Background Down's syndrome (DS) is characterized by several immunological defects, especially regarding T cell compartment. DS is considered the best example of accelerated ageing in humans. Direct observations of the thymus have shown that in DS this organ undergoes severe histological and morphological changes. However, no data on its capacity to generate T cells are present in the literature. Here, using a new technology based upon real time PCR, we have investigated the capacity of the thymus to produce and release newly generated T lymphocytes (the so called "recent thymic emigrants", RTE) in children with DS.
Background
Down's syndrome (DS) is characterized by several immunological defects, especially regarding T cell compartment. DS is considered the best example of accelerated ageing in humans. Direct observations of the thymus have shown that in DS this organ undergoes severe histological and morphological changes. However, no data on its capacity to generate T cells are present in the literature. Here, using a new technology based upon real time PCR, we have investigated the capacity of the thymus to produce and release newly generated T lymphocytes (the so called "recent thymic emigrants", RTE) in children with DS.Methods We studied 8 children affected by DS, aged 2–7 years, compared with 8 age- and sex-matched healthy controls. Flow cytometry was used to determine different lymphocytes subsets. Real time PCR with the Taqman system was used to quantify the amount of RTE,  i.e.  peripheral blood lymphocytes that express the T cell receptor rearrangement excision circles (TREC).
Methods
We studied 8 children affected by DS, aged 2–7 years, compared with 8 age- and sex-matched healthy controls. Flow cytometry was used to determine different lymphocytes subsets. Real time PCR with the Taqman system was used to quantify the amount of RTE,  i.e.  peripheral blood lymphocytes that express the T cell receptor rearrangement excision circles (TREC).i.e.Results In comparison with control children, those with DS had a significant lower number of TREC+ peripheral blood cells. Moreover, in DS children but not in controls, a strong negative correlation between age and the levels of TREC+ cells was found.
Results
In comparison with control children, those with DS had a significant lower number of TREC+ peripheral blood cells. Moreover, in DS children but not in controls, a strong negative correlation between age and the levels of TREC+ cells was found.Conclusions The direct measure of thymic output indicates that the impairment of the organ results in a reduced production of newly generated T cells. This observation could suggest that cytokines able to modulate thymic function, such as interleukins, could be useful to improve the functionality of the organ and to treat the immunodeficiency present in DS subjects.
Conclusions
The direct measure of thymic output indicates that the impairment of the organ results in a reduced production of newly generated T cells. This observation could suggest that cytokines able to modulate thymic function, such as interleukins, could be useful to improve the functionality of the organ and to treat the immunodeficiency present in DS subjects.

Introduction Down's Syndrome (DS) is the most common chromosomal abnormality in humans, that occurs in 1 out of every 800–1,000 births. It is an autosomal disorder resulting by triplication of chromosome 21. Many characteristics are commonly seen in DS, including some degree of intellectual impairment, that varies widely from individual to individual, heart defects, hypotonia, hyperuricemia, and the development of Alzheimer disease-type neuropathology beginning at about 40 years of age [ 1 ]. DS subjects also present alterations of the immune system which are similar to those of aged people, including increased susceptibility to bacterial and viral infection and to the onset of different types of haematological malignancies, along with a high frequency of autoantibodies. Alterations of B lymphocytes, T cell subsets, and natural killer cells, defective phagocytosis and chemotaxis of polymorphonuclear leukocytes and interleukin-2 production by activated T cells were also reported [ 2 - 7 ]. Quantitative studies of peripheral blood T lymphocytes reveal a reduction, often quite small, in the percentage and/or absolute number of T lymphocytes, although normal proportions or numbers of T and B lymphocytes in DS children have also been reported [ 8 ]. Several studies have focused their attention on the role of the thymus, and have described a variety of structural and anatomic alterations present in DS [ 9 ]. Few data, if any, exist on the direct measure of thymic functionality in terms of production of newly generated T cells. Accordingly, the aim of this study was to measure the capability of the thymus of DS children to produce new T lymphocytes, and to analyze how such capability changes with age. For this reason, we have quantified the so called "recent thymic emigrants" (RTE), that are the main contributors to the naïve T-cell pool, and are characterized by the presence in the nucleus of a circular, episomal DNA molecule called TREC ( T  cell receptor  R earrangement  E xcision  C ircles), generated during the intrathymic rearrangement of the α-chain locus of the T cell receptor (TCR). Genes for the δ-chain of the TCR are distributed within the genomic region that codifies for the α-chain, and are removed in two steps during the recombination of Vα with Jα. Thus, a thymocyte that starts to rearrange the α-chain produces the first TREC, called signal-joint (sj)-TREC, then proliferates three or four times, and finally completes the rearrangement of Vα with Jα, so producing the second TREC, called coding-joint (cj)-TREC. The removal of genes for the δ-chain from the α region does not imply their elimination, as such DNA remains into the nucleus as a circle that is not able to replicate. As a consequence, when a cell undergoes a division, TREC are passed only to one of the two daughter cells. During the following cell cycles, TREC are then diluted into the population that origins from the first cell. Several data indicated that the percentage of TREC+ cells is a marker of thymic activity, that TREC+ cells are almost present within the subset of virgin T lymphocytes, and that their number consistently declines with age [ 10 ].
Introduction
Down's Syndrome (DS) is the most common chromosomal abnormality in humans, that occurs in 1 out of every 800–1,000 births. It is an autosomal disorder resulting by triplication of chromosome 21. Many characteristics are commonly seen in DS, including some degree of intellectual impairment, that varies widely from individual to individual, heart defects, hypotonia, hyperuricemia, and the development of Alzheimer disease-type neuropathology beginning at about 40 years of age [ 1 ].DS subjects also present alterations of the immune system which are similar to those of aged people, including increased susceptibility to bacterial and viral infection and to the onset of different types of haematological malignancies, along with a high frequency of autoantibodies. Alterations of B lymphocytes, T cell subsets, and natural killer cells, defective phagocytosis and chemotaxis of polymorphonuclear leukocytes and interleukin-2 production by activated T cells were also reported [ 2 - 7 ]. Quantitative studies of peripheral blood T lymphocytes reveal a reduction, often quite small, in the percentage and/or absolute number of T lymphocytes, although normal proportions or numbers of T and B lymphocytes in DS children have also been reported [ 8 ].Several studies have focused their attention on the role of the thymus, and have described a variety of structural and anatomic alterations present in DS [ 9 ]. Few data, if any, exist on the direct measure of thymic functionality in terms of production of newly generated T cells. Accordingly, the aim of this study was to measure the capability of the thymus of DS children to produce new T lymphocytes, and to analyze how such capability changes with age. For this reason, we have quantified the so called "recent thymic emigrants" (RTE), that are the main contributors to the naïve T-cell pool, and are characterized by the presence in the nucleus of a circular, episomal DNA molecule called TREC ( T  cell receptor  R earrangement  E xcision  C ircles), generated during the intrathymic rearrangement of the α-chain locus of the T cell receptor (TCR).Genes for the δ-chain of the TCR are distributed within the genomic region that codifies for the α-chain, and are removed in two steps during the recombination of Vα with Jα. Thus, a thymocyte that starts to rearrange the α-chain produces the first TREC, called signal-joint (sj)-TREC, then proliferates three or four times, and finally completes the rearrangement of Vα with Jα, so producing the second TREC, called coding-joint (cj)-TREC. The removal of genes for the δ-chain from the α region does not imply their elimination, as such DNA remains into the nucleus as a circle that is not able to replicate. As a consequence, when a cell undergoes a division, TREC are passed only to one of the two daughter cells. During the following cell cycles, TREC are then diluted into the population that origins from the first cell. Several data indicated that the percentage of TREC+ cells is a marker of thymic activity, that TREC+ cells are almost present within the subset of virgin T lymphocytes, and that their number consistently declines with age [ 10 ].Materials and Methods Subjects We could analyze blood samples from 8 children with DS (with a mean age of 4.62 years, range: 2–7 years), 7 males and 1 females. As control group, we studied 8 children with a mean age of 4.75 years (range, 2–8 years), 7 males and 1 female. All subjects were in good conditions, and had no acute or chronic disease affecting the immune system. Written informed consent was obtained by their parents, according to the Italian laws. Isolation of PBMC from blood Peripheral blood mononuclear cells (PBMC) were separated from a minimum of 8 mL freshly collected blood according to standard procedures. Analysis of the phenotype of peripheral blood lymphocytes Blood samples were stained with different monoclonal antibodies for the cytometric analysis, as described [ 11 ]. The quantification of the main subpopulations present among lymphocytes (electronically selected on the basis of an electronic gate put in the region of lymphocytes) was performed by flow cytometry using a CyFlow ML instrument (Partec, Münster, Germany), according to standard procedures [ 11 ]. DNA extraction DNA was extracted from 5 × 10 6  PBMC using the QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer's protocols and stored in sterile water at -20°C until use. Quantification of percentage of sjTREC positive PBMC by a Real Time PCR approach The percentage of PBMC containing sjTREC was measured by an original method that has been recently developed (patent pending) using a Real Time PCR approach. This assay was performed by two parallel polymerase chain reactions (PCR), that quantify sjTREC or nuclear DNA (nDNA) in a given sample, carried out in two different reaction tubes, but in the same plate, in order to have similar reaction conditions. In the first reaction, we quantified sjTREC using a mix that consisted of: Supermix Biorad 1X, primers for sjTREC 500 pmol and we added 3 μl of the sample in each tube. The sjTREC primers we used were: sjTREC Dir (5'-CAC ATC CCT TTC AAC CAT GCT-3') and sjTREC Rev (5'-GCC AGC TGC AGG GTT TAG G-3'). TaqMan probe for sjTREC (5'-FAM-ACA CCT CTG GTT TTT GTA AAG GTG CCC ACT – BLACK HOLE-3') was included in the reaction mixture at the concentration of 200 nM, as a real time detector for the amplified product. One cycle of denaturation (95°C for 6 min) was performed, followed by 50 cycles of amplification (95°C for 30 sec, 58°C for 1 min 30 sec). The same approach was used to quantify nDNA, which was required to obtain the number of cells present in the sample. In this case, primers GenDir (5'-GGC TCT GTG AGG GAT ATA AAG ACA-3') and GenRev (5'-CCA ACC ACC CGA GCA ACT AAT CT-3'), designed on FasL gene sequence, present in two copies in the human genome (one in each chromosome 1), were used at the concentration of 600 and 400 nM respectively, and the TaqMan probe GenProbe (5' TexasRed – CTG TTC CGT TTC CTG CCG GTG C – BlackHole Quencher2 3') was included in the reaction mixture at the concentration of 300 nM. One cycle of denaturation (95°C for 3 min 30 sec) was performed, followed by 45 cycles of amplification (95°C for 30 sec, 60°C for 35 sec). All of the aforementioned primers and probes have been designed using the program "primer3", available on the internet address:  . The length of amplified fragments is 104 pb for nDNA and 101 pb for sjTREC. PCR was performed by using an iCycler Thermal cycler (BioRad, Hercules, CA, USA), that monitors changes in the fluorescence spectrum of each reaction tube during the annealing phase. The fluorescence signal was processed using the "Real-time detection system iCycler iQ" software, that calculates the threshold and the threshold cycles of each sample. All reactions were carried out in triplicate. To optimize the precision of the assay, whose results derive from the comparison between the threshold cycles of two different real time PCR reactions, we have developed a new approach. Indeed, the regions used as template for the two amplifications ( i.e. , those of sjTREC and nDNA) were purified and cloned tail to tail in a vector (pGEM-11Z, from Promega), to have a ratio of 1:1 of the molecules used as reference. The nDNA region used has been excised as a SacI-ApaI fragment and cloned in the pGEMT easy vector (Promega), obtaining the pGEM11Z-nDNA vector. The sjTREC region used has been excised as a PstI-SacI fragment and cloned in the same vector, obtaining the pSJ vector we used. The plasmid has been purified, quantified with the spetrofotometer and linearized using SacI. Then a series of reactions has been performed for nDNA and sjTREC with serial dilution of the standard. All the times the threshold cycles of nDNA and sjTREC were the same, proving the ratio 1:1 of the two fragments. Then, serial known dilutions of this vector, amplified in triplicate, were included in each PCR run to generate a standard curve from which the relative copy number of either sjTREC or nDNA present in the unknown samples was determined. The measured values for sjTREC and nDNA were always within the range of the standard curve, whose correlation coefficient was always >0.990. The values of sjTREC and nDNA present in each sample were calculated using the mean of the threshold cycles of the three replicates. Then, the percentage of PBMC containing sjTREC in each sample was simply obtained from the ratio between the relative values of sjTREC (obtained dividing for 3 the result given by the iCycler, as we used 3 μl of DNA of the sample and 1 μl of standard to quantify sjTREC) and nDNA obtained (obtained  versus  the same vector), multiplied by 2 (as two copies of the nuclear gene are present in a cell).
Materials and Methods
Subjects We could analyze blood samples from 8 children with DS (with a mean age of 4.62 years, range: 2–7 years), 7 males and 1 females. As control group, we studied 8 children with a mean age of 4.75 years (range, 2–8 years), 7 males and 1 female. All subjects were in good conditions, and had no acute or chronic disease affecting the immune system. Written informed consent was obtained by their parents, according to the Italian laws.
Subjects
We could analyze blood samples from 8 children with DS (with a mean age of 4.62 years, range: 2–7 years), 7 males and 1 females. As control group, we studied 8 children with a mean age of 4.75 years (range, 2–8 years), 7 males and 1 female. All subjects were in good conditions, and had no acute or chronic disease affecting the immune system. Written informed consent was obtained by their parents, according to the Italian laws.Isolation of PBMC from blood Peripheral blood mononuclear cells (PBMC) were separated from a minimum of 8 mL freshly collected blood according to standard procedures.
Isolation of PBMC from blood
Peripheral blood mononuclear cells (PBMC) were separated from a minimum of 8 mL freshly collected blood according to standard procedures.Analysis of the phenotype of peripheral blood lymphocytes Blood samples were stained with different monoclonal antibodies for the cytometric analysis, as described [ 11 ]. The quantification of the main subpopulations present among lymphocytes (electronically selected on the basis of an electronic gate put in the region of lymphocytes) was performed by flow cytometry using a CyFlow ML instrument (Partec, Münster, Germany), according to standard procedures [ 11 ].
Analysis of the phenotype of peripheral blood lymphocytes
Blood samples were stained with different monoclonal antibodies for the cytometric analysis, as described [ 11 ]. The quantification of the main subpopulations present among lymphocytes (electronically selected on the basis of an electronic gate put in the region of lymphocytes) was performed by flow cytometry using a CyFlow ML instrument (Partec, Münster, Germany), according to standard procedures [ 11 ].DNA extraction DNA was extracted from 5 × 10 6  PBMC using the QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer's protocols and stored in sterile water at -20°C until use.
DNA extraction
DNA was extracted from 5 × 10 6  PBMC using the QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer's protocols and stored in sterile water at -20°C until use.Quantification of percentage of sjTREC positive PBMC by a Real Time PCR approach The percentage of PBMC containing sjTREC was measured by an original method that has been recently developed (patent pending) using a Real Time PCR approach. This assay was performed by two parallel polymerase chain reactions (PCR), that quantify sjTREC or nuclear DNA (nDNA) in a given sample, carried out in two different reaction tubes, but in the same plate, in order to have similar reaction conditions. In the first reaction, we quantified sjTREC using a mix that consisted of: Supermix Biorad 1X, primers for sjTREC 500 pmol and we added 3 μl of the sample in each tube. The sjTREC primers we used were: sjTREC Dir (5'-CAC ATC CCT TTC AAC CAT GCT-3') and sjTREC Rev (5'-GCC AGC TGC AGG GTT TAG G-3'). TaqMan probe for sjTREC (5'-FAM-ACA CCT CTG GTT TTT GTA AAG GTG CCC ACT – BLACK HOLE-3') was included in the reaction mixture at the concentration of 200 nM, as a real time detector for the amplified product. One cycle of denaturation (95°C for 6 min) was performed, followed by 50 cycles of amplification (95°C for 30 sec, 58°C for 1 min 30 sec). The same approach was used to quantify nDNA, which was required to obtain the number of cells present in the sample. In this case, primers GenDir (5'-GGC TCT GTG AGG GAT ATA AAG ACA-3') and GenRev (5'-CCA ACC ACC CGA GCA ACT AAT CT-3'), designed on FasL gene sequence, present in two copies in the human genome (one in each chromosome 1), were used at the concentration of 600 and 400 nM respectively, and the TaqMan probe GenProbe (5' TexasRed – CTG TTC CGT TTC CTG CCG GTG C – BlackHole Quencher2 3') was included in the reaction mixture at the concentration of 300 nM. One cycle of denaturation (95°C for 3 min 30 sec) was performed, followed by 45 cycles of amplification (95°C for 30 sec, 60°C for 35 sec). All of the aforementioned primers and probes have been designed using the program "primer3", available on the internet address:  . The length of amplified fragments is 104 pb for nDNA and 101 pb for sjTREC. PCR was performed by using an iCycler Thermal cycler (BioRad, Hercules, CA, USA), that monitors changes in the fluorescence spectrum of each reaction tube during the annealing phase. The fluorescence signal was processed using the "Real-time detection system iCycler iQ" software, that calculates the threshold and the threshold cycles of each sample. All reactions were carried out in triplicate. To optimize the precision of the assay, whose results derive from the comparison between the threshold cycles of two different real time PCR reactions, we have developed a new approach. Indeed, the regions used as template for the two amplifications ( i.e. , those of sjTREC and nDNA) were purified and cloned tail to tail in a vector (pGEM-11Z, from Promega), to have a ratio of 1:1 of the molecules used as reference. The nDNA region used has been excised as a SacI-ApaI fragment and cloned in the pGEMT easy vector (Promega), obtaining the pGEM11Z-nDNA vector. The sjTREC region used has been excised as a PstI-SacI fragment and cloned in the same vector, obtaining the pSJ vector we used. The plasmid has been purified, quantified with the spetrofotometer and linearized using SacI. Then a series of reactions has been performed for nDNA and sjTREC with serial dilution of the standard. All the times the threshold cycles of nDNA and sjTREC were the same, proving the ratio 1:1 of the two fragments. Then, serial known dilutions of this vector, amplified in triplicate, were included in each PCR run to generate a standard curve from which the relative copy number of either sjTREC or nDNA present in the unknown samples was determined. The measured values for sjTREC and nDNA were always within the range of the standard curve, whose correlation coefficient was always >0.990. The values of sjTREC and nDNA present in each sample were calculated using the mean of the threshold cycles of the three replicates. Then, the percentage of PBMC containing sjTREC in each sample was simply obtained from the ratio between the relative values of sjTREC (obtained dividing for 3 the result given by the iCycler, as we used 3 μl of DNA of the sample and 1 μl of standard to quantify sjTREC) and nDNA obtained (obtained  versus  the same vector), multiplied by 2 (as two copies of the nuclear gene are present in a cell).
Quantification of percentage of sjTREC positive PBMC by a Real Time PCR approach
The percentage of PBMC containing sjTREC was measured by an original method that has been recently developed (patent pending) using a Real Time PCR approach. This assay was performed by two parallel polymerase chain reactions (PCR), that quantify sjTREC or nuclear DNA (nDNA) in a given sample, carried out in two different reaction tubes, but in the same plate, in order to have similar reaction conditions.In the first reaction, we quantified sjTREC using a mix that consisted of: Supermix Biorad 1X, primers for sjTREC 500 pmol and we added 3 μl of the sample in each tube. The sjTREC primers we used were: sjTREC Dir (5'-CAC ATC CCT TTC AAC CAT GCT-3') and sjTREC Rev (5'-GCC AGC TGC AGG GTT TAG G-3'). TaqMan probe for sjTREC (5'-FAM-ACA CCT CTG GTT TTT GTA AAG GTG CCC ACT – BLACK HOLE-3') was included in the reaction mixture at the concentration of 200 nM, as a real time detector for the amplified product. One cycle of denaturation (95°C for 6 min) was performed, followed by 50 cycles of amplification (95°C for 30 sec, 58°C for 1 min 30 sec). The same approach was used to quantify nDNA, which was required to obtain the number of cells present in the sample. In this case, primers GenDir (5'-GGC TCT GTG AGG GAT ATA AAG ACA-3') and GenRev (5'-CCA ACC ACC CGA GCA ACT AAT CT-3'), designed on FasL gene sequence, present in two copies in the human genome (one in each chromosome 1), were used at the concentration of 600 and 400 nM respectively, and the TaqMan probe GenProbe (5' TexasRed – CTG TTC CGT TTC CTG CCG GTG C – BlackHole Quencher2 3') was included in the reaction mixture at the concentration of 300 nM. One cycle of denaturation (95°C for 3 min 30 sec) was performed, followed by 45 cycles of amplification (95°C for 30 sec, 60°C for 35 sec).All of the aforementioned primers and probes have been designed using the program "primer3", available on the internet address:  . The length of amplified fragments is 104 pb for nDNA and 101 pb for sjTREC.PCR was performed by using an iCycler Thermal cycler (BioRad, Hercules, CA, USA), that monitors changes in the fluorescence spectrum of each reaction tube during the annealing phase. The fluorescence signal was processed using the "Real-time detection system iCycler iQ" software, that calculates the threshold and the threshold cycles of each sample. All reactions were carried out in triplicate.To optimize the precision of the assay, whose results derive from the comparison between the threshold cycles of two different real time PCR reactions, we have developed a new approach. Indeed, the regions used as template for the two amplifications ( i.e. , those of sjTREC and nDNA) were purified and cloned tail to tail in a vector (pGEM-11Z, from Promega), to have a ratio of 1:1 of the molecules used as reference.i.e.The nDNA region used has been excised as a SacI-ApaI fragment and cloned in the pGEMT easy vector (Promega), obtaining the pGEM11Z-nDNA vector. The sjTREC region used has been excised as a PstI-SacI fragment and cloned in the same vector, obtaining the pSJ vector we used. The plasmid has been purified, quantified with the spetrofotometer and linearized using SacI. Then a series of reactions has been performed for nDNA and sjTREC with serial dilution of the standard. All the times the threshold cycles of nDNA and sjTREC were the same, proving the ratio 1:1 of the two fragments.Then, serial known dilutions of this vector, amplified in triplicate, were included in each PCR run to generate a standard curve from which the relative copy number of either sjTREC or nDNA present in the unknown samples was determined. The measured values for sjTREC and nDNA were always within the range of the standard curve, whose correlation coefficient was always >0.990. The values of sjTREC and nDNA present in each sample were calculated using the mean of the threshold cycles of the three replicates.Then, the percentage of PBMC containing sjTREC in each sample was simply obtained from the ratio between the relative values of sjTREC (obtained dividing for 3 the result given by the iCycler, as we used 3 μl of DNA of the sample and 1 μl of standard to quantify sjTREC) and nDNA obtained (obtained  versus  the same vector), multiplied by 2 (as two copies of the nuclear gene are present in a cell).versusResults Peripheral blood cell phenotype in DS Down's syndrome is characterized by severe immunological alterations, which mainly affect the T cell compartment and are often regarded as signs of accelerated ageing. Numerous reports suggested that thymic retention of T cells or maturation defects might be the cause of the observed alterations in the T cell compartment. Accordingly, we first checked the presence of possible changes in the phenotype of peripheral blood lymphocytes from DS children. As shown in Figure  1 , we found that, in comparison with karyotypically normal healthy controls, DS children had less T helper cells and more cytotoxic T lymphocytes or cells expressing markers related to NK activity. This indicates that the population we analyzed can well represent the immunological situation present in DS children. Figure 1 Phenotypic analysis of peripheral blood lymphocytes in patients with Down's syndrome (DS) and healthy subjects. Data are referred to 8 individuals per group. Asterisk indicates a statistically significant difference (p < 0.05). Quantitative analysis of cells expressing TREC We then studied directly the capacity of the thymus to produce new T lymphocytes, by the analysis of TREC+ cells. Figure  2  shows a typical example of real time PCR assay for quantification of the amount of TREC per cell. It is to note that the threshold cycle,  i.e.  the cycle of PCR in which the fluorescent signal deriving from the amplification of the DNA becomes evident, is quite different in DS children and controls. Indeed, such cycle is much lower in control children, indicating the presence of a higher number of TREC. Figure 2 Representative example of real time PCR for the quantification of TREC in a group of 3 children with Down's syndrome (DS) and in 4 healthy controls (each measure is performed in triplicate). Note how the threshold cycle is different in the two groups. The percentage of TREC+ lymphocytes in children affected by Down's syndrome and in controls is reported in Figure  3 . A statistically significant difference was present between the two groups (p = 0.007), indicating that DS is characterized by a lower thymic output. Figure 3 DS children have less TREC+ lymphocytes than healthy controls, as shown in this box-and-whiskers graphics. The boxes extend from the 25 th  percentile (x [25] ) to the 75 th  percentile (x [75] ) [ i.e. , the interquartile range (IQ)]; lines inside boxes represent median values. Lines emerging from boxes ( i.e. , the whiskers) extend to the upper and lower adjacent values. The upper adjacent value is defined as the largest data point ≤x [75] +1.5xIQ, and the lower adjacent value is defined as the smallest data point ≥x [25] -1.5xIQ. Note that no outliers are present in the two groups. Correlation between age and TREC Finally, we have investigated the correlation between age and TREC levels in the two groups. As reported in Figure  4 , it is noteworthy that while control children did not display any age-related change (in the age range 2–8 years), those with DS had a significant age-related decrease in the number of TREC+ cells. This indicates that in DS, in contrast with control children, major changes in thymic output occur in the very first years of life. Figure 4 Correlation between age and TREC levels in DS (squares) and control (triangles) children. In the year range 2–8, the correlation was significant in DS but not in control children.
Results
Peripheral blood cell phenotype in DS Down's syndrome is characterized by severe immunological alterations, which mainly affect the T cell compartment and are often regarded as signs of accelerated ageing. Numerous reports suggested that thymic retention of T cells or maturation defects might be the cause of the observed alterations in the T cell compartment. Accordingly, we first checked the presence of possible changes in the phenotype of peripheral blood lymphocytes from DS children. As shown in Figure  1 , we found that, in comparison with karyotypically normal healthy controls, DS children had less T helper cells and more cytotoxic T lymphocytes or cells expressing markers related to NK activity. This indicates that the population we analyzed can well represent the immunological situation present in DS children. Figure 1 Phenotypic analysis of peripheral blood lymphocytes in patients with Down's syndrome (DS) and healthy subjects. Data are referred to 8 individuals per group. Asterisk indicates a statistically significant difference (p < 0.05).
Peripheral blood cell phenotype in DS
Down's syndrome is characterized by severe immunological alterations, which mainly affect the T cell compartment and are often regarded as signs of accelerated ageing. Numerous reports suggested that thymic retention of T cells or maturation defects might be the cause of the observed alterations in the T cell compartment. Accordingly, we first checked the presence of possible changes in the phenotype of peripheral blood lymphocytes from DS children. As shown in Figure  1 , we found that, in comparison with karyotypically normal healthy controls, DS children had less T helper cells and more cytotoxic T lymphocytes or cells expressing markers related to NK activity. This indicates that the population we analyzed can well represent the immunological situation present in DS children.Quantitative analysis of cells expressing TREC We then studied directly the capacity of the thymus to produce new T lymphocytes, by the analysis of TREC+ cells. Figure  2  shows a typical example of real time PCR assay for quantification of the amount of TREC per cell. It is to note that the threshold cycle,  i.e.  the cycle of PCR in which the fluorescent signal deriving from the amplification of the DNA becomes evident, is quite different in DS children and controls. Indeed, such cycle is much lower in control children, indicating the presence of a higher number of TREC. Figure 2 Representative example of real time PCR for the quantification of TREC in a group of 3 children with Down's syndrome (DS) and in 4 healthy controls (each measure is performed in triplicate). Note how the threshold cycle is different in the two groups. The percentage of TREC+ lymphocytes in children affected by Down's syndrome and in controls is reported in Figure  3 . A statistically significant difference was present between the two groups (p = 0.007), indicating that DS is characterized by a lower thymic output. Figure 3 DS children have less TREC+ lymphocytes than healthy controls, as shown in this box-and-whiskers graphics. The boxes extend from the 25 th  percentile (x [25] ) to the 75 th  percentile (x [75] ) [ i.e. , the interquartile range (IQ)]; lines inside boxes represent median values. Lines emerging from boxes ( i.e. , the whiskers) extend to the upper and lower adjacent values. The upper adjacent value is defined as the largest data point ≤x [75] +1.5xIQ, and the lower adjacent value is defined as the smallest data point ≥x [25] -1.5xIQ. Note that no outliers are present in the two groups.
Quantitative analysis of cells expressing TREC
We then studied directly the capacity of the thymus to produce new T lymphocytes, by the analysis of TREC+ cells. Figure  2  shows a typical example of real time PCR assay for quantification of the amount of TREC per cell. It is to note that the threshold cycle,  i.e.  the cycle of PCR in which the fluorescent signal deriving from the amplification of the DNA becomes evident, is quite different in DS children and controls. Indeed, such cycle is much lower in control children, indicating the presence of a higher number of TREC.i.e.The percentage of TREC+ lymphocytes in children affected by Down's syndrome and in controls is reported in Figure  3 . A statistically significant difference was present between the two groups (p = 0.007), indicating that DS is characterized by a lower thymic output.Correlation between age and TREC Finally, we have investigated the correlation between age and TREC levels in the two groups. As reported in Figure  4 , it is noteworthy that while control children did not display any age-related change (in the age range 2–8 years), those with DS had a significant age-related decrease in the number of TREC+ cells. This indicates that in DS, in contrast with control children, major changes in thymic output occur in the very first years of life. Figure 4 Correlation between age and TREC levels in DS (squares) and control (triangles) children. In the year range 2–8, the correlation was significant in DS but not in control children.
Correlation between age and TREC
Finally, we have investigated the correlation between age and TREC levels in the two groups. As reported in Figure  4 , it is noteworthy that while control children did not display any age-related change (in the age range 2–8 years), those with DS had a significant age-related decrease in the number of TREC+ cells. This indicates that in DS, in contrast with control children, major changes in thymic output occur in the very first years of life.Discussion Immunological ageing is part of a continuum of developmental processes, encompassing complex reorganizational events, compensatory mechanisms and qualitative alterations in the functionality of several systems and organs. Among those organs that undergo major changes with ageing, thymus plays a special role [ 12 ]. The thymus is a central lymphoid organ that is the primary site of T-cell maturation and development. Shortly after birth, the thymus undergoes a life long process of involution whereby the organ is replaced by adipose tissue. The result is a reduction in the number of constituent thymocytes with age, a consequent shrinking of the thymus and a decline in the output of newly generated T lymphocytes [ 13 ]. Aged peripheral T-cell pool is characterized by the accumulation of T-cell capable of limited replication [ 14 ]. Since an efficient immune response is based upon the expansion of antigen-specific clones, the consequence of a qualitative and quantitative impairment of the system is an increased susceptibility to infections or cancer. Several groups, including ours, have studied the immune system during human ageing, using different models, including the one that represents the best example of successful ageing,  i.e.  healthy centenarians, and conditions of accelerated ageing, such as DS [ 2 - 6 , 15 ]. In DS subjects, the proportion of T-helper cells is decreased, resulting in a decreased ratio of helper/cytotoxic cells, and we could observe this phenomenon also in the patients here described. Furthermore, peripheral blood T cells have a decreased number of cells expressing the T-cell receptor-αβ (TCRαβ) complex, elevated numbers expressing TCRγδ and a decreased proportion of CD4+, CD45RA+ naïve T cells, along with a high number of cells with NK phenotype, suggesting that the DS thymus is inefficient in the release of functionally mature T cells [ 16 ]. An age-dependence in the proliferative response to phytohemagglutinin (PHA) of DS lymphocytes, but not of lymphocytes from healthy individuals, was observed. Allogeneic mixed lymphocyte proliferative responses are decreased, as are PHA-induced interleukin-2 production and cytotoxic T-lymphocyte activity [ 7 ]. All of the above mentioned alterations have a common substrate,  i.e.  the dysfunction of thymus. Indeed, defects in the capacity to produce thymic hormones has been described several years ago [ 17 , 18 ]. Furthermore, anatomic studies gave further evidence that changes in the T-lymphocyte system derive from structural abnormalities of the thymus. In comparison to age-matched controls, thymuses from infants with DS from 1 day to 15 months of age have marked lymphoid depletion, with a thin cortex and poor corticomedullary demarcation. The Hassall corpuscles are increased in size and frequently cystic. The presence of lower proportions of cells bearing high levels of the TCR-αβ complex and of CD3, a signal-transducing complex for the TCR, in thymuses of children with DS and the increase in the proportions of cells with these markers with age are indicative of delayed maturation of T cells within the thymus [ 9 ]. In addition, DS thymuses contain elevated levels of IFN-γ and TNF-α mRNA expressing cells, and there is mast cell hyperplasia and overexpression of class I MHC, CD18 and ICAM-1 [ 19 ]. DS thymocytes also have a greater than normal sensitivity to inhibition of IL-4-induced proliferation by IFN-γ and TNF-α [ 20 ]. Taken together, these findings indicate an abnormal thymocyte maturation and cytokine dysregulation in the DS thymus, possibly initiated by gene dose-related increased sensitivity to IFN-γ and to overexpression of CD18 (LFA-1β). In conclusion, in this paper we show that the aforementioned alterations of the thymus result in the reduced production and output of newly generated lymphocytes, that can be directly measured by the assay we have developed and used. The quantitative analysis of TREC+ cells in the periphery is a relatively new and sensitive marker of thymic functionality, which is able to provide information on the status of the organ in different pathological conditions, and on its capability to generate new T cells [ 21 - 25 ]. A markedly reduced capacity of the thymus to produce RTE is present in the DS children we have studied. Such observation, if confirmed in a higher number of cases, could be useful to develop novel strategies to treat the immunodeficiency typical of this syndrome, based for example on the use of cytokines such as interleukin-2 or interleukin-7, which is capable of maintaining or restoring an efficient thymic output [ 26 ].
Discussion
Immunological ageing is part of a continuum of developmental processes, encompassing complex reorganizational events, compensatory mechanisms and qualitative alterations in the functionality of several systems and organs. Among those organs that undergo major changes with ageing, thymus plays a special role [ 12 ]. The thymus is a central lymphoid organ that is the primary site of T-cell maturation and development. Shortly after birth, the thymus undergoes a life long process of involution whereby the organ is replaced by adipose tissue. The result is a reduction in the number of constituent thymocytes with age, a consequent shrinking of the thymus and a decline in the output of newly generated T lymphocytes [ 13 ].Aged peripheral T-cell pool is characterized by the accumulation of T-cell capable of limited replication [ 14 ]. Since an efficient immune response is based upon the expansion of antigen-specific clones, the consequence of a qualitative and quantitative impairment of the system is an increased susceptibility to infections or cancer. Several groups, including ours, have studied the immune system during human ageing, using different models, including the one that represents the best example of successful ageing,  i.e.  healthy centenarians, and conditions of accelerated ageing, such as DS [ 2 - 6 , 15 ].i.e.In DS subjects, the proportion of T-helper cells is decreased, resulting in a decreased ratio of helper/cytotoxic cells, and we could observe this phenomenon also in the patients here described. Furthermore, peripheral blood T cells have a decreased number of cells expressing the T-cell receptor-αβ (TCRαβ) complex, elevated numbers expressing TCRγδ and a decreased proportion of CD4+, CD45RA+ naïve T cells, along with a high number of cells with NK phenotype, suggesting that the DS thymus is inefficient in the release of functionally mature T cells [ 16 ]. An age-dependence in the proliferative response to phytohemagglutinin (PHA) of DS lymphocytes, but not of lymphocytes from healthy individuals, was observed. Allogeneic mixed lymphocyte proliferative responses are decreased, as are PHA-induced interleukin-2 production and cytotoxic T-lymphocyte activity [ 7 ].All of the above mentioned alterations have a common substrate,  i.e.  the dysfunction of thymus. Indeed, defects in the capacity to produce thymic hormones has been described several years ago [ 17 , 18 ]. Furthermore, anatomic studies gave further evidence that changes in the T-lymphocyte system derive from structural abnormalities of the thymus. In comparison to age-matched controls, thymuses from infants with DS from 1 day to 15 months of age have marked lymphoid depletion, with a thin cortex and poor corticomedullary demarcation. The Hassall corpuscles are increased in size and frequently cystic. The presence of lower proportions of cells bearing high levels of the TCR-αβ complex and of CD3, a signal-transducing complex for the TCR, in thymuses of children with DS and the increase in the proportions of cells with these markers with age are indicative of delayed maturation of T cells within the thymus [ 9 ]. In addition, DS thymuses contain elevated levels of IFN-γ and TNF-α mRNA expressing cells, and there is mast cell hyperplasia and overexpression of class I MHC, CD18 and ICAM-1 [ 19 ]. DS thymocytes also have a greater than normal sensitivity to inhibition of IL-4-induced proliferation by IFN-γ and TNF-α [ 20 ]. Taken together, these findings indicate an abnormal thymocyte maturation and cytokine dysregulation in the DS thymus, possibly initiated by gene dose-related increased sensitivity to IFN-γ and to overexpression of CD18 (LFA-1β).i.e.In conclusion, in this paper we show that the aforementioned alterations of the thymus result in the reduced production and output of newly generated lymphocytes, that can be directly measured by the assay we have developed and used. The quantitative analysis of TREC+ cells in the periphery is a relatively new and sensitive marker of thymic functionality, which is able to provide information on the status of the organ in different pathological conditions, and on its capability to generate new T cells [ 21 - 25 ]. A markedly reduced capacity of the thymus to produce RTE is present in the DS children we have studied. Such observation, if confirmed in a higher number of cases, could be useful to develop novel strategies to treat the immunodeficiency typical of this syndrome, based for example on the use of cytokines such as interleukin-2 or interleukin-7, which is capable of maintaining or restoring an efficient thymic output [ 26 ].


Background Hz-2V infection of female  Helicoverpa zea  moths is manifested as insects that are either sterile "agonadal" individuals with malformed reproductive tissues or fertile asymptomatic carriers which are capable of transmitting virus on to their progeny. Virus infected progeny arising from eggs laid by asymptomatic carrier females may themselves be either sterile agonadals or asymptomatic carriers.
Background
Hz-2V infection of female  Helicoverpa zea  moths is manifested as insects that are either sterile "agonadal" individuals with malformed reproductive tissues or fertile asymptomatic carriers which are capable of transmitting virus on to their progeny. Virus infected progeny arising from eggs laid by asymptomatic carrier females may themselves be either sterile agonadals or asymptomatic carriers.Helicoverpa zeaResults By injecting virus into female moths, a correlation was established between virus doses administered to the females and the levels of resulting asymptomatic and sterile progeny.
Results
By injecting virus into female moths, a correlation was established between virus doses administered to the females and the levels of resulting asymptomatic and sterile progeny.Conclusions The results of these experiments indicate that high virus doses produced a higher level of agonadal progeny and lower doses produced higher levels of asymptomatic carriers.
Conclusions
The results of these experiments indicate that high virus doses produced a higher level of agonadal progeny and lower doses produced higher levels of asymptomatic carriers.

Background The insect virus, Hz-2V originally named gonad-specific virus (GSV) [ 1 ] was first identified in moths from a colony of corn earworms,  Helicoverpa zea  originating at the USDA-ARS in Stoneville, MS [ 1 , 2 ]. Insects infected with this virus were found to have malformed and missing reproductive tissues and were sterile, a condition that has been referred to as "agonadal". The examination of infected moths revealed that this virus replicated in a variety of male and female reproductive tissues including the common and lateral oviducts. Hence the tropism and replication of the virus is not specific to gonadal tissues. This rod shaped, enveloped, DNA virus has been more appropriately named Hz-2V since it resembles Hz-1V in size, pathology  in vitro  and in genome structure and size [ 3 - 5 ]. While examining progeny from eggs laid by infected female moths, Hamm  et al . [ 2 ] identified individuals that appeared healthy and were capable of transmitting Hz-2V to their progeny. Using PCR analysis Lupiani  et al . [ 6 ], were able to detect viral DNA sequences in feral corn earworms from wild populations that appeared healthy. These apparently healthy, infected moths are asymptomatic carriers of Hz-2V. The ability of this virus to persist in these asymptomatic carriers is a key feature of the biology of this virus. Since productive replication of Hz-2V results in the gross malformation of reproductive tissues and sterility of infected adult moths, persistence in asymptomatic carrier moths allows the virus to be maintained in insect populations such as the Stoneville colony. Hamm  et al . [ 2 ] presented evidence from experimental matings involving asymptomatic female moths and uninfected males that showed the proportion of agonadal progeny arising from eggs laid on successive oviposition days increased rapidly with each oviposition day, suggesting a change in viral activity in the asymptomatic female. They proposed that the outcome of virus infection in progeny was related to virus dose, such that eggs laid on early oviposition days received a low virus dose resulting in more asymptomatic virus carrier moths, whereas those arising from later oviposition days received a high virus dose and developed into agonadal moths. These findings indicate that Hz-2V is able to exist in a persistent or latent state in some corn earworms and become induced into productive replication at a specific time in the development of the insect. During their experiments, Hamm  et al . [ 2 ] were unable to accurately determine and control the virus dose female moths received and they were unable to directly detect females that were asymptomatic carriers of the virus. Raina  et al . [ 7 ] showed that it was possible to inject Hz-2V into healthy female corn earworm moths, and upon mating with healthy male moths, produce asymptomatic carrier and agonadal progeny. They found that about half of all of the progeny produced by females that were infected with a moderate virus dose exhibited the agonadal condition and that about 90% of the remaining apparently healthy progeny actually carried viral DNA sequences detectable by PCR. This data suggests that adult females can be injected with virus to experimentally produce females that mimic the asymptomatic carrier females described by Hamm  et al . [ 2 ]. In this study we have used the approach of injecting virus into healthy female moths to examine the relationship between virus dose and the level of infected, agonadal and asymptomatic carrier progeny insects hatching from eggs laid on successive ovipostion days. The results presented here demonstrate that virus dose affects both the level of infected progeny and the kind of infection found in insects hatching from eggs laid by virus infected females, indicating a direct correlation between virus dose received by females and the level of infected progeny they produce. Also demonstrated here is the fact that for each virus dose, as the level of agonadal insects hatching from eggs laid on successive oviposition days increase, the level of asymptomatic carrier progeny decreases.
Background
The insect virus, Hz-2V originally named gonad-specific virus (GSV) [ 1 ] was first identified in moths from a colony of corn earworms,  Helicoverpa zea  originating at the USDA-ARS in Stoneville, MS [ 1 , 2 ]. Insects infected with this virus were found to have malformed and missing reproductive tissues and were sterile, a condition that has been referred to as "agonadal". The examination of infected moths revealed that this virus replicated in a variety of male and female reproductive tissues including the common and lateral oviducts. Hence the tropism and replication of the virus is not specific to gonadal tissues. This rod shaped, enveloped, DNA virus has been more appropriately named Hz-2V since it resembles Hz-1V in size, pathology  in vitro  and in genome structure and size [ 3 - 5 ].Helicoverpa zeain vitroWhile examining progeny from eggs laid by infected female moths, Hamm  et al . [ 2 ] identified individuals that appeared healthy and were capable of transmitting Hz-2V to their progeny. Using PCR analysis Lupiani  et al . [ 6 ], were able to detect viral DNA sequences in feral corn earworms from wild populations that appeared healthy. These apparently healthy, infected moths are asymptomatic carriers of Hz-2V. The ability of this virus to persist in these asymptomatic carriers is a key feature of the biology of this virus. Since productive replication of Hz-2V results in the gross malformation of reproductive tissues and sterility of infected adult moths, persistence in asymptomatic carrier moths allows the virus to be maintained in insect populations such as the Stoneville colony.et alet alHamm  et al . [ 2 ] presented evidence from experimental matings involving asymptomatic female moths and uninfected males that showed the proportion of agonadal progeny arising from eggs laid on successive oviposition days increased rapidly with each oviposition day, suggesting a change in viral activity in the asymptomatic female. They proposed that the outcome of virus infection in progeny was related to virus dose, such that eggs laid on early oviposition days received a low virus dose resulting in more asymptomatic virus carrier moths, whereas those arising from later oviposition days received a high virus dose and developed into agonadal moths. These findings indicate that Hz-2V is able to exist in a persistent or latent state in some corn earworms and become induced into productive replication at a specific time in the development of the insect. During their experiments, Hamm  et al . [ 2 ] were unable to accurately determine and control the virus dose female moths received and they were unable to directly detect females that were asymptomatic carriers of the virus.et alet alRaina  et al . [ 7 ] showed that it was possible to inject Hz-2V into healthy female corn earworm moths, and upon mating with healthy male moths, produce asymptomatic carrier and agonadal progeny. They found that about half of all of the progeny produced by females that were infected with a moderate virus dose exhibited the agonadal condition and that about 90% of the remaining apparently healthy progeny actually carried viral DNA sequences detectable by PCR. This data suggests that adult females can be injected with virus to experimentally produce females that mimic the asymptomatic carrier females described by Hamm  et al . [ 2 ].et alet alIn this study we have used the approach of injecting virus into healthy female moths to examine the relationship between virus dose and the level of infected, agonadal and asymptomatic carrier progeny insects hatching from eggs laid on successive ovipostion days. The results presented here demonstrate that virus dose affects both the level of infected progeny and the kind of infection found in insects hatching from eggs laid by virus infected females, indicating a direct correlation between virus dose received by females and the level of infected progeny they produce. Also demonstrated here is the fact that for each virus dose, as the level of agonadal insects hatching from eggs laid on successive oviposition days increase, the level of asymptomatic carrier progeny decreases.Results A total of 1856 progeny moths resulting from approximately116 eggs laid on each of the first four oviposition days by females infected with 2 × 10 5 , 2 × 10 6 , 2 × 10 7 , or 2 × 10 8  TCID 50  units of Hz-2V were dissected and the reproductive tissues examined for signs of virus pathology. The PCR products of DNA samples from reproductive tissues of all apparently healthy progeny moths were examined for the presence of Hz-2V DNA via slot blot hybridization (figure  1 ), and the size of the PCR products of representative samples was determined by agarose gel electrophoresis. The results of agarose gel electrophoresis of PCR products from representative samples of agonadal, asymptomatic carriers and apparently healthy moths are shown in figure  2 . Figure 1 Slot blot hybridization results of DNA extracted from reproductive tissues of corn earworm moths. DNA was extracted, amplified via PCR, transferred onto a nylon membrane, and hybridized with a DIG-labeled viral DNA probe. Dark blots are indicative of DNA from asymptomatic carrier moths (As). Blots of DNA samples from insects from the healthy colony (H) and from insects that were determined to be apparently healthy (Ah) were blank or very light. Figure 2 Agarose gel of PCR products from DNA extracted from the reproductive tissues of corn earworm moths. The first lane is from a sample containing purified Hz-2V DNA (V). Lanes 2 denotes a sample from normal, healthy moth (H) from our insect colony. Lane 3 contains a DNA sample from an apparently healthy (AH) progeny moth arising from and infected female. Lanes 4–8 contain DNA samples extracted from asymptomatic progeny corn earworm moths (AS). Moths that had reproductive tissues that appeared to be normal but tested positive for Hz-2V DNA by PCR analyses were considered asymptomatic carriers of the virus. For each virus dose tested the number of agonadal moths, asymptomatic carriers, infected individuals (the sum of agonadal and asymptomatic carriers), and uninfected progeny moths hatching from eggs laid on each oviposition day was recorded. The analysis of these results showed that the percentage of total infected progeny (asymptomatic carriers and agonadal moths) at all doses tested increased with each successive oviposition day, and the level of infected progeny increased as virus dose increased from 2 × 10 5  to 2 × 10 8  TCID 50  units (figure  3 ). For individuals hatching from eggs on oviposition day one, the highest percentage of infected progeny (approximately 80%) was produced by females infected with the two highest virus dose (2 × 10 7  and 2 × 10 8 ), whereas the lowest percentage (about 60%) was produced by females infected with the lowest doses of virus (2 × 10 5  and 2 × 10 6  TCID 50 ). Figure 3 Mean percentages of infected (agonadal and asymptomatic carriers) progeny arising from eggs laid by female moths infected with 2 × 10 5 , 2 × 10 6 , 2 × 10 7 , or 2 × 10 8  TCID 50  units of Hz-2V. Virus infected progeny moths arising from eggs laid on each oviposition day by females infected with different virus doses were divided into agonadal and asymptomatic carriers and these results are presented in figure  4 . No agonadal insects arose from eggs laid on oviposition day one by females infected at the lowest virus doses, whereas approximately 15% of the progeny females from eggs laid at this time by females infected at the two highest doses were agonadal. At all of the viruses doses tested, between 70 and 90% of the individuals hatching from oviposition day one eggs were asymptomatic carriers (figure  4 ). Figure 4 Mean percentages of all (male and female) agonadal (AG) and asymptomatic carrier (AS) F1 moths arising from eggs laid by females infected with 2 × 10 5 , 2 × 10 6 , 2 × 10 7 , or 2 × 10 8  TCID 50  units of Hz-2V. For all F1 insects hatching from eggs laid on day two, (figure  4 ) the percentage of agonadal moths increased with increasing virus dose and the percentage of asymptomatic carriers at each dose declined (figure  4 ). The highest number of agonadal moths (approximately 70%) hatching from eggs laid on day two came from females that received the highest virus dose. At the two lowest doses the level of agonadals hatching from day two eggs was between 5 and 20%. At all doses almost 100% of the eggs laid on days three and four gave rise to agonadal moths. In order to better illustrate the relationship between the two types of infections and to emphasize the effects of virus dose upon the proportions of asymptomatic and agonadal infections, percentages of asymptomatic carriers and agonadal progeny for only the highest and lowest dose are presented in figure  5 . The trend in the two types of infected progeny insects follows the same general pattern for both virus doses relative to oviposition day. That is, at both doses the percentage of agonadal progeny increases with ovipostion day as the percentage of infected insects that are asymptomatic carriers of Hz-2V decreases. At the highest dose, the proportion of agonadal insects starts out higher (~ 10%) on the first oviposition day than that of agonadal progeny of females infected at the lowest dose (0%), and rises more quickly to ~ 70% of the progeny from eggs laid on oviposition day two. This is compared to only about 5% of the progeny arising from day two eggs laid by females infected at the lowest dose. Interestingly the reverse is the case for asymptomatic progeny hatching from oviposition day one eggs. Whereas approximately 90% of the infected oviposition day one individuals from females infected at the lowest virus does are asymptomatic only about 10% of the individuals from females infected at the highest dose are asymptomatic. Figure 5 Mean percentages of all (male and female) agonadal (AG) and asymptomatic carrier (AS) F1 moths arising from eggs laid by females infected at the highest and lowest doses of Hz-2V.
Results
A total of 1856 progeny moths resulting from approximately116 eggs laid on each of the first four oviposition days by females infected with 2 × 10 5 , 2 × 10 6 , 2 × 10 7 , or 2 × 10 8  TCID 50  units of Hz-2V were dissected and the reproductive tissues examined for signs of virus pathology. The PCR products of DNA samples from reproductive tissues of all apparently healthy progeny moths were examined for the presence of Hz-2V DNA via slot blot hybridization (figure  1 ), and the size of the PCR products of representative samples was determined by agarose gel electrophoresis. The results of agarose gel electrophoresis of PCR products from representative samples of agonadal, asymptomatic carriers and apparently healthy moths are shown in figure  2 .Moths that had reproductive tissues that appeared to be normal but tested positive for Hz-2V DNA by PCR analyses were considered asymptomatic carriers of the virus. For each virus dose tested the number of agonadal moths, asymptomatic carriers, infected individuals (the sum of agonadal and asymptomatic carriers), and uninfected progeny moths hatching from eggs laid on each oviposition day was recorded.The analysis of these results showed that the percentage of total infected progeny (asymptomatic carriers and agonadal moths) at all doses tested increased with each successive oviposition day, and the level of infected progeny increased as virus dose increased from 2 × 10 5  to 2 × 10 8  TCID 50  units (figure  3 ). For individuals hatching from eggs on oviposition day one, the highest percentage of infected progeny (approximately 80%) was produced by females infected with the two highest virus dose (2 × 10 7  and 2 × 10 8 ), whereas the lowest percentage (about 60%) was produced by females infected with the lowest doses of virus (2 × 10 5  and 2 × 10 6  TCID 50 ).Virus infected progeny moths arising from eggs laid on each oviposition day by females infected with different virus doses were divided into agonadal and asymptomatic carriers and these results are presented in figure  4 . No agonadal insects arose from eggs laid on oviposition day one by females infected at the lowest virus doses, whereas approximately 15% of the progeny females from eggs laid at this time by females infected at the two highest doses were agonadal. At all of the viruses doses tested, between 70 and 90% of the individuals hatching from oviposition day one eggs were asymptomatic carriers (figure  4 ).For all F1 insects hatching from eggs laid on day two, (figure  4 ) the percentage of agonadal moths increased with increasing virus dose and the percentage of asymptomatic carriers at each dose declined (figure  4 ). The highest number of agonadal moths (approximately 70%) hatching from eggs laid on day two came from females that received the highest virus dose. At the two lowest doses the level of agonadals hatching from day two eggs was between 5 and 20%. At all doses almost 100% of the eggs laid on days three and four gave rise to agonadal moths.In order to better illustrate the relationship between the two types of infections and to emphasize the effects of virus dose upon the proportions of asymptomatic and agonadal infections, percentages of asymptomatic carriers and agonadal progeny for only the highest and lowest dose are presented in figure  5 . The trend in the two types of infected progeny insects follows the same general pattern for both virus doses relative to oviposition day. That is, at both doses the percentage of agonadal progeny increases with ovipostion day as the percentage of infected insects that are asymptomatic carriers of Hz-2V decreases. At the highest dose, the proportion of agonadal insects starts out higher (~ 10%) on the first oviposition day than that of agonadal progeny of females infected at the lowest dose (0%), and rises more quickly to ~ 70% of the progeny from eggs laid on oviposition day two. This is compared to only about 5% of the progeny arising from day two eggs laid by females infected at the lowest dose. Interestingly the reverse is the case for asymptomatic progeny hatching from oviposition day one eggs. Whereas approximately 90% of the infected oviposition day one individuals from females infected at the lowest virus does are asymptomatic only about 10% of the individuals from females infected at the highest dose are asymptomatic.Discussion Injecting Hz-2V into female moths results in experimentally infected insects that resemble asymptomatic females and females that have become infected with the virus during copulation, not unlike the females infected during mass-matings by infected males in transmission experiments conducted by Hamm  et al . [ 2 ]. These infected moths appear healthy, are fertile, and can transmit the virus to progeny that result from mating. Some of the progeny moths arising from these infected females do not carry any detectable Hz-2V DNA sequences, others are sterile with malformed reproductive tissues, and still others are fertile, asymptomatic carriers of the virus. This variety of infections suggests that the virus is not initially present in all of the eggs laid by infected females, but is transmitted transovarially to some of the eggs at sometime time prior to oviposition. This idea is important in that it suggests that the dose or titer of virus transmitted from the parent female moth to the developing oocytes is not constant, and that the virus dose that each oocyte receives determines the outcome of the infection when these progeny insects mature into adult moths. The precise molecular mechanism that determines which infected individuals become agonadal and which will maintain the virus in the population as asymptomatic carriers has yet to be determined. The results presented in figure  4 , demonstrate that the percentage of agonadal progeny resulting from eggs laid on oviposition day one by female moths infected with Hz-2V increased as the dose of Hz-2V used to infect female moths increased. Progeny arising from eggs laid on oviposition day two also exhibited this correlation between virus dose and percent agonadal progeny. This indicates that the titer of the virus present in the experimentally infected female moths determines the amount of virus that is transmitted to eggs, and is directly correlated to the percentage of agonadal progeny arising from eggs laid by the infected females. As the dose of Hz-2V used to infect a female moth is increased, a corresponding increase is observed in total agonadal progeny arising from all eggs laid by the infected female. The percentage of agonadal progeny also increases with each successive oviposition day, approaching 100% agonadal progeny by day three at all virus doses tested, and all progeny moths arising from eggs laid on oviposition day four in all groups were agonadal. Based on the correlation between virus titer and percent agonadal progeny observed in these experiments, the increase in agonadal progeny per oviposition day is likely due to an increase in the titer of virus transmitted to the eggs, suggesting that the titer of virus increases in the parent female moths with each successive oviposition day. Studies of Hz-2V replication  in vitro  revealed a rapid increase in virus titer by 24 hours post infection in Tn-368 and Ld-652Y cells [ 4 , 5 ]. Hz-2V replication  in vivo  in the epithelial cells of agonadal female oviduct tissue has been described previously by Rallis and Burand [ 8 ]. The level of detectable virus in these tissues increased dramatically between 8 days post pupation (dpp), measured from the day the last larval exuviae was shed, and 10 dpp. It is likely that the large increase in virus over a 24 hour cycle observed  in vitro  also occurs  in vivo , resulting in a significant daily increase in the titer of Hz-2V in these experimentally infected female moths. Although the precise site of virus replication in these experimentally infected females is not known, the increase in virus titer in these individuals almost certainly results in an increase in virus being transmitted to the progeny with each successive oviposition day and ultimately in the patterns of infection reported here. If, as we have proposed, low virus doses result in asymptomatic carrier moths, and high virus doses produce agonadal progeny, then asymptomatic carrier progeny would likely arise from eggs produced on the earliest oviposition days and decrease with each day, as the virus titer in the egg-laying female moth increases. In fact, the percentage of asymptomatic carrier progeny in these experiments does decrease with each successive oviposition day to 0% by day four. The percent asymptomatic carriers is highest in progeny that receive the lowest virus dose, specifically progeny from oviposition day one and progeny arising from the parent female moths that were experimentally infected with the lowest dose of virus. This is directly opposite of what is observed for agonadal progeny, which is at its highest level at the highest virus dose, specifically on the later oviposition days (days three or greater) and in progeny arising from parent female moths that were infected with the highest virus dose. Interestingly, the lowest percentage of asymptomatic carrier progeny arose from eggs laid by the group of female moths that received the highest virus dose of Hz-2V (figure.  3 ). These data suggest that the virus dose transmitted by infected female moths to their developing eggs determines whether the progeny develop the agonadal condition or become asymptomatic carriers of Hz-2V. The results presented here clearly show that there is a direct correlation between virus dose and the relative percentage of agonadal and asymptomatic progeny. That is, increasing the virus dose causes an increase in the percentage of agonadal progeny, but a decrease in the percentage of asymptomatic progeny. At the present time, it is unknown how the development of an infected individual into an agonadal adult or an asymptomatic carrier is regulated. It is likely that a minimum titer of Hz-2V is needed at a key point in larval development to produce a viral factor(s) within the larval tissues at a threshold level required to reprogram the development and differentiation of the reproductive tissues into the agonadal structures. If this threshold is equaled or exceeded at this point in development, the progeny will exhibit the agonadal condition. However, if this threshold level is not attained, then the reproductive tissues are not reprogrammed and the infected insect becomes an apparently healthy, fertile, asymptomatic carrier of Hz-2V.
Discussion
Injecting Hz-2V into female moths results in experimentally infected insects that resemble asymptomatic females and females that have become infected with the virus during copulation, not unlike the females infected during mass-matings by infected males in transmission experiments conducted by Hamm  et al . [ 2 ]. These infected moths appear healthy, are fertile, and can transmit the virus to progeny that result from mating. Some of the progeny moths arising from these infected females do not carry any detectable Hz-2V DNA sequences, others are sterile with malformed reproductive tissues, and still others are fertile, asymptomatic carriers of the virus. This variety of infections suggests that the virus is not initially present in all of the eggs laid by infected females, but is transmitted transovarially to some of the eggs at sometime time prior to oviposition. This idea is important in that it suggests that the dose or titer of virus transmitted from the parent female moth to the developing oocytes is not constant, and that the virus dose that each oocyte receives determines the outcome of the infection when these progeny insects mature into adult moths. The precise molecular mechanism that determines which infected individuals become agonadal and which will maintain the virus in the population as asymptomatic carriers has yet to be determined.et alThe results presented in figure  4 , demonstrate that the percentage of agonadal progeny resulting from eggs laid on oviposition day one by female moths infected with Hz-2V increased as the dose of Hz-2V used to infect female moths increased. Progeny arising from eggs laid on oviposition day two also exhibited this correlation between virus dose and percent agonadal progeny. This indicates that the titer of the virus present in the experimentally infected female moths determines the amount of virus that is transmitted to eggs, and is directly correlated to the percentage of agonadal progeny arising from eggs laid by the infected females. As the dose of Hz-2V used to infect a female moth is increased, a corresponding increase is observed in total agonadal progeny arising from all eggs laid by the infected female.The percentage of agonadal progeny also increases with each successive oviposition day, approaching 100% agonadal progeny by day three at all virus doses tested, and all progeny moths arising from eggs laid on oviposition day four in all groups were agonadal. Based on the correlation between virus titer and percent agonadal progeny observed in these experiments, the increase in agonadal progeny per oviposition day is likely due to an increase in the titer of virus transmitted to the eggs, suggesting that the titer of virus increases in the parent female moths with each successive oviposition day.Studies of Hz-2V replication  in vitro  revealed a rapid increase in virus titer by 24 hours post infection in Tn-368 and Ld-652Y cells [ 4 , 5 ]. Hz-2V replication  in vivo  in the epithelial cells of agonadal female oviduct tissue has been described previously by Rallis and Burand [ 8 ]. The level of detectable virus in these tissues increased dramatically between 8 days post pupation (dpp), measured from the day the last larval exuviae was shed, and 10 dpp. It is likely that the large increase in virus over a 24 hour cycle observed  in vitro  also occurs  in vivo , resulting in a significant daily increase in the titer of Hz-2V in these experimentally infected female moths. Although the precise site of virus replication in these experimentally infected females is not known, the increase in virus titer in these individuals almost certainly results in an increase in virus being transmitted to the progeny with each successive oviposition day and ultimately in the patterns of infection reported here.in vitroin vivoin vitroin vivoIf, as we have proposed, low virus doses result in asymptomatic carrier moths, and high virus doses produce agonadal progeny, then asymptomatic carrier progeny would likely arise from eggs produced on the earliest oviposition days and decrease with each day, as the virus titer in the egg-laying female moth increases. In fact, the percentage of asymptomatic carrier progeny in these experiments does decrease with each successive oviposition day to 0% by day four. The percent asymptomatic carriers is highest in progeny that receive the lowest virus dose, specifically progeny from oviposition day one and progeny arising from the parent female moths that were experimentally infected with the lowest dose of virus. This is directly opposite of what is observed for agonadal progeny, which is at its highest level at the highest virus dose, specifically on the later oviposition days (days three or greater) and in progeny arising from parent female moths that were infected with the highest virus dose. Interestingly, the lowest percentage of asymptomatic carrier progeny arose from eggs laid by the group of female moths that received the highest virus dose of Hz-2V (figure.  3 ). These data suggest that the virus dose transmitted by infected female moths to their developing eggs determines whether the progeny develop the agonadal condition or become asymptomatic carriers of Hz-2V.The results presented here clearly show that there is a direct correlation between virus dose and the relative percentage of agonadal and asymptomatic progeny. That is, increasing the virus dose causes an increase in the percentage of agonadal progeny, but a decrease in the percentage of asymptomatic progeny. At the present time, it is unknown how the development of an infected individual into an agonadal adult or an asymptomatic carrier is regulated. It is likely that a minimum titer of Hz-2V is needed at a key point in larval development to produce a viral factor(s) within the larval tissues at a threshold level required to reprogram the development and differentiation of the reproductive tissues into the agonadal structures. If this threshold is equaled or exceeded at this point in development, the progeny will exhibit the agonadal condition. However, if this threshold level is not attained, then the reproductive tissues are not reprogrammed and the infected insect becomes an apparently healthy, fertile, asymptomatic carrier of Hz-2V.Conclusions The evolution of Hz-2V infection in  H. zea  has resulted in the ability of the virus to produce two different types of infections in the insects that enable the virus to replicate to high titers in the reprogrammed reproductive tissues in sterile agonadal moths, while maintaining itself in a population in asymptomatic carrier moths. This replication strategy appears to be essential for the continued existence of Hz-2V, since the development of the sterile, agonadal condition in  all  infected moths would lead to the extinction of the insect host, and the possibly the virus as well. The production of asymptomatic carrier moths ensures that some fertile, infected moths exist that can mate and produce infected progeny, enabling an Hz-2V-infected population to sustain itself, as in the case of the Stoneville colony.
Conclusions
The evolution of Hz-2V infection in  H. zea  has resulted in the ability of the virus to produce two different types of infections in the insects that enable the virus to replicate to high titers in the reprogrammed reproductive tissues in sterile agonadal moths, while maintaining itself in a population in asymptomatic carrier moths. This replication strategy appears to be essential for the continued existence of Hz-2V, since the development of the sterile, agonadal condition in  all  infected moths would lead to the extinction of the insect host, and the possibly the virus as well. The production of asymptomatic carrier moths ensures that some fertile, infected moths exist that can mate and produce infected progeny, enabling an Hz-2V-infected population to sustain itself, as in the case of the Stoneville colony.H. zeaallMethods Source of insects and virus Corn earworm larvae used to start a laboratory colony of healthy  H. zea  were obtained from the USDA-ARS in Stoneville, MS. Insects were reared on artificial diet and maintained as outlined previously [ 9 ]. Hz-2V for infecting female moths was prepared as described previously and purified via sucrose gradient centrifugation [ 4 ]. Injection of adults Newly emerged adult female moths were prepared and injected with Hz-2V as outlined by Rallis and Burand [ 8 ]. The female moths were divided into four dose groups, and 9 or 10 insects were infected with Hz-2V at one of the following concentrations of 2 × 10 5 , 2 × 10 6 , 2 × 10 7 , and 2 × 10 8  TCID 50  units. TCID50 assays Tn368 cells were cultured as per Burand & Lu [ 4 ] and 100 ul of cell culture medium containing 8 × 10 4  Tn368 cells were seeded into each well of a 96-well plate. Between 6 and 13 serial dilutions were made from each virus sample assayed and 10 or 20 wells were plated with 10 ul for each dilution. Plates were incubated at 27°C for 3 to 4 days and examined for the appearance of cytopathic effect (CPE). The numbers of wells with CPE were counted and the TCID 50  calculated [ 9 ]. DNA extraction and purification of viral DNA DNA was extracted from the reproductive tissues of adult moths by first homogenizing dissected tissues in 200 ul of TE buffer (10 mM Tris, pH 7.4, 1 mM EDTA, pH 8.0) followed by a 2-minute incubation in a boiling water bath. The homogenate was then chilled on ice, after which Ribonuclease A (10 ug/ul) was added to each sample, which was then incubated at room temperature for 15 min. The samples were then clarified by centrifugation at 15,600 × g for 2 min. Viral DNA used as template for PCR reactions was extracted from purified virus using 1% SDS in TE containing 1 mg/ml Protease K as outlined by Burand and Lu [ 4 ]. PCR amplification of viral DNA sequences Two sets of primers were used to amplify Hz-2V genomic DNA to prepare a probe for use in slot blot analysis of insect reproductive tissues. The first set (P4-1, 5'-GCACGATTCGTAATGTTC-3'; and P4-2, 5'-GCACACCTATCAATCACC-3') was designed to amplify a 434 bp sequence of the Hz-2V genome [ 6 ]. PCR reactions using P4-1 and P4-2 primers were brought to a final volume of 20 ul using the Bioneer AccuPower ®  PCR reagent premix kit with 1 unit of Taq DNA polymerase. Each reaction was carried out in10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl 2  and 40 mM KCl, containing 250 uM of each of the four dNTP's, with 100 pM of P4-1 forward and P4-2 reverse primers, and 10 ng of purified viral DNA as template. These primer set and reaction conditions were also used to amplify viral DNA sequences in approximately 100 ng of DNA from reproductive tissues of moths thought to be asymptomatic carriers of Hz-2V. The second set of primers (P4-3, 5'-GCTGTGCTGTACAAGTGC-3'; and P4-4, 5'-CCCTTGACGATCCCTTTTG-3') was designed to amplify a 350 bp region directly interior to that of the P4-1 and P4-2 amplified sequence. These primers were used to generate a DIG-labeled probe for Hz-2V to be used in slot blot hybridization assays. PCR reactions for production of the DIG-labeled probe were carried out in a final volume of 50 ul using the Boehringer Manheim DIG High Prime DNA Labeling and Detection Kit, with 1X concentrations of Taq Polymerase buffer (100 mM Tris-HCl pH 8.0, 500 mM KCL pH 8.3, and 25 mM MgCL 2 ), 100 pM of both P4-3 and P4-4 primers, a hexanucleotide mixture containing DIG-labeled dUTP (2 mM dATP, dCTP, dGTP, 1.3 mM dTTP, and 0.7 mM alkali labile DIG-11 dUTP pH 7.0), and 100 pM of Hz-2V genomic DNA. The DIG-labeled PCR product was purified on a 0.8% agarose gel using the Qiagen gel electrophoresis purification kit. Both PCR reactions for amplification of the viral DNA in tissue samples and for the production of the viral DNA probe consisted of 30 cycles of a DNA denaturation step at 95°C for 1 min., a primer annealing step for 1 min. at 55°C, and a 1 min. primer extension step at 72°C. Detection of a viral DNA sequence by slot blotting To prepare the DNA for slot blot analysis, 15 ul of the P4-1 and P4-2 PCR amplified DNA from insect samples was denatured by incubating with NaOH (0.4 M)/ EDTA (10 mM, pH 8.2) at 100°C for 10 min., then applied to a Hybon-N+ membrane prewashed with 500 ul 5X SSC buffer (0.6 M NaCl, 60 mM Na citrate pH 7.0) in a Manifold II slot blotter (Schleicher & Schuell). After applying the DNA, the membrane was baked at 88°C for 2 hrs under vacuum and prehybridized for 6 hrs. at 42°C in 50% formamide prehybridization buffer (5X SSC, 0.1% (w/v) N-laurylsarcosine, 1% (w/v) Na 2 -Dodecylsulfate, 2% Blocking reagent (Boehringer-Manheim), and 50% Formamide). Slot blots were hybridized with 150 ng DIG-labeled Hz-2V probe at 37°C for 12–14 hrs. Following washing, chemiluminescent detection was carried out as recommended by the DIG High Prime Labeling and Detection Kit Manual for DNA Hybridization (Boehringer Mannheim). Analysis of PCR products by agarose gel electrophoresis In order to confirm that the PCR products that hybridized to the viral DNA probe contained an amplified DNA fragment of the appropriate size (434 bp), representative samples were analyzed by electrophoresis on 0.8% agarose gels with 0.5X TBE buffer at 100 volts for approximately 1 hr, then stained with EtBr to visualize DNA bands under ultraviolet light.
Methods
Source of insects and virus Corn earworm larvae used to start a laboratory colony of healthy  H. zea  were obtained from the USDA-ARS in Stoneville, MS. Insects were reared on artificial diet and maintained as outlined previously [ 9 ]. Hz-2V for infecting female moths was prepared as described previously and purified via sucrose gradient centrifugation [ 4 ].
Source of insects and virus
Corn earworm larvae used to start a laboratory colony of healthy  H. zea  were obtained from the USDA-ARS in Stoneville, MS. Insects were reared on artificial diet and maintained as outlined previously [ 9 ].H. zeaHz-2V for infecting female moths was prepared as described previously and purified via sucrose gradient centrifugation [ 4 ].Injection of adults Newly emerged adult female moths were prepared and injected with Hz-2V as outlined by Rallis and Burand [ 8 ]. The female moths were divided into four dose groups, and 9 or 10 insects were infected with Hz-2V at one of the following concentrations of 2 × 10 5 , 2 × 10 6 , 2 × 10 7 , and 2 × 10 8  TCID 50  units.
Injection of adults
Newly emerged adult female moths were prepared and injected with Hz-2V as outlined by Rallis and Burand [ 8 ]. The female moths were divided into four dose groups, and 9 or 10 insects were infected with Hz-2V at one of the following concentrations of 2 × 10 5 , 2 × 10 6 , 2 × 10 7 , and 2 × 10 8  TCID 50  units.TCID50 assays Tn368 cells were cultured as per Burand & Lu [ 4 ] and 100 ul of cell culture medium containing 8 × 10 4  Tn368 cells were seeded into each well of a 96-well plate. Between 6 and 13 serial dilutions were made from each virus sample assayed and 10 or 20 wells were plated with 10 ul for each dilution. Plates were incubated at 27°C for 3 to 4 days and examined for the appearance of cytopathic effect (CPE). The numbers of wells with CPE were counted and the TCID 50  calculated [ 9 ].
TCID50 assays
Tn368 cells were cultured as per Burand & Lu [ 4 ] and 100 ul of cell culture medium containing 8 × 10 4  Tn368 cells were seeded into each well of a 96-well plate. Between 6 and 13 serial dilutions were made from each virus sample assayed and 10 or 20 wells were plated with 10 ul for each dilution. Plates were incubated at 27°C for 3 to 4 days and examined for the appearance of cytopathic effect (CPE). The numbers of wells with CPE were counted and the TCID 50  calculated [ 9 ].DNA extraction and purification of viral DNA DNA was extracted from the reproductive tissues of adult moths by first homogenizing dissected tissues in 200 ul of TE buffer (10 mM Tris, pH 7.4, 1 mM EDTA, pH 8.0) followed by a 2-minute incubation in a boiling water bath. The homogenate was then chilled on ice, after which Ribonuclease A (10 ug/ul) was added to each sample, which was then incubated at room temperature for 15 min. The samples were then clarified by centrifugation at 15,600 × g for 2 min. Viral DNA used as template for PCR reactions was extracted from purified virus using 1% SDS in TE containing 1 mg/ml Protease K as outlined by Burand and Lu [ 4 ].
DNA extraction and purification of viral DNA
DNA was extracted from the reproductive tissues of adult moths by first homogenizing dissected tissues in 200 ul of TE buffer (10 mM Tris, pH 7.4, 1 mM EDTA, pH 8.0) followed by a 2-minute incubation in a boiling water bath. The homogenate was then chilled on ice, after which Ribonuclease A (10 ug/ul) was added to each sample, which was then incubated at room temperature for 15 min. The samples were then clarified by centrifugation at 15,600 × g for 2 min.Viral DNA used as template for PCR reactions was extracted from purified virus using 1% SDS in TE containing 1 mg/ml Protease K as outlined by Burand and Lu [ 4 ].PCR amplification of viral DNA sequences Two sets of primers were used to amplify Hz-2V genomic DNA to prepare a probe for use in slot blot analysis of insect reproductive tissues. The first set (P4-1, 5'-GCACGATTCGTAATGTTC-3'; and P4-2, 5'-GCACACCTATCAATCACC-3') was designed to amplify a 434 bp sequence of the Hz-2V genome [ 6 ]. PCR reactions using P4-1 and P4-2 primers were brought to a final volume of 20 ul using the Bioneer AccuPower ®  PCR reagent premix kit with 1 unit of Taq DNA polymerase. Each reaction was carried out in10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl 2  and 40 mM KCl, containing 250 uM of each of the four dNTP's, with 100 pM of P4-1 forward and P4-2 reverse primers, and 10 ng of purified viral DNA as template. These primer set and reaction conditions were also used to amplify viral DNA sequences in approximately 100 ng of DNA from reproductive tissues of moths thought to be asymptomatic carriers of Hz-2V. The second set of primers (P4-3, 5'-GCTGTGCTGTACAAGTGC-3'; and P4-4, 5'-CCCTTGACGATCCCTTTTG-3') was designed to amplify a 350 bp region directly interior to that of the P4-1 and P4-2 amplified sequence. These primers were used to generate a DIG-labeled probe for Hz-2V to be used in slot blot hybridization assays. PCR reactions for production of the DIG-labeled probe were carried out in a final volume of 50 ul using the Boehringer Manheim DIG High Prime DNA Labeling and Detection Kit, with 1X concentrations of Taq Polymerase buffer (100 mM Tris-HCl pH 8.0, 500 mM KCL pH 8.3, and 25 mM MgCL 2 ), 100 pM of both P4-3 and P4-4 primers, a hexanucleotide mixture containing DIG-labeled dUTP (2 mM dATP, dCTP, dGTP, 1.3 mM dTTP, and 0.7 mM alkali labile DIG-11 dUTP pH 7.0), and 100 pM of Hz-2V genomic DNA. The DIG-labeled PCR product was purified on a 0.8% agarose gel using the Qiagen gel electrophoresis purification kit. Both PCR reactions for amplification of the viral DNA in tissue samples and for the production of the viral DNA probe consisted of 30 cycles of a DNA denaturation step at 95°C for 1 min., a primer annealing step for 1 min. at 55°C, and a 1 min. primer extension step at 72°C.
PCR amplification of viral DNA sequences
Two sets of primers were used to amplify Hz-2V genomic DNA to prepare a probe for use in slot blot analysis of insect reproductive tissues. The first set (P4-1, 5'-GCACGATTCGTAATGTTC-3'; and P4-2, 5'-GCACACCTATCAATCACC-3') was designed to amplify a 434 bp sequence of the Hz-2V genome [ 6 ]. PCR reactions using P4-1 and P4-2 primers were brought to a final volume of 20 ul using the Bioneer AccuPower ®  PCR reagent premix kit with 1 unit of Taq DNA polymerase. Each reaction was carried out in10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl 2  and 40 mM KCl, containing 250 uM of each of the four dNTP's, with 100 pM of P4-1 forward and P4-2 reverse primers, and 10 ng of purified viral DNA as template. These primer set and reaction conditions were also used to amplify viral DNA sequences in approximately 100 ng of DNA from reproductive tissues of moths thought to be asymptomatic carriers of Hz-2V.The second set of primers (P4-3, 5'-GCTGTGCTGTACAAGTGC-3'; and P4-4, 5'-CCCTTGACGATCCCTTTTG-3') was designed to amplify a 350 bp region directly interior to that of the P4-1 and P4-2 amplified sequence. These primers were used to generate a DIG-labeled probe for Hz-2V to be used in slot blot hybridization assays. PCR reactions for production of the DIG-labeled probe were carried out in a final volume of 50 ul using the Boehringer Manheim DIG High Prime DNA Labeling and Detection Kit, with 1X concentrations of Taq Polymerase buffer (100 mM Tris-HCl pH 8.0, 500 mM KCL pH 8.3, and 25 mM MgCL 2 ), 100 pM of both P4-3 and P4-4 primers, a hexanucleotide mixture containing DIG-labeled dUTP (2 mM dATP, dCTP, dGTP, 1.3 mM dTTP, and 0.7 mM alkali labile DIG-11 dUTP pH 7.0), and 100 pM of Hz-2V genomic DNA. The DIG-labeled PCR product was purified on a 0.8% agarose gel using the Qiagen gel electrophoresis purification kit.Both PCR reactions for amplification of the viral DNA in tissue samples and for the production of the viral DNA probe consisted of 30 cycles of a DNA denaturation step at 95°C for 1 min., a primer annealing step for 1 min. at 55°C, and a 1 min. primer extension step at 72°C.Detection of a viral DNA sequence by slot blotting To prepare the DNA for slot blot analysis, 15 ul of the P4-1 and P4-2 PCR amplified DNA from insect samples was denatured by incubating with NaOH (0.4 M)/ EDTA (10 mM, pH 8.2) at 100°C for 10 min., then applied to a Hybon-N+ membrane prewashed with 500 ul 5X SSC buffer (0.6 M NaCl, 60 mM Na citrate pH 7.0) in a Manifold II slot blotter (Schleicher & Schuell). After applying the DNA, the membrane was baked at 88°C for 2 hrs under vacuum and prehybridized for 6 hrs. at 42°C in 50% formamide prehybridization buffer (5X SSC, 0.1% (w/v) N-laurylsarcosine, 1% (w/v) Na 2 -Dodecylsulfate, 2% Blocking reagent (Boehringer-Manheim), and 50% Formamide). Slot blots were hybridized with 150 ng DIG-labeled Hz-2V probe at 37°C for 12–14 hrs. Following washing, chemiluminescent detection was carried out as recommended by the DIG High Prime Labeling and Detection Kit Manual for DNA Hybridization (Boehringer Mannheim).
Detection of a viral DNA sequence by slot blotting
To prepare the DNA for slot blot analysis, 15 ul of the P4-1 and P4-2 PCR amplified DNA from insect samples was denatured by incubating with NaOH (0.4 M)/ EDTA (10 mM, pH 8.2) at 100°C for 10 min., then applied to a Hybon-N+ membrane prewashed with 500 ul 5X SSC buffer (0.6 M NaCl, 60 mM Na citrate pH 7.0) in a Manifold II slot blotter (Schleicher & Schuell). After applying the DNA, the membrane was baked at 88°C for 2 hrs under vacuum and prehybridized for 6 hrs. at 42°C in 50% formamide prehybridization buffer (5X SSC, 0.1% (w/v) N-laurylsarcosine, 1% (w/v) Na 2 -Dodecylsulfate, 2% Blocking reagent (Boehringer-Manheim), and 50% Formamide). Slot blots were hybridized with 150 ng DIG-labeled Hz-2V probe at 37°C for 12–14 hrs. Following washing, chemiluminescent detection was carried out as recommended by the DIG High Prime Labeling and Detection Kit Manual for DNA Hybridization (Boehringer Mannheim).Analysis of PCR products by agarose gel electrophoresis In order to confirm that the PCR products that hybridized to the viral DNA probe contained an amplified DNA fragment of the appropriate size (434 bp), representative samples were analyzed by electrophoresis on 0.8% agarose gels with 0.5X TBE buffer at 100 volts for approximately 1 hr, then stained with EtBr to visualize DNA bands under ultraviolet light.
Analysis of PCR products by agarose gel electrophoresis
In order to confirm that the PCR products that hybridized to the viral DNA probe contained an amplified DNA fragment of the appropriate size (434 bp), representative samples were analyzed by electrophoresis on 0.8% agarose gels with 0.5X TBE buffer at 100 volts for approximately 1 hr, then stained with EtBr to visualize DNA bands under ultraviolet light.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions CPR participated in the design of the study, carried out the work with the insects, coordinated the project and assisted in the molecular analysis and drafting of the manuscript. JPB conceived the study, designed and supervised the experimental work and drafted the manuscript. All authors read and approved the final manuscript.
Authors' contributions
CPR participated in the design of the study, carried out the work with the insects, coordinated the project and assisted in the molecular analysis and drafting of the manuscript. JPB conceived the study, designed and supervised the experimental work and drafted the manuscript. All authors read and approved the final manuscript.