified in the current study. The BA strains had three G protein lengths of 312, 315 and 319 amino acids reflecting the 60-nucleotide duplication in the G protein gene. Similarly, G protein lengths of 312 [START_REF] Zlateva | Genetic variability and molecular evolution of the human respiratory syncytial virus subgroup B attachment G protein[END_REF], 315 [START_REF] Trento | Major changes in the G protein of human respiratory syncytial virus isolates introduced by a duplication of 60 nucleotides[END_REF], 317 [START_REF] Zlateva | Genetic variability and molecular evolution of the human respiratory syncytial virus subgroup B attachment G protein[END_REF] and 319 [START_REF] Kuroiwa | A phylogenetic study of human respiratory syncytial viruses group A and B strains isolated in two cities in Japan from 1980-2002[END_REF][START_REF] Zlateva | Genetic variability and molecular evolution of the human respiratory syncytial virus subgroup B attachment G protein[END_REF] have been previously reported. Analysis of the duplicated regions of the Irish HRSV B isolates revealed substitutions in the amino acid sequences when compared to the originally described BA viruses, supporting the view that mutations have accumulated over time and that positive selection influenced the evolution of HRSV [START_REF] Trento | Natural history of human respiratory syncytial virus inferred from phylogenetic Page 33 of 35 John Wiley & Sons Journal of Medical Virology 29 analysis of the attachment (G) glycoprotein with a 60-nucleotide duplication[END_REF]. The BA genotype has been reported in several countries worldwide since its emergence, Japan [START_REF] Kuroiwa | A phylogenetic study of human respiratory syncytial viruses group A and B strains isolated in two cities in Japan from 1980-2002[END_REF][START_REF] Nagai | Nosocomial outbreak of respiratory syncytial virus subgroup B variants with the 60 nucleotides-duplicated G protein gene[END_REF][START_REF] Sato | Molecular epidemiology of respiratory syncytial virus infections among children with acute respiratory symptoms in a community over three seasons[END_REF], China [START_REF] Zhang | Genetic Variability of Respiratory Syncytial Viruses Prevalent in Southwestern China from 2006 -2009: Emergence of RSV BA as Dominant Strains[END_REF], Kenya [START_REF] Scott | Molecular epidemiology of respiratory syncytial virus in Kilifi district, Kenya[END_REF], Belgium [START_REF] Zlateva | Genetic variability and molecular evolution of the human respiratory syncytial virus subgroup B attachment G protein[END_REF], Canada, Brazil the United Kingdom and the United States [START_REF] Trento | Natural history of human respiratory syncytial virus inferred from phylogenetic Page 33 of 35 John Wiley & Sons Journal of Medical Virology 29 analysis of the attachment (G) glycoprotein with a 60-nucleotide duplication[END_REF]. This rapid and global spread implies that the BA genotype of the HRSV B virus may have a selective advantage over other circulating strains. It can be hypothesised that the 60-nucleotide duplication in this genotype may result in an antigenic change in the G protein and avoidance of host immune responses which would result in a selective advantage. However, it is possible that there may be mutations elsewhere in the genome that have conferred on these viruses more replicative capacity than other HRSV B genotypes. The frequency and pattern of glycosylation sites identified in this study was different between the HRSV A and HRSV B subgroups. One to four potential (three in the majority of isolates) Nglycosylation sites were identified in Irish HRSV A isolates while two to three potential sites were identified in the HRSV B isolates. More extensive N-glycosylation among the HRSV A subgroup of isolates may influence antibody binding to the protein [Cane et al., 1991]. This in turn, may explain the greater antigenic variability observed for the G glycoprotein [Garcia- [START_REF] Garcia-Barreno | Marked differences in the antigenic structure of human respiratory syncytial virus F and G glycoproteins[END_REF]]. The predicted O-glycosylation sites in the C-terminal region of the G protein were identified in both HRSV A and HRSV B isolates where there were 25 to 34 and 40 to 43 identified, respectively. The number of sites identified in the HRSV A isolates in this study are very similar to the number identified in HRSV A isolates in another study in Germany which varied from 22 to 34 [START_REF] Reiche | Genetic variability of group A human respiratory syncytial virus strains circulating in Germany from 1998 to 2007[END_REF]. Furthermore, the greater number of potential sites observed in the HRSV B BA genotype isolates may be due to the 60-nucleotide duplication in the G protein where 16 to 19 potential sites were identified among the duplicated region alone. Similarly, these additional O-glycosylation residues have also been identified in the duplicated region in a previous study [START_REF] Zlateva | Genetic variability and molecular evolution of the human respiratory syncytial virus subgroup B attachment G protein[END_REF]. Therefore, these additional Olinked glycosylation residues in the BA genotype may influence the expression of some of the antigenic epitopes by either masking the antigenic sites or contributing to antibody recognition, thus giving these strains an evolutionary advantage over other HRSV subgroup B viruses. In The average dS/dN ratios for the HRSV A and HRSV B isolates in this study were 1.59 and 2.04, respectively, which implied neutral selection pressure on the variable region of the G protein for genotypes GA2, GA5 and BA in this study. Ratios of greater than or equal to one (>=1) demonstrate a high abundance of synonymous mutations [START_REF] Reiche | Genetic variability of group A human respiratory syncytial virus strains circulating in Germany from 1998 to 2007[END_REF].

However, when the individual genotypes of the RSV A isolates were compared with each other, the average dS/dN ratios were calculated to be 1.64 and 3.13 for GA2 and GA5 isolates, respectively. A study in Germany found that there was neutral selection pressure on the GA5 isolates which were found to have an average dS/dN value of 4.34 similar to a dS/dN of 3.13 in the current study, but a positive selection pressure on the GA2 isolates with a dS/dN of 1.18 [START_REF] Reiche | Genetic variability of group A human respiratory syncytial virus strains circulating in Germany from 1998 to 2007[END_REF]. However, in another study in India, the average dN/dS ratio was 1.16 and the dS/dN ratio was 1.37 for their HRSV subgroup A isolates and they report that these values suggest positive and negative selective pressures, respectively [START_REF] Parveen | Genetic variability in the G protein gene of group A and B respiratory syncytial viruses from India[END_REF].

These results are similar to the results of isolates in the present study as the dS/dN ratios appear to be lower for the GA2 isolates (1.64) than those of GA5 isolates. The observation that a dS/dN of 1.16 was evidence of positive selection [START_REF] Reiche | Genetic variability of group A human respiratory syncytial virus strains circulating in Germany from 1998 to 2007[END_REF]] but a dS/dN of 1.37 was evidence of neutral selection [START_REF] Parveen | Genetic variability in the G protein gene of group A and B respiratory syncytial viruses from India[END_REF] implies that the dS/dN of 1.64 for the GA2 isolates in this study indicated neutral selection. In this study, the dS/dN for the HRSV B isolates was 2.04. No reports were found to compare this value with for other HRSV B, genotype BA samples in other countries but this dS/dN value does suggest that there was a neutral selection pressure in these isolate as the value is greater than one. However, due to the

Figure 1 .Figure 3 .

 13 Figure 1. Distribution of HRSV subgroups in each winter season. 143x101mm (96 x 96 DPI)

5

  Data on HRSV infection based on GP and hospital sentinel surveillance programmes in the Republic of Ireland is published weekly by the Health Protection Surveillance (HPSC) (http://www.ndsc.ie/hpsc/A-Z/Respiratory/Influenza/SeasonalInfluenza/InfluenzaSurveillanceReports). This information shows that HRSV infection occurs predominantly in children less than five years of age and in a seasonal pattern, mainly between October and March each year. An active surveillance programme in the adult haematopoetic stem cell transplant facility in St James's Hospital targets immunocompromised haematology patients with the aim of early identification of influenza A and B viruses, HRSV, parainfluenza viruses types 1 to 4 and adenovirus. There is no information published regarding the molecular characterisation of HRSV in the Republic of Ireland. Therefore, this study investigated the distribution of HRSV subgroups A and B virus infections among adult patients with haematological malignancy attending St. James's Hospital over between November 2004 and March 2009. Furthermore, phylogenetic analysis was performed to determine the different genotypes of HRSV in this cohort during those five winter seasons.

  to the intensive care unit (ICU). None of them died of HRSV lower respiratory tract infection.Human respiratory syncytial virus (HRSV) subgroupsOf these 60 isolates, 31 (52%) belonged to HRSV subgroup A and 29 (48%) belonged to HRSV subgroup B. An overview of the distribution of HRSV subgroups over the five seasons is shown in Figure 1. HRSV subgroup B predominated in 2004/2005 and 2006/2007 while in the 2005/2006 and 2007/2008 seasons HRSV A were more prevalent. The 2008/2009 season showed an even distribution of isolates of both subgroups.

  level among the Irish HRSV A strains. Differences of 8% were observed among the genotype GA2 strains at both nucleotide and amino acid level. Genotype GA5 strains showed as much as 6% nucleotide and amino acid differences.The phylogenetic tree also demonstrated that both of the HRSV A genotypes co-circulated in each of the epidemic seasons with the exception of the 2008/2009 season, where all of the HRSV A isolates belonged to genotype GA2 (RSV 51, RSV 54, RSV 57, RSV 59 and RSV 60) (Figure 2). In 2004/2005, of the six HRSV A isolates, one (RSV 13) belonged to genotype GA2 and five (RSV 2, RSV 3, RSV 5, RSV 7 and RSV 15) to GA5. The 2005/2006 season had five (RSV 20, RSV 22, RSV 27, RSV 28 and RSV 30) isolates that were genotype GA5 and one (RSV 24) was GA2. The 2006/2007 season, showed an even distribution of isolates between the two genotypes GA2 and GA5, with two (RSV 31 and RSV 35) belonging to GA5 and two (RSV 37 and RSV 39) belonging to GA2. The 2007/2008 season also showed an even distribution between the two genotypes, with five (RSV 41, RSV 43, RSV 44, RSV 48 and RSV 49) identified as genotype GA2 and five (RSV 42, RSV45, RSV 46, RSV 47 and RSV 50) in genotype GA5 cluster. The

  the known HRSV B genotypes (Figure3). The reference strain Ken/109/02 (GenBank AY524573) was used an outgroup in the phylogenetic tree. In this study, all of the HRSV subgroup B isolates examined belonged to the newly identified genotype, genotype BA.

  The same program also predicted 40 to 43 serine and threonine residues to be potentially Oglycosylated with the score predictors (G scores) of between 0.5 and 0.8 in the deduced 94 to 101 amino acids of group B strains.

  , the C-terminal region of the G protein was examined in all isolates collected over five seasons between 2004/2005 and 2008/2009 to obtain important phylogenetic and antigenic information of the circulating HRSV viruses in Ireland. Phylogenetic analysis of HRSV positive isolates in this study revealed that the HRSV A subgroup occurred at a slightly higher frequency over the five winter seasons from 2004/2005 to 2008/2009, with HRSV A and B at 52% and 48%, respectively. It was observed that HRSV B predominated in two of the five seasons (2004/2005 and 2006/2007) investigated in this study, although HRSV A was the only subgroup found in the 2005/2006 and 2007/2008 seasons when it was the only HRSV subgroup detected. During the 2008/2009 season an even distribution of HRSV A and HRSV B isolates was observed. Although shifts in HRSV subgroup dominance have been reported worldwide, HRSV

  circulating inIreland between 2004Ireland between /2005Ireland between and 2008Ireland between /2009. The GA5 genotype predominated at 55% compared to the GA2 genotype (45%) in the HRSV subgroup A isolates examined. The identification of GA2 and GA5 genotypes in this study is consistent with the results of long-term studies from Europe and other geographic areas. In countries like Sweden, Belgium, Uruguay, Argentina and the United States genotypes GA2 and GA5 and, at a lower frequency GA7, have

  conclusion, the number and location of the glycosylation sites in the G protein of the HRSV virus may cause a long lasting circulation of certain genotypes by avoiding the neutralization by pre-existing antibodies in the host. Further work is however necessary to understand the functional importance of glycosylation sites in the G protein of HRSV which may be important changes in virulence and immune responses to the HRSV virus in future vaccination studies.

  used in this study, further work on a larger sample number from other population groups is needed to confirm this value. Further worldwide data on the dS/dN values of this genotype would also be beneficial in determining the true selection pressure on this genotypes.The use of pre-emptive aerosolised ribavirin treatment in this study appeared to be effective in preventing progression from upper to lower respiratory tract infection with HRSV. Most samples were collected from adults with HRSV upper respiratory tract infection. Those patients at high risk of progression to HRSV pneumonia were treated pre-emptively with aerosolised ribavirin 2g every 8 hours for 5 days. None of this cohort progressed to lower respiratory tract infection, supporting findings of a previous study (Boeckh M et al Clinical Infectious Diseases 2007; 44(2); 245-9 Randomized controlled multicenter trial of aerosolized ribavirin for respiratory syncytial virus upper respiratory tract infection in hematopoietic cell transplant recipients). However, three patients who presented to hospital with HRSV pneumonia were admitted to the intensive care unit (ICU) and survived following treatment with intravenous ribavirin and supportive care. Although all the samples examined in this study were collected from adult patients, these infections were community-acquired reflecting strains circulating in the community, including among children who are recognised as potential sources of HRSV. The molecular epidemiology of the HRSV isolates circulating in Ireland, based on this study, only reflected isolates from a Dublin hospital from the 2004/2005 to 2008/2009 seasons. The adult HSCT facility in St James's Hospital is a national unit with at least 50% of patients referred from outside of Dublin. HRSV A viruses were observed to be marginally predominant over HRSV B in Ireland over five winter season from 2004/2005 to 2008/2009 and both the HRSV subgroups were observed to be cocirculating in each of the seasons, except in 2008/2009. Two genotypes,GA2 and GA5 were identified among the HRSV A isolates and have been reported to be circulating in Europe for more than 20 years[START_REF] Rafiefard | Molecular epidemiology of respiratory syncytial virus (RSV) of group A in Stockholm, Sweden, between 1965 and 2003[END_REF]. All of the HRSV B isolates belonged to genotype BA. Phylogenetic analysis of the C-terminal regions of the G protein of the two HRSV subgroups has shown that closely related strains of HRSV can be found in different parts of the world at different times and the same genotypes have been circulating for a long time.

John Wiley & SonsJournal of Medical Virology

John Wiley & Sons Journal of Medical Virology John Wiley & Sons Journal of Medical Virology John Wiley & Sons Journal of Medical Virology John Wiley & Sons Journal of Medical Virology John Wiley & Sons Journal of Medical Virology John Wiley & Sons Journal of Medical Virology John Wiley & Sons Journal of Medical Virology John Wiley & Sons Journal of Medical Virology John Wiley & Sons Journal of Medical Virology John Wiley & Sons Journal of Medical Virology John Wiley & Sons Journal of Medical Virology John Wiley & Sons Journal of Medical Virology John Wiley & Sons Journal of Medical Virology John Wiley & Sons Journal of Medical Virology John Wiley & Sons Journal of Medical Virology


Zornitsa ; Mladenova
email: zornitsavmbg@yahoo.com

Miren Iturriza-Gomara

Mathew D Esona

James Gray

Neli Korsun

Genetic characterization of Bulgarian rotavirus isolates and detection of 1 rotavirus variants: challenges for the rotavirus vaccine program? 2 3

Keywords: rotavirus, genotype, mutant, rotavirus vaccine

Annually 20-70% of all hospital admissions and 20% of fatal diarrhea cases among 2 children less than 5 years of age occur due to severe rotavirus diarrhea. Universal 3 immunization is the major strategy aimed at controlling rotavirus infection. The main

analysis revealed strain diversity and circulation of different rotavirus variants belonging to 7 a single genotype. A mutated G4P [START_REF] Trojnar | Johne R The first complete genome sequence of a chicken group A 9 rotavirus indicates independent evolution of mammalian and avian strains[END_REF] strain with the insertion of an asparagine residue in 8 position 76; G2, G9 and G1 variants with amino acid substitutions in the antigenic regions 9

A, B and/or C were all identified in this study in the absence of an immunization program. 10 Rotavirus strain surveillance in both the pre-and post-vaccine eras is of increasing 11 importance in order to assess the effectiveness of the rotavirus vaccines for protection 12 against disease associated with a diverse population of rotavirus strains. [START_REF] Schumann | Evidence of interspecies transmission and 14 reassortment among avian group A rotaviruses[END_REF] 14 Key words: rotavirus, genotype, mutant, rotavirus vaccine 15 rotaviruses. Rotavirus genotypes G1P [START_REF] Trojnar | Johne R The first complete genome sequence of a chicken group A 9 rotavirus indicates independent evolution of mammalian and avian strains[END_REF] G2P [4], G3P [START_REF] Trojnar | Johne R The first complete genome sequence of a chicken group A 9 rotavirus indicates independent evolution of mammalian and avian strains[END_REF], G4P [START_REF] Trojnar | Johne R The first complete genome sequence of a chicken group A 9 rotavirus indicates independent evolution of mammalian and avian strains[END_REF] and G9P [START_REF] Trojnar | Johne R The first complete genome sequence of a chicken group A 9 rotavirus indicates independent evolution of mammalian and avian strains[END_REF] are 2 associated most frequently with diarrhea in children. However, other uncommon G and P 3 types (for example G5, G6, G8, G10, G12, P [START_REF] Iturriza-Gomara | Rotavirus genotyping: keeping up with an 10 evolving population of human rotaviruses[END_REF], P [11] and P [14]) have been reported in a 4 variety of locations worldwide [START_REF] Desselberger | Rotavirus epidemiology and 20 surveillance[END_REF]Santos and Hoshino, 2005]. 5 Some of these genotypes such as G5P [START_REF] Trojnar | Johne R The first complete genome sequence of a chicken group A 9 rotavirus indicates independent evolution of mammalian and avian strains[END_REF] in Brazil, G8P [6] in Malawi, and G10P [11] in 6

India are important causes of diarrhea in countries where they are found [Alfieri et al., 7 1996;[START_REF] Cunliffe | Rotavirus G and P types in children with acute diarrhea in 17 Blantyre, Malawi, from 1997 to 1998: predominance of novel P[6]G8 strains[END_REF][START_REF] Iturriza-Gomara | Rotavirus genotyping: keeping up with an 10 evolving population of human rotaviruses[END_REF], and are likely to emerge by 8 zoonotic introduction. Zoonotic transmission and reassortment with human adapted strains 9 play a major role in the emergence and spread of rotavirus strains in the human population 10 for the G and P genotypes using primers and protocols described previously [Gouvea et G4P [START_REF] Trojnar | Johne R The first complete genome sequence of a chicken group A 9 rotavirus indicates independent evolution of mammalian and avian strains[END_REF], five G2P [4], three G9P [START_REF] Trojnar | Johne R The first complete genome sequence of a chicken group A 9 rotavirus indicates independent evolution of mammalian and avian strains[END_REF] and seven G1P [START_REF] Trojnar | Johne R The first complete genome sequence of a chicken group A 9 rotavirus indicates independent evolution of mammalian and avian strains[END_REF], representing four of the five globally 10 common strains in circulation were selected at random for the present study (Table 1). The 11 rotavirus strains selected for investigation were collected from children under the age of 5 12 years. [START_REF] Schumann | Evidence of interspecies transmission and 14 reassortment among avian group A rotaviruses[END_REF]

14

VP7 and VP4 characterization by sequence analysis: 15

The extracted dsRNA of each of the 19 selected strains was denatured at 97 o C for 5 min 16 and RT-PCR was carried out using a One-Step RT-PCR kit (Qiagen, Valencia, CA) 17 according to manufacturer's instructions. Forward and reverse primers described 18 previously were used for the amplification of the gene encoding VP7 and the VP8* portion 19 of the VP4 gene segment [START_REF] Gouvea | 19 Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool 20 specimens[END_REF][START_REF] Gentsch | Identification of group A rotavirus gene 4 types by Polymerase Chain Reaction[END_REF] belonged to cluster II together with G1 rotaviruses from Europe, Latin America and Asia. 17

These strains showed a similarity level of 99% with an G1 strain from Paraguay 18 (Py03SR286), isolated in 2003 and was also similar to a G1 strain SI-201 detected in 19

Slovenia three years later. The amino acid (aa) substitutions of the Bulgarian and the 20 reference G1 strain WA are shown in Table 2. 21 for VP7 sequence analysis. The data revealed that the Bulgarian G2 strains belonged to 4 one, highly homogenous cluster (95-99% similarity at the nt and aa levels; Figure 2). The 5 comparison with the reference G2 strain DS-1 showed that all Bulgarian G2 rotaviruses 6 sequenced displayed aa changes, similar to that described for G2 strains isolated since the 7 mid-1990's -two aa changes within antigenic region A (aa 87 and aa 96), and additional aa 8 substitution in position aa 213 in antigenic region C (Table 2). 9 10 respectively. They showed 98-99% and 100% homology at the nt and deduced amino acid 16 (aa) level, respectivelly. All 6 strains in this cluster possessed an insertion of three nts 17 (ATA) which led to the insertion of a single asparagine residue at position 76 in the aa 18 sequence in relation to prototype strain ST-3 (Table 2). Three additional aa substitutions, 19 one in position 79 close to the antigenic region A and one in the region B (aa 143 and aa 20 145) were also observed. 21

Genetic diversity among G4 isolates 11

The fourth strain, BG1385/2007, shared 85-86% and 94-97% homology at the nt and aa 22 levels, respectively, with the other three Bulgarian G4 strains described above and other 23 was closely related to a Hungarian G4 human rotavirus detected in 2004, Hu/BP1125. The 2 phylogenetic analysis indicated that these two strains formed a separate lineage from those 3 previously described, and denominated lineage IX following the classification of Stupka et 4 al., [2009]. The differentiation of the new lineage IX was supported by nt and aa similarity 5 distances (data not shown). Neither G4 strain in this new lineage possessed the insertion at 6 position 76, and differed from the reference strain ST3 in seven aa positions: aa 74, aa 75 7 and aa 79 in close proximity to antigenic region A; three within region A; aa 87, aa 90, and 8 aa 96 and aa 145 in the antigenic region B (Table 2). 9 10

Genetic diversity among G9 isolates 11

The genes encoding VP7 of three Bulgarian G9 rotaviruses (one from the rotavirus season 12 2005/2006 and two from the season 2006/2007) were sequenced. The Bulgarian G9 strains 13 were closely related to each other with nt identity of 96-98%, but distantly related to G9 14 prototype strains WI61 and 116E isolated from USA and India (85-86% nt similarity). 15 Detailed analysis clearly demonstrated that all Bulgarian G9s belonged to lineage ІІІ, sub-16 lineage d and were genetically similar to G9 strains isolated recently in Europe, Asia, 17 America and Australia (Figure 4). The aa substitutions of the Bulgarian G9s and the 18 reference G9 strain WI61 are shown in Table 2. 19 20

Genetic diversity among P[8] genotype 21

The partial VP4 gene (VP8*) of nine rotavirus strains with a P [START_REF] Trojnar | Johne R The first complete genome sequence of a chicken group A 9 rotavirus indicates independent evolution of mammalian and avian strains[END_REF] August 2008 showed significant morbidity attributable to rotavirus infection in the country, 5 with an incidence of rotavirus diarrhea of between 28 and 43%. Strain characterization 6

showed that 95% of the strains were of the same rotavirus genotypes distributed 7 worldwide, and an annual shift in the predominant rotavirus strain between each of the four 8 rotavirus seasons, defined as starting in September of one calendar year though to August 9 of the following year [Mladenova et al., 2010]. To gain insight into the relationships 10 between the prevalent rotavirus strains in Bulgaria and the common rotaviruses circulating 11 worldwide, 19 previously genotyped rotavirus strains (seven G1P [START_REF] Trojnar | Johne R The first complete genome sequence of a chicken group A 9 rotavirus indicates independent evolution of mammalian and avian strains[END_REF] strains, five G2P [4], 12 four G4P [START_REF] Trojnar | Johne R The first complete genome sequence of a chicken group A 9 rotavirus indicates independent evolution of mammalian and avian strains[END_REF], and three G9P [START_REF] Trojnar | Johne R The first complete genome sequence of a chicken group A 9 rotavirus indicates independent evolution of mammalian and avian strains[END_REF] ) collected during these four rotavirus seasons were 13 selected at random and subjected to sequence and phylogenetic analysis of the VP7 gene 14 segment (G genotype). In addition, the partial VP4 gene (VP8*) of 9 rotavirus strains with 15 P [START_REF] Trojnar | Johne R The first complete genome sequence of a chicken group A 9 rotavirus indicates independent evolution of mammalian and avian strains[END_REF] genotype (two with G4 genotype, one with G2 genotype, three with G1 specificity, 16 and all three G9 rotavirus strains), were also sequenced. The phylogenetic analyses showed 17 that the Bulgarian rotaviruses with G1, G2, G4 and G9 specificities displayed considerable 18 sequence similarity with the rotavirus lineages of rotavirus strains detected in the last 10 extremely frequent events because rotavirus RNA-dependent RNA polymerase is error-2 prone with a mutation rate of 5х10 -5 per nt per replication [START_REF] Blackhall | Genetic stability of a porcine rotavirus RNA 11 segment during repeated plaque isolation[END_REF]. Some 3 point mutations are highly conserved and are inherited by the rotavirus progeny. The most 4 significant are those mutations within the antigenic regions which may lead to the 5 generation of antibody escape mutants. Such antibody escape mutants may lead to the 6 emergence "epidemics" and infect large proportions of the populations causing symptoms 7 even in individuals who may have previously been primed either through naturally 8 acquired wild time rotavirus infections or though vaccination. Furthermore, the emergence 9 and accumulation of nt mutations followed by amino acid transitions in uncommon human-10 animal reassortants and zoonotic rotavirus strains could drive the adaptation of these strains 11 in the human host and their transformation from sporadic to epidemic/ pandemic strains. 12

The rapid dissemination of G9 rotaviruses in the human population in the early 1990s may 13 have been the result of both genetic shift between animal G9P [6] and human rotavirus 14 strain with P [START_REF] Trojnar | Johne R The first complete genome sequence of a chicken group A 9 rotavirus indicates independent evolution of mammalian and avian strains[END_REF] specificity and the continuous adaptation of the newly emerged strain to 15 the human host as a result of genetic drift. Thus, after a decade of sporadic infections with 16 no, or limited, onward transmission, the G9 genotype evolved, acquired a replicative 17 advantage and became one of the five most prevalent human genotypes transmitted 18 worldwide [Santos and Hoshino, 2005]. 19 20 In Bulgaria, G4P [START_REF] Trojnar | Johne R The first complete genome sequence of a chicken group A 9 rotavirus indicates independent evolution of mammalian and avian strains[END_REF] rotavirus strains were the dominant rotavirus G-P combination 21 Nicaragua [Bucardo et al., 2005]. The detailed analysis revealed that this unique insertion 6 together with several other minor mutations led to modification of the secondary structure 7 of the VP7 protein in these human G4 strains, and this change, was probably the trigger for 8 the large outbreak described in Nicaragua in which a high attack rate was observed in 9 adults suggested lack of protection conferred by previous rotavirus infections [Bucardo et 10 al., 2007]. Unfortunately, a G4 isolate, which circulated during 2004/2005 rotavirus season, 11 when the G4 was detected as a dominant strain, was not available for genetic 12 characterization. Sequence analysis of such isolates might have shed some light on whether 13 the predominance of G4P [START_REF] Trojnar | Johne R The first complete genome sequence of a chicken group A 9 rotavirus indicates independent evolution of mammalian and avian strains[END_REF] in 2004/2005 was related to the introduction of the strain 14 possessing the insertion or with any other genetic changes which may have also correlated 15 with the emergence of an antibody escape mutant, including the emergence of the novel 16 genetic lineage described in this paper. 17

The G4 rotavirus strain BG1385 showed the closest genetic relatedness with the human 18 rotavirus BP1125/2004 found in Hungary and thought to be of potential zoonotic origin 19 based on its genetic constellation -G4-P[6]-GGII-B, respectively VP7-VP4-VP6- NSP4 20 [Banyai et al., 2009]. The VP7 gene of these two strain showed high degree of similarity of 21 nt and aa similarity (96% and 97% respectively), and only four aa substitution were found 22 between the two strains, all outside the antigenic regions (aa 64, aa82, aa162 and aa 171).. 23 The VP4 associated with the Bulgarian stain was however a P [START_REF] Trojnar | Johne R The first complete genome sequence of a chicken group A 9 rotavirus indicates independent evolution of mammalian and avian strains[END_REF], which may indicate that 1 this strain may have undergone reassortment with a human strains, but elucidating its likely 2 origin and evolution will require further work. The comparison with the reference G2 strains S2 and DS-1 showed that all Bulgarian G2 8 rotaviruses sequenced, displayed two aa changes at positions aa 87 (Ala-Thr) and aa 96 9

Figure 1 .Figure 2 .Figure 3 .Figure 4 .

 1234 Figure 1. Phylogenetic tree based on partial VP7 gene (nt 201-771) of G1 rotavirus isolates constructed using Neighbor-joining method of MEGA4. The sequence nomenclatures show the origin, name and laboratory number, and season of circulation (where data are available). Bulgarian G1 strains are marked by the symbol ■. On the right, the clustering of the strains into lineages is represented.

Introduction 1 Human 20 21

 120 group A rotaviruses have been recognized as the most important etiological agents 2 of severe diarrhea in infants and young children throughout the world. The WHO estimates 3 that annually 20-70% of all hospitalizations and 20% of fatal diarrhea cases in children <5 4 years of age occur due to severe rotavirus infection [Parashar et al.; 2006; Estes and 5 Kapikian, 2007]. Rotavirus vaccination is recognized currently as the best strategy for the 6 global control and prevention of severe rotavirus illnesses in young children, as 7 recommended by WHO. As vaccination becomes used more widely, the knowledge of 8 strain distribution pre-and post-vaccine introduction becomes of increasing importance in 9 order to monitor any possible changes in strain distribution and assess vaccine efficacy 10 against co-circulating and potential emerging rotavirus strains. 11 12 Rotaviruses are classified as a genus within the Reoviridae family and contain a genome of 13 11 segments of double-stranded RNA which encode 6 structural (VP1-VP4 and VP6, VP7) 14 and 6 non-structural (NSP1-NSP6) proteins [Estes and Kapikian, 2007]. The VP7 and VP4 15 gene segments code for two outer capsid proteins, a glycoprotein (G) and a protease-16 sensitive protein (P) that define genotypes/serotypes and form the basis for classifying 17 group A rotaviruses into G and P types [Estes and Kapikian, 2007]. The VP7 and VP4 18 coded proteins elicit neutralizing antibodies after natural rotavirus infection or rotavirus 19 vaccination. To date, 23 different G genotypes and 32 P genotypes have been described [Matthijnssens 22 et al., 2008; Schumann et al., 2009; Solberg et al., 2009; Trojnar et al., 2009; Ursu et al., 23 et al., 2009], indicating extensive genomic diversity within group A 1

[ 3 From

 3 Iturriza-Gómara et al., 2001; Martella et al., 2010]. Another mechanism important for the 11 generation of strain diversity, which could lead to emergence of antibody escape mutants, 12 is the accumulation of point mutations [Estes and Kapikian, 2007]. 13 14 National and international strain surveillance networks have been established worldwide to 15 document the prevalence of local strains and to monitor for possible emergence of 16 uncommon G and P genotypes prior to the introduction of rotavirus vaccination and during 17 the implementation of vaccination programs. In this context, the Bulgarian Rotavirus 18 Surveillance Network was established in order to investigate the rotavirus epidemiology 19 and strain diversity in the country in pre-and post-vaccine eras. In previous studies it was 20 demonstrated high incidence of rotavirus gastroenteritis among hospitalized children less 21 than 5 years of age and co-circulation of common and uncommon rotavirus G-P genotype 22 combinations [Mladenova et al., 2010; Tcheremenskaia et al., 2007]. The objective of the 23 January 2005 to August 2008, a total of 1015 rotavirus-positive stool samples were 4 collected from children less than 9 years of age and adults, who presented with acute 5 diarrhea at nine different hospitals in Bulgaria [Mladenova et al., 2010]. After genotyping 6

author 2 F 2

 22 ; Iturriza-Gomara et al., 20 2000; Iturriza-Gomara et al., 2001; Iturriza-Gomara et al., 2004; Simmonds et al., 2008]. 21 After denaturation of the dsRNA, reverse transcription (RT) of each gene from each sample 22 was carried out for 30 min at 50 o C, followed by 15 min at 95 o C to inactivate the reverse 23 the DNA polymerase. The cDNA was then subjected to 30 cycles 1 of PCR in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, Foster City, 2 CA) using the following conditions: 60 sec at 94 o C; 60 sec at 50 o C; 60 sec at 72 o C, 3 followed by a single 7 min extension at 72 o C and a 4 o C cooling step. Amplicons were 4 purified with the QIAquick PCR purification kit (Qiagen, Hilden, Germany) according to 5 the manufacturer's protocol. Sequencing of each amplicon was performed with the same 6 consensus primers used for RT-PCR, using a Big Dye Terminator cycle sequencing Ready 7 kit v3.1 (Perkin-Elmer, Applied Biosystems). Automated separation and base-calling of 8 cycle sequencing products was carried out using an ABI 3100 sequencer (Applied 9 Biosystems). The VP7 and VP4 gene derived sequences are freely available from the 10 //www.clcbio.com/index.php?id=1220]. The rotavirus genotype was confirmed by 16 alignment of each of the Bulgarian sequences with sequences published in the GenBank 17 database using BLAST search [http://www.ncbi.nlm.nih.gov]. Dendrograms were 18 constructed after multiple alignments using ClustalX and neighbor-joining or maximum 19 parsimony method listed in the MEGA 4 analytical package [Tamura et al., 2007] and 1000 20 bootstrap replicates were used for statistical evaluation. Bootstrap values of >80 were used 21 to define genetic lineages among clusters of sequences. Genetic distances were calculated 22 two-parameter method. The accession numbers and strains used for this 1 study are shown in Appendix 1. Phylogenetic analysis was conducted on the following G1 Bulgarian rotaviruses: 3 BG149/2005, BG1053/2007, BG1120/2007, BG1153/2007, BG1670/2007, BG45/2008 and 4 BG389/2008. The phylogenetic tree, based on the classification described by Phan et al. 5 [2007], revealed that Bulgarian G1 rotaviruses belonged to two different lineages (Figure 6 1). Four of the Bulgarian G1 rotaviruses that circulated during different rotavirus seasons -7 BG149/2005, BG1120/2007, BG1670/2007 and BG389/2008 were grouped within lineage 8 I, together with G1 strains detected worldwide during last ten years. The similarity in 9 nucleotide (nt) and deduced amino acid (aa) sequences of these rotaviruses varied in range 10 of 94-99% and 97-100%, respectively. Based on the deduced aa sequence, strains 97'SH19, 11 CMH026, Dhaka 3-03, ChZ678 and two Bulgarian strains BG149/2005 and BG1670/2007 12 had a substitution at position 94 in antigenic region A, while the Japanese isolate 7154J and 13 Bulgarian strains BG1120/2007 and BG389/2008 possess aa change in antigenic region B 14 (aa 147). 15 The remaining three Bulgarian G1s (BG1053/2007, BG1153/2007 and BG45/2008) 16

1 A

 1 total of five G2 strains, representing different seasons of circulation (including 2 BG80/2006, BG915/2007, BG1643/2007, BG1726/2007, and BG683/2008) were selected 3

Four

  Bulgarian G4 strains (BG170/2006, BG1385/2007, BG623/2008 and BG773/2008) 12 were analyzed through sequencing of the VP7 encoding gene (Figure 3). Strains 13 BG170/2006, BG623/2008 and BG773/2008 clustered in lineage I, together with human 14 strains Py99355, J-4614 and RUS-Nov08-3260 detected in Paraguay, Japan and Russia, 15

  G4 rotaviruses presented in the GenBank database to date. This strain 1

1 2

 1 The study described above is the first detailed molecular genetic investigation of rotavirus 3 strain diversity in Bulgaria. Rotavirus surveillance in Bulgaria between January 2005 and 4

  19 years worldwide. Furthermore, the sequence data revealed co-circulation of different 20 rotavirus variants of a single genotype with nt and aa changes in antigenic regions Amain driving forces of rotavirus evolution is genetic drift. Point mutations are 1

(

  65.8%) in the 2004/2005 rotavirus season, but had low prevalence of 0.3-4.3% throughout 22 the next three seasons until their re-emergence in August 2008. The sequence data provided 23 G4 strains detected in 2005/2006 and the newly-introduced strain in 1 2007/2008 were genetic variants with an aa insertion close to antigenic region A. The first 2 G4P[8] mutants with such insertion were reported in Argentina in 1998, Uruguay in 1999 3 and Brazil from 2000 to 2004. A year later, in 2005, this strain was responsible for a large 4 outbreak of acute gastroenteritis affecting over 64,000 individuals and causing 56 deaths in 5

3 G2

 3 rotaviruses were the second genotype found most commonly during the four-year study 4 period in Bulgaria. Their incidence throughout the different rotavirus seasons varied from a 5 sporadic spread during seasons 2004/2005 and 2005/2006 to an epidemic scale in 6 2006/2007, with an incidence of 49.3% among patients with confirmed rotavirus diarrhea. 7

( 19 A 4 5 1 (

 1941 Asp-Asn) in the antigenic region A. The same aa substitution at position aa 96 was 10 reported by Iturriza-Gomara and colleagues[Iturriza-Gómara et al., 2001] who linkedit 11 with the failure to react with G2-specific monoclonal antibodies. In another study based on 12 the antigenic and genetic variation of the VP7 gene of human G2 rotaviruses isolated in 13 various African countries, two strains which displayed the same aa change and an 14 additional substitution in antigenic region C, have not reacted with either IC10 or S2-2G10 15 G2-specific monoclonal antibodies[START_REF] Page | Antigenic and genetic characterization of serotype G2 human 4 rotavirus strains from the African continent[END_REF]. Therefore, the aa substitutions 16 in one or more antigenic regions in the newly emerging G2 rotavirus variant could explain 17 the evasion of pre-existing immunity. 18 total of three G9 and seven G1 strains were investigated in order to characterise 20 Bulgarian G9 and G1 rotaviruses by sequencing of their VP7 genome segments and to 21 compare them with other G9 and G1 rotaviruses circulating in different countries. The 22 results showed the co-circulation of variants in Bulgaria similar to those which have been 23 parts of the world during the last few years [Banyai et al., 2009; 1 Gentch et al., 2009]. Overall, these data support the likelihood that the introduction of new 2 genetic variants in Bulgaria may be facilitated by current globalization, by transnational 3 and transcontinental transport, trade and migration.In summary, a decision on the introduction of rotavirus vaccine into the routine vaccination 6 schedule in Bulgaria is pending. Rotavirus molecular genetic and epidemiological 7 investigations provide baseline data for the prevalence and strain diversity of rotaviruses in 8 Bulgaria in the pre-vaccine era. Active surveillance and detailed genetic characterization of 9 the common rotavirus strains in the pre-vaccine era are extremely important in order to 10 gather and update information of the currently circulating variants and to identify candidate 11 virus strains suitable for the next generation of rotavirus vaccine. The monitoring of the 12 new variants of the common rotavirus strains in the post-vaccine era, which will have 13 emerged spontaneously or selected under the vaccine pressure, will contribute to assessing 14 their epidemic potential, global spread and impact on the global rotavirus vaccine program. 15 M, Cubitt D, Desselberger U, Gray J. 2001. Amino acid substitution 1 within the VP7 protein of G2 rotavirus strains associated with failure to serotype. J Clin ; ICB2185 (AF192267); Arg/P23 (AY115859); CMP114 (DQ683511); K X58439); for G9 analysis: G9-SP5.6-9 (EU15197); AHP66 (AB364375); GUP30 2 (AB364374); G9-SP5.0-9 (EU159192); ITA-BIA2 (EF150330); SP3-9 (EF159955);

Table 2 .

 2 List of the 19 Bulgarian rotavirus strains selected for analysis. The common amino acid substitutions in different lineages are bolded

	Rotavirus strain ID	Year/place of collection	PCR genoty pe	Sequence genotype	Reference strain	Key aa polymorphism
	BG149/2005	May 2005/Sofia-region	G1	lineage I	WA	N94S; D97E; S146N;
						M217T; I218V
	BG1053/2007 July 2007/Sofia	G1	lineage II	WA	D97E; I218V
	BG1120/2007 August 2007/Sofia	G1	lineage I	WA	D97E; S146N; L147F;
						M217T; I218V
	BG1153/2007 August 2007/Sofia	G1	lineage II	WA	D97E; I218V
	BG1670/2007 November 2007/ Sofia	G1	lineage I	WA	T91N; N94S; G96D;
						D97E; S146N;
						M217T; I218V
	BG45/2008	December 2007/Pleven	G1	lineage II	WA	D97E; I218V
	BG389/2008	May 2008/Sofia	G1	lineage I	WA	D97E; S146N; L147F;
						M217T; I218V
	BG80/2006	January 2006/Sofia	G2		DS-1	A87T; D96N; N213D
	BG915/2007	June 2007/Sofia	G2		DS-1	A87T; D96N; N213D
	BG1643/2007 November 2007/Sofia-region G2		DS-1	A87T; D96N; N213D
	BG1726/2007 August 2007/Varna	G2		DS-1	A87T; D96N; N213D
	BG683/2008	August 2008/Sofia-region	G2		DS-1	A87T; D96N; N213D
	BG170/2006	March 2006/Pleven-region	G4	lineage I	ST3	-76N; F79S; R143K;
						V145T
	BG1385/2007 September 2007/Sofia-region G4	lineage IX	ST3	D74N; N75D; F79S;
						S87T; P90Q; T96N;
						V145A
	BG623/2008	July 2008/Sofia-region	G4	lineage I	ST3	-76N; F79S; R143K;
						V145T
	BG773/2008	September 2008/Sofia-region G4	lineage I	ST3	-76N; F79S; T96N;
						R143K; V145A
	BG167/2005	October 2005/Sofia	G9	lineage III,	WI61	A87T; N100D;
				sub-lineage d		K150E; T208I;
						A220T
	BG4/2007	January 2007/Sofia	G9	lineage III,	WI61	A87T; G94R; N100D;
				sub-lineage d		K150E; T208I;
						A220T
	BG825/2007	June 2007/Sofia-region	G9	lineage III,	WI61	A87T; N100D;
				sub-lineage d		K150E; T208I;
						A220T

  al., 7 1990; Gentsch et al., 1992; Iturriza-Gomara et al., 2000; Iturriza-Gomara et al., 2001; 8 Iturriza-Gomara et al., 2004; Simmonds et al., 2008], 19 rotavirus strains comprising four 9

Microbiol 39:3796-3798.

John Wiley & Sons

Journal of Medical Virology

John Wiley & Sons

Journal of Medical Virology


Pardo

A Basu

A Mandal

L Pardo

Hypothesis testing for two discrete populations based on the Hellinger distance

Keywords: Minimum Hellinger distance estimator, Empty cell penalty, Asymptotic distributions

Hypothesis testing for two discrete populations based on the Hellinger distance

Introduction

Let X 1 ; : : : ; X m1 and Y 1 ; : : : ; Y m2 be two independent random samples from two discrete populations X and Y with common support X = fx 0 ; x 1 ; : : :g and probability mass functions f 1 (x) = Pr 1 (X = x) and f 2 (x) = Pr 2 (Y = x); x 2 X , i 2 R, i = 1; 2; respectively: We denote by n 1 (x) (n 2 (x)) the number of elements in the sample X 1 ; : : : ; X m1 (Y 1 ; : : : ; Y m2 ) that coincide with x 2 X . We are interested in performing tests of hypothesis involving both 1 and 2 . In order to keep the exposition short and notation simple, we have assumed 1 and 2 to be scalar parameters in this paper. The multiparameter cases can be handled by extensions of essentially the same ideas. In this paper, therefore, we will restrict ourselves to the problem of testing

H 0 : 1 = 2 (1)
on the basis of some new statistics introduced in this paper. The test statistics considered here are based on the Hellinger distance between two di¤erent probability vectors. The unknown parameters are estimated by minimizing the Hellinger distance between the data and the model probability vectors, or a penalized version of it.

The statistics are introduced in Section 2; their asymptotic distributions are also derived in this section. The performance of the proposed tests are demonstrated numerically in Section 3.

Hellinger Distance: Estimation and Testing

The (twice) squared Hellinger distance between the probability vectors d i = (d i (x 0 ); : : : ; d i (x j ); : : :) = n i (x 0 ) m i ; : : : ; n i (x j ) m i ; : : : ; i = 1; 2;

(2) and f i = (f i (x 0 ); : : : ; f i (x j ); : : :) ; i = 1; 2;

(3) is given by

HD d i ; f i = 2 1 X j=0 d 1=2 i (x j ) f 1=2 i (x j ) 2 ; i = 1; 2; (4)
and the minimum Hellinger distance estimator of i is de…ned as the value b i H of R satisfying

b i H = arg min i HD d i ; f i : (5)
See [START_REF] Beran | Minimum Hellinger distance estimates for parametric models[END_REF], [START_REF] Simpson | Minimum Hellinger distance estimation for analysis of count data[END_REF][START_REF] Simpson | Hellinger deviance tests: E¢ ciency, breakdown points and examples[END_REF] and [START_REF] Basu | Minimum distance estimation: the approach using density based distances[END_REF] for more details on this method of estimation. It has been empirically observed that the minimum Hellinger distance estimator often performs poorly in small samples -compared to the maximum likelihood estimator -when the data generating distribution is correctly speci…ed by the parametric model. To avoid this problem, one of the suggestions is to use the penalized Hellinger distance (e.g. [START_REF] Harris | Hellinger distance as a penalized log likelihood[END_REF]; [START_REF] Basu | Tests of hypothesis in discrete models based on the penalized Hellinger distance[END_REF][START_REF] Basu | Penalized minimum disparity methods for multinomial models[END_REF]. In our context the penalized Hellinger distance between the probability vectors d i , de…ned in (2), and f i ; de…ned in [START_REF] Basu | Minimum distance estimation: the approach using density based distances[END_REF], is given by

P HD d i ; f i = 1 X j2Ai d 1=2 i (x j ) f 1=2 i (x j ) 2 + h 1 X j2A C i f i (x j ) ; (6)
where h is a real, positive number and

A i = fj : d i (x j ) > 0g and A C i = fj : d i (x j ) = 0g :
As in the de…nition given in [START_REF] Bishop | Discrete Multivariate Analysis: Theory and Practice[END_REF], the minimum penalized Hellinger distance estimator of i ; i = 1; 2; is given by b

i P H = arg min i P HD d i ; f i : (7)
While the penalized Hellinger distance is de…ned for any real, positive h, values around h = 1 appear to be preferable for small sample e¢ ciency; for the penalized distances h = 1 will be our default value. The rationale of this choice is that h = 1 makes the weight on the empty cells identical with that applied by likelihood based methods. Note that h = 2 generates the ordinary Hellinger distance.

As the probability of the empty cells eventually goes to zero it is intuitive that the results based on the ordinary and penalized Hellinger distance will provide equivalent asymptotic results. [START_REF] Mandal | Minimum Hellinger Distance Inference and the Empty Cell Penalty: Asymptotic Results[END_REF] prove that for any …xed h, the methods based on the ordinary Hellinger distance and the penalized Hellinger distance have the same asymptotic inference properties in the one sample problem.

Based on ( 5) and ( 6) it seems natural to consider test statistics of the type

HD f b 1 H ; f b 2 H ( 8
)
and

HD f b 1 P H ; f b 2 P H (9)
for testing the null hypothesis in [START_REF] Basu | Penalized minimum disparity methods for multinomial models[END_REF]. The idea of considering test statistics of the type ( 8) and ( 9) has been used before in the literature. [START_REF] Kupperman | Further application to information theory to multivariate analysis and statistical inference[END_REF] considered, for the …rst time, the test statistic

2 m 1 m 2 m 1 + m 2 D Kull (f b 1 ; f b 2 ) ; (10)
where

D Kull (f b 1 ; f b 2 ) is the Kullback-Leibler divergence between f b 1 and f b 2 : Its expression is given by D Kull (f b 1 ; f b 2 ) = 1 X j=0 f b 1 (x j ) log f b 1 (x j ) f b 2 (x j ) :
For more details about Kullback-Leibler divergence see [START_REF] Kullback | Kullback information[END_REF]. The symbols b 1 and b 2 represent the maximum likelihood estimators of 1 and 2 ; respectively. [START_REF] Kupperman | Further application to information theory to multivariate analysis and statistical inference[END_REF] established that the asymptotic distribution of the test statistic given in [START_REF] Lindsay | E¢ ciency versus robustness: the case for minimum Hellinger distance and related methods[END_REF] is a chi-square with one degree of freedom. This result was extended by Salicru et al (1994) by considering the family of -divergence test statistics

2 m 1 m 2 m 1 + m 2 D (f b 1 ; f b 2 )
where D (f b 1 ; f b 2 ) is the phi-divergence or phi-disparity between f b 1 and f b 2 : Its expression is given by,

D (f b 1 ; f b 2 ) = 1 X j=0 f b 2 (x j ) f b 1 (x j ) f b 2 (x j ) ! ; 2 (11)
where is the class of all convex functions (x) ; x 0; such that; (1) = 0 and 00 (1) 6 = 0: In (11) we shall assume the conventions 0 (0=0) = 0 and 0 (p=0) = p lim u!1 (u) =u; for p > 0: Let 2 be di¤erentiable at x = 1; then the function (x) (x) 0 (1) (x 1) also belongs to and has the additional property that 0 (1) = 0: This property, together with the convexity, implies that (x) 0; for any x 0:

Further, D f b 1 ; f b 2 = D f b 1 ; f b 2 :
In particular if we replace (x) = 4 p x 1 2 (x + 1) in [START_REF] Mandal | Minimum Hellinger Distance Inference and the Empty Cell Penalty: Asymptotic Results[END_REF], we get

D (f b 1 ; f b 2 ) = HD f b 1 ; f b 2 :
For more details about -divergences see [START_REF] Pardo | Statistical Inference Based on Divergence Measures[END_REF] and [START_REF] Lindsay | E¢ ciency versus robustness: the case for minimum Hellinger distance and related methods[END_REF]. These divergences have also been referred to as disparities in the literature. Also see [START_REF] Sarkar | On disparity based robust tests for two discrete populations[END_REF] who considered a linear combination of divergences with weights proportional to their sample sizes in constructing an overall divergence involving two independent samples to test statistical hypotheses of the type given in equation ( 1).

The joint likelihood function based on X 1 ; : : : ; X m1 and Y 1 ; : : : ; Y m2 is given by

L ( 1 ; 2 ) = 1 Q j=0 f 1 (x j ) m1(xj ) f 2 (x j ) m2(xj )
and if we denote by e the maximum likelihood estimate of the common parameter under the hypothesis 1 = 2 ; the log likelihood ratio test statistic is given by

LRT = 2 h log L b 1 ; b 2 log L e ; e i (12)
whose asymptotic distribution is a chi-square with one degree of freedom. Now, we denote,

d = 1 m 1 + m 2 n 1 (x 0 )
; n 2 (x 0 ); : : : ; n 1 (x j ); n 2 (x j ); : : :

and

f 1; 2 = 1 m 1 + m 2 m 1 f 1 (x 0 ); m 2 f 2 (x 0 ); : : : ; m 1 f 1 (x j ); m 2 f 2 (x j ); : : : :
It is easy to see that the log likelihood ratio statistic in ( 12) is identical to

LRT = 2 (m 1 + m 2 ) D Kull d ; f b 1; b 2 D Kull d ; f e ; e (14)
and

LRT = 2 (m 1 + m 2 ) D Kull f b 1; b 2 ; f e ; e + o p (1) : (15)
The last equality follows using the same arguments that in Bishop (1975, page 525). Formula ( 14) suggests the consideration of the following test statistic based on Hellinger distance in order to test the null hypothesis in (1):

2 (m 1 + m 2 ) HD d ; f b 1 H ; b 2 H HD d ; f e H ; e H ;
where b i H was de…ned in ( 5) and e H is de…ned by e H = arg min HD d ; f ; :

Based on minimum penalized Hellinger distance estimators we can consider

2 (m 1 + m 2 ) P HD d ; f b 1 P H ; b 2 P H P HD d ; f e P H ; e P H ;
where b i P H was de…ned in [START_REF] Kullback | Kullback information[END_REF]. This statistic has been considered by Simpson in (1989). Finally formula [START_REF] Sen | Large Sample Methods in Statistics[END_REF] suggest to us the test statistic

2 (m 1 + m 2 ) HD f b 1 H ; b 2 H ; f e H ; e H :
In the following Theorem we present the asymptotic distribution of the six test statistics introduced here.

Theorem 1 Let each of the sample sizes m 1 and m 2 go to in…nity at a rate such that the limiting value of m 1 (m 1 +m 2 ) 1 belongs to the open interval (0; 1). Then each of the following test statistics

T H = 2 m 1 m 2 m 1 + m 2 HD f b 1 H ; f b 2 H ; T P H = 2 m 1 m 2 m 1 + m 2 HD f b 1 P H ; f b 2 P H ; S H = 2 (m 1 + m 2 ) HD d ; f b 1 H ; b
HD f b 1 H ; f b 2 H = 1 2 b 1 H b 2 H 2 I ( 1 ) + o p b 1 H 1 2 + o p b 1 H 2 2 :
By [START_REF] Simpson | Minimum Hellinger distance estimation for analysis of count data[END_REF] we know that

p m 1 b 1 H 1 L ! m1!1 N 0; I 1 ( 1 ) p m 2 b 2 H 1 L ! m2!1 N 0; I 1 ( 1 ) : Therefore, r m 1 m 2 m 1 + m 2 b 1 H b 2 H L ! m1;m2!1 N 0; I 1 ( 1 )
and

T H = 2 m 1 m 2 m 1 + m 2 HD f b 1 H ; f b 2 H L ! m1;m2!1
p m 1 b 1 P H 1 L ! m1!1 N 0; I 1 ( 1 ) p m 2 b 2 P H 1 L ! m2!1 N 0; I 1 ( 1 ) : A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT Therefore, T P H = 2 m 1 m 2 m 1 + m 2 HD f b 1 P H ; f b 2 P H L ! m1;m2!1 2 1 :
We denote 1 = ( 1 ; 1 )

T . If we consider the function g ( 1 ; 2 ) = 1 2 the null hypothesis (1) can be written by g ( 1 ; 2 ) = 0: We denote by B = @g @ = 1

= (1; 1): It is well known (see, for instance, Sen and Singer 1993) that

p n( e b ) = I 1 F ( 1 )B T BI 1 F ( 1 )B T 1 B p n( b 1 ) + o p (1); (16
I F ( 1 ) = m1+m2 m1 I ( 1 ) 0 0 m1+m2 m2 I ( 1 )

:

The following results are routine extensions of the approach of [START_REF] Sarkar | On disparity based robust tests for two discrete populations[END_REF]:

S H = p m 1 + m 2 b H e H T I F ( 1 ) p m 1 + m 2 b H e H + o p (1)
; Putting the above together with equation ( 16), we have

p m 1 + m 2 b = p m 1 + m 2 b H + o p (1)
p m 1 + m 2 ( e H b H ) = I 1 F ( 1 )B T BI 1 F ( 1 )B T 1 B p m 1 + m 2 ( b H 1 ) + o p (1): Now S H = p m 1 + m 2 b H e H T I F ( 1 ) p m 1 + m 2 b H e H + o p (1) = p m 1 + m 2 ( b H 1 ) T B T BI 1 F ( 1 )B T 1 BI 1 F ( 1 )I F ( 1 )I 1 F ( 1 )B T BI 1 F ( 1 )B T 1 B p m 1 + m 2 ( b H 1 ) + o p (1) = p m 1 + m 2 ( b H 1 ) T B T BI 1 F ( 1 )B T 1 B p m 1 + m 2 ( b H 1 ) + o p (1) :
Taking into account that

p m 1 + m 2 ( b H 1 ) L ! m1;m2!1 N 0;I F ( 1 ) 1
and using Ser ‡ing (1980), Theorem 4.4.4, we have

S H L ! m1;m2!1 2 1 :
The asymptotic distribution of S P H follows the same steps that the proof of S H taking into account that p

m 1 + m 2 ( b H 1 ) = p m 1 + m 2 ( b P H 1 ) + o p (1)
: Finally the asymptotic distributions of T H and T P H follow in a similar way because a second Taylor expansion gives,

T H = p m 1 + m 2 b H e H T I F ( 1 ) p m 1 + m 2 b H e H + o p (1)
;

T P H = p m 1 + m 2 b P H e P H T I F ( 1 ) p m 1 + m 2 b P H e P H + o p (1) :
The techniques presented here for discrete models apply, in principle, to continuous models as well. However, this may require additional accessories, such as kernel density estimation, for the densities to be compatible when constructing the divergences. This makes the approach considerably more complicated. We hope to take up the issue of continuous models in a separate, future paper.

Simulation Study

To investigate the performance of the tests developed in the previous section, we present here the results of an extensive simulation study based on the Poisson distribution. In this connection it is useful to check whether the test statistics T H ; T P H , T H and T P H have simpli…ed expressions under the Poisson model. We note that a direct calculation gives

HD f 1 ; f 2 = 4 1 exp 1 + 2 2 exp p 1 2
where f i , i = 1; 2 represents the probability vector of a Poisson random variable with parameter i . Some straightforward algebra based on the above gives

T H = 8 m 1 m 2 m 1 + m 2 1 exp b 1 H + b 2 H 2 ! exp q b 1 H b 2 H ! ; T P H = 8 m 1 m 2 m 1 + m 2 1 exp b 1 P H + b 2 P H 2 ! exp q b 1 P H b 2 P H ! ; T H = 8 ( m 1 1 exp b 1 H + e H 2 ! exp q b 1 H e H ! +m 2 1 exp b 2 H + e H 2 ! exp q b 2 H e H !) ; A C C E P T E D M A N U S C

!)

: )f 1 + f 1c mixture, where our target parameter is 1 and 100 % data are coming from a contaminating population with parameter 1c . The second sample data are generated from the f 2 distribution. Assuming that the samples come from pure Poisson distributions with densities f 1 and f 2 respectively, 1 ; 2 unknown, we are interested in testing H 0 : 1 = 2 against the alternative that they are not equal. (We will use the " " symbol for the unknown values of the parameters involved in the hypotheses, and the " " symbol to denote the actual distributions from which the data have been generated.) All the

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

tests are performed at 5% level of signi…cance at common values of m 1 and m 2 chosen as 20, 30, 50, 75, 100 and 150. All the results are based on 10000 replications. Assuming binomial rejection frequencies, the estimate of the standard deviation given a probability estimate p may be computed from [p(1 p)=10000] 1=2 . Hence the error will be no greater than [0:5(1 0:5)=10000] 1=2 = 0:005.

In Figure 1, the histograms of the six test statistics and the likelihood ratio test statistic are plotted with the theoretical curve -the 2 (1) density -overlaid. Here the sample sizes for the both populations are 100, = 0, 1 = 2 and the common value of the parameter is 5. Although there are …ne di¤erences between the statistics, it is clear from the …gure that all the test statistics approximate their asymptotic null distribution quite well. Seven tables are constructed and presented here. In the …rst table, the observed levels (the proportion of statistics exceeding the chi-square critical value) of all the six tests at all the six sample sizes are presented, together with the corresponding levels of the likelihood ratio test. Here = 0, so both samples represent pure Poisson data. While the observed levels of the likelihood ratio test match the nominal level very closely, most of the other tests are somewhat liberal (S P H being the exception). Except for S P H the observed probabilities of rejection are higher (quite substantially for some small samples) than the nominal level. The penalty seems to have a major e¤ect, however, and the penalized statistics appear to generate observed levels which are signi…cantly closer to the nominal levels compared to the ordinary ones. The statistic S P H is very conservative for small samples, but for sample sizes of 75 or larger and S P H appear to quite close to the likelihood ratio test.

In Table 2, the e¤ect of the contaminant on the level is studied. Here 1 = 2 = 5, but = 0:05, so that the …rst sample is generated by a mixture of two Poissons, with the contaminating smaller component having a mean of 15. It is clearly seen that all the six statistics based on the Hellinger and penalized Hellinger distances largely discount the contaminating component, but the e¤ect of the latter on the likelihood ratio test is quite disastrous. Note that the observed levels become worse for all the methods as the sample size increases, since …xed amounts of contamination have greater impact in larger samples.

In Table 3 we look at the power of the tests when both samples represent pure Poisson data, but the …rst sample comes from a distribution with mean 5, and the second comes from a distribution with mean 6. The powers of all the statistics are quite competitive with those of the likelihood ratio test. Some of the tests have higher observed power than the likelihood ratio test, an artifact In Table 4, we study the power of the tests under contamination. The values of 1 and 2 are the same as in Table 3, but the …rst sample comes from a mixture of Poissons, with the contaminating component having a mean of 15 and a weight of 5%. This leads to a severe loss in the power in the likelihood ratio test, but all the other tests hold their levels pretty well.

The empirical critical values of the tests at 5% level of signi…cance are given in Table 5. The values of 1 and 2 are taken to be 5 and equal zero. The theoretical chi-square critical value in this case is 3.8415. The empirical critical values of all the tests (except S P H ) are higher than their theoretical critical values, although the degree of in ‡ation is much smaller for the penalized distances. The high power of some of the statistics in Table actually due to the true critical values being much higher than that of the chi-square density with 1 degrees of freedom. All the empirical critical values approach the theoretical value as the sample size increases; this happens much faster for the penalized distances.

In Table 6 and 7 we have used the same data as was used in Table 3 and 4 respectively. But now the empirical critical values (as presented in Table 5) have been used to determine the observed power instead of the theoretical critical values. When 1 = 5, 2 = 6 and = 0 we can see that the powers of all the tests are very close to those of the likelihood ratio test and for 1 = 5, 2 = 6, = 0:05 and 1c = 15 all the tests perform much better than the likelihood ratio test.

On the whole, all the six proposed tests appear to do quite well in terms of their ability to hold their levels and powers under contamination. Considering the entire evidence, T P H and T P H appear to be the most desirable statistics in terms of their closeness to the likelihood ratio test, attained power, and robustness against contaminations.

It may take more extensive studies to determine whether the amount of penalty applied here (h = 1) is optimal in terms of the desirable properties, or whether another choice can do better. However, the choice of h = 1 makes the method identical to the likelihood ratio test in terms of their treatment of the empty cells. In addition, a choice of h = 1=2 (results not reproduced here for brevity) does not appear to produce any appreciable change in the results produced here. Thus we believe the choice h = 1 is a sensible choice for our purpose.

2 1 :

 21 By Mandal et al. (2008) we have

) where b = b 1 ; b 2 T

 2 is the unrestricted maximum likelihood estimator, e = e ; e T is maximum likelihood estimator restricted to the null hypothesis and

; and p m 1 + m 2 e = p m 1 +

 11 m 2 e H + o p (1):

Figure 1 :

 1 Figure 1: Histogram s of the six test statistics and the LRT where m 1 =m 2= 100, 1= 2 and "=0

2 H

 2 HD d ; f e H ; e H ; S P H = 2 (m 1 + m 2 ) P HD d ; f b 1

	P H ; b 2 P H

P HD d ; f e P H ; e P H ; T H = 2 (m 1 + m 2 ) HD f b 1 H ; b 2 H ; f e H ; e H ; T P H = 2 (m 1 + m 2 ) HD f b 1 P H ; b 2 P H ; f e P H ; e P H ; asymptotically has a chi-square distribution with one degree of freedom under the null hypothesis. Here b H = b 1 H ; b 2 H T is the unrestricted minimizer of HD d ; f 1; 2 over 1 ; 2 2 , while e H = e H ; e H T represents the minimizer under the null hypothesis (1). The corresponding minimizers of the penalized Hellinger distance are denoted by b P H = b 1 P H ; b 2 P H T and e P H = e P H ; e P H T . Proof. Assume that the null hypothesis given in equation (1) is true. Let I( 1 ) be the Fisher information, i.e. I( 1 ) = E h @ @ 1 log f 1 (X) i 2 . A second order Taylor expansion of HD f b 1 H ; f b 2 H (see page 443 in Pardo 2006) gives

Table 1 :

 1 Comparison of the observed levels of the six tests and the LRT at nominal level 0:05 where 1 = 2 = 5 and = 0.

	m 1 = m 2	T H	T P H	S H	S P H	T H	T P H	LRT
	20	0.0612 0.0532 0.0742 0.0350 0.0650 0.0557 0.0473
	30	0.0655 0.0563 0.0786 0.0396 0.0685 0.0586 0.0520
	50	0.0586 0.0527 0.0672 0.0427 0.0596 0.0539 0.0487
	75	0.0603 0.0566 0.0666 0.0480 0.0613 0.0573 0.0509
	100	0.0597 0.0555 0.0683 0.0503 0.0608 0.0565 0.0527
	150	0.0582 0.0535 0.0627 0.0505 0.0588 0.0542 0.0508

Table 2 :

 2 Comparison of the observed levels of the six tests and the LRT at nominal level 0:05 where 1 = 2 = 5, = 0:05 and 1c = 15.

	m 1 = m 2	T H	T P H	S H	S P H	T H	T P H	LRT
	20	0.0839 0.0700 0.0911 0.0429 0.0905 0.0747 0.1476
	30	0.0802 0.0715 0.0863 0.0484 0.0842 0.0749 0.1797
	50	0.0853 0.0784 0.0891 0.0567 0.0871 0.0802 0.2286
	75	0.0913 0.0867 0.0919 0.0698 0.0934 0.0884 0.2982
	100	0.1006 0.0940 0.0999 0.0787 0.1026 0.0952 0.3673
	150	0.1138 0.1019 0.1105 0.0874 0.1154 0.1033 0.4688

Table 3 :

 3 Comparison of the observed powers of the six tests and the LRT at nominal level 0:05 where 1 = 5; 2 = 6 and = 0.

	m 1 = m 2	T H	T P H	S H	S P H	T H	T P H	LRT
	20	0.2853 0.2686 0.3124 0.2088 0.2934 0.2773 0.2706
	30	0.3989 0.3927 0.4301 0.3363 0.4056 0.3994 0.3920
	50	0.5695 0.5696 0.5931 0.5325 0.5746 0.5736 0.5733
	75	0.7364 0.7419 0.7524 0.7209 0.7394 0.7435 0.7450
	100	0.8487 0.8524 0.8595 0.8408 0.8501 0.8532 0.8559
	150	0.9590 0.9615 0.9624 0.9586 0.9592 0.9615 0.9616
	of their higher observed levels at the null under the true model.

Table 4 :

 4 Comparison of the observed powers of the six tests and the LRT at nominal level 0:05 where 1 = 5; 2 = 6, = 0:05 and 1c = 15.

	m 1 = m 2	T H	T P H	S H	S P H	T H	T P H	LRT
	20	0.2611 0.2391 0.2773 0.1772 0.2714 0.2476 0.1539
	30	0.3438 0.3260 0.3588 0.2646 0.3516 0.3337 0.1831
	50	0.4724 0.4632 0.4804 0.4085 0.4771 0.4683 0.2325
	75	0.5952 0.5960 0.5972 0.5556 0.5981 0.5992 0.2883
	100	0.7162 0.7312 0.7126 0.7008 0.7193 0.7331 0.3566
	150	0.8556 0.8682 0.8496 0.8522 0.8570 0.8697 0.4561

Table 5 :

 5 Comparison of empirical critical values of the six tests and the LRT at nominal level 0:05 where 1 = 2 = 5, and = 0 (here the chi-square critical value is 3.8415).

	m 1 = m 2	T H	T P H	S H	S P H	T H	T P H	LRT
	20	4.4331 3.9548 4.8967 3.3039 4.5926 4.0533 3.7896
	30	4.2640 3.9608 4.6493 3.4807 4.3635 4.0349 3.7185
	50	4.2050 4.0283 4.5314 3.6427 4.2470 4.0652 3.8910
	75	4.1069 3.9385 4.3513 3.7271 4.1474 3.9683 3.8795
	100	4.0501 3.9225 4.2609 3.7786 4.0795 3.9484 3.8265
	150	3.9963 3.8587 4.1468 3.7674 4.0136 3.8699 3.8159

Table 6 :

 6 Comparison of the observed powers of the six tests and the LRT using the empirical critical values at nominal level 0:05 where 1 = 5; 2 = 6, = 0.

	m 1 = m 2	T H	T P H	S H	S P H	T H	T P H	LRT
	20	0.2404 0.2587 0.2344 0.2584 0.2401 0.2596 0.2745
	30	0.3580 0.3812 0.3567 0.3783 0.3569 0.3811 0.4032
	50	0.5353 0.5509 0.5327 0.5562 0.5367 0.5513 0.5688
	75	0.7140 0.7331 0.7124 0.7303 0.7137 0.7333 0.7411
	100	0.8359 0.8471 0.8351 0.8452 0.8359 0.8466 0.8571
	150	0.9559 0.9613 0.9559 0.9604 0.9560 0.9613 0.9624

Table 7 :

 7 Comparison of the observed powers of the six tests and the LRT using the empirical critical values at nominal level 0:05 where 1 = 5; 2 = 6, = 0:05 and 1c = 15.

	m 1 = m 2	T H	T P H	S H	S P H	T H	T P H	LRT
	20	0.2161 0.2311 0.2028 0.2192 0.2170 0.2325 0.1565
	30	0.3108 0.3158 0.2953 0.2993 0.3105 0.3166 0.1894
	50	0.4419 0.4440 0.4234 0.4292 0.4439 0.4462 0.2300
	75	0.5740 0.5876 0.5545 0.5671 0.5747 0.5884 0.2856
	100	0.6998 0.7240 0.6829 0.7060 0.7011 0.7247 0.3574
	150	0.8480 0.8673 0.8317 0.8563 0.8488 0.8678 0.4581

Acknowledgements: This work was partially supported by Grant MTM2009-10072. The authors would like to thank the referees for critically reading the paper and making useful suggestions.