Persistence of the rotavirus A genome in mesenteric lymph nodes of cattle raised on farms

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1 Journal of General Virology (2015), 96, DOI /vir Persistence of the rotavirus A genome in mesenteric lymph nodes of cattle raised on farms Hiromichi Mitake, 1 Naoto Ito, 1,2 Kota Okadera, 1 Kazuma Okada, 1 Kento Nakagawa, 1 Tomomi Tanaka, 3 Kiyohito Katsuragi, 3 Kasumi Kasahara, 3 Toshihide Nihongi, 3 Shoji Sakurai, 4 Hiroshi Tsunemitsu 1,5 and Makoto Sugiyama 1,2 Correspondence Makoto Sugiyama sugiyama@gifu-u.ac.jp 1 The United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu , Japan 2 Laboratory of Zoonotic Diseases, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu , Japan 3 Fukui Prefectural Livestock Hygiene Service Center, Obatake, Fukui , Japan 4 Gifu City Meat Hygiene Inspection Center, Sakaigawa, Gifu , Japan 5 Dairy Hygiene Research Division, National Institute of Animal Health, 4 Hitsujigaoka, Toyohira, Sapporo, Hokkaido , Japan Received 29 March 2015 Accepted 18 May 2015 Previous studies revealed that rotavirus A (RVA) is present in not only the small intestine but also various organs. It was reported that RVA persisted in mesenteric lymph nodes (MLNs) in experimental models. However, there have been no reports focused on RVA in MLNs of animals under natural conditions. In this study, in order to investigate the persistence of the RVA genome in MLNs in cattle under natural conditions, reverse transcription-semi-nested PCR was carried out to detect RVA genomes in the MLNs from 17 calves that had been subjected to autopsy examinations. RVA genomes were detected in MLNs from 10 (60 %) of the 17 autopsied calves. MLNs from 170 healthy adult cattle that had been slaughtered were also examined; 15 (,10 %) of the 170 cattle had RVA genomes in their MLNs, indicating that RNA genomes are found frequently in MLNs of cattle under natural conditions. Genetic analyses revealed that RVAs in MLNs were classified as G and/or P genotypes generally prevalent in bovines. Basically, the strains in intestinal contents were genetically identical to those in MLNs from individual cattle, suggesting that bovine RVAs have the ability to spread from the intestine to MLNs. Furthermore, amongst RVA-positive cattle, six of 10 autopsied calves and 12 of 15 healthy adult cattle were negative for the virus in the intestinal contents, indicating that bovine RVA genomes can persist in MLNs after viral clearance in the digestive tract. INTRODUCTION Rotavirus A (RVA) is a major cause of severe diarrhoea in infants and young mammalian animals worldwide (Estes & Greenberg, 2013). RVA is responsible for an estimated deaths annually in children v5 years of age (Tate et al., 2012). RVA is a species of the genus Rotavirus, within the family Reoviridae, and its genome consists of 11 segments of dsrna encased in a triple-layered capsid (Matthijnssens et al., 2012). These segments encode six structural proteins (VP1 VP4, VP6 and VP7) and five or six non-structural proteins (NSP1 NSP5/6). Amongst these proteins, the outer capsid proteins VP7 and VP4 One supplementary table is available with the online Supplementary Material. independently elicit the production of neutralization antibodies. In a dual-classification system, RVAs are classified into 27 G and 37 P genotypes based on nucleotide sequences of the VP7 and VP4 genes, respectively (Matthijnssens et al., 2011; Trojnar et al., 2013). Amongst these, G6, G8 and G10 genotypes and P[1], P[5] and P[11] genotypes are known to be most prevalent in cattle (Dhama et al., 2009; Estes & Greenberg, 2013; Martella et al., 2010). RVA infection has been generally considered to be restricted to the small intestine. However, there has been an increasing number of reports of infectious RVA and the viral antigen being detected in sera and various organs, such as mesenteric lymph nodes (MLNs), lungs and livers in humans and animals (Blutt et al., 2003; Crawford et al., 2006; Dharakul G 2015 The Authors Printed in Great Britain

2 Persistence of the RVA genome in MLNs of cattle et al., 1988; Fenaux et al., 2006; Lynch et al., 2003; Mossel & Ramig, 2003). In experimental animal models, RVA has been detected not only in the gastrointestinal tract but also in extra-intestinal organs, indicating that the virus is able to spread systemically from the gastrointestinal tract (Crawford et al., 2006; Fenaux et al., 2006; Kim et al., 2011; Mossel & Ramig, 2003; Park et al., 2013, 2014). Notably, a previous study using a mouse model showed that RVA strain RRV persists longer in the MLNs than in the small intestine (Fenaux et al., 2006).Also,previousstudies on the dynamics of RVA in extra-intestinal organs in calves under experimental conditions showed that MLNs had the highest viral RNA copy number amongst extra-intestinal organs (Kim et al., 2011;Parket al., 2013, 2014). It was also reported that viral RNA copy numbers in MLNs were comparable to or sometimes higher than those in faeces through the duration of experimental infection for 14 days. These studies raised the possibility that RVA persists in MLNs for a long period. However, there has been no report on RVA in MLNs of animals under natural conditions. To evaluate whether the RVA genome persists in MLNs of cattle raised on farms, we examined MLNs and intestinal contents from calves and adult cattle for RVA genomes by reverse transcription (RT)-semi-nested PCR. The results indicated that the detection rate of RVA genomes is higher in MLNs than in intestinal contents. Notably, there were no RVA-positive cattle in which the viral genome was not detected in MLNs. These observations indicated that the RVA genome persists in MLNs of cattle after primary infection in the gut under natural conditions. RESULTS Detection of the RVA genome in MLNs and intestinal contents The specimens collected from cattle were initially screened for the presence of the RVA genome by detection of VP4 genes using RT-semi-nested PCR. RVA VP4 genes were detected not only in intestinal contents but also in MLNs from autopsied calves without diarrhoea and from healthy fattening cattle. RVA VP4 genes were detected in 10 (60%) of the MLN samples, one (5%) of the small intestine samples and three (*20%) of the rectal stool samples collected from 17 autopsied calves (Table 1). Of those, six calves were positive for the RVA VP4 gene only in MLNs. There were no VP4-positive calves without the detection of the RVA genome in MLNs (Table 2). In 170 healthy adult cattle, RVA VP4 genes were detected in 15 (10%) of the MLN samples and three (2%) of the rectal stool samples (Table 1). The detection of RVA in the stools of healthy adult cattle signified an inapparent infection. Also, 12 of 15 VP4-positive healthy adult cattle were positive only in MLNs (Table 3). Taken together, the results showed that the detection rate of the RVA VP4 gene was higher in MLNs than in the digestive tract in both autopsied calves and healthy fattening cattle (Table 1). Characterization of VP4 and VP7 segments of the detected strains from autopsied calves and healthy fattening cattle To analyse the genetic properties of the detected strains in MLNs and intestinal contents, sequence analyses of their VP4 and VP7 genes were performed. For P and G genotyping, the partial nucleotide sequences of VP4 ( bp, corresponding to nt in the VP4 gene of the bovine NCDV strain) and VP7 (512 bp, corresponding to nt in the VP7 gene of the bovine NCDV strain) segments of the strains detected in this study were determined; all strains were tested except for the strain from the healthy adult cow GSN334. BLAST analyses using the partial VP4 and VP7 genes determined for the detected strains revealed nucleotide identities of % and % with known RVA strains, respectively. As a result, the strains detected in this study were assigned to five different G genotypes (G6, G8, G10, G15 and G21) and three different P genotypes (P[11], P[14] and P[29]) (Tables 2 and 3). These genotypes of all strains detected in the MLNs and intestinal contents are generally prevalent in bovines (Abe et al., 2009; Ghosh et al., 2008; Martella et al., 2010; Matthijnssens et al., 2009). Comparison of nucleotide sequences of VP4 and VP7 genes of strains coincidently detected in MLNs and intestinal contents from the same cattle To examine the genetic relationships between the strains detected in MLNs and intestinal contents from individual Table 1. Detection rate of RVA VP4 genes Cattle No. of cattle sampled Detection rate [% (no. of positive samples)] MLNs Small intestine contents Rectal stool Autopsied calves (10) 6 (1) 18 (3) Fattening cattle (15) NC 2 (3) NC, Not collected

3 H. Mitake and others Table 2. Detection of the RVA genome, and combination of G and P genotypes of detected strains in autopsied calves Calf Age (months) Genotype MLNs Small intestine contents Rectal stool SS * HI KK MK G6P[11] G8P[14] MK G10P[11] G10P[11] KK MK G6P[11] G6P[11] MK G10P[11] TC G10P[11] MK KK G6P[11] KK G15P[29] KS G10P[11] MK G6P[11] G6P[11] TC MS SB G8P[14] *Negative for RT-semi-nested PCR targeting the VP4 gene. faeces for a long period: the diarrhoeal stool samples collected at 38 days of age and the normal stool samples collected at 46 and 83 days of age were all positive for the RVA VP4 gene by RT-semi-nested PCR. BLAST analyses revealed that all of the specimens contained an RVA strain classified to the same G10P[11] genotype as that of the strains detected in MLNs and rectal stool samples after death (Table 2). Comparative analyses between these G10P[11] strains, including the strains detected in MLNs and rectal stool samples after death, showed 100 and % homology in the nucleotide sequences of the partial VP4 ( bp, corresponding to nt in the VP4 gene of the bovine NCDV strain) and VP7 (512 bp, corresponding to nt in the VP7 gene of the bovine NCDV strain) genes, respectively. Only 1 nt difference in the partial VP7 gene was observed between the strain detected in a faecal specimen from the calf at 83 days of age and others. DISCUSSION The presence of RVA in MLNs has so far been reported in experimental animal models (Brown & Offit, 1998; Crawford et al., 2006; Dharakul et al., 1988; Fenaux et al., 2006; Kim et al., 2011; Mossel & Ramig, 2003; Park et al., cattle, the partial nucleotide sequences of the VP4 and VP7 genes were compared. There were four autopsied calves and three healthy fatting cattle for which RVA VP4 genes were detected in both MLNs and intestinal contents (Tables 2 and 3). For three autopsied calves (MK3536, MK2092 and MK3545) and three healthy fattening cows (GSM331, GSA063 and GSE178), RVA strains detected in MLNs belonged to the same G and P genotypes as those of the strains detected in intestinal contents of individual animals. The partial nucleotide sequences of VP4 and VP7 genes of these strains detected in MLNs were individually identical to the strains detected in intestinal contents, except for the strains from the calf MK3545. For the strains from MK3545, the partial VP4 and VP7 nucleotide sequences of the strain in MLNs shared 100 and 99.8% identity with those of the strain detected in the small intestine, respectively. These results indicated that the strains in MLNs from six of the seven cattle were genetically identical to the strains in the digestive tract. However, in one case (MK2098) a genotype difference was observed between the strain detected in MLNs (G6P[11]) and that in the rectal stool sample (G8P[14]) (Table 2). Detection of RVA in faecal specimens from calf MK3536 before death, and sequence analyses of VP7 and VP4 genes One of the autopsied calves that was positive for RVA in MLNs, MK3536, was found to have excreted RVA in Table 3. Specimens in which the RVA genome was detected, and combination of G and (possible) P genotypes of RVA strains detected in 15 VP4-positive healthy fattening cattle Cow Age (months) Genotype MLNs Rectal stool GSM G21P[29] G21P[29] GSG G21P[29] * GSI G6P[11] GSA G15P[29] G15P[29] GSC G10P[11] GSE G21P[29] G21P[29] GSH G15P[29] GSF G8P[29] GSJ G21P[29] GSB G6P[11] GSE G21P[29] GSN GXDP[29]d GSD G21P[29] GSK G15P[29] GSL G15P[29] *Negative for RT-semi-nested PCR targeting the VP4 gene. DNot determined as the VP7 gene was not detected by RT-nested PCR. dpossible genotype because the lengths of the partial VP4 sequences determined were too short to meet Rotavirus Classification Working Group requirements for genotyping Journal of General Virology 96

4 Persistence of the RVA genome in MLNs of cattle 2013, 2014). Infectious RVA and/or viral antigens have been detected in MLNs in experimentally infected mice, cattle and piglets. In this study, RVA RNAs were detected in MLNs from cattle raised on farms in Japan using RTsemi-nested PCR. The high detection rate of RVA genomes in MLNs from autopsied calves (Table 1) indicated that the presence of the RVA genome in MLNs is likely to be seen frequently in calves under natural conditions. In addition, RVA genomes were also detected in MLNs from some healthy adult cattle (Table 1), indicating that the presence of the RVA genome in MLNs is likely to be observed not only in calves, but also in adult cattle. Genetic analyses showed that all strains for which the genome was detected in MLNs from autopsied calves and healthy adult cattle were classified as G and/or P (possible) genotypes generally prevalent in bovines (Tables 2 and 3). Also, in most of the cattle that had RVA genomes in both MLNs and intestinal contents, the strains in intestinal contents were genetically identical to those in MLNs. These observations suggest that bovine RVAs have the ability to spread to MLNs of cattle from the digestive tract. Interestingly, there are a few reports of humans and animals with extended excretion of RVA (Goto et al., 1986; Miyazaki et al., 2012; Mukhopadhya et al., 2013; Richardson et al., 1998). Those studies raised the possibility that RVA persists in the digestive tract. In this study, a calf (MK3536) with extended excretion of RVA was found. High homologies in the partial nucleotide sequences of VP4 and VP7 genes between RVA strains from MK3536 indicated that these strains should be identical virus, suggesting that the RVA had persistently infected the calf for 2 months from diarrhoea to death. Importantly, MLNs from calf MK3536 also harboured the genome of the G10P[11] RVA strain (Table 2). Thus, these observations indicated the possibility that if RVA infects the digestive tract then this will result in the presence of the viral genome in MLNs. Notably, in most of the RVA-positive cattle, RVA genomes were detected in the MLNs even though the virus was not detected in intestinal contents (Tables 2 and 3). Although we were not able to exclude the possibility that the negative result for PCR in intestinal contents could be due to low viral titre in the digestive tract, the data are consistent with the idea that viral genomes become present in MLNs at a late stage of infection. Furthermore, a previous study using a mouse model showed that replication of the simian RRV strain persists longer in the MLNs than in the digestive tract (Fenaux et al., 2006). Consistent with this, our data indicated the possibility that viral genomes can persist in MLNs after viral clearance in the gut under natural conditions, implying that RVAs had persisted in MLNs of cattle for prolonged periods. In this study, we showed the presence of the RVA genome in MLNs from cattle under natural conditions. Our data also indicated that persistence of the RVA genome in MLNs is likely to occur frequently under natural conditions. We believe that these observations enhance our understanding of the mechanism of persistence of RVA in nature. METHODS Collection of MLNs and intestinal contents from autopsied calves and healthy adult cows. MLNs, small intestine contents and rectal stools were collected from 17 calves ( months of age) in cattle farms in Fukui Prefecture, Japan from 2012 to These calves had been subjected to autopsy examinations and diagnosed as having diseases other than RVA infection. MLNs were aseptically extirpated from calves and gut contents were sampled last to avoid cross-contamination. MLNs and rectal contents were also collected from healthy adult fattening cattle ( months of age) that were slaughtered at a municipal slaughterhouse in Gifu City, Japan from 2013 to The MLNs collected in the slaughterhouse were placed in 100% ethanol and flamed to prevent cross-contamination before sample preparation. All samples were subjected to 20% dilution or homogenization in PBS and clarified by centrifugation at 750 g for 10 min. The supernatants were collected and stored at 280 uc until use. Faecal specimens from a calf. We were able to obtain faecal specimens from one of the autopsied calves before death. The calf (MK3536), raised on a beef cattle farm in Fukui Prefecture, Japan, had diarrhoea at 38 days of age and recovered from diarrhoea after 4 days in December The calf then showed symptoms of pneumonia from 54 days of age. The calf eventually died at 92 days of age in February 2012 without recovery of respiratory symptoms. Three faecal specimens were collected from the calf before death and stored at 280 uc for another epidemiological surveillance: diarrhoeal faeces at 38 days of age, and normal faeces at 46 and 83 days of age. These faecal specimens were processed as described above. RNA extraction, RT, and nested and semi-nested PCR. Viral RNA was extracted from suspensions and homogenates using a QIAamp Viral RNA Mini kit (Qiagen). Synthesis of the cdna was performed using a PrimeScript II 1st Strand cdna Synthesis kit (TaKaRa Bio) with random hexanucleotides (TaKaRa Bio) as primers. Genomic RNAs were heated at 95 uc for 5 min and immediately chilled on ice before carrying out the RT reaction. For detection of RVA, the cdnas were amplified by an outer PCR with the primers VP4-HeadF and VP4-1094R2, and by a semi-nested PCR with the primers VP4-HeadF and VP4-887R, which were designed in the conserved region of the VP4 gene (Abe et al., 2009). The PCR conditions were the same as those described previously (Abe et al., 2009). VP7 and other regions of VP4 genes were then amplified by (semi-) nested PCR and directly sequenced with inner PCR primers for further analyses (Table S1, available in the online Supplementary Material). Outer PCR was performed with an initial denaturation step at 95 uc for 5 min, followed by 40 cycles of 95 uc for 30 s, 45 uc for 45 s and 68 uc for 1 min, and a final extension at 72 uc for 5 min. The cycle conditions for inner PCR were 30 cycles of 95 uc for 45 s, 45 uc for 45 s and 68 uc for 1 min. DNA sequencing and genetic analyses. The second PCR products were purified with a NucleoSpin Extract II (Macherey-Nagel) and sequenced with a BigDye Terminator version 3.1 Cycle Sequencing kit (Applied Biosystems) on an ABI Prism 3100 DNA analyser (Applied Biosystems). The second PCR primers were also used as sequencing primers. The sequences were assembled, edited and analysed using ApE (A plasmid Editor; wayned/ape/) version The nucleotide sequences of VP4 and VP7 genes determined were compared with those of reference strains available from GenBank

5 H. Mitake and others Genotype assignment. The G and P genotypes of the strains detected in this study were determined according to the guidelines for genotype classification established by the Rotavirus Classification Working Group (RCWG) (Matthijnssens et al., 2008). As we were not able to obtain the length of the partial VP4 sequences of the strain from one of the adult cows (GSN334) to meet RCWG requirements for genotyping, the P genotype of the strain was estimated. In addition, the G genotype of the strain from the adult cow GSN334 was not determined due to the VP7 gene of the strain not being detected by RT-nested PCR. Comparison of partial VP4 and VP7 nucleotide sequences of the detected strains from individual cattle. In cases where the same G and P genotypes of RVA strains were detected in both MLNs and intestinal contents from the same cattle, the partial VP7 and VP4 nucleotide sequences of the strains were compared using MEGA5 (Tamura et al., 2011). Briefly, the partial nucleotide sequences determined for each VP4 and VP7 of the strains were aligned using CLUSTAL W. Nucleotide identities between the strains detected in MLNs and in intestinal contents were then calculated using the p distance parameter. In addition, partial VP4 and VP7 nucleotide sequences of the strains detected in stool samples from a calf (MK3536) were compared with the corresponding strain detected in MLNs of the calf as described above. ACKNOWLEDGEMENTS The authors wish to thank the staff at Fukui Prefectural Livestock Hygiene Service Center and Gifu City Meat Hygiene Inspection Center for assistance with collection of specimens. We are also grateful to Dr G. W Moseley, University of Melbourne, Australia, for critical reading of the manuscript. This work was supported by the Japan Society for the Promotion of Science (KAKENHI Grant ). REFERENCES Abe, M., Ito, N., Morikawa, S., Takasu, M., Murase, T., Kawashima, T., Kawai, Y., Kohara, J. & Sugiyama, M. (2009). 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