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Conceived and designed the experiments: KAS ROD JB JWL RJM TMC. Performed the experiments: KAS SL JB JMB LMC TMC. Analyzed the data: KAS JWL RJM TMC JB ROD. Contributed reagents/materials/analysis tools: JB JMB ROD LMC. Wrote the paper: KAS JWL ROD JB RJM TMC.
Highly pathogenic H5N1 avian influenza viruses have caused major disease outbreaks in domestic and free-living birds with transmission to humans resulting in 59% mortality amongst 564 cases. The mutation of the amino acid at position 627 of the viral polymerase basic-2 protein (PB2) from glutamic acid (E) in avian isolates to lysine (K) in human isolates is frequently found, but it is not known if this change affects the fitness and pathogenicity of the virus in birds. We show here that horizontal transmission of A/Vietnam/1203/2004 H5N1 (VN/1203) virus in chickens and ducks was not affected by the change of K to E at PB2-627. All chickens died between 21 to 48 hours post infection (pi), while 70% of the ducks survived infection. Virus replication was detected in chickens within 12 hours pi and reached peak titers in spleen, lung and brain between 18 to 24 hours for both viruses. Viral antigen in chickens was predominantly in the endothelium, while in ducks it was present in multiple cell types, including neurons, myocardium, skeletal muscle and connective tissues. Virus replicated to a high titer in chicken thrombocytes and caused upregulation of TLR3 and several cell adhesion molecules, which may explain the rapid virus dissemination and location of viral antigen in endothelium. Virus replication in ducks reached peak values between 2 and 4 days pi in spleen, lung and brain tissues and in contrast to infection in chickens, thrombocytes were not involved. In addition, infection of chickens with low pathogenic VN/1203 caused neuropathology, with E at position PB2-627 causing significantly higher infection rates than K, indicating that it enhances virulence in chickens.
Since 2003, highly pathogenic (HP) H5N1 avian influenza viruses (AIV) of the Asian lineage have caused major outbreaks in ducks, swans and geese and other free-living birds as well as in backyard flocks of chickens and ducks and commercial poultry [1]. By early-2012, H5N1 viruses have infected around 578 humans almost entirely by direct contact with infected birds resulting in a case fatality rate of approximately 59% [2].
Several virus-specific factors seem important in successful interspecies transmission of H5N1 from birds to humans [3]. In particular, functional adaptation of the viral ribonucleoprotein complex appears to be significant since sequence analysis of H5N1 isolates from humans and other mammals indicate that it is under considerable positive selection [4]–[7]. One of the changes in the polymerase basic-2 protein (PB2) protein commonly associated with adaptation of a bird-derived HP H5N1 virus to mammalian species is the change of glutamic acid (E) to lysine (K) at position 627 [8]–[10]. Estimates of the importance of the E to K change depend on the experimental model used. In the mouse model, PB2-627K is significantly more pathogenic than PB2-627E whereas in ferrets there was little difference. Not all human H5N1 isolates from lethal cases have the E to K substitution. Although in the absence of PB2-627K, the change in PB2-701 from D to N change is frequently present [11], and has also been associated with transmission from avian to mammalian hosts [12]. The importance and function of the substitution of E to K for the in vivo pathogenicity has not been fully elucidated. Hatta et al. [8] examined the pathogenicity of human isolates with PB2-627K obtained from the 1997 Hong Kong outbreak and found that the change of K to E attenuated the infection in mice. The PB2-627K virus spread faster than PB2-627E in infected mice, perhaps overwhelming the host defense [13]. Furthermore, selection for PB2-627K and PB2-701N occurs in humans infected with avian-origin viruses [14]. Similarly, in vitro studies showed that the influenza polymerase complex requires K, rather than E, for efficient activity in primate cells [15], [16].
The threat to public health posed by H5N1 viruses with an E to K mutation at PB2-627 may have increased since the Qinghai Lake (China) outbreak in free-living birds. Virtually all viruses isolated from this outbreak in free-living birds and poultry including chickens, domestic ducks and geese also had PB2-627K [17]. These H5N1 viruses, characterized by the presence of the clade 2.2 HA gene [18], spread to Southern Asia, Africa and Europe and almost invariably contain PB2-627K [19].
In contrast to the body of studies in mammalian species there are relatively few studies comparing the pathogenesis of H5N1 AIV in ducks and chickens or assessing the contribution of specific amino acids to the pathogenicity in avian species. Infection of ducks with different H5N1 isolates showed differences in virus replication and pathogenicity [19]–[23]. Differences in pathogenicity in chickens have been linked to NP, PB1 and PB2 genes [24]–[25] and within the NP gene a change from A to K at position 184 increased virus titers and reduced the mean time to death [26]. Hulse-Post et al. [27] examined highly virulent large plaque and nonpathogenic small plaque isolates purified from A/Vietnam/1203/2004 H5N1 (VN/1203) for sequence differences. In ducks, the pathogenicity was associated with differences in PB1 and PA.
One of the major differences between chickens and ducks infected with HP H5N1 viruses is the difference in total mortality and the mean time to death [21], [28]–[30]. Recently, Barber et al. [31] suggested that the absence of RIG-I in chickens makes them more susceptible to HP H5N1 infection and may explain the greater pathogenicity seen in chickens compared to ducks which express RIG-I. H5N1 replication in endothelial cells of infected chickens has been associated with high mortality and shortened mean time to death as compared to ducks [25], [29]. Furthermore, we have previously observed that inflammatory cytokine mRNAs are increased significantly within 24 hours post infection (pi) [32]. We speculated that a rapid overwhelming inflammatory response may play a role in the pathogenesis in chickens. Sterz and Weiss [33]–[34] had previously shown that AIV replicates in chicken thrombocytes in vivo and in vitro. Chicken and duck thrombocytes are highly phagocytic [34]–[37], express low levels of CD154 [38] and after in vitro stimulation with LPS can rapidly increase IL-1β, IL-6 and IL-12 mRNA expression [39]–40. Furthermore, chicken thrombocytes release high levels of functional IL-6 following LPS stimulation (Schat, unpublished data).
In this study, we used reverse genetics to generate HP VN/1203 viruses that contain either PB2-627K or PB2-627E. We also produced isogenic low pathogenic (LP) VN/1203 viruses that lacked the hemagglutinin (HA) cleavage site for HP. To our knowledge studies comparing the pathogenicity of such viruses have not been reported in chickens or ducks. Here we report that the switch from K to E at PB2-627 of HP VN/1203 does not influence the pathogenesis in ducks and chickens. However, the PB2-627E mutation in LP VN/1203 increased virulence relative to PB2-627K. Furthermore, we present evidence to support our hypothesis that thrombocytes may play a major role in the pathogenesis of HP H5N1 in chickens, but not in ducks.
To determine if the change of PB2-627 from E to K in VN/1203 influences horizontal transmission and/or pathogenicity, we infected chickens and ducks with HP VN/1203/K and HP VN/1203/E viruses. On the following day, naïve birds were placed in cohabitation with the inoculated animals. Infection was measured by survival to 14 days pi, isolation of virus from swabs and tissues, and seroconversion.
Chickens infected with HP VN/1203/K or HP VN/1203/E viruses died within 18–24 hours pi. There were no significant differences in replication between the two viruses. Virus was isolated from all organs and oral and cloacal swabs at the time of clinical disease or death and most of the swabs collected at 24 hours pi were positive (Table 1). Virus was not isolated from chickens that subsequently survived until 14 days pi nor were they seropositive, indicating they did not receive an infectious dose. Contact transmission from infected chickens was not established at all for HP VN/1203/E and at a low level with HP VN/1203/K with only 3/14 birds positive (Table 1). All inoculated and contact-challenged ducks became infected as determined by disease or seroconversion in survivors.
To determine the virulence for ducks and the extent of horizontal spread to contact ducks and chickens, ducks were infected with 105.8 and 106.0 EID50 of HP VN/1203/E and HP VN/1203/K, respectively. Inoculation with either virus resulted in similar rates of clinical disease in ducks and in chickens, resulting in euthanasia or death in some ducks and all chickens. Virus was readily isolated from oral swabs from all inoculated ducks (Table 2) and all surviving ducks were seropositive. Both viruses were transmitted to 100% of contact ducks. Ducks infected with HP VN/1203/E transmitted the infection to contact ducks more efficiently than HP VN/1203/K infected animals, as evidenced by virus isolation from oral swabs collected after 5 days in cohabitation (p<0.05, two-tailed Fisher's exact test) (Table 2). The mortality rates and all other data were not significantly different between the two contact-infected duck groups. All contact infected chickens died or were euthanized between 5 and 7 days post contact with both viruses.
The clinical disease and pathology at the time of death were essentially similar in chickens infected with the HP VN/1203/E and VN/1203/K viruses. Establishment of infection caused mortality or severe depression between 21 and 46 hours pi. The morbidity periods were very short, with some chickens requiring euthanasia within 3 hours of the initial manifestation of clinical signs. There was an absence of gross pathology. Microscopically, there were small foci of necrosis, most noticeably in the red pulp of the spleen, but also in the lung interstitium and lamina propria of the intestine. Tissues of infected birds were assessed for viral antigen by immunohistochemistry at time of euthanasia. High levels of antigen expression was detected mainly in the endothelium (Figure 1A, C, E), indicating that viral replication occurred in all vascularised tissues. Antigen was particularly dense in rich vascular networks, such as in the spleen, lung, glomerular tufts and lamina propria of intestinal villi. Dense antigen was also present in inflammatory foci in the submucosa of various tissues, such as intestine, respiratory airways, liver and kidney. The parenchyma of organs contained low to moderate levels of antigen, and this was most frequent in myocardium (Figure 1E), but also in small foci in the brain, lung, liver, pancreas and kidney tubules.
There were also no noticeable differences in the clinical disease and pathology between ducks infected with the HP VN/1203/E and VN/1203/K viruses. Gross lesions in ducks were often absent or subtle and included mild pale streaking on the heart, pale or red spots on the pancreas and mildly increased fluid in the body cavities. Microscopic lesions included acute, diffuse, mild to severe cardiomyopathy, diffuse mild to moderate non-suppurative encephalitis and acute focal necrosis of the pancreas. Influenza virus antigen was detected in the myocardium, in foci of neurons in the brain, in skeletal and smooth muscle, necrotic foci in the pancreas (Table 3) and in air sac epithelium. Antigen was rare in lung interstitium and kidney tubules. The pathology observed in contact-infected ducks was similar to that in inoculated ducks.
To determine whether the PB2-627 amino acid had changed during the course of infection in vivo we sequenced the corresponding region of viruses recovered from two diseased (for HP VN/1203/E and HP VN/1203/K viruses) contact ducks. In both instances the same sequence as the inoculated virus was obtained suggesting that this position was not undergoing rapid host-driven selection in vivo.
In order to determine the influence of amino acid substitution at PB2-627 on the pathogenesis of infection, chickens and ducks were inoculated with virus and euthanased at intervals for sample collection. Two experiments were conducted in chickens, both with similar dose and route of inoculation, but sampled at different time points. In the first experiment chickens were inoculated with 106.0 TCID50 of either virus. Neither HP VN/1203/E nor HP VN/1203/K were isolated from spleen, lung, and brain tissue samples as well as cloacal and oral swabs collected at 6 and 12 hours pi, however, all specimens collected at 24 hours pi were positive. In the second experiment, chickens infected with 106.0TCID50 of HP VN/1203/E had virus in different organs at 12 hours pi in contrast with birds infected with the same dose of HP VN/1203/K (Figure 2). At 18 hrs pi, titers in the different organs and swabs were not significantly different between the two viruses. All tissue samples and swabs contained infectious virus by the time clinical disease developed (21–24 hours pi), with titers between 102 to 108 TCID50/mL (Figure 2).
Antigen was not detected in any of the chickens at 6 and 12 hours pi, but was present in all birds sampled at or after 18 hours pi (data not shown). There was no difference in antigen levels between the two viruses at these time points and at time of clinical disease.
With both HP VN/1203/E and HP VN/1203/K virus antigen was detected in duck tissues with peak levels found between 3 and 4 days pi, with a decline in antigen levels by day 6 (Table 3). There was no apparent difference in the tissue tropism between the two viruses with heart and brain most frequently positive. There appeared to be a higher overall prevalence of heart and brain infection in ducks infected with HP VN/1203/E, but the overall differences with HP VN/1203/K were not significant.
Virus was isolated from all ducks in both virus groups at 3 and 4 days pi, but not all organs were positive. Titers in the spleen, lung, brain and cloacal and oral swabs at the different time points are presented in Figure 3. Although there were minor differences between the two virus groups all time points, the differences were not statistically significant. Taken together, these findings suggested that in this model there are no significant differences in the kinetics of virus infection and virus distribution in the major organ systems of chickens and ducks as a result of the change of amino acid at position 627 of PB2.
The virulence of wild-type HP VN/1203 virus in chickens was overwhelming and precluded detection of moderate differences between PB2-627K and PB2-627E infections. Therefore, we developed isogenic viruses with a deletion of the multibasic cleavage site of the HA; i.e. LP VN/1203/E and LP VN/1203/K. To confirm that the
LP VN/1203/K and LP VN/1203/E viruses were indeed of low pathogenicity, we inoculated chickens (n=6) with 106.1 EID50 of either virus and evaluated the response for 14 days. Three of the 6 birds infected with LP VN/1203/E developed ataxia and torticollis and were euthanased, one 8 days pi and the other two 13 days pi. These 3 birds had non-suppurative encephalitis and dermatitis, as described below, and virus antigen associated with lesions. While 5/6 chickens seroconverted after infection with LP VN/1203/K, none showed clinical signs (Table 4). The remaining birds of both groups were euthanased and sampled at 14 days pi; although they survived apparently healthy, many had microscopic lesions in brain and skin. Characteristic lesions that were associated with antigen included focal glial nodules with the presence of mononuclear cell perivascular cuffing in the brain and focal dermal and subdermal necrosis, particularly in the comb, and folliculitis. In the absence of viral antigen in such lesions were considered highly likely as indicative of prior influenza virus replication. Such lesions were present in the remaining three chickens that were euthanased at 14 days pi for LP VN/1203/E and in 1 of 5 chickens surviving to 14 days pi for LP VN/1203/K (Table 4). Viral antigen was present in single neurons in glial nodules in the brain (Figure 1B), and at the stratum granulosum of epidermis of skin (Figure 1D) and feather follicles. The incidence of histopathologic lesions in the brain and skin were significantly higher in birds infected with LP VN/1203/E than with LP VN/1203/K (p<0.05, two-tailed Fisher's exact test, Table 4). All birds that survived until termination developed HI antibodies except one bird infected with LP/VN1203/K. However, the antibodies failed to completely clear the virus based on immunohistochemistry and did not prevent the development of clinical disease in the case of the two birds with nervous symptoms at 13 days pi. To confirm that both LP VN/1203/E and LP VN/1203/K viruses replicated in vivo, oral secretions collected at 6 days pi were analyzed by RT-PCR; viral RNA was detected in both groups of chickens.
Chickens infected with 106.1 EID50 of LP VN/1203/E and LP VN1203/K were sacrificed at 1, 2, 3, 4, and 5 days pi and organs were examined for the presence of viral antigen by immunohistochemistry and virus isolation by inoculation of 9- to 10-day old embryonated chicken eggs. None of the chickens showed clinical signs, although some sampled at 4 and 5 days pi had lesions, including glial nodules in the brain and mild, focal necrosis in the dermis. Overall, virus isolation rates were higher for LP VN/1203/E than for LP VN/1203/K. The difference was significant at 3 days pi (p<0.05, two-tailed Fisher's exact test, Table 5).
Viral antigen was detected in neurons and glial cells (with or without associated lesions), in single myocardial fibers (Figure 1F), kidney tubular epithelium, single cells in dermis, connective tissues (follicle sheath, periosteum), nasal gland and feather pulp. Definitive antigen in endothelium, as seen with the HP AIV infections, was not found. Interestingly, LP VN/1203/E caused a higher incidence of virus-antigen positive tissues than LP VN/1203/K starting at 3 days pi. The difference between the total number of antigen positive tissues of all birds combined for 3–5 days pi is significantly higher for LP VN/1203/E (14/18) than LP VN/1203/K (3/18) (p<0.05, two-tailed Fisher's exact test).
Sequence analysis confirmed that the virus obtained from the bird with neurological signs at 8 days pi and from the four birds that we viral-antigen positive still retained the HA cleavage site associated with the LP form as well as the original E at PB2-627.
In conclusion, these studies indicated that there are differences associated with infection in chickens between viruses with E versus K at PB2-627, but that these changes can only be demonstrated when birds were infected with the LP viruses.
Previous studies by Sterz and Weiss [33]–[34] suggested that AIV can replicate in thrombocytes, we therefore examined whether thrombocytes obtained from infected chickens and ducks contained infectious virus. Thrombocytes from all chickens contained infectious virus at 18 and 21–24 hours pi with either HP VN/1203/E or HP VN/1203/K, and at 12 hours pi for 5/6 chickens infected with HP VN/1203/E (Figure 2). In ducks, thrombocyte preparations from only some of the animals contained infectious virus but in contrast with the findings in chickens the titers were extremely low (≤30 TCID50, data not shown). In contrast, thrombocytes from chickens infected with LP VN/1203/E and LP VN/1203/K were negative for virus isolation except for one sample from the LP VN/1203/E group taken at 4 days pi.
Both HP VN/1203/E and VN/1203/K viruses could be isolated at high levels from chicken thrombocytes suggesting that AIV might replicate in these cells or serve as a vehicle for the spread virus in the vasculature. To quantify the viral RNA in thrombocytes, we analyzed purified cells by real-time RT-PCR using primers specific for viral genomic RNA. Chicken thrombocytes collected at 18 hours pi with HP VN/1203/K or HP VN/1203/E had Ct values of 18.6 and 17.0 for genomic RNA and 17.8 and 16.2 for cRNA specific cDNA reactions, respectively. In contrast thrombocytes from uninfected birds had Ct values of >40 for both primers.
The profound transcriptome changes observed in thrombocytes collected at 18 hours pi suggested that HP VN/1203/K and HP VN/1203/E may have initiated replication in chicken thrombocytes or progenitor megakaryocytes. Table 6 shows the complete list of significant changes, defined as ≥2-fold increase in transcription relative to uninfected controls. Analysis of the TLR receptor signaling pathway indicated significant upregulation of TLR3, which can be triggered by dsRNA, and involve STAT1, IRF7, and IFNAR2. In addition several cell adhesion molecules (SELE, VCAM1, CD274), SOCS 1 and MX were also highly upregulated in thrombocytes.
These results showed a major difference in the response of thrombocytes between chickens and ducks inoculated with HP AIV and between chickens inoculated with HP versus LP viruses. The microarray results further support the postulated importance of thrombocytes for the pathogenesis and pathology of HP virus in chickens.
Several previous studies have compared the pathogenicity of different HP H5N1 isolates in chickens and ducks. Most of these isolates caused peracute mortality in chickens often without clear pathology [30], [41]–[42]. The pathogenicity in domestic ducks varied from 0 to 100% depending on the isolate [19], [22]–[23], [43]–[44], age at infection [45]–[46], and virus dose [47]–[48]. The pathogenicity determinants for these viruses have only been partially elucidated. In addition to the HA cleavage site, the PA, PB1, PB2 and NP genes contribute to differences in the pathogenicity of HP H5N1 viruses in chickens and ducks [24]–[27], [49]–[50]. In contrast to the many studies showing the importance of the K versus E at PB2-627 in mammalian species, there is a lack of information on this change for the pathogenesis in chickens and ducks. Our studies show that infection with HP VN/1203/K and HP VN/1203/E are both lethal in chickens, causing rapid death. The absence of antibodies in chickens that survived inoculation with 104.5 to 105.2 EID50 confirmed a previous report indicating that the minimum infectious dose of wild type Asian lineage H5N1 viruses for chickens is relatively high, 103.4 EID50 for ID50, and almost identical to the lethal dose [47]. HP VN/1203/K and HP VN/1203/E were also equally pathogenic for white Pekin ducks with 25–55% disease in inoculated and contact-infected ducks, respectively, using a moderate dose of 100 EID50 and 5-week-old ducks. Virus isolation from the oral swabs obtained from the contact-infected ducks suggested subtle differences in replication between the two viruses, but the subsequent time course experiment to evaluate the pathogenesis in ducks failed to reveal significant differences between the two viruses. Clearly, the clade 2.2 viruses of the Qinghai Lake outbreak with PB2-627K can be as pathogenic as viruses with PB2-627E and spread horizontally just as readily as other H5N1 viruses among birds [17], [19], [51]–[62].
Infection of chickens with 105.9 EID50 of HP VN/1203/K and HP VN/1203/E showed that virus was being shed at low levels as early as 12 hours pi and HP VN1203/E but not HP VN1203/K was isolated from respiratory and systemic site samples. Antigen from both viruses was abundant in these organs as early as 18 hours pi reflecting rapid replication leading to fatal outcomes between 21 and 24 hours pi. The rapid systemic dissemination of virus in chickens is consistent with earlier reports [25], [42] and contrasts with the slower dissemination of the HP VN/1203/E and HP VN/1203/K viruses in ducks.
In addition to the difference between chickens and ducks in extrapulmonary virus spread and replication kinetics, two other major differences were observed. First, the distribution of viral antigen in tissues was different between the two species. Vascular endothelial cells were positive for viral antigen at 18 hours pi with HP VN/1203/E and HP VN/1203/K in the tissues examined from infected chickens as has been reported by others [25], [29]–[34], [42], [63]. In contrast endothelial expression of viral antigen in ducks was rare [29]. The second major contrast between chickens and ducks was the striking difference in the virus titers in thrombocytes. The high virus titers associated with the chicken thrombocytes as well as the detection of viral RNA clearly indicated the presence of virus in the purified cell preparations. Minor contamination with other monocytes and heterophils cannot be excluded but seems unlikely. We cultured thrombocytes for 24 hours and found that the cultures consisted of >95% thrombocytes (Schat and Guo, unpublished data). It is currently unknown whether the HP AIV was phagocytosed by the thrombocytes or that the virus entrance was receptor-mediated. The finding that duck thrombocytes were basically negative for virus between 1 and 6 days pi is of interest in view of the differences in pathology and viral dissemination.
The question of whether the pathogenicity in chickens is influenced by the change of K to E at PB2-627 could not be resolved in studies using the wild-type HP VN/1203 virus pair due to the overwhelming pathology in this species. However, LP virus counterparts with a deletion of the HA multibasic cleavage site clearly showed that the presence of E at PB2-627 had a significant impact on the pathology. LP VN/1203/E did spread systemically, but at low levels and without causing high lethality, as reported previously [64], and in contrast to its HP counterpart, was not detected in thrombocytes. Absence of virus in thrombocytes may allow for a more effective host antiviral response. The mechanisms mediating increased pathogenicity in LP VN/1203/E as compared to LP VN/1203/K are not clear. Sequence analysis of virus re-isolated from inoculated and contact-infected animals confirmed that the HA and PB2 gene had not changed and were identical to the inoculated viruses. Pillai et al. [65] showed that infection of chickens with a diverse collection of LP AIV isolates caused minimal lesions. However, Mundt et al. [66] found some mild lesions in some organs including the cerebrum after infection with North-American lineage viruses such as A/chicken/Pennsylvania/13609/93 (H5N2) and A/chicken/Texas/167280-4/02, suggesting that systemic infection can occur with LP AIV. The use of HP AIV isolates in which the HA cleavage site has been mutated to the LP cleavage site may be advantageous to further identify virulence factors for the Asian H5N1 strains.
Involvement of thrombocytes in the infection is also suggested by the microarray data. TLR3 and several of the genes involved in the TLR signaling pathway were significantly upregulated during infection. Moreover, SOCS1, which can regulate TLR-mediated signal transduction [67] was also strongly upregulated. Le Goffic et al. [68] showed that influenza A viruses activate TLR3-dependent proinflammatory responses in epithelial cells, leading to increased levels of IFN-β production [69]. We have recently reported that HP VN/1203/K, but not LP VN/1203/K, caused a strong upregulation of IL-6, IL-12, IFNα, IFNβ, IFNγ and IFNλ mRNA at 24 hours pi [32]. It is certainly feasible that infection of thrombocytes plays a major role in the rapid upregulation of the proinflammatory cytokines. It is also of interest to note that several cell adhesion molecules were upregulated. Some of these molecules such as VCAM1 and selectin E may bind to neutrophils leading to the formation of micro-thrombi which are often associated with HPAIV infection [41]. Clearly, additional studies are needed to further elucidate the role of thrombocytes in the pathogenesis of HP AIV in chickens. Moreover, the correlation of viral load differences in chicken and duck thrombocytes with differential species-specific susceptibility between these animals suggests a possible role in pathogenesis.
In conclusion, our studies indicated that HP viruses with PB2-627E may be marginally more pathogenic in ducks than viruses with PB2-627K, but such differences could not be demonstrated in chickens due to the rapid onset of mortality. However, the LP VN/1203/E was certainly more pathogenic in chickens than its PB2-627K counterpart. Finally, our studies indicate that thrombocytes may play a role in the pathogenesis of HP Asian H5N1 AIV infections.
This study was carried out in strict accordance with the recommendations in the National Health and Medical Research Council's Australian Code of Practice for the Care and Use of Animals for Scientific Purposes 7th edition. All experimental protocols were approved by the CSIRO Australian Animal Health Laboratory Animal Ethics Committee (Approval numbers AEC 1152, 1174, 1300 and 1301) at the Australian Animal Health Laboratory (Bureau of Animal Welfare Scientific Procedures Premises Licence SPPL 113). All efforts were made to minimize suffering.
Four-week-old, straight-run broilers were obtained from a commercial producer. Five-week-old White Pekin ducks were obtained from Luv-a-Duck (Victoria, Australia). All birds were identified individually with leg bands and were assigned to the different treatment groups for each experiment using a randomization tool (www.graphpad.com). Birds were housed in Microbiological Containment level 3 Zoonotic rooms with the temperature set at 22°C and the airflow set at approximately 15 air changes/hour [23].
HP VN/1203/K was derived from the parental wild-type virus by standard reverse genetics methods [70]. Mutations at positions 627 of the PB2 were generated by standard oligonucleotide mutagenesis methods [71] to generate HP VN/1203/E. An additional mutation was generated in both VN/1203/K and VN/1203/E by deleting 4 basic amino acids in the HA cleavage site. These LP viruses are referred to as LP VN/1203/K and LP VN/1203/E, respectively. All viruses were propagated by two passages in 10-day-old embryonated specific-pathogen-free chicken eggs and stored at −80°C until use. Virus stocks were titrated by endpoint dilution to determine the EID50. Following propagation, the full genomes of rescued viruses were sequenced to confirm the presence of the appropriate wild type or mutant sequence.
Birds were inoculated by placing one or two drops of virus in each eye and nostril, and instilling the remainder into the oral cavity. Virus titers were confirmed by back titration in 9- to 10-day-old embryonated chicken eggs.
Pharyngeal and cloacal swabs were collected into 2 mL of phosphate-buffered saline containing penicillin, streptomycin and gentamycin. After euthanasia, fragments of spleen, brain, lung and heart tissues were frozen at −80°C until processing for virus isolation. For some experiments, small amounts of 1.0 mm silicon carbide beads (Daintree Scientific, St Helens, Tasmania, Australia) were added to the collection tubes with phosphate-buffered saline.
Thrombocytes were prepared as described by Scott and Owens [39]. Cells were resuspended in 0.2 ml of HBSS and stored at −80°C until use. The dilution factor of plasma in the final cell preparation was estimated to be between 5 and 6 log10.
Tissue samples were ground by mortar and pestle using sterile sand or pulverized using a MP Fast-Prep24 instrument (MP Biomedicals, Inc, Solon, OH) when collected with silicon carbide beads and diluted in PBS to 10% w/v homogenates. Virus isolations for the transmission experiment in chickens were performed by inoculating 10-day-old embryonated chicken eggs. Allantoic fluids from all eggs were examined for the presence of HA using chicken red blood cells [72]. Tissue extracts and fluids from swabs obtained from birds inoculated with HP AIV virus were titrated directly in Vero cells as described [23] except that low dilutions of samples causing non-specific cytopathic effects (CPE) in cell culture were tested for HA if higher dilutions did not show specific CPE. The limit of detection of HPAI virus in Vero cells was 1.25log10 TCID50/mL.
Blood samples were obtained from all experimental birds prior to challenge and from survivors at termination. Sera were stored at −20°C until used for the hemagglutination inhibition test [72].
Tissues were collected and processed for histology and immunohistochemistry as described by Bingham et al. [23].
Total RNA was extracted from animal tissues using an RNA isolation kit (RNEasy, Qiagen, Doncaster, Australia) according to the manufacturer's instructions. A viral RNA extraction kit, QIAamp Viral RNA Mini Kit, (Qiagen) was used to extract RNA from oral and cloacal swabs according to the manufacturer's instructions. RT-PCR of the HA cleavage site was conducted using the oligonucleotide primers Fwd 5′-TGTCAAGAAAGGGGACTC-3′ and Rev 5′-CTTTGTCTGCAGCGTACC-3′. RT-PCR of the PB2 gene was conducted using the primers Fwd 5′-CCAAAGATTAAACCCCATGCATCA-3′ and Rev 5′-ACATATGGTCTCGTATTAGTAGAAACAAGGTCGTTT-3′ All samples were subjected to electrophoresis and amplicons were visualised and photographed (Kodak Digital Science, Rochester, NY).
PCR products were purified using a QIAquick® PCR Purification Kit (Qiagen, Germany) according to the manufacturer's instructions. DNA sequencing was performed by the sequencing facility at CSIRO-AAHL using an ABI Prism 377 automated DNA sequencer (Applied Biosystems) and the oligonucleotide primers shown above. Nucleotide sequence data and the translated amino acid sequence were subjected to sequence alignments using clone manager 9 (Version 9.0, Scientific and Educational Software).
Real-time RT-PCR to verify the presence of H5N1 was performed to determine if virus was replicating in the thrombocytes, according to the method of Heine et al. [73], using the modifications described below. RNA was isolated from the same samples analyzed in the microarray assay, using the Meridian total RNA isolation kit (Cartagen, Seattle, WA). RNA was converted to cDNA using the Quantitect reverse transcription kit (Qiagen), in two separate reverse transcription reactions with either the forward primer AGCAAAAGCAGGTAGATGTTGAA which anneals to viral HA genomic RNA, or the reverse primer AGTAGAAACAAGGTAGTTTTTTACTCC which anneals to HA cRNA. Following reverse transcription each cDNA sample was subjected to real-time PCR to verify the presence of H5N1 viral RNA and cRNA as described [73]. The thermal cycling parameters consisted of an initial Taq polymerase hot start activation at 95°C for 10 min, followed by 45 cycles of 15 sec at 94°C and 1 min at 60°C. Assays were performed on an AB7900HT real time PCR system (Applied Biosystems) and analyzed using the associated sequence detection software version 2.3 (Applied Biosystems).
Total RNA for all samples was isolated using the Meridian total RNA isolation kit (Cartagen, Seattle, WA). One microgram of total RNA was reverse transcribed into cDNA and indirectly labeled with Cy3 using the NimbleGen One-Color DNA labeling kit (Roche, Basel, Switzerland). Each Cy3 labeled cell culture sample was individually hybridized to the NimbleGen whole genome chicken array (designed in-house) and hybridized according to manufactures instructions. The custom array design has been deposited into ArrayExpress (accession number A-MEXP-2133) and all data is MIAME compliant and has been deposited in ArrayExpress (accession number E-MEXP-3432). Arrays were scanned at 2.0 microns on a NimbleGen MS 200 scanner. Raw data were extracted using NimbleScan software v2.1 (Roche). All arrays were subjected to quality control measures before being included in the analysis. The signal intensities from all arrays were normalized using Robust Multiple-chip Analysis (RMA) [74]. Subsequent statistical tests were carried out using Genespring version 7.2 (Silicon Genetics, Santa Clara, CA) to determine all genes differentially regulated at a false discovery rate (FDR) of p=0.05. Pathway analysis was conducted using Genowiz Version 220.127.116.11 (Ocimum Biosciences, Gaithersburg, MD).
To determine pathology, viral shedding and transmission, four-week-old broilers or 5-week-old ducks were infected with HP VN/1203/K or HP VN/1203/E. One day later age-matched uninfected chickens or ducks were added to each infected group. Oral and cloacal swabs were collected daily from all birds. Birds were euthanized prior to the development of severe clinical illness and tissue and blood samples were collected for virus isolation, immunohistochemistry and histology. Sera were collected from all surviving birds at the trial termination, 14 days pi.
To study the early development of infection and disease, groups of chickens and ducks were inoculated with HP VN/1203/K and HP VN/1203/E and individual birds were euthanased and sampled at defined time intervals. Chickens were euthanized at 6, 12, 18 and 24 hours pi. Infected ducks were euthanized at 1, 2, 3, 4 and 6 days pi. At each time point, oral and cloacal swabs, spleen, lung and brain tissues and thrombocytes were collected for virus isolation. In addition, tissues were collected for immunohistochemistry and histopathology. Uninfected control birds from the same batch were euthanized at the start of the experiments (chickens) or towards the end (ducks).
Because both of these HP viruses caused disease and or mortality in chickens within 24 hrs, we used LP VN/1203/K and LP VN/1203/E to determine if the presence of E versus K at PB2-627 influences the pathogenicity of infection in chickens. In a pilot trial, infected chickens were observed for 14 days pi to confirm that these viruses were low pathogenicity. Oral swabs were collected from these chickens at 6 days pi and analyzed by real-time RT-PCR. Surviving birds were bled at 14 days pi for serum collection and euthanized. Samples from different organs were collected for immunohistochemistry and histopathology. To further study the pathogenesis of infection with both low pathogenic viruses, chickens were infected and euthanized at 1, 2, 3, 4 and 5 days pi and samples were collected for virus isolation, immunohistochemistry and histology. Uninfected control birds from the same batch were euthanized simultaneously with infected chickens at 1 and 5 days pi.
Where appropriate, the two-tailed Fisher's exact test was used to determine whether numerical differences between virus isolation rates, pathology and immunohistochemistry are significant. Significant differences for all tests were declared at P<0.05.
We thank the National Institute of Hygiene and Epidemiology, Hanoi, Ministry of Health, Vietnam, for providing virus samples. We thank Dayna Johnson, Tim Hancock, Jessica Klippel and Rachel Robinson for excellent care of the experimental animals, and Jean Payne, Manabu Yamada, Jenni Rookes, Diane Green, and Gail Russell for conducting histological procedures. The views expressed are those of the authors and do not reflect the policies of the Centers for Disease Control and Prevention, Atlanta, Georgia USA.
Competing Interests: The authors have declared that no competing interests exist.
Funding: Funding was provided by the Commonwealth Scientific and Industrial Research Organisation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text:
Standard maintenance requires your septic tank be pumped once every 2-3 years. Not doing so will result in sludge particles overflowing on your lawn.
Welcome to Pro Rooter & Septic
We offer free estimates and same day service to everyone in the Central Valley. We're licensed, bonded and insured for all plumbing matters residential and commercial. If you're experiencing a plumbing emergency please don't hesitate to call.
Family owned and operated for 30+ years.
Pro Rooter & Septic is a family owned and locally based company. Our first priority is making sure every customer has a great experience from start to finish. We're available 24 hours a day, 7 days a week and respond to every call quickly and efficiently to save you time and money. Save 10% when you mention what you like about our Facebook, Twitter or website page. Pro Rooter & Septic is prepared to handle all of your plumbing issues. Our skilled technicians strive to maintain their reputation for high quality workmanship and customer service. It is the goal of each technician to build a positive relationship with each customer, in hopes that the customer will continue to choose the technician as their go to Plumber. Fresno residents look no further Pro Rooter & Septic for all of your plumbing issues and concerns with your present plumbing system. Our professional plumbing technicians can manage any and all plumbing services including: remodeling or additions to a bathroom, drain lines, repair or installation of a gas line, remodeling a kitchen, rooter service, repairs and installation of a water heater, in addition to many other services. At Pro Rooter & Septic we understand that your time is very valuable, which is why our professional plumbers are available to assist you with any necessary plumbing service 24 hours a day, 7 days a week.
For years our company has maintained a reputation for being the premier Plumbers in Fresno. We are known for high quality plumbing services that ensure 100% customer satisfaction. If you are in need of emergency plumbing repairs, installations, or replacements we can do it all. Our plumbing services are available to both commercial and residential locations. We dispatch only skilled and experienced plumbing technicians to your home to resolve your plumbing issues, such as leaks, pipe and drain cleaning, repairs for tankless water heaters and installation of fixtures. No matter whether you choose a conventional tank unit, or upgrade to a tankless model, you can take careful precautions to get the most out of your hot water usage and reduce the amount of your monthly water bill. By lowering the thermostat on your water heater to 120 degrees you can assist in the prevention of scalding and additional heating costs. By calling the reliable and professionals at Pro Rooter & Septic, you can guarantee that they will deliver the quality repairs, installations, and replacement services that Fresno residents have come to expect. If you suspect any issues regarding your home's plumbing don't wait for the issue to reach a point where you will be required to pay the high costs for additional and unnecessary repairs. Our team of professionally skilled plumbing technicians offer homeowners and commercial business owners a large variety of plumbing services that go beyond the scope of just emergency.
If you're experiencing recurring clogs, your main drainage system may be damaged. It can also be a sign of old age or root intrusion.
Sewage (wastewater) can be very harmful to the environment and create a health risk for animals and humans. It is very important to keep your sewer system in good working order.
Is your toilet leaking, backed up or overflowing? You name it, we can fix it, from your water valves to your vacuum breaker and bowls themselves.
Faucets are needed for almost every plumbing appliance in the average home. We can repair and/or replace any faucet regardless if it's inside, outside, sink or shower.
A grease trap or interceptor is the device between drain lines and sanitary sewers. This trap is designed to intercept fats, oils and grease from municipal sewer systems.
Pro Rooter is equipped to handle any garbage disposal trouble you have in your home or commercial property. From installation to repair, we can solve your issue quickly.
This service essentially requires the removal and replacement of any and all hot and cold water pipes currently installed in your home or office.
Gas leaks usually occur from wear and tear, swapping out flexible gas connector or recent work done by someone not so experienced. Either way, we can help.
If you are noticing a strange odor, loss in water pressure or water hardness we suggest contacting us for a free estimate.
If you're unable to fix a clog hydro jetting is a great alternative. When snaking your line just won't cut it, call us and we'll bring the big guns.
Please take a minute to view our work.View Full Portfolio
Sewer Projects
Leach Line...
Septic Gallery
Plumbing Equipment
We are available 24 hours per day, 7 days per week, including holidays to correct any plumbing issues.
Pro Rooter & Septic is a family owned and operated company. We have been in the plumbing and septic industry for more than 30 years. Pro Rooter & Septic has locations throughout California's Central Valley, making it easy for us to quickly respond to any call and provide around the clock emergency service. Our first priority is making sure every customer has a great experience from start to finish. We are licensed, insured and to correct any plumbing or septic issue ranging from clogged drains to broken pipes and septic tank backflow. We're available 24 hours a day, 7 days a week. Our professional plumbing, septic and drain cleaning technicians are experienced and trained to efficiently address any residential, commercial, municipal, industrial or agricultural issue you're experiencing. Call us for a quote or a free estimate today.
" When sludge started coming from our shower drain we called Pro Rooter & Septic immediately. A friendly plumber showed up 30 minutes later and pumped our septic tank. The entire process was less than 2 hours. "
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We love our customers as much as they love us. Call today and find out why we're your neighbors "go to" plumber in the Central Valley.
We provide service to many county districts.

subset: commoncrawl


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COLUMBIA, S.C. -- Hello, from Williams-Brice Stadium. It feels like my home away from home.
You know who else feels like that? Auburn. The Tigers strolled, check that, strutted onto the field and walked right through South Carolina's warmups. Leading the charge was running back Michael Dyer, who was singing along to some song buzzing in his earphones.
A few Tigers walked all the way to the goal posts and started hollering something. It seemed to be all in fun and most of the Gamecocks players didn't pay them any mind, but Auburn is looking to get some sort of edge before game time.
Dyer should be a major focal point of the South Carolina defense and he wanted to make sure those players got a good look at him before kickoff.
South Carolina will be honoring the Wounded Warrior Project's "Believe in Heroes" campaign with their custom Under Armour uniforms. The uniforms feature sweet camo design on the jerseys and pants. The last names of the players on the backs of the jerseys will be replaced by the "core value" embellishment – Duty, Honor, Courage, Commitment, Integrity, Country, or Service.
It is an absolutely gorgeous day here. It's not too hot or too cold. It's just about perfect.


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text:
Canberra Rex Hotel & Serviced Apartments - Braddon
- Bar/Lounge
- Dining venue
A restaurant and free self parking are featured at this hotel along with dry cleaning/laundry. Go for a dip in the indoor pool during your stay. The bar/lounge is a perfect place to have a drink.
Knowledgeable staff are available at the front desk 24 hours-a-day and can provide tour/ticket assistance. Free WiFi is provided.
Canberra Rex Hotel & Serviced Apartments - Braddon
Hotel Info: 1-877-214-4288from $131
- Studio Suite
- Standard Room, 1 Queen Bed
- Easy Access Room
- Spa Suite With Balcony
- King Room
- Summer Deal!
- Twin Room
- Deluxe Double Room, 1 King Bed
- Triple Room
- Apartment, 2 Bedrooms, 2 Bathrooms
- Family Room
- Deluxe Twin Room, 2 Single Beds
- King Suite
- Apartment, 2 Bedrooms
- Apartment, 1 Bedroom
- King Room with Balcony
- Crown Suite
- Sauna Suite
- Steam Suite
Also popular with travelers from the USA
At a glance
Key facts
Hotel size
- This hotel has 149 rooms
- This hotel is arranged over 4 floors
- Check-in time starts at 2 PM
- Check-out time is 10 AM
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
- Minimum check-in age is 18
Travelling with others
- Pets not allowed (service dogs only)
- Free WiFi in public areas
- Free WiFi in rooms
- Free self parking (limited)
Other information
- Smoke-free property
- Resort fee included
In the hotel
- Restaurant
- Bar/lounge
- Room service (during limited hours)
- Indoor pool
- Free use of nearby fitness center
- Conference space
- Conference center
- 24-hour front desk
- Tours/ticket assistance
- Dry cleaning/laundry service
- Laundry facilities
- Free newspapers in lobby
- Luggage storage
- Wedding services
- Year Built 1960
- Elevator/lift
- Safe-deposit box at front desk
- Accessible bathroom
- In-room accessibility
- Roll-in shower
In the room
- Air conditioning
- Iron/ironing board
- Private bathroom
- Shower only
- Free toiletries
- Hair dryer
- Free WiFi
- Daily housekeeping
Special features
DiningMillies Restaurant - This restaurant serves breakfast and dinner. Guests can enjoy drinks at the bar. Reservations are required. Open select days.
Canberra Rex Hotel & Serviced Apartments - Braddon's small print
Also known as
- Canberra Hotel Rex
- Canberra Rex Hotel Serviced Apartments
- Rex Hotel Serviced Apartments
- Canberra Rex Serviced
- Rex Serviced
- Canberra Rex
- Canberra Rex Hotel
- Hotel Rex
- Hotel Rex Canberra
- Rex Canberra
- Rex Canberra Hotel
- Rex Hotel
- Rex Hotel Canberra
The name on the credit card used at check-in to pay for incidentals must be the primary name on the guestroom reservation.
Mandatory fees
Cash deposit: AUD 150.00 per stay
The charges below are included in the total price displayed:
- Resort fee
Optional extras
Late check-out can be arranged for an extra charge of AUD 60.00
Rollaway beds are available for AUD 30.00 per day
Cribs/Infant beds are available for AUD 15.00 per day
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.
Recent reviews
Good 3.6 /5 from 300 reviews
Check in was great, very friendly staff. upon checking into the room it was found all power points on one wall did not work, this included the tv, fridge,kettle. Contacted reception and the manager on duty came to investigate.There were major renovations to the hotel at the time and this was the reason only some of the power was working in the room. The manager kindly offered to upgrade the room to an excutive room from the deluxe room I had booked which I was extremly happy with and more than compensated for the problems with the first room. I highly recomend the excutive rooms much nicer than a deluxe room. Well done to the staff for rectifying this one and only problem I had at Canberra Rex Hotel. The buffet breakfast was excellent!
It was value for money - but there are a few issues that make me understand why the cost is low. e.g. You need to be quite flexible to use the shower (or be under 5 foot tall) The combination of bed and pillow left me with a neck ache for the next couple of days - which is unusual as i've normally been fine with a variety of different hotels beds. In all, there was nothing particularly bad about the hotel but it wouldn't be my first preference if there were alternatives.
The bed was very comfortable, but decor and bathroom needed updating. Renovations going on at the moment. Works were underway on the floor above us, sounded like a jackhammer, an incredibly loud noise starting at 7:30am! We received a letter in our room on day 2 of our 4 day stay, informing us there would be noise and that we could be moved to a room further away from it if we wished. Why we weren't put in another room at the start, I am not sure. We should have been informed of this situation at check-in. There were two of us, but only one breakfast was included. There was no mention of this on the website as far as I remember. Parking was an issue on Saturday night, there was ONE space available underneath the apartment side of the hotel. But again, nothing was explained how to get to it at check-in or that if no parks were available that you would have to park at Fenner Hall across the road. The restaurant staff were very nice at breakfast. A nice place for coffee/dining is Lonsdale Street...walking distance from the hotel.
Staff very friendly. Experience was as we expected. Would stay again.
beds were too small


text:
Perhaps I should have checked the archives, but I'll ask anyway.
Is there really any adverse effect on the navigational systems or accidental triggering of warning mechanisms if some one were to use their phone?
(I once heard someone say that airlines tell passengers that to merely get them to use the air phones but I have to really know.)


text:
Users offer outsourcing services with ASP deal
Application service provider Esoft Global has launched a package to allow its users to offer outsourcing services themselves.
Accountancy firm Redwood...
Application service provider Esoft Global has launched a package to allow its users to offer outsourcing services themselves.
Accountancy firm Redwood used the Esoft service to set up a new outsourcing division for small- and medium-sized businesses. The Web-based bureau service covers financial and management accounts and is based on a financial application from OpenAccounts.
Your exclusive guide to CIO trends
A collection of our most popular articles for IT leaders from the first few months of 2016, including: - Corporate giants recruit digitally-minded outsiders to drive transformation - Analytics platforms to drive strategy in 2016 - Next generation: The changing role of IT leaders.
By submitting your email address, you agree to receive emails regarding relevant topic offers from TechTarget and its partners. You can withdraw your consent at any time. Contact TechTarget at 275 Grove Street, Newton, MA.
You also agree that your personal information may be transferred and processed in the United States, and that you have read and agree to the Terms of Use and the Privacy Policy.
Redwood can run a firm's entire accounts department or offer support for business planning. Customers simply need a PC or thin client with an internet connection.
Redwood's outsourcing service is managed remotely from a central datacentre by Esoft and OpenAccounts as part of a $7.5m (£5.35m) agreement between the two suppliers. Esoft claimed the service can be set up within three days.
Redwood's managing director Graham Hoggard said: "Our aim is to persuade clients to outsource the whole of their financial function for a fraction of the cost required to employ a financial director and their back-up team."
Prices for the service range from £75 to £300 per user, depending on the range of desktop features, such as Microsoft Exchange, offered with the service.
Esoft already has 30 alliances with software suppliers, including financials supplier Great Plains and customer relationship management supplier Epicor. Esoft is now keen to target larger companies, particularly in the telecoms sector.
Enjoy the benefits of CW+ membership, learn more and join.


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text:
Motel 6 Manhattan, Manhattan
- Free parking
Main amenities
- 87 smoke-free guestrooms
- Outdoor pool
- WiFi in the lobby
- 24-hour front desk
- Coffee/tea in a common area
- Air conditioning
- Daily housekeeping
- Self-serve laundry
For families
- Private bathroom
- Television
- Daily housekeeping
- Laundry facilities
- In-room climate control (air conditioning)
- Cable TV service
What's around
- Flint Hills Discovery Center (0.6 mi / 0.9 km)
- Memorial Stadium (1.7 mi / 2.7 km)
- Kansas State University (1.7 mi / 2.7 km)
- Ahearn Field House (1.7 mi / 2.7 km)
- Sunset Zoological Park (2.4 mi / 3.9 km)
- Manhattan, KS (MHK-Manhattan Regional) 17-min drive; 9.8 miles/15.7 km
- Free self parking
- RV, bus, and truck parking
Motel 6 Manhattan, Manhattan
Hotel Info: 020 3027 9803from £35
- Standard Room, 2 Double Beds, Non Smoking, Refrigerator & Microwave
- Standard Room, 1 Double Bed, Accessible, Non Smoking (Roll-In Shower)
- Standard Room, 1 Queen Bed, Non Smoking
- Standard Room, 1 Queen Bed, Non Smoking, Refrigerator & Microwave
- Standard Room, 2 Double Beds, Non Smoking
Also popular with travellers from the UK
At a glance
Key facts
Hotel size
- This hotel has 87 rooms
- This hotel is arranged over 2 floors
- Check-in time starts at 3 PM
- Check-out time is noon
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
- Minimum check-in age is 18
Travelling with others
- Children (17 years old and younger) stay free when occupying the parent or guardian's room, using existing bedding
- Pets stay free
- 2 per room
- WiFi in public areas *
- WiFi in rooms *
- Free self parking
- RV/bus/truck parking
Other information
- Smoke-free property
In the hotel
- Coffee/tea in a common area
- Outdoor pool
- 24-hour front desk
- Laundry facilities
- Number of buildings/towers - 3
- Designated smoking areas
- In-room accessibility
- Roll-in shower
In the room
- In-room climate control (air conditioning)
- Air conditioning
- Private bathroom
- Cable TV channels
- WiFi (surcharge)
- Free local calls
- Daily housekeeping
- Access via exterior corridors
Motel 6 Manhattan, Manhattan's small print
Also known as
- Motel 6 Manhattan
- 6 Manhattan
- Manhattan Motel Six
- Motel Six Manhattan
- Motel Manhattan
- Motel 6 Manhattan Hotel Manhattan
- Manhattan Motel 6
This property does not have elevators.
Optional extras
WiFi is available in guestrooms for USD 2.99 for 24 hours (rates may vary)
WiFi is available in public areas for USD 2.99 per night (rates may vary)
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.
Recent reviews
2.4 /5 from 101 reviews
Very poorly maintained motel. Floor boards in bathroom need to be replaced, bathroom door too damaged to close, mattress was in really bad shape, unable to lock the window (room was on ground floor).
It was fine. You get what you pay for. We didn't do much but sleep in the hotel room so it worked for that. The bedding was questionable (we brought our own). The shower was poor, no water pressure, very little heat, curtain was questionable. I did appreciate that the room wasn't carpeted, that made me feel like it was a little more clean.
Room was good except that we were emailed us we would be on a ground floor, I told them we would except two beds to avoid stairs, instead they said they couldn't do it and we had the King size room with about 15 steps and no bannister, I walked to the front of the hotel where there was a bannister each day, balance is an issue and I use a cane, very dangerous situation if my husband hadn't been with me.
Not happy since when I tried to pay cash for the room, the amount was charged to my daughters card who reserved the room. Tried to fix it, but agent said it could not be switched to be able to pay cash, it is already on your daughters credit card. I called previous to our stay and the agent told me that would not be a problem, my card would not be charged. Obviously that person was wrong or the person who checked in my parents was not qualified to check a person in adequately!
Great value. Clean room.
Is the description of this hotel not correct? Tell us


text:
Get an overview of this hotelBed & breakfast in Collinas with free breakfast and outdoor pool
Situated in a rural location, this bed & breakfast is within 1 mi (2 km) of Genna Maria Archaeological Area and Genna Maria Archaeological Museum. Sa Corona Arrubia Museum is 3 mi (4.9 km) away.
Property Features
Along with an outdoor pool, this bed & breakfast has meeting rooms and a 24-hour front desk. Free continental breakfast and free WiFi in public areas are also provided. Additionally, an arcade/game room, a library, and express check-in are onsite.
Room Amenities
All 3 rooms feature comforts like Frette Italian sheets and down comforters, while conveniences include living rooms and dining areas. Other amenities available to guests include free WiFi, furnished balconies, and private yards.Information missing or incorrect? Tell us!Opens in a new window
Hotel Amenities
Hotel Amenities
A complimentary breakfast is offered each morning. High-speed wireless Internet access is complimentary. This Collinas bed & breakfast also offers an outdoor pool, a library, and a terrace.
Villa Serena has designated areas for smoking.
- Luggage storage
- Beach umbrellas
- Free RV, bus, truck parking
- Express check-in
- Designated smoking areas
- Fireplace in lobby
- Arcade/game room
- Free newspapers in lobby
- Picnic area
- Express check-out
- 24-hour front desk
- Total number of rooms - 3
- Number of floors - 2
- Barbecue grill(s)
- Free breakfast
- Front desk (limited hours)
- Tours/ticket assistance
- Free WiFi
- Library
- Meeting rooms
- Garden
- Terrace
- Safe-deposit box at front desk
- Number of meeting rooms - 1
- Free long-term parking
- Number of buildings/towers - 1
- Outdoor pool
Family Friendly Amenities
- Family Hotel
- Outdoor pool
- Separate living room
- Free Wi-Fi
- Arcade/game room
- Playground on site
- Separate dining area
Available in all rooms: Free WiFi
Available in some public areas: Free WiFi
Free long-term parking, Free RV, bus, truck parking
Room Amenities
- Free newspaper
- Coffee/tea maker
- Daily housekeeping
- Private bathroom
- Yard
- Separate sitting area
- Free bottled water
- Bidet
- Shower only
- Separate dining area
- Down comforter
- Furnished balcony
- Hair dryer
- Iron/ironing board (on request)
- Separate living room
- Free WiFi
- Frette Italian sheets
Where to Eat
A complimentary continental breakfast is served each morning between 7:30 AM and 9:00 AM.
Nearby Things to Do
The recreational activities listed below are available either on site or nearby; fees may apply.
- Playground on site
Hotel Policies
Check-in time starts at 1 PM
Check-in time ends at 10:00 PM
Check-out time is 2:30 PM
- Pets stay free
You need to know
Extra-person charges may apply and vary depending on hotel policy.
Government-issued photo identification and a cash deposit are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
Cash Deposit Required
Pets allowed (no fees)
- Resort fee
Hotel Name
- Villa Serena B&B
- Villa Serena B&B Collinas
- Villa Serena Collinas
We should mention
All guests, including children, must be present at check-in and show their government-issued photo ID card or passport. Cash transactions at this property cannot exceed EUR 2999.99, due to national regulations. For further details, please contact the property using information in the booking confirmation. A resort fee is included in the total price displayed. Children 3 years old and younger stay free when occupying the parent or guardian's room, using existing bedding.


text:
15 Parks near Edmond, Oklahoma
Guthrie, Oklahoma 9.33 miles NE
The Village, Oklahoma 12.75 miles SW
Nichols Hills, Oklahoma 13.37 miles SW
Spencer, Oklahoma 15.86 miles SE
Edmond, Oklahoma (map) | (405) 216-7470
Sites: 50 | Latest Rate: $29.00 | 16 Reviews
|Clean Restrooms:
|Clean Showers:
Apr 2016 - $29.00
This park is hard to rate. The pull thru is FHU. The lake is nice. The playground is too. Its only 5 or 6 miles north of rt. 44. The back in sights are nice. The pull thru hookups were convenient. The downside is that the pull thrus ate uphill. If you hit it just right, you are relatively level but if it rains, you can see where mud runs down through the sight. If you miss the right turn for the pull thru road you have to go around and.drive it again. The road is narrow. I might look elsewhere next time although this park provided everything we needed. - Tammy7
Jul 2015 - $20.00
We came upon Arcadia lake upon the suggestion of another RV park that was full, which turned out to be a good thing. This is a beautiful park with well paved roads and a beautiful lake. The staff was extremely helpful and courteous. Park was exceptionally clean and the bathrooms were also well maintained. The showers were a perfect temperature. We moved to a lakeside spot after everyone left on Monday and paid $19 for the premium spot. Like others have said, the only downfall is the lack of hydrants. We filled ours tank before going in so it was not a problem. - PaleRider
Oct 2014 - $18.00
Hidden secret in the metropolitan area North of Oklahoma City. There are two parks on this lake, and someone at the park staff said that if they were camping, they would use the Nothern part first, which I believe is off of 15th ave. The Freeway offramp, on old route 66, is tricky to find. Exit too soon and you will have 20 minutes through a round about road construction to find your way back, so plot your course carefully. Beautifully wooded sites with large pull throughs, and some that have a lake view. You will experience large AWAC Air Force Planes coming over the park on their way to land at Tinker Air Force Base in Oklahoma City but the air traffic stopped about 10:30 in the evening. They seemed to be pretty low. At the electric sites, the water was extremely far away and I noticed some of the campers had very long hoses in which to connect up, so be advised. Very peaceful for being so close to a major city and we would camp here again. We did not have sewer hookups but a nice dump station was available on the way out. Some sites not level but ours was great with lots of elbow room. - elduque
Guthrie, Oklahoma (map) | (405) 282-3557
Sites: 63 | Latest Rate: $30.00 | 12 Reviews
|Clean Restrooms:
|Clean Showers:
Jul 2016 - $30.00
This park is easy to access from the highway. Just 5 miles south of Guthrie, it is convenient and reasonable. Not a lot of amenities (no cable, pool, Wi-Fi) but the park is quiet and sites are fairly large so you don't feel like you are right on top of your neighbor. Roads in the park are potholed, but they were working on them when we left. Would stay again. - MbMurchison
Apr 2013 - $30.00
Park is basic, but clean. Unfortunately it had rained and the RV sites in the new area were very muddy. Everything worked as it should. It's OK for an over night stop. - MovinOnDownTheRoad
Sep 2012 - $1.00
My husband has stayed in several RV parks over the years. I have been to some of them with him. This park is basic, but very clean. We had no problems with the facilities or the management. The lady who runs the park was always very friendly and helped us anytime we needed it. Laundry facilities were clean. Trash is always picked up. There is a mini convenience store on site that sells basics as well as hot pizza. I stayed there quite a bit and have no complaints...and I am picky! The location is not in the middle of town so there is privacy. You are close to shopping and lakes. I would recommend this park to someone looking for a decent place for a decent rate...we paid $380 for the month. - PipelinerWife
Guthrie, Oklahoma (map) | (405) 282-4478
Sites: 71 | Latest Rate: $18.00 | 62 Reviews
|Clean Restrooms:
|Clean Showers:
Jul 2016 - $18.00
Once again we were so happy to stay at Cedar Valley. We always call ahead and reserve a spot as we are traveling through Oklahoma. This RV park is about 6 miles off of I35, but is quiet, safe and extremely clean. Nothing negative to say. We are very pleased and will stay here every chance we get. - LoboFromTX
Apr 2016 - $18.00
Probably only 15 sites available as it is full of workers. That being said the workers must be making good money because they have some very nice rigs. The only problem is the diesel noise at 5 to 6 in the morning especially when they let it run for a half hour. Price is right but no picnic table. - russ1102
Jan 2016 - $18.00
We had PA rate and ok for what we paid. Shower rooms clean and adequate. Had laundry available as well. Park seemed a bit run down. Lots of longer term residents here - local construction perhaps. We had a pull through up near the office. Rows of cedar trees have been trimmed up to allow more room to maneuver but could be a bit tight for rigs larger than 40'. Concrete pads are a bit too narrow. Wouldn't have paid full price to stay here but worth it for $18 as we hadn't yet de-winterized our rig and needed showers and laundry. - Nautical Wheelers
12115 N I-35 Service Rd Oklahoma City, Oklahoma 73131 (map) | (405) 478-0278
Sites: 80 | Latest Rate: $35.00 | 12 Reviews
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|Clean Showers:
Jun 2016 - $35.00
Park is very close to the freeway, and our space was the first one as you pull in. Needless to say, it wasn't a very quite stay. Also, their website says they give an AARP discount. When asked for the discount, we were told...it's $35. I guess because there was NCAA Softball in town, they weren't honoring their posted discounts. It's a shame, because this park is NOT worth $35/night. Regardless, we'll be reporting them to AARP as fraudulent advertisement. To their credit, they did provide a 30A/50A adapter. - DesignoSLK
Mar 2016 - $33.00
Dated facilites. Messy grounds with abandoned limo, rvs, cars, etc scattered about. Pads are slopped and cracked...hard to level the MH. Mostly full-timers with 2-3 vehicles per site. - foxrose
Nov 2015 - $33.00
Was initially disappointed that none of the pull thrus close to the front of the park were available but the back-in site assigned far back in the park had its advantages. Nice concrete pad. Our site was not underneath any trees. Farther from road noise. Water, electric, wifi worked good. No picnic table. No fenced dog park. Dogs picked up quite a few burrs when being walked. A few unleashed dogs in the back of the park. Biggest problem being so far back in the park was that the dumpsters, laundry and rest rooms are all far away in the front of the park. - Markk
Guthrie, Oklahoma (map) | (405) 282-8831
Sites: 10 | Latest Rate: $35.00 | 5 Reviews
|Clean Restrooms:
|Clean Showers:
May 2015 - $35.00
The one pro is that they recently mowed and there weren't any grass burs. The rates have gone up to $35 and all you get is W/E/S. My 35' RV hit the curb getting in to the narrow site. I agree with the previous reviews. Expensive for an overnight stay. Probably will just overnight in the a parking lot in Edmond if in the area again. - ETX
Jul 2012 - $30.00
Lots of sticker-burrs. Picking them out of clothes and dogs for a month. Stayed there 2 nights because it is owned by a good friend of a relative of mine. Old. Bare sites. Concrete pads. Almost had a disaster. Left two dogs in RV with a/c's on. 107' outside. Came back in two hours and no power. 50 amp kicked off. Dogs were hurting bad. Turns out it was an old CB that would not reset. We took cord to other side and was ok. Be careful if you stay here. - charlie mike
Aug 2009 - $27.00
This is an okay stop for overnight. There are only 10 sites and they operate on a first come first serve basis. The pads are narrow and it is difficult to stay on the concrete and get the slides out without hitting the utilities on the left side. The space between sites is generous. There is good WI-FI, and lots of TV channels. The pool seems nice, however, we didn't use it. There is a free continental breakfast in the morning. - Kastle
Oklahoma City, Oklahoma (map) | (405) 475-5514
Sites: 152 | Latest Rate: $44.00 | 224 Reviews
|Clean Restrooms:
|Clean Showers:
Sep 2016 - $44.00
Very attractive layout. Wide spacing between sites. Good pool and spa and large store. Problems are with basic services. Wifi is terrible and bathrooms dirty and not well maintained. Only 2 showers for each sex and one shower in each is broken. Pet friendly and plenty of places to walk.
Would not stay here again. - Satisfying Retirement
Sep 2016 - $50.00
I was somewhat disappointed in this CG. I was impressed when I pulled in but then ... Wi-fi was down. My site was a pull-thru cement pad, but a bit steep before it leveled off. The concrete was covered in dried mud from a storm the week before with a lot of ants. I washed the mud from the area around my trailer so that I wasn't tracking the red dirt into my rig. They trimmed the grass on the second day of my stay but no one bothered to clean up the remaining dirt. I hooked up to their cable but the signal was fuzzy. Over the air stations were much better and they were all in HD. One expects more when paying premium prices for a site, but this place doesn't deliver. Laundry was inexpensive at $1.50 wash and the same to dry. - jnateg
Aug 2016 - $49.00
We stay here visiting family. This is a nice clean park. Staff are very nice and helpful. There are a good number of long term residence but they are aligned along the perimeter of the park and all are well kept and orderly. Sites are concrete except the perimeter sites and some of the new sites that have just been added which are gravel. There are a couple of sites that although are concrete are quite unLevel front to rear. Wi-fi is available but comes and goes. I know others use it but we don't so it does not effect my rating nor do rest rooms but I will leave comments on them. This park is a short distance off I-35 and I-44 runs along the North side of the park. There is some noise but it has never bothered us. They have a swimming pool which is well maintained and a miniature golf course. Our grandkids have spent the night with us there and there was no additional charge for them. Most sites are pull through and most utility connections are along the center line of the trailers. This is a comfortable and secure feeling park. - BWS
Buncombe Creek, Oklahoma (map) | (580) 564-2901
Sites: 54 | Latest Rate: $15.00 | 1 Reviews
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|Clean Showers:
Sep 2005 - $15.00
One of several Corps of Engineer campgrounds on Lake Texoma. Off the beaten path so it is more secluded and quieter than the more developed parks. Some of the waterfront sites are elevated above the lake with nice views. All sites are heavily wooded with lots of shade. Large gravel pads with picnic tables and fire rings. No sewer hookups but there is a dump station. Showers are pretty basic but clean. Some sites have a very long water run so bring an extra hose. Short hiking trail and group picnic pavillion. No developed swimming area, but two boat ramps and boats can be moored at waterfront sites. - rscraig
Oklahoma City, Oklahoma (map) | (405) 787-5992
Sites: 126 | Latest Rate: $40.00 | 109 Reviews
|Clean Restrooms:
|Clean Showers:
Jul 2016 - $40.00
Our one night stay turned into 5 days due to RV issues. Wonderful place to get stuck! Quiet park. Level gravel sites, great dog walking area. Indoor pool, weight room and beauty salon onsite. Clean laundry facilities. The campground also holds church services in their community room on Sunday morning. The staff is helpful and friendly, with great advice on local sightseeing. Easy access on/off highway. Close to restaurant, shopping, sightseeing. Would definitely stay again! - MbMurchison
Jul 2016 - $40.00
CG was gravel, site fairly level. Cable was pathetic, water and electric sufficient. Indoor pool but didn't use it. Our site was SO close to our neighbor, we could pass tea bags. The saving redemption was our driver's side was next to their driver's side. Good navigation roads. Staff was wonderful. Close to I-40 and there was noise. Residents were quiet. Some stored, junk RVs are in the back. A bunch of good restaurants are across I-40 (Wild Wings, Texas Roadhouse). Would stay again for another two nights. - SD75mac
Jul 2016 - $40.00
We spent last night here. Luckily, this was a one night stay kind of place. We did have a tree on our site, and the site was level with full hookups. It's basically a parking lot with many full-timers. WiFi wasn't great and the park is right off the highway. The full-timers close to us decided to make a lot of noise last night at 11 PM. One of the three trucks on their site fired up his truck for at least 45 minutes. We did not use the indoor pool or hot tub, as they didn't look appealing to us. I went inside to turn in our card this morning and there were no muffins to be found as advertised. I felt sorry for the bison they have corralled up in a fairly small area for that herd. They were not roaming the property as advertised. The park isn't as the pictures depict it to be and it was overpriced. We will skip staying in Oklahoma City next trip. - Droolie
Oklahoma City, Oklahoma (map) | (405) 789-2103
Sites: 102 | Latest Rate: $31.00 | 50 Reviews
|Clean Restrooms:
|Clean Showers:
Aug 2016 - $31.00
A very fine park. A good value for the cost. - W Davidson
Mar 2016 - $32.00
Overpriced. Park in the middle of an industrial area. Construction on Council Rd made it noisy and hard to get to with the snarled traffic.
We picked this one based on a review that said you could not hear the freeway noise. It was very prevalent in our site, which was in the back of the park.
Dont plan on staying here if you want to stay hooked up unless you are a relatively small RV and don't need or want to go anywhere. - Drakelos
Dec 2015 - $32.00
We only stay at this park because it's convenient to visit relatives that live near by. The park is run down and in need of some pepping up. My biggest complaint is that the water hookup is underground and very inconvenient to connect the hose.
Lot spacing is nearby with a sunny/open exposure. Site surface is gravel. - gobigred
Oklahoma City, Oklahoma (map) | (405) 736-1013
Sites: 45 | Latest Rate: $23.00 | 5 Reviews
|Clean Restrooms:
|Clean Showers:
Sep 2015 - $23.00
This RV Park has taken a turn for the worst!!! Stayed here approximately 5 years ago and it was clean and pleasant, now it's one of the nastiest parks I have ever seen. 90% of the park is full of permanent residents, around 1/3rd of them are what could be best described as no income pests. They live in dilapidated trailers and have no problem going door to door asking for handouts. There seems to be no park rules that are enforced, even though they are posted. We stayed reluctantly for a few days and boy what an experience that was. The only hookup that seemed to work well was the 50 amp service, water connection leaked at the valve, sewer line was broke off below ground level and covered with a broken cinder block, dogs tied up outside all day and night some even running loose, feces everywhere. This park has so much potential if someone would just spend a little time and money taking care of the little things. The location is fantastic, a little tight getting in but just minutes from anything Oklahoma City has to offer. I would suggest staying at one of the other RV parks in the area for a better piece of mind (One Scary Place)... - Lone Wolf
Jun 2015 - $23.00
We did not stay, park very run down, no room to get into a site due to permanent residents cars lining the narrow rd. We were just stopping for the night at 10:30 pm and had to wake a resident up to move their car parked on the corner so we could get out of the park. Better off on the side of the road if you have to rest. - Kbtx
Apr 2009 - $23.00
This is a small, tight rv park. Not big enough for large rigs. The road going in is full of pot holes but the interior roads are good and paved. There are no children under 16 allowed. The lots have grass and shade. Right next to Denny's. The WiFi is very poor. Weekly rates are $115 including electric and monthly is $350 including electric. The residents are mostly working full timers. I will stay here again. - Capn Kenny and Nancy Sallee


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2013 - Мy Green Community
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1) Only 44 out of 145 (30%) local governments in the area of central Serbia and Vojvodina, have the strategy of environmental protection.
2) The lack of strategy grows with a reduction in the level of development. 11 out of the 23 municipalities that belong to the first group of the most developed have the strategy, while only 8 local governments out of the 46 municipalities, in the fourth group (least developed),have adopted it.


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|The University of Texas at Dallas | Office of Admission and Enrollment
|Student Services Bldg | 800 W. Campbell Rd, Richardson, TX 75080-3021


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Assaggi Osteria
$20 or $40 to Spend on Italian Cuisine
90% recommendedin LivingSocial customer ratings
This charming osteria offers inviting outdoor seating, an elegant atmosphere, and fare recognized by Washingtonian magazine as some of the best in McLean in 2011 and 2012:
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• Enjoy house-made pastas, sustainably fished seafood, and local produce
A Chef with Decades of Experience
Chef Domenico Cornacchia started cooking in Italian kitchens at the tender age of 12. You'll taste the artistry in standout dishes such as spaghetti with Manila clams and fresh chili peppers; risotto with mushrooms and truffles; and tender veal braised in white wine, thyme, and caramelized onions.
PAID VALUE EXPIRES ON September 30, 2021
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Mabledon Court Hotel
Address10-11 Mabledon Place, Bloomsbury LondonSee nearby area
For rates, enter dates/guests
Description provided by property
Mabledon Court Hotel is less than 600 metres from London Euston, King's Cross and St Pancras International rail stations. It has free Wi-Fi, a lift and a guest lounge. The British Museum is just over a 10-minute walk from the hotel.
The Mabledon Court Hotel is in the Bloomsbury district of London. The British Library is only a 3-minute walk away.
The en suite rooms have TVs, telephones, hairdryers and tea/coffee. Some double rooms have garden views.
A Full cooked English breakfast is served and includes sausage, tomato, baked beans and eggs. A lighter continental breakfast is also on offer and consists of cereals, yoghurt, toast and jam.
Services & facilities
- Non-smoking throughout
- Non-smoking rooms
Guest services
- 24-hour front desk
Bureau de Change/ATM
- Currency exchange
Wi-Fi/internet access
- Free WiFi
Nearby places
British Library
Consisting of low-slung red-brick terraces and fronted by a large plaza featuring an oversized statue of Sir Isaac Newton, Colin St John...
St Pancras Station & Hotel
Looking at the jaw-dropping Gothic splendour of St Pancras, it's hard to believe that the 1873 Midland Grand Hotel languished empty for...
King's Cross Station
With its clean lines and the simple arches of its twin train sheds, you might be forgiven for thinking that King's Cross is a more...
One of London's most exciting cultural venues, this was the birthplace of modern British dance; it still concentrates on challenging and...
North Sea Fish Restaurant
The North Sea sets out to cook fresh fish and potatoes – a simple ambition in which it succeeds admirably. Look forward to jumbo-sized...


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Business Shrink: Take an Introspective Approach
Episode 21
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Leading a Values Based Business (8/5/06)
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Stress Free Performance Appraisals (8/19/06)
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