The objective of this study was to explore the influence factors of hospitalization costs of treating colorectal cancer in China. And the study provides new estimates on hospitalization costs and length of hospital stay for patients with colorectal cancer in China.Data for inpatient hospitalization associated with colorectal cancer were obtained from a 3-tier hospital in Guangdong Province and were analyzed post hoc. We conducted descriptive statistical methods, Wilcoxon rank–sum tests (for 2 groups) and the Kruskal–Wallis test (for more than 2 groups) to analyze the hospitalization costs of treating colorectal cancer.P = .039) and insurance status (P < .001). The mean hospitalization costs were < 16,279.58. Mean hospitalization costs were different among the UEBMI, the URBMI and the Unspecified .The analysis included 8021 patients . The overall mean length of hospital stay was 11.35 days. Over the 5 years, the mean length of hospital stay showed a small decrease from 12.22 days in 2012 to 10.69 days in 2016, while per-day costs showed a trend of increase between 2012 and 2015 (increase from < 1190.94 to < 1382.50). The mean length of hospital stay was statistically significant difference was found for sexes (The study showed that hospitalization costs increase were associated with a small decreasing length of hospital stay and increasing per-day hospitalization costs. Moreover, the proportion of the hospitalization costs reimbursed by insurances increased. For inpatients with UEBMI, it possibly lead to over treatment and the medical expense rise which result in medical resources waste and significant society costs. The rising hospitalization costs may lead to a remarkably increased financial burden in the future in China. The research of hospitalization costs is one of the key points of social medical and sanitary resources.Health insurance play an important role in medicine service in every country and the type of health insurance may be important to the cancer patient survival. Because health insurance influences the quality of health care.Research on the behavior of patients with different types of medical insurance has already been studied. For example, Gravelle et al2This retrospective analysis identified inpatients with at colorectal cancer between 2012 and 2016. We used the database from affiliated hospital of Guangdong Medical University, a third-grade class-A hospital in Guangdong Province in China. The hospital medical service covers about 30 million people from Guangdong Province, Guangxi Province, and Hainan Province. This database could provide a sufficient sample size for this analysis.Patient data collected included demographics, diagnosis, International Classification of Diseases (ICD), insurance type, length of hospital stay, and hospitalization costs.We used the SPSS (version 20.0) for statistical analysis and the significance level was set at α = 0.05. The baseline characteristics of inpatients with colorectal cancer were analyzed by descriptive statistical methods. Wilcoxon rank–sum tests (for 2 groups) and the Kruskal–Wallis test (for more than 2 groups) were used to account for non-normally distributed data.33.1Overall, there were 8021 patients with colorectal cancer requiring hospitalization during 2012 to 2016. Of the total, the patients’ mean age was 60.04 ± 13.37. The males and females comprised 59.46%  and 40.54% , respectively. There are approximately 30.81% were covered by the UEBMI and about 58.00% were covered by the URBMI, respectively. The rest, about 11.20%, paid all by themselves without insurance. The numbers of inpatient have shown a rising trend over the 5 years (increase from 1402 to 1865). (Table 3.2P = .039) and statistically significant difference was found for insurance status (P < .001). The mean length of hospital stay among the UEBMI was 11.96 days, while the mean length of hospital stay for the URBMI was 10.87 days.The overall mean length of hospital stay was 11.35 days. The mean length of hospital stay was not similar across sexes (P = .049). Mean hospitalization costs were different among the UEBMI, the URBMI and Unspecified . , and they were statistically significantly different . In the meantime, the estimated mean hospitalization costs showed an increase of almost 7.9% from 2012 to 2015. But a decrease occurred between 2015 and 2016 . In the other hand, test costs revealed a trend of fluctuations. (Table 4P = .039) and insurance status (P < .001). The mean hospitalization costs were ¥16,279.58. Mean hospitalization costs were different among the UEBMI, the URBMI and Unspecified (P < .001).This retrospective database study analyzed data from 2012–2016 to estimate hospitalization costs and length of hospital stay due to colorectal cancer. The mean length of hospital stay was 11.35 days. Over the 5 years, the mean length of hospital stay revealed a slight decrease from 12.22 days in 2012 to 10.69 days in 2016, while per-day costs showed a trend of increase between 2012 and 2015 (increase from ¥1190.94 to ¥1382.50). The mean length of hospital stay was statistically significant difference was found for sexes  and statistically significant difference was found for insurance status (P < .001). The mean hospitalization costs by the UEBMI was higher than that by the URBMI. Moreover, the proportion of insurance reimbursement by the UEBMI is more than that by the URBMI. Medical insurance helps people to overcome barriers of treatment, but different medical insurances potentially bring overtreatment and resources waste in a way.In different insurance types, the mean length of hospital stay was not similar across sexes  and computed tomography (CT) which is quiet important for doctors to evaluate disease and prognosis. Moreover, the treatment costs increased year by year. It revealed that values of certain medical services, such as skills and labor of medical staff, increased gradually.Therefore, improving the quality of diagnosis and treatment, reducing the medical expenses by controlling the drug costs, and shortening the length of hospital stay can reduce the heavy financial burden of patients with colorectal cancer. On the other hand, clinical pathway management is an effective and effective way to manage the medical expenses of hospitalized patients.An important advantage of our study is that it used recent hospital data to estimate on hospitalization costs and length of hospital stay due to colorectal cancer. The study can provide details that can be used in economic evaluation. It also has some limitations included single hospital and small sample. Also, subgroups were just analyzed by sex and insurance type. But it was not analyzed subgroups based on age or stage. In addition, inflation is not considered.5Our study used recent and cross-sectional data to provide more accurate estimates on the hospitalization costs of colorectal cancer in China. The study showed that hospitalization costs of the UEBMI was higher than the URBMI, and the length of hospital day of the UEBMI was longer than the URBMI. The proportion of the hospitalization costs reimbursed by health insurances increased. For inpatients with UEBMI, it possibly lead to over treatment and the medical expense rise which result in medical resources waste and significant society costs. In the case of China, the hospitalization costs remain unaffordable for most inpatients with colorectal cancer, especially for inpatients without health insurance. It also showed that drug costs and test costs play important roles in hospitalization costs. Reducing the cost of drugs and test can make more patients receive better treatment. This study fails to clarify how to maximize benefit of inpatients with colorectal cancer. Therefore, we still have to make further study.The authors would like to thank the Guangdong Medical University, China and Affiliated Hospital of Guangdong Medical University, China for providing database and other research support. We also would like to thank for setting up and managing the database dedicated to the collection of such a large amount of data.Conceptualization: Zhong-Fang Zhang.Data curation: Li-Zhong Liang.Formal analysis: Zhen-Yi Huang.Funding acquisition: Li-Zhong Liang, Zhong-Fang Zhang, Qilian Liang.Investigation: Hui-Jie Zhang, Shao-Ang Cheng, Xiao-Xia Peng.Methodology: Zhen-Yi Huang.Project administration: Qilian Liang.Resources: Li-zhong Liang, Hui-Jie Zhang, Xiao-Xia Peng.Software: Hui-Jie Zhang, Shao-Ang Cheng, Xiao-Xia Peng.Supervision: Zhong-Fang Zhang, Shao-Ang Cheng.Validation: Zhong-Fang Zhang.Visualization: Hui-Jie Zhang, Shao-ang Cheng, Xiao-xia Peng.Writing – original draft: Gao-Le Yuan.Writing – review & editing: Zhong-Fang Zhang, Qilian Liang.Qilian Liang orcid: 0000-0003-3245-1239.


As the first line of host defense, the innate immune system utilizes germline-encoded receptors named pattern-recognition receptors (PRRs) to detect invading pathogens. PRRs recognize conserved molecular structures of pathogens known as pathogen-associated molecular patterns (PAMPs) to initiate immune responses that counteract pathogen infection. The immunostimulatory feature of exogenous nucleic acids, such as viral DNA and RNA, has been known for more than half a century, but the mechanism by which they function as an immune stimulant remained unclear for a long time. The past two decades have witnessed tremendous progress in understanding the signaling mechanisms of innate immune networks and established the retinoic acid inducible gene-I (RIG-I)/melanoma differentiation associated gene 5 (MDA5)–mitochondrial antiviral-signaling protein (MAVS) axis and cyclic GMP-AMP synthase (cGAS)–stimulator of interferon genes (STING) axis as the major sensing pathways for cytosolic RNA and DNA, respectively . HowevercGAS binds to cytosolic double-stranded DNA (dsDNA) from various sources, including bacteria, DNA viruses, and retroviruses, in a sequence-independent but length-dependent manner , 2. FollThe question of whether cGAS and STING are also engaged in antiviral responses to RNA viruses has been asked since the very beginning. A quick answer to this question would be yes because studies have shown that deficiency of cGAS or STING in cells or mice greatly facilitated replication of several RNA viruses, such as vesicular stomatitis virus (VSV), Sendai virus (SeV), dengue virus (DENV), and West Nile virus (WNV) –11. HoweSTING was found to interact with RIG-I and MAVS , 16, whiDNA virus infection leads to quick ubiquitination and phosphorylation of STING, which is required for its trafficking and subsequent degradation in the perinuclear lysosomal compartment , 20. HowConsidering the central role of cGAS and STING in the innate DNA sensing pathway, it’s not surprising to find that many DNA viruses have evolved effective strategies to antagonize the function of cGAS and STING in order to facilitate their replication in host cells  Fig 1)..Fig 1). Emerging evidence also indicates that RNA viruses have developed their own strategies to antagonize cGAS and STING activity . The nonAlthough it is clear that cGAS is essential in cytosolic recognition of DNA viruses, its role in restricting RNA virus infection is still inconclusive. On the other hand, it is now apparent that STING is required for host responses against both DNA and RNA viruses. Studies of mechanisms employed by STING to restrict RNA virus infection have only just begun. The study which unraveled STING’s function in protein translation inhibition has shed light on our understanding of how the cGAS–STING pathway functions in RNA virus restriction . However


In this study, novel composite titanium-based metal-organic framework (MOF) beads were synthesized from titanium based metal organic framework MIL-125 and chitosan (CS) and used to remove Pb(II) from wastewater. The MIL-125-CS beads were prepared by combining the titanium-based MIL-125 MOF and chitosan using a template-free solvothermal approach under ambient conditions. The surface and elemental properties of these beads were analyzed using scanning electron microscopy, Fourier transform infrared and X-ray photoelectron spectroscopies, as well as thermal gravimetric analysis. Moreover, a series of experiments designed to determine the influences of factors such as initial Pb(II) concentration, pH, reaction time and adsorption temperature was conducted. Notably, it was found that the adsorption of Pb(II) onto the MIL-125-CS beads reached equilibrium in 180 min to a level of 407.50 mg/g at ambient temperature. In addition, kinetic and equilibrium experiments provided data that were fit to the Langmuir isotherm model and pseudo-second-order kinetics. Furthermore, reusability tests showed that MIL-125-CS retained 85% of its Pb(II)-removal capacity after five reuse cycles. All in all, we believe that the developed MIL-125-CS beads are a promising adsorbent material for the remediation of environmental water polluted by heavy metal ions. With the development of modern industry, the standards of living have been continuously improving. However, this has also led to many environmental problems, including the heavy metal pollution of water, which creates risks for human health, the environment and ecological systems . Most spWith the rapid development of new materials, metal-organic frameworks (MOFs) have received an increasing amount of attention in recent years . MOFs ar2-MIL-125, which exhibits good sorption-isotherm-model behavior and high water capacity for an adsorbent-heat-transformation system [The development of titanium-based MIL-125 represents an important breakthrough in the use of metal nodes as functional moieties [n system . Althougn system .Chitosan (CS) is a natural and completely biodegradable polymeric material that is very attractive to researchers because of its versatile chemical and physical properties. Chitin is the raw material for chitosan and is produced from chitinous solid waste from the food industry . ChitosaIn this study, we synthesized MIL-125 using the hydrothermal-solvent method and mixed it with chitosan to form solidified beads in a sodium tripolyphosphate solution. The synthesized MIL-125-CS beads contained carboxyl and hydroxyl groups derived from chitosan, and its bead structure facilitated the separation from water in subsequent experiments. Furthermore, chitosan wraps on the surface of MIL-125 were found to enhance the stability of the beads in water. Finally, the abilities of the MIL-125-CS beads to adsorb Pb(II) from polluted water were tested. Analysis of the Pb(II) levels revealed that the beads exhibited good adsorption capacities and solid-liquid separation characteristics. In addition, filtration or centrifugation was not required during the recovery of the MIL-125-CS beads.SEM pictures of MIL-125, the MIL-125-CS beads and Pb(II) loaded on MIL-125-CS (MIL-125-CS-Pb) are displayed in −1 corresponded to the O–H stretching vibrations, while the band at 1860 cm−1 was ascribed to the carboxyl C=O moiety. Similarly, the bands at 3360 cm−1 in both spectra shown in −1 corresponded to the C–H stretching vibrations based on MIL-125-CS, while those at 1162 and 1064 cm−1 were assigned to the C–C stretching vibrations, proving that MIL-125-CS contained MIL-125. In addition, the band 1210 cm−1 was ascribed to the O-Ti-O vibration [−1 (-NH2 bending mode) reflected the interaction between the Fe3+ ion and -NH2 group [FTIR spectra of MIL-125, MIL-125-CS and MIL-125-CS-Pb are shown in ibration  . FurtheH2 group . MoreoveH2 group . Reporte7/2, further verifying that Pb(II) was loaded on MIL-125-CS and suggesting that lead carbonate and lead oxide were possibly formed during the adsorption process [XPS was used to investigate the chemical compositions of MIL-125-CS and MIL-125-CS-Pb. In particular, Ti, C, O, N and Pb were found to be present in the adsorbent exposed to Pb(II). The XPS survey, Pb 4f and C 1s spectra of MIL-125-CS and Pb(II)-loaded MIL-125-CS are shown  process . In cont2 powder that was not easy to crack at higher temperatures (>500 °C) [2 residue [TGA examines the stabilities and compositions of the materials being studied, in this case MIL-125 or MIL-125-CS, through programmed temperature changes. >500 °C) . The mai residue .2, and this similarity was due to the fact that CS and MIL-125 have polymerized and that the structure of MIL-125 had not changed.The XRD patterns of pure MIL-125 and MIL-125-CS are shown in tq increases rapidly with the increasing adsorption time during the initial stages of adsorption, which was ascribable to unoccupied MIL-125-CS. However, the adsorption of Pb(II) into the MIL-125-CS beads gradually reached equilibrium within 180 min, as evidenced by the fact that no obvious adsorption-capacity changes were noticed after 180 min. Consequently, a contact time of 180 min was applied to the following experiments.Contact time experiments were conducted in the time range of 5–300 min during the Pb(II) removal using the MIL-125-CS beads, and the respective adsorption capacities were determined for different adsorption times. As can be observed in Additionally, we studied the kinetics of the adsorption of Pb(II) by the MIL-125-CS beads. Experiments were conducted in which MIL-125-CS beads (0.5 g) were added to 20-mL aliquots of Pb(II) solutions with different initial concentrations (10–1000 mg/g). During the analysis of the adsorption data, we assumed either a pseudo-first-order or pseudo-second-order kinetics model, the specific rate equations of which are:k1 (min−1) and k2 (g mg−1 min−1) are the adsorption rate constants from the two kinetics models, respectively. In these equations, R2 = 0.9999) better than the pseudo-first-order model (R2= 0.97978). In addition, the pseudo-second-order-calculated eq value of 102.04 mg/g is in good agreement with the experimentally-determined value of 99.94 mg/g. Hence, we concluded that the Pb(II) adsorption onto the MIL-125-CS beads follows the pseudo-second-order kinetics, which is consistent with the chemical-adsorption process [The rate constant data for the adsorption process fit the pseudo-second-order kinetic model ( process .eq for the Pb(II) adsorption was observed to increase from 40.1 ± 0.57–101.75 ± 0.67 mg/g, as the pH increased from 2–6, respectively. Therefore, it is clearly more beneficial to remove Pb(II) with MIL-125-CS at a higher pH. This phenomenon was attributed to the prolific H+ ions in the solution that compete with Pb(II) for loading onto the MIL-125-CS beads [−) and ferric (Fe2+) ions reacting to form a precipitate. Finally, we conclude that at low values, the pH had a significant impact on the Pb(II)-adsorption process, with a maximum eq observed at pH 6. As a result, subsequent tests were carried out at pH 6, at which hydrolysis and Pb(II) sediment formation can be avoided.Additionally, we investigated the effect of the initial Pb(II)-solution pH on the adsorption by the MIL-125-CS beads and summarize the results in CS beads . At the The effect of the temperature on the Pb(II)-adsorption capacity of the MIL-125-CS beads is illustrated in eC/eq were used to determine the changes in Gibbs free energy , enthalpy  and entropy .These quantities are related by the following formula:In order to provide insight into the mechanism of the adsorption process, the changes in T (K) indicates the adsorption temperature and represents the common gas constant. Moreover, ΔG was used to establish if the adsorption process is spontaneous and if the thermodynamic temperature is conducive to the Pb(II) loading on the MIL-125-CS beads. Notably, ΔH was affected by temperature, with the positive values confirming the spontaneous nature of the experimental process [In this equation,  process . These vC0 values.Batch experiments were carried out in order to determine the impact of the Pb(II) concentration on the adsorption capacity of the MIL-125-CS beads. As illustrated in qmax (mg/g) indicates the maximum removal capacity for Pb(II) loaded onto the MIL-125-CS beads, b is the Langmuir constant, FK (mg/g) is the Freundlich adsorption constant and n is the Freundlich adsorption constant that is related to the adsorption intensity. These values are listed in Equilibrium adsorption isotherms are an efficient means of confirming the mechanism associated with the adsorption process of an adsorbent. In this study, we employed the isotherm model to analyze the adsorption-equilibrium curve for Pb(II) interacting with the MIL-125-CS beads. The Langmuir and Freundlich isotherm models are described by the following formulas :(4)Ceqe=LR is a dimensionless separation factor that can be determined from the Langmuir model according to the following formula:LR values  and MIL-125 (94.72 mg·g−1). The results also indicated that MIL-125 and the chitosan beads played the same significant role during the adsorption of Pb(II), especially when combined in the composite MIL-125-CS beads. Consequently, the MIL-125-CS beads were used as the adsorbent in subsequent experiments.Comparison experiments involving the absorption of Pb(II) on MIL-125, the chitosan beads and the MIL-125-CS beads revealed that MIL-125-CS exhibited a In order to investigate the recycling characteristics of the MIL-125-CS beads, 0.1 mol/L NaOH (desorbent) were added to a mixture of the MIL-125-CS beads in the Pb(II) solution . FollowiThe structure stability of MIL-125 in a water environment with a pH range of 2–7 was investigated by FTIR and XRD analyses. As can be seen from the FTIR spectra shown in a,b, no o12H28O4Ti, 95%), ferric chloride hexahydrate (FeCl3·6H2O), benzene-1,4-dicarboxylicacid (BDC), N,N-dimethylformamide (DMF), sodium tripolyphosphate (Na5P3O10) solution and methanol (CH3OH) were provided by Aladdin Reagent Co., Ltd. , while chitosan with a viscosity of 100–200 mpa.s and a degree of deacetylation ≥95% was furnished by Aladdin Reagent Co., Ltd. . Deionized water was prepared by our laboratory. All reagents were used without further purification.Titanium isopropoxide (C12H28O4Ti (14.64 g) and BDC (13.38 mL) were added to a mixture of methanol (24 mL) and DMF (216 mL) and stirred at room temperature until a homogeneous solution was obtained. Then, the mixed solution was placed in a Teflon-lined stainless steel autoclave and heated at 80 °C for 48 h. The reaction was cooled to room temperature and collected by filtration, after which the white precipitate was washed three times with DMF to remove the remaining unreacted titanium isopropoxide from the porous framework. The resulting solid was washed several times with methanol and dried at 80 °C under vacuum for 5 h, and the prepared white solid powder was triturated for subsequent use.MIL-125(Ti) was formed according to a previously-reported method . C12H28O3 (1.35 g) was mixed with DI water (50 mL) and stirred until evenly dispersed. Then, sodium chitosan (1.5 g) was evenly added to the solution. Finally, MIL-125 (1.5 g) was added into the above solution until well mixed. This mixed solution was added dropwise into a pre-prepared 200 mL solution of 3% Na5P3O10 with constant stirring at room temperature for 2 h. The synthesized MIL-125-CS beads were collected and washed three times with DI water. These beads were then comprehensively characterized using multiple physicochemical techniques to confirm the formation of MIL-125-CS.The synthesis of the MIL-125-CS beads is depicted schematically in The surface morphologies of the beads were analyzed using scanning electron microscopy . The functional groups and elemental composition of the adsorbent were monitored by Fourier transform infrared (FTIR) spectroscopy  and X-ray photoelectron spectroscopy . Additionally, thermal gravimetric analysis (TGA) was performed using a thermal gravimetric analyzer .A 0.5-g aliquot of MIL-125-CS was added to 20 mL of a 200-mg/L Pb(II) solution in a 100-mL conical flask. A set of initial Pb(II) concentrations (100–1000 mg/L) was used in the following experiments. In order to study the effect of pH on the adsorption reaction, the pH of the Pb(II) solution was adjusted to 2–7 with HCl (0.1 M) or NaOH (0.1 M). The flask with the adsorbent and Pb(II) solution was shaken at 150 rpm, while maintaining a temperature of 298.2 K during the adsorption process. After the adsorption, the solution was poured into a small brown bottle, and the post-adsorption concentration of Pb(II) was determined.eq, mg/g) of MIL-125-CS was determined by Equation (7):m (g) is the weight of MIL-125-CS, C0 (mg/L) is the initial concentration of Pb(II), V (L) is the volume of the Pb(II) solution and eC (mg/L) is the Pb(II) concentration at equilibrium.The removal capacity (tq (mg/g) for MIL-125-CS at time t was calculated according to Equation (8):tC (mg/L) indicates the resulting concentration of Pb(II).The value of R) for the elimination of Pb(II) from the polluted water by MIL-125-CS was calculated using Equation (9):The rate , aqueous Pb(II) solutions  were treated with each adsorbent (0.5 g).5P3O10 solution to form beads under ambient conditions. The synthetic polymer beads were analyzed using SEM, FTIR, XPS and TGA, and a thorough study of the surface characteristics of MIL-125-CS confirmed its successful preparation. Furthermore, the formed beads, as novel adsorbents, were used to adsorb Pb(II) from simulated wastewater. A series of adsorption kinetic studies, adsorption-isotherm modeling and thermodynamic studies led us to conclude that the adsorption process was spontaneous. Notably, 180 min were required to reach equilibrium, eventually leading to a qmax of the attached Pb(II) of 406.5 mg/g at a pH of 6. After batch experiments and regeneration testing, we finally concluded that the MIL-125-CS beads were an effective and reusable material for the adsorption of Pb(II) from polluted water.In this study, we hydrothermally prepared MIL-125 following a literature procedure. Then, we mixed it with chitosan and dropped it into a Na


Respiratory viral infections are a leading cause of mortality worldwide. As many as 40% of patients hospitalized with influenza-like illness are reported to be infected with more than one type of virus. However, it is not clear whether these infections are more severe than single viral infections. Mathematical models can be used to help us understand the dynamics of respiratory viral coinfections and their impact on the severity of the illness. Most models of viral infections use ordinary differential equations (ODE) that reproduce the average behavior of the infection, however, they might be inaccurate in predicting certain events because of the stochastic nature of viral replication cycle. Stochastic simulations of single virus infections have shown that there is an extinction probability that depends on the size of the initial viral inoculum and parameters that describe virus-cell interactions. Thus the coinfection dynamics predicted by the ODE might be difficult to observe in reality.In this work, a continuous-time Markov chain (CTMC) model is formulated to investigate probabilistic outcomes of coinfections. This CTMC model is based on our previous coinfection model, expressed in terms of a system of ordinary differential equations. Using the Gillespie method for stochastic simulation, we examine whether stochastic effects early in the infection can alter which virus dominates the infection.We derive extinction probabilities for each virus individually as well as for the infection as a whole. We find that unlike the prediction of the ODE model, for similar initial growth rates stochasticity allows for a slower growing virus to out-compete a faster growing virus.The online version of this article (10.1186/s12859-019-2793-6) contains supplementary material, which is available to authorized users. With the advent of molecular diagnostic techniques, respiratory tract specimens from patients with influenza-like illness (ILI) are now being recognized as having multiple viruses –4. ArounCoinfection can be better understood using mathematical modeling. While mathematical modeling of single virus infections at the cellular level has proven crucial for finding answers where laboratory experiments are impossible, impractical or expensive –23, littFor example, stochastic simulations of single virus infections have shown that there is an extinction probability that depends on the size of the initial viral inoculum and parameters that describe virus-cell interactions . SimilarOf particular interest for stochastic models is the probability of extinction, a feature that ODE models cannot capture. In stochastic models, analytic expression of extinction probability is formulated by keeping track of the number of infected cells , the numIn this work, we implement a stochastic counterpart of our previously published ODE coinfection model , in the T ≈ T0). As a result, the states become decoupled making the stochastic events independent of each other. In addition, each event produces progeny over a lifetime which is also independent of the lifetime of all other events. More details on how to derive a branching process from a CTMC can be found in [Stochastic extinction is most relevant during the initial stage of infection. At this stage the number of target cells is small. We can consider that the target cells are constant or equal to the initial number of target cells  probability mass function. If a time-homogeneous CTMC is a branching process, the only absorbing state is ith reaction is defined as ai such that a1=β1TV1,a2=β2TV2, a3=k1E1,a4=k2E2, a5=δ1I1,a6=δ2I2, a7=p1I1,a8=p2I2, a9=c1V1,a10=c2V2, then the probability that the ith reaction is the next reaction is given by If the rate of the nmax is the number of transitions involved in the model and is equal to 10. The time of the next reaction is a random variable with distribution and Substituting the expressions for mentclass2pt{minimAlthough the general solution of this expression is intractable, the CTMC assumption of independent events means that the functional equation of at state , so we cV1, initiates a process with E1, or infected cell, I1 and so on. Eq.  ×107, respectively. From the distributions , so we will systematically alter p for one virus to alter its growth rate.Since growth rate determines which virus is the stronger competitor , we inver=1×10−1×102. We count the number of times, out of 1000 simulated infections, a particular virus has a higher viral titer peak than the other virus. The results are shown in Fig. For ease of interpretation, we define the relative viral production rate always have a higher peak viral load  for both viruses overlaps for a wide range of relative viral production, indicating that the viruses can have similar peak viral loads.In Fig. 0, although the time of peak decreases as the relative viral production rate increases. This is because the viral production rate of virus 1 is increased over its baseline value causing an earlier time of peak. This drives the earlier time of peak of virus 2, which is the weaker competitor in this case. The decline in time of viral peak is not as sharp in the CTMC model since stochasticity can temper the effect of the increased production rate of virus 2 by allowing virus 1 to still have an opportunity to infect some cells.The time of viral peak also shows some differences between the ODE and CTMC models. For the ODE model, the time of viral peak is similar for both viruses when the relative viral production rate is greater than 10Finally, we compare the predicted duration of coinfection variation for ODE and stochastic models , when the coinfection is initiated with a single virus of each type Fig. . Note thThe dynamics of coinfection were previously modeled deterministically in several studies , 25, 29.The ODE coinfection model resulted in a simple rule for determining which virus would be dominant in a coinfection — the virus with a higher growth rate. Replication of the slower-growing virus is suppressed due to lack of accessibility to target cells. This simple rule suggests that we can easily determine which viruses will be suppressed in coinfections. For example, application of the ODE model to several respiratory viruses indicated that parainfluenza virus (PIV) replication is substantially reduced during coinfection with other respiratory viruses , suggestStochasticity also impacts our ability to use viral interference as a possible mechanism for treating or preventing more serious infections. If we cannot guarantee that a fast-growing virus will suppress growth of a slow-growing virus, then this strategy might be risky. For example, some have suggested using defective interfering particles (DIPs) as a possible method for blocking infections –55. DIPsWhile our extension of the simple coinfection model has provided insight into how stochasticity might affect coinfections, this simple model does not capture all biological processes during the infection. More complex ODE models that include cell regeneration  and supeWhile ODE models are useful for giving a broad picture of possible dynamical behaviors of infection, in reality each infection is distinct with disease outcome dependent on early stochastic events. This is particular important when considering interactions between viruses during coinfection since stochasticity can drive one or both viruses to extinction before the infection has time to take hold. Our models show that for viral coinfections, this sometimes leads to a less fit virus out-competing a more fit virus.Ω. The states of the full system are defined as The previously  proposedn et al. . Figure T is the number of susceptible target cells, E1 and E2 are the number of eclipse cells or non-infectious infected cells, I1 and I2 are the number of active infectious cells and V1 and V2 are the number of virus particles. Viruses of each type infect the target cells, which are limited, at infection rates, β1 and β2. Each type of infected cell transitions into eclipse phases where viruses undertake intracellular processes for progeny virus production. After the time durations of I1 and I2, which produce viruses at production rates p1 and p2 over their lifespans of c1 and c2. Thus the number of transitions involved in the model is 10 with respective transition rates (propensities) defined in Table where It has been shown that, the stochastic representations of chemical reactions converge to the differential equations as the number of particles goes to infinity when we can assume that the probability of a reaction depends on the density of the reactants –59. We m8. Due to the increased simulation efficiency with some accuracy trade off, Gillespie tau-leap algorithms are getting more attention. In the tau-leap method a small time interval is chosen such that the number of times each transition occurs in this interval is drawn from a Poisson distribution with mean equal to the expected number of transitions during that interval. The time step is fixed for the most basic tau-leap method. However, the time step should be small enough that the rate at which transitions occur remains approximately constant during the fixed interval. For numerical implementation of the CTMC model, we use the Gillespie tau-leaping method with time step equal to 10−3 day. The transitions involved in the stochastic process and their rates are summarized in Table 50/mL, PFU/mL or EID50/mL rather than as the total number of virus in the host, while the CTMC model uses discrete values for each of its state variables. In order to convert viral concentration measurements to the number of infectious virus particles, studies have used a conversion factor, α [α, previous estimates suggest that 1 TCID50/mL of nasal wash corresponds to 1×102−1×105 [4−3×105[α equal to 1×102 to convert the concentrations of virus into numbers of virus particles according to the method mentioned in [The direct method popularized by Gillespie , in geneactor, α , 62, 63.ioned in .Table 2Additional file1Pdf file containing additional figures.


With the native, C. cylindracea population frond number decreased irrespectively of pressure level. High propagule pressure did not increase stolon length and single plant size decreased due to the effects of intra- and interspecific competition. Native biomass decreased with increasing C. cylindracea propagule pressure. These results indicate that higher propagule pressure may fail in enhancing C. cylindracea invasion success in habitats colonized by the native species, and they suggest that biotic resistance and propagule pressure co-regulate the invasion process. These findings emphasize the need to preserve/restore native seaweed populations and may help to design effective management actions to prevent further C. cylindracea spread.Propagule pressure is considered a major driver of plant invasion success. Great propagule pressure would enable invasive species to colonize new areas overcoming the resistance of native species. Many highly invasive aquatic macrophytes regenerate from vegetative propagules, but few studies have experimentally investigated the importance of propagule pressure and biotic resistance, and their interaction, in determining invasion success. By manipulating both recipient habitat and the input of vegetative propagules of the invasive seaweed C Numerous factors may individually affect the establishment of a NNS outside its native range4. Understanding how these factors interact each other to determine the outcome of the invasion process is one of the main goals of ecology, and it is also highly relevant to improving our ability to manage invasive species.The invasion of environments by non-native plant species (NNS) is an increasing threat to biodiversity and functioning of ecosystems globally7. A large propagule input theoretically would enhance the probability of successful invasion enabling an invader to overcome difficulties related to demographic, environmental or genetic stochasticity of a new location10. Relatively few experimental studies, however, have investigated the relationship between propagule pressure and invasion success, and for many plant species it is still unclear whether they may actually gain increasing benefits from increasing propagule pressure12. Moreover, the importance of the interaction between propagule pressure and local factors that can affect plant invasion15 has been rarely tested, especially in aquatic environments20, probably because of the difficulty in measuring and directly controlling the number of propagules introduced in these environments.The number of propagules  of a NNS entering the new environment, also termed propagule pressure, is considered a major factor of plant invasion success in a wide range of ecosystems21. For example, the presence of native species capable of exploiting available key resources  may inhibit the establishment or limit the spread of an invader once it has established23 (biotic resistance hypothesis). Native congeneric species are expected to be more resistant to NNS invasion based on the conjecture that closely related species have similar resource requirements24. However, once the invader has established, the impact on the native congeneric may be strong foraging on the same resources. On the other hand, increasing number of studies indicates that in stressful habitats native species may facilitate, for example by physically trapping propagules within the leaf canopy, stabilizing substrates or ameliorating abiotic stresses, rather than compete with NNS25, which is in direct opposition of predictions of biotic resistance26 (facilitation hypothesis). Therefore, in environments where the resistance of resident species is strong, a much higher input of propagules should be necessary to a NNS to maximize its colonization success28. Instead, in environments where the resistance of resident species is weak, or interactions between resident and NNS are facilitative, only a few propagules should be sufficient to a non-native species to ensure successful invasion11.Evidence from studies conducted predominantly in terrestrial environments suggests that the nature of interactions between newly arriving species and those already established may play an important role in controlling plant invasion30. Among aquatic macrophytes with this feature there are some marine clonal macroalgae32 (seaweeds), but for most of these species the role of vegetative propagule pressure in determining the colonization of novel habitats remains to be elucidated. The invasion of marine habitats by non-native seaweeds can cause ecological perturbations in local communities and loss of biodiversity with consequent negative impact on ecosystem composition, functioning and services to human society33. Understanding the role of vegetative propagule pressure and biotic resistance is crucial not only to improve our knowledge on invasion processes but also to formulate effective strategies to reduce the risk of new marine habitat invasions and the chance of spread of already introduced species, i.e., expend more efforts in restoring and maintaining resident native vegetation if invasion resistance is more important or limiting the release of more propagules if propagule pressure is determinant.The invasion success of many highly invasive macrophytes inhabiting aquatic environments is related to the ability to regenerate from detached plant fragments (vegetative propagules) that are passively transported to new locations, in addition to reproduce sexuallyCaulerpa cylindracea Sonder, since it is a well cited example of successful invasive clonal seaweed. As model native, we used a congeneric clonal species, Caulerpa prolifera Forssk J.V. Lamouroux. Caulerpa cylindracea is a green seaweed native of Australia that has rapidly spread in the Mediterranean basin during the last decades by invading a variety of habitats, including soft-bottoms and areas formerly occupied by seagrasses, with negative consequences on native primary producers36. The ability of drifting C. cylindracea vegetative propagules to establish and grow in nature has been documented37, but quantitative data on propagule pressure are not available. Field observations and experimental studies suggest that canopy-forming algal species may either inhibit or facilitate, depending on the species, the invasion of C. cylindracea, while turfs-forming algae may generally facilitate its spread40. Disturbances may also promote the spread of the species providing new substrate for colonization while the effect of propagule input seems to have only weak effects on the cover of C. cylindracea40. C. prolifera is a canopy-forming alga that can co-occur with C. cylindracea. This species is considered not invasive in Mediterranean assemblages, but observations have shown that it had replaced co-occurring native seagrasses in degraded environments41. Since C. cylindracea and C. prolifera are similar, both functionally and architecturally, we assumed they may compete in situations where resources are limited.Here, we assessed in mesocosm the individual and combined effects of vegetative propagule pressure (number of propagules per introduction event) and the resistance of a native resident species on the invasion success of a non-native clonal seaweed. In addition, in a separate mesocosm experiment we examined the impact of different levels of propagule pressure  of the invasive species on the performance of the native species. As our non-native species model, we chose C. prolifera in the recipient habitat would reduce the colonization success of C. cylindracea (biotic resistance hypothesis), (2) higher propagule pressure would be necessary to C. cylindracea to successfully invade a habitat colonized by C. prolifera (i.e. the effect of propagule pressure on invasion success would be modulated by biotic resistance), and (3) the establishment of C. cylindracea would reduce the performance of C. prolifera and this effect would be greater at higher propagule pressure.Specifically, we tested the following hypotheses: (1) the establishment of the native species C. cylindracea propagule in all the mesocosms, thus the greatest probability of invasion was reached irrespective of treatments in both the experiments. At the end of the first experiment, the majority of the established propagules colonized the mesocosm. At population level, total stolon length was substantially affected by the interaction between propagule pressure and native presence treatment  on the growth and intensity of plant-plant interactions of an invasive seaweed in mesocosm. This experimental approach was chosen as it allows to control not only propagule number and size, but also receiving habitat conditions, minimizing the effects of potential confounding factors such differences in propagule supply and microhabitat characteristics that make it difficult to evaluate the effective role of propagule pressure in invasion success in natural marine habitats.Our study focuses on still poorly investigated aspects of the invasion of marine environments by non-native clonal macrophytes, i.e., the role of vegetative propagule pressure and its interaction with biotic resistance in determining the outcome of invasion, and the impact of vegetative propagule pressure on the performance of resident species. Ecologists have manipulated propagule supply (i.e. seeds and vegetative propagule) both in the field and in mesocosm to study the invasibility of terrestrial plant communities for decadesC. prolifera in the receiving habitat may influence the establishment probability of the non-native clonal seaweed C. cylindracea. Indeed, even only a single propagule of C. cylindracea may successfully establish a new patch irrespective of the absence/presence of the native.Our results demonstrate that neither vegetative propagule pressure nor the presence of the native congeneric C. prolifera reduced the horizontal spread of C. cylindracea population only with high propagule pressure. RII for interspecific interaction obtained for all plant growth variables of C. cylindracea grown in the presence of C. prolifera and with high propagule pressure indicated a significant competitive effect of C. prolifera on C. cylindracea.Our first hypothesis (biotic resistance hypothesis) was not fully supported. The presence of C. cylindracea to successfully invade a habitat colonized by C. prolifera) was not supported. With high propagule pressure and in the absence of the native, the total extension of C. cylindracea populations increased, attaining surprisingly high values (up to 178 m of stolon and 4400 fronds m−2), but at the expense of a reduced growth of individual plants. Under this condition, there was only intraspecific interaction, and the slightly negative values of RII for the interaction on all plant growth variables indicated negligible competition between conspecifics. In contrast, in the presence of the native the spread of C. cylindracea populations did not increase, and the highly negative values of RII for intraspecific interaction on all plant growth variables indicated that the outcome of interaction between conspecifics shifted from neutral to intense competition. However, these latter RII values are based on the response of the invader to conspecifics but also incorporated the effect of the native. Therefore, the jointly effects of intraspecific competition and interspecific competition due to resource  limitation possibly cancelled out the positive effects of increased propagule pressure on C. cylindracea spread. These findings are in contrast with results of previous studies conducted on other invasive clonal macrophytes, which have found that with increased propagule pressure the effect of intraspecific interaction shifted from competition in open habitats to facilitation in vegetated habitats19. Instead, they are in agreement with studies on some non-clonal species showing that increased propagule pressure may not overcome communities with high resistance15. Our results are also in agreement with the lack of consistent effects of high propagule pressure on the growth of C. cylindracea observed in patches of the seagrass Posidonia oceanica L. Delile using higher numbers (5 and 10) of planted manipulated algal fragments20.Our second hypothesis (higher propagule is necessary to C. cylindracea reduces the performance of C. prolifera) was supported. The establishment of even only a single propagule of C. cylindracea had a considerable impact on biomass production of the native C. prolifera, resulting in a consistent reduction of biomass (up to 50%) compared to control, and the impact increased with increasing propagule pressure. Although our study was not designed to compare the competitive ability of the two species, the greater biomass reduction observed in a single C. prolifera individual compared to that observed in a C. cylindracea one when grown in the presence of one individual of the other species, suggests that this latter species could have higher competitive superiority on the congeneric. This is consistent with a previous study suggesting that C. cylindracea would be the favoured species in the outcome of the competition with another congeneric, Caulerpa taxifolia (Vahl) C. Agardh, invasive in the Mediterranean Sea43.Our third hypothesis  of C. cylindracea may favour the colonization of bare substrate patches but the same propagule input may not be sufficient to maximize the invasion success of areas colonized by a native competitor. However, the loss of native vegetation due to disturbances by releasing plants from interspecific competition could locally promote the spread of C. cylindracea, in accordance with the large invasion of disturbed areas described in most previous field studies40. Our findings may have relevant ecological and management implications. Firstly, in most previous field and observational studies comparing the invasibility of different habitats by clonal seaweeds, the interaction between vegetative propagule pressure and biotic resistance has formally not been investigated. Failure to include this factor can make it difficult to determine if differences in invasibility across habitats are effectively due to differential susceptibility to invasion (or resistance of resident communities) or rather result from variations in propagule input49. Even when the invasion extent of two different habitats is similar, the mechanisms of invasion may differ; for example, in one habitat  the invader might have established under low propagule pressure while in the other one  the invader might have established under high propagule pressure.Overall, the results of the present study provide new experimental evidence of the high invasion potential and spread of C. prolifera, high propagule pressure may contribute little to enhance invasion success. Under these conditions, strategies such as minimizing propagule release or eradicating already established propagules of the invasive species could not provide benefits, and managers should thus expend efforts on conserving existing algal communities and restoring degraded ones to reduce the chance of establishment and growth. On the other hand, in habitats where propagule pressure may be important, as our results suggest for open/disturbed habitats, managers should focus on minimizing the risk of new propagule introductions to limit the spread of the invasive species.From a management point of view, the approach required to prevent or control seaweed invasion would differ according to the nature and intensity of interaction between the invasive and the native species in the recipient habitat. For example, in habitats where the effects of intra- and interspecific competition are important as observed here in the presence of the congener 50. The first experiment examined the main and interactive effects of propagule pressure and native species presence on the colonization success (establishment and spread) of the non-native invasive C. cylindracea. The second experiment assessed the impact of different levels of propagule pressure of C. cylindracea on the clonal growth of the native.We conducted two separate mesocosm experiments in an outdoor aquaculture system set up on a coastal dune area at the INVE Aquaculture Research Center of Rosignano Solvay , and equipped following a protocol established for successfully growing marine plantsC. cylindracea were collected along the shoreline after a storm while C. prolifera fragments were collected from a bed near to the Aquaculture Center. All plant material was transported in seawater from the sampling site to the laboratory for plant measurements (stolon length and number of erect fronds). To standardize propagule size, we selected propagules of C. cylindracea of similar size . Fragments of C. prolifera were gently washed free of any adhering sediment particle and manually cut into fragments of equal size to that of selected C. cylindracea propagules. A sample of selected propagules and fragments (n = 10) was dried at 60 °C to constant mass and weighed to determine initial plant biomass (0.062 ± 0.012 g dry weight (dw) for C. prolifera and 0.041 ± g dw for C. cylindracea) while the remaining ones were left to acclimate to tank conditions for three days before their use.Before the start of the experiments (March 2015), plastic growth containers (240 mm diameter × 220 mm height), filled with natural sediment (carbonate sand) collected at 0.5 m depth near the Aquaculture Centre (North Western Mediterranean Sea), were equally distributed in two tanks (10000 L) full of natural seawater. The sediment was previously carefully mixed and sieved (0.2 mm). During the experimental period, continuous seawater flux maintained a constant level of water (1.5 m) above the containers (thereafter referred as mesocosms) in each of the two tanks. Seawater temperature ranged from 16 to 27.8 °C, pH was 8.2 and salinity varied between 38 and 38.2 throughout the experiments. In April 2015, vegetative propagules of C. prolifera and six mesocosms were left unplanted in each of two tanks. After C. prolifera establishment (3 days), each mesocosm was artificially invaded by adding one propagule  or three propagules  of C. cylindracea. There were therefore four treatments: one propagule or three propagules of C. cylindracea in bare habitat thus in the absence of interspecific interaction, and one propagule or three propagules of C. cylindracea planted with the native species C. prolifera thus in the presence of interspecific interaction. There were three replicates per treatment in each of the two tanks, in total 24 mesocosms.For the first experiment, six mesocosms were planted with a single stolon fragment of C. prolifera in each of two tanks. After C. prolifera established, the mesocosms were randomly attributed to one of the following treatments: no exposure to the invader C. cylindracea , low pressure  or high pressure . There were three replicates per treatment in each of the two tanks, 18 mesocosms in total.For the second experiment, nine mesocosms were planted with a single fragment of C. prolifera fragments were planted in centre of the mesocosms and C. cylindracea propagules were placed in randomly chosen positions at the edge of the C. prolifera fragment. The mesocosms were randomly assigned to new positions in each of the two tanks every month to minimize biases caused by variations across the different areas within the tank. The experiments lasted for six months to cover the whole growing season of C. cylindracea in the Mediterranean Sea51. After this period, all plants were carefully harvested, washed with seawater to remove sand and transported to the laboratory for clonal growth measurements . All the plants were then dried to constant mass at 60 °C and weighed to determine total biomass. Growth measures as well as total biomass were calculated both at population level (mesocosm) and individual level (single plant).In both the experiments, C. cylindracea established (rooted in the substrate) in each mesocosm was recorded twice per week for the first two months to estimate the probability of successful invasion, expressed as proportion of mesocosms in each treatment with one or more established clones of C. cylindracea at the end of the first two months  with categorical predictor variables for fixed effects of native congeneric  and propagule pressure , and random effect of tank position  were separately performed for each selected response variable measured at both individual level and population level to test for the main and interactive effects of propagule pressure and native species presence.52, and the effect of propagule pressure and tank position on the RII for interspecific interaction based on stolon length, number of fronds and biomass data of C. cylindracea. The intraspecific RII was calculated as follows:high is the mean growth measure of a C. cylindracea propagule grown with high propagule pressure and Ylow is that measure with low propagule pressure. The index was calculated for each of the two conditions, C. prolifera absent and present. The interspecific RII was calculated as follows:with is the mean growth measure of a C. cylindracea propagule with C. prolifera and Ywithout is that measure without C. prolifera. The index was calculated for each of the two propagule pressure conditions, low and high propagule pressure. RII has defined limits , and is negative when competition prevails, positive when there is prevalence of facilitation and zero when there is no interaction. For each of the calculated RII values significant departures of mean RII values from zero were assessed by using one-sample mean t-test  on data tanks pooled.Separate two-way ANOVAs were used to assess the effect of native species and tank position on the index of relative intensity of intraspecific interaction, RIIC. cylindracea) and random effect of tank position (a and b) were performed separately for total stolon length and biomass of C. prolifera to evaluate the impact of propagule invasion on the native performance.In the second experiment, two-way ANOVAs with categorical predictor variables for fixed effects of propagule pressure  transformed to meet the assumption of homogeneity of variance and normality. When significant main effects were found in the ANOVAs, the means were compared using the Student-Newman-Keuls  test. All ANOVA analyses were conducted with the software—GMAV version 5.0 for Windows54.Prior to all analyses, data were assessed for normality and homogeneity of variance using Shapiro-Wilk test and Cochran’s C tests (α = 0.05), respectively. In the second experiment, data on the stolon length and biomas


TAP intervention costs captured alongside the randomized controlled study included labor of program staff, mileage, supplies and materials. Staff time costs were converted to $US 2017 by multiplying hours spent by the appropriate wage rate plus fringe benefits; mileage was costed using the federal reimbursement rate. Research costs were excluded to approximate real world implementation. Costs varied based on number of visits required but mean component costs per dyad were: occupational therapist training ($133.50), home visit time ($527.57), travel ($718.02), work outside of intervention delivery ($57.14), program screening for eligibility ($3.73), supervision for quality assurance ($250.23), activity supplies ($51.64), and program materials ($29.32). Findings will be compared to a 2008 TAP pilot study post hoc cost analysis to identify how broader scale implementation impacts intervention costs. Opportunities to streamline program delivery will be discussed as well as potential reimbursement mechanisms via Medicare part B, Medicare Advantage, and Medicaid waiver programs.


Canine circovirus (CanineCV) was detected, together with canine parvovirus (CPV), in samples from an outbreak of fatal gastroenteritis in dogs in Argentina. We obtained the full-length genome of this recently discovered virus by overlapping PCR, designated strain UBA-Baires. Sequence analysis revealed a highly conserved genome but also showed several unique mutations in amino acids from the capsid protein that have not been previously reported. Phylogenetic analysis shows that this strain is more closely related to European strains than to viruses detected in North America or Asia. Although the pathogenic role of CanineCV in dogs is still unclear, this study highlights the importance of CanineCV as a coinfecting virus in disease development. To our knowledge, this is the first report of the involvement of CanineCV in severe clinical disease in dogs in South America. Our results expand our information on the geographical extent of this virus and contribute to the understanding of its role in disease. Since tthy dogs –6. Lesio related .canine parvovirus (CPV), but also canine distemper virus, canine coronavirus, and canine influenza virus is present in CanineCV positive dogs with clinical symptoms at rates between 50 to 100% [A number of case-control studies have been published, most of them describing significant association between diarrhea and CanineCV detection –11. The  to 100% , 6, 8–11 to 100% , 15.Circoviridae, genus circovirus; they are small, non-enveloped virus with a circular single strand DNA genome of around 2000 bp [cap gene sequence has been proposed very recently [Circoviruses belong to the family  2000 bp . The genrecently .In this work, we present, to our knowledge, the first report of a complete full-length CanineCV genome sequence from South America.This study did not involve any animal experiments. Tissue samples were collected during necropsy of dead animals for laboratory analysis, with consent from the dog’s owner.Severe disease outbreaks occurred in a mixed-breed colony in Buenos Aires between 2014 and 2015, in a cascade fashion. The age of affected dogs ranged from 50 days to one year, belonging to different breeds. The attending veterinarian described the situation as a highly contagious disease of very rapid onset and development, characterized by anorexia, vomiting and hemorrhagic diarrhea lasting from three to 7 days. In total, 18 puppies died, and the rest recovered within a week. All dogs had been properly vaccinated against CPV, canine distemper virus and infectious canine hepatitis virus. Fresh tissues obtained from pancreas, liver, kidney, lung, lymph nodes, spleen, large intestine and stomach belonging to three dead animals were submitted to the Virology Department of the Faculty of Veterinary Sciences at the University of Buenos Aires for testing. Samples belonging to surviving animals or to the other dead dogs were not available.For genomic/Rev 533 was utilized to amplify a 533 bp fragment from the Rep gene for diagnostic purposes, after which positive samples were further tested with other primer sets in order to cover the entire genome for sequencing purposes . One hundred ng of DNA were used in a PCR reaction using 25 pmoles of each primer set , 1X reacpurposes .for and 555rev primers; this fragment includes a MboII restriction site identified by Buonavoglia et al. which allows the differentiation of CPV-2c strains from other CPV strains [Samples were additionally tested for CPV. Two μL (10 ng) of extracted DNA were used in a PCR reaction that amplifies a 583 bp fragment of the VP2 gene using 555 strains . The res strains .In order to determine the full-length genome of the CanineCV strain from Argentina, a positive sample was tested with the additional primer sets listed in http://tree.bio.ed.ac.uk/software/figtree/).For phylogenetic analysis of the complete CanineCV genome sequence, we set up a matrix that included the 22 different complete CanineCV sequences deposited so far in GenBank. Details of selected sequences are shown in For genomic/Rev 533. Then, individual organs were further screened with the same primer set finding that all tissues , except for the stomach, were positive as well. DNA from one of these tissues (lymph node) was randomly selected for further analysis with the remaining primer sets in order to assemble the entire CanineCV genome sequence, which was concluded to be of 2063 bp . However, four aa changes within the Cap gene and one within the Rep gene were found, which have not been previously reported for other CanineCV sequences being unique to strain UBA-Baires  and a capsid protein (Cap), which are 303 and 270 aa long, respectively. The 5’ intergenic region, located between the start codons and encompassing 135 nt, contains a conserved stem-loop structure and a 9 nt sequence (TAGTATTAC) for initiation of rolling cycle replication. The overall nt identity of strain UBA-Baires ranged from 98% (strain Bari/411-13) to 85% (isolate UCD3-478). When compared to other CanineCV, both Rep and Cap ORFs were, as expected, highly conserved including sequences related to rolling circle replication  and dNTP binding (GCGKS) in the non-structural gene , suggesting that, until sequences from more extent geographical areas are analyzed, these variable regions need to be taken cautiously. Point mutations in the cap gene have been described for PCV2 and are a potential source of virus evolution, especially due to selective pressure caused by vaccination [Although virus isolation was not attempted since it was proved unsuccessful in previous reports , the fulCanineCV . Only oncination . In the cination . In addicination .canine influenza virus, canine parainfluenza virus, and canine respiratory coronavirus)[In the case of PCV2, coinfection with other pathogens has been associated with different clinical outcomes . Specialonavirus). In our onavirus). Surprisonavirus). Again, onavirus) suggestiTo our knowledge, this is the first report of a complete genome sequence of CanineCV associated with disease from Latin America. A very recent paper published by Weber and col. characterizing the dog’s virome found CanineCV in the sera from seven healthy dogs from Brazil . Phyloge


Zoonotic diseases are the infectious diseases that can be transmitted to human beings and vice versa from animals either directly or indirectly. These diseases can be caused by a range of organisms including bacteria, parasites, viruses and fungi. Viral diseases are highly infectious and capable of causing pandemics as evidenced by outbreaks of diseases like Ebola, Middle East Respiratory Syndrome, West Nile, SARS-Corona, Nipah, Hendra, Avian influenza and Swine influenza.have been reported in Australia. West Nile fever has spread to many newer territories across continents during recent years.Many viruses affecting equines are also important human pathogens. Diseases like Eastern equine encephalitis (EEE), Western equine encephalitis (WEE), and Venezuelan-equine encephalitis (VEE) are highly infectious and can be disseminated as aerosols. A large number of horses and human cases of VEE with fatal encephalitis have continuously occurred in Venezuela and Colombia. Vesicular stomatitis (VS) is prevalent in horses in North America and has zoonotic potential causing encephalitis in children. Hendra virus (HeV) causes respiratory and neurological disease and death in man and horses. Since its first outbreak in 1994, 53 disease incidents It has been described in Africa, Europe, South Asia, Oceania and North America. Japanese encephalitis has expanded horizons from Asia to western Pacific region including the eastern Indonesian archipelago, Papua New Guinea and Australia. Rabies is rare in horses but still a public health concern being a fatal disease. Equine influenza is historically not known to affect humans but many scientists have mixed opinions. Equine viral diseases of zoonotic importance and their impact on animal and human health have been elaborated in this article.Equine viral diseases though restricted to certain geographical areas have huge impact on equine and human health. Diseases like West Nile fever, Hendra, VS, VEE, EEE, JE, Rabies have the potential for spread and ability to cause disease in human. Equine influenza is historically not known to affect humans but some experimental and observational evidence show that H3N8 influenza virus has infected man. Despite our pursuit of understanding the complexity of the vector-host-pathogen mediating disease transmission, it is not possible to make generalized predictions concerning the degree of impact of disease emergence. A targeted, multidisciplinary effort is required to understand the risk factors for zoonosis and apply the interventions necessary to control it. Out of etc .The pathetc . Zoonotietc , 5. Incretc -8. Vesicetc . Infectietc .West Niletc . Rabies etc -15. Rabietc  and donketc . Middle etc -20. EquiIn this article, an overview of viral diseases common to equines and human beings along with a focus on their impact is presented. The information will be useful to increase awareness about these diseases and guide devising effective strategies for the prevention and control of these infections.1.1Flavivirus belonging to the family Flaviviridae [West Nile virus (WNV) is a zoonotic iviridae . The viriviridae -27, equiiviridae -30 and biviridae -34.1). In North America, more than 1.8 million people have been infected, with over 12,852 reported cases of encephalitis or meningitis and 1,308 deaths from 1999 to 2010 [WNV was first isolated and identified in 1937 from a woman presented with mild febrile illness in the Nile district of Uganda . It has  to 2010 . The mor to 2010 . WNV in  to 2010 -43 bat [ to 2010  and man  to 2010 , 45-47.  to 2010 -52.Horses and human are the main hosts. Animals other than horses may be susceptible to WNV, but rarely become ill. Antibodies have been found in serum samples from bats, horse, dogs, cats, racoons, opossums, squirrels, domestic rabbits, eastern striped skunks, cows, sheep, deer and pigs -60. The Culex mosquitoes are the vectors and passerine birds are the vertebrate reservoirs in enzootic transmission cycles. The virus is carried in the salivary glands of infected mosquitoes and transmitted to susceptible birds during blood-sucking. Competent bird reservoirs sustain an infectious viraemia for 1 to 4 days subsequent to exposure, and then develop life-long immunity. Horses, human and most other mammals rarely develop the infectious levels of viraemia and are dead-end hosts. Few cases in human have been spread through blood transfusions, organ transplants, breast feeding and during pregnancy [Ornithodoros maritimus, Argas hermanni and Hyalomma marginatum and the virus has also been isolated from other species of hard ticks in Africa, Europe and Asia [Oeciacus hirundinis) have been implicated as vectors in Austria [WNV is amplified by continuous transmission cycles between mosquitoes and birds. Generally regnancy . The ticand Asia , 63, 64. Austria . In most Austria -67. The  Austria . There cThere is no effective treatment for clinical WNV infection in humans, horses or any other animal. Vaccines are available for control of WNV in horses in the USA -70. Vacc1.2Henipavirus of family Paramyxoviridae, order Mononegavirales containing two members, Hendra and Nipah viruses [Hendra virus (HeV) is a rare, emerging zoonotic virus. It causes respiratory and neurological disease and death in man and horses . HeV is  viruses .The pote viruses . HeV was viruses . Up to 2 viruses .Eidolon, family Pteropodidae, have been found serologically positive for HeV antibodies indicating it’s prevalence in Africa [Pteropodidae, particularly the species belonging to the Pteropus are the natural hosts for HeV. There are no visible disease symptoms in fruit bats. The transmission route is likely through contamination of pasture or feed by infected uterine fluids or fetal tissues from bats [HeV has been documented to infect horses, humans, dogs and flying foxes naturally -77. Expen Africa . The HeVn Africa . Fruit brom bats , 87. Horrom bats . The virrom bats .Infections in horses range from asymptomatic infection to fatal respiratory and neurological syndromes. For fatal cases, the course of illness takes an average of two days. Symptoms of infection in horses are not distinctly different from other respiratory and neurological illnesses of horses. The outbreaks in horses occur one to two weeks before illness in humans which could trigger prevention measures to prevent associated outbreaks in humans. The incubation period in horses varies between 4 and 16 days. The mortality rate in horses is about 75% .There are currently no drugs available to treat HeV infection. Symptomatic treatment with supportive care is the main approach to managing the infection in people. HeV vaccine for horses, EquivacHeV  has been developed . The vac1.3Vesiculovirus in family Rhabdoviridae [Vesicular stomatitis is a viral disease which primarily affects cattle, horses, and swine. It occurs in enzootic and epizootic forms in the tropical and subtropical areas . The disoviridae . The viroviridae .Vesiculovirus caused encephalitis outbreaks in different states of India leading to mortalities in children [2).The disease has been reported across continents in Belize, Bolivia, Brazil, Colombia, Costa Rica, Ecuador, El Salvador, Guatemala, Honduras, Mexico, Nicaragua, Pakistan, Panama, Peru, USA and Venezuela , 92. Outchildren . Isfahanchildren , 97. TheClinical disease has been observed in cattle, horses, pigs and camels whereas sheep, goats and llamas tend to be resistant. White-tailed deer and numerous species of small mammals in the tropics are considered as wild hosts. Many species, including cervids, nonhuman primates, rodents, birds, dogs, antelope, and bats have shown serological evidence of infection -100. ExpThe virus is zoonotic and causes flu-like symptoms characterized by fever, chills, nausea, vomiting, headache, retrobulbar pain, myalgia, sub-sternal pain, malaise, pharyngitis, conjunctivitis, and lymphadenitis in humans. Vesicular lesions may be present in the pharynx, buccal mucosa, or tongue -106. EncLutzomyia sp. (sand fly) is proven biologic vectors. Black flies (Simulidae) are the most likely biologic insect vector in USA. Other insects may also act as mechanical vectors. Saliva, exudates and epithelium from open vesicles are sources of virus. Plants and soil are also suspected as the source of virus.The transmission is more likely by trans-cutaneous or transmucosal route. The virus can be transmitted through direct contact with infected animals having lesions of the disease or by blood-feeding insects. In endemic areas, Horses of all ages appear equally susceptible  but lesi1.4Eastern equine encephalitis (EEE) commonly called triple E or, sleeping sickness is a rare but serious viral disease affecting horses and man. The disease is transmitted through mosquitoes and man and horses are dead-end hosts .Alphavirus of the family Togaviridae. It is closely related to Venezuelan equine encephalitis (VEE) virus and Western equine encephalitis (WEE) virus [3). EEEV infection in horses is often fatal. The human cases were identified first time in 1938 in the north-eastern United States. Thirty children died of encephalitis in this outbreak. The fatality rate in humans was 35%. The outbreaks of the disease also occurred in horses simultaneously in the same regions. A total of 19 human cases of the disease were reported in children between 1970-2010 in Massachusetts and New Hampshire [EEEV belongs to the genus E) virus . This viE) virus . The virE) virus . The serE) virus , 120. Coampshire . As per ampshire . In 2007ampshire , 123. EiCuliseta melanura and Cs. morsitans species are primarily involved. Transmission of EEEV to mammals occurs via other mosquitoes which are primarily mammalian feeders and called as bridge vectors [Alternate infection of birds and mosquitoes maintains these viruses in nature.  vectors . Infecte vectors . Most pe vectors . The ner vectors .There is no cure for EEE. Severe illnesses are treated by supportive therapy consisting of corticosteroids, anticonvulsants, intravenous fluids, tracheal intubation, and antipyretics. Vaccines containing killed virus are used for prevention of the disease. These vaccinations are usually given as combination vaccines, most commonly with WEE, VEE, and tetanus. Elimination of mosquito breeding sites and use of insect repellents may help in control of the disease.1.5Alphavirus, family Togaviridae. The VEE virus complex is composed of six subtypes (I–VI); Subtype I includes five antigenic variants (AB–F), of which variants 1-AB and 1-C are associated with epizootics in equines and concurrent epidemics in humans [4).Venezuelan equine encephalitis (VEE) is an arbovirus infection transmitted by mosquitoes. VEE viruses (VEEV) are classified in the genus n humans . The epin humans . The enzn humans . CountriMelanoconion subgenus (Culex cedecci). Epizootic VEEV strains (I-AB and I- C) are transmitted by several mosquito vectors  to equids [The primary vectors for the bird or rodent-mosquito life cycle are members of the o equids .Infections with VEE virus (VEEV) may present, in both humans and horses, as either encephalitic disease or as simply a febrile disease without profound neurologic signs. Horses may die after a very acute course, even without any neurologic signs, but mortality in humans is generally low. Horses are not dead-end hosts for VEEV epizootic strains like they are for EEEV and WEEV. Horses are, in fact, the key reservoir species for the epizootic strains of VEEV that cause clinical disease in both horses and humans .Epizootic subtypes highly pathogenic to equines, can spread rapidly through large populations. Equines are the primary animal species and serve as amplifying hosts for epizootic VEE virus strains. Blood-sucking insects feed on infected horses, pick up this virus and transmit to other animals or humans. Animal like cattle, swine, and dogs, can become infected, but they neither show the signs of the disease nor contribute to spread . AerosolVEE can cause disease in equines including horses, mules, donkeys and zebras. Cattle, swine, chickens and dogs have been shown to seroconvert after epizootics; mortality has been observed in domesticated rabbits, dogs, goats and sheep. Humans also can contract this disease. Epidemics of VEE involving tens of thousands of humans have been reported. The mortality rates in equines during epizootics have been 19-83% while 4-14% in human beings associated with neurological disease It usually causes influenza like symptoms in adults, but in children and horses it can cause severe encephalitis. Equines may suddenly die or exhibit progressive central nervous system disorders. Infections with VEEV may present, in both humans and horses, as either encephalitic disease or as simply a febrile disease without profound neurologic signs. Horses may die after a very acute course, even without any neurologic signs, but mortality in humans is generally low. Young and immune compromised horses are most likely to develop clinical signs . It causThe epizootic VEE was initially limited to northern and western South America in Venezuela, Colombia, Ecuador, Peru and Trinidad, but the epizootics have been reported in years from 1969 to 1972 in Guatemala, Nicaragua, El Salvador, Honduras, Costa Rica, Belize, Mexico, and the United States of America due to variant 1-AB. Epizootics caused by I-AB or I-C virus have not occurred in North America and Mexico after 1972. Equine and human epizootic VEE viruses were subtype 1-C from Venezuela in 1993, 1995 and 1996 and Colombia in 1995 . In 1960Treatment of viral encephalitis is supportive, as there are no specific antiviral therapies. The two VEE vaccines, a modified-live vaccine (TC-83) and an inactivated adjuvant vaccine, have been used in field. Horses were vaccinated with TC-83 vaccine during outbreak in Mexico and Texas in 1971 as equine vaccine was not available but it is still in use for humans working with VEE. Formalin-inactivated virulent VEE virus vaccines are not recommended for use in equids due to risk of residual virulence .1.6Alphavirus of the family Togaviridae which is maintained between birds and mosquitoes, occasionally causing disease in humans and equids [Culex and Culiseta. It is a recombinant between Sindbis and Eastern equine encephalitis like viruses [Culex tarsalis mosquitoes and birds. WEE has several subtypes consisting Sindbis, Aura, Ft. Morgan and Y 62–33. WEEV previously isolated in the south and eastern USA has been shown to belong to the HJ virus serogroup.Western equine encephalitis (WEE) is an uncommon viral illness of horses and human. WEE virus (WEEV) is an d equids , 136. Th viruses -139. It  viruses . WEEV isHorses and humans are often referred to as “dead-end” hosts as the virus does not build to high enough levels in blood to infect other mosquitoes . Most pee.g., sudden onset of fever, headache, nausea, vomiting, anorexia and malaise [Geographically, WEEV exists throughout uine deaths were estimated in central America and northern portions of South America, Mexico and Canada. In the US, WEEV exist in the western two third of the country. Outbreaks of the disease have been recorded since 1847. In 1930 about 6000 horses and mules were infected leading to about 50% mortalities in California. The largest epidemic was recorded in 1937 and 1938 in USA and Canada. In 1938 outbreak an estimated 264000 equids were infected with a morbidity of 21.4% . In the  malaise . In someIn horses, infections with WEEV begin with fever, inappetence and lethargy, progressing to various degrees of excitability and then drowsiness, ultimately leading to paresis, seizures and coma in 5-10 day course of the disease . The WEEThere is no treatment for WEE other than supportive care. Formalin-inactivated whole viral vaccines for EEE, WEE, and VEE are commercially available in mono-, bi-, or trivalent form. Previously non vaccinated adult horses require booster. For adult horses in temperate climates, an annual vaccine within 4 wk of the start of the arbovirus season is recommended. However, for horses that travel between areas affected by the virus, 2 or even 3 times vaccination in a year is recommended. Mares should be vaccinated 3–4 wk before foaling to induce colostral antibody.1.7Flavivirus of the family Flaviviridae [Ardeidae (herons and egrets) are natural maintenance reservoir and generate high viraemias upon infection without clinical disease. The disease is transmitted principally by Culex spp. (Culex tritaeniorhynchus) as it has a wide host range including birds, horses, swine and humans. Pigs act as important amplifiers of the virus producing high viraemias [Japanese encephalitis (JE) is caused by JE virus belonging to genus iviridae . There iiviridae . The viriviridae , 147. Itiviridae , 149. JEiviridae , 151. Hoiraemias , 153. Otiraemias .5). In horses three syndromic manifestations have been described. Transitory type syndrome, lethargic type syndrome and hyper-excitability type syndrome [Sporadic clinical cases of JE in horses have been reported in various countries including Japan , Hong Kosyndrome . The symsyndrome .In human beings, most infections are asymptomatic but can manifest as severe encephalitis, and with neurological sequelae in survivors. Japanese encephalitis tends to be a childhood disease in endemic areas but all ages can be affected in a naive population. As per the estimates 68000 clinical cases occur globally each year, with approximately 13600 to 20400 deaths . The fir1.8Lyssavirus, family Rhabdoviridae. The disease is important being zoonotic and highly fatal. All mammals are susceptible, but some species like dogs, jackals, coyotes, wolves, foxes, skunks, mongooses, and raccoons and bats act as reservoir hosts. Rabies cases have been reported across the globe in more than 150 countries. The worldwide cases of human rabies are estimated to be 55,000 or more every year with annual human mortalities of about 31,000 and 24,000 in Asia and Africa, respectively [Rabies is caused by the rabies virus, a neurotropic virus in the genus ectively , 175. A Rabies is relatively rare in horses and usually less than 100 cases are reported in the United States every year , 177. A Exposure to rabies is less common in people handling equines compared to small animals or wildlife. Vaccines are available for prevention of the disease in horses but there has been documented evidence of illness even in vaccinated horses . For pre1.9Equine influenza is historically not known to affect humans but many scientists have mixed opinions. Xie and co-workers  have revEquine viral diseases though restricted to certain geographical areas have huge impact on equine and human health. Many of these diseases have complex life cycles and require vectors for spread, while others can be contracted directly through aerosols. In some cases silent carriers like bats, pigs and passerine birds can contribute towards amplification of organisms and their spread to newer territories. A number of zoonotic diseases like West Nile fever, Hendra, VS, VEE, EEE, JE, Rabies have the potential for spread and ability to cause disease in human. Equine influenza is historically not known to affect humans but some scientists could find considerable experimental and observational evidence where H3N8 influenza virus has infected man. Despite our pursuit of understanding the complexity of the vector-host-pathogen mediating disease transmission, it is not possible to make generalized predictions concerning the degree of impact of disease emergence. The complex character and non-straightway behaviour of the human-natural systems in which host-pathogen systems are nested make specific prevalence of disease emergence or epidemics inherently difficult to predict. A targeted, multidisciplinary effort is required to understand the risk factors for zoonosis and apply the interventions necessary to control it.


Generally, it is difficult to observe IAV particles in postmortem samples from patients with seasonal influenza. In fact, only a few viral antigens are detected in bronchial epithelial cells from autopsied lung sections. Previously, we detected many viral antigens in AEC-IIs from the lung. This was because the majority of A/H1N1/pdm09 in the lung tissue harbored an aspartic acid-to-glycine substitution at position 222 (D222G) of the hemagglutinin protein. A/H1N1/pdm09 harboring the D222G substitution has a receptor-binding preference for α-2,3-linked sialic acids expressed on human AECs and infects them in the same way as H5N1 and H7N9 avian IAVs. Here, we report the first successful observation of virus particles, not only in AEC-IIs, but also in Ms/Mϕs and Neus, using electron microscopy. The finding of a M/Mϕ harboring numerous virus particles within vesicles and at the cell surface suggests that Ms/Mϕs are involved in the pathogenesis of IAV primary pneumonia. A/H1N1 2009 pandemic influenza virus (A/H1N1/pdm09) was first identified as a novel pandemic influenza A virus (IAV) in 2009. Previously, we reported that many viral antigens were detected in type II alveolar epithelial cells (AEC-IIs) within autopsied lung tissue from a patient with A/H1N1/pdm09 pneumonia. It is important to identify the association between the virus and host cells to elucidate the pathogenesis of IAV pneumonia. To investigate the distribution of virus particles and morphological changes in host cells, the autopsied lung specimens from this patient were examined using transmission electron microscopy (TEM) and a novel scanning electron microscopy (SEM) method. We focused on AEC-IIs as viral antigen-positive cells and on monocytes/macrophages (Ms/Mϕs) and neutrophils (Neus) as innate immune cells. We identified virus particles and intranuclear dense tubules, which are associated with matrix 1 (M1) proteins from IAV. Large-scale two-dimensional observation was enabled by digitally “stitching” together contiguous SEM images. A single whole-cell analysis using a serial section array (SSA)-SEM identified virus particles in vesicles within the cytoplasm and/or around the surfaces of AEC-IIs, Ms/Mϕs, and Neus; however, intranuclear dense tubules were found only in AEC-IIs. Computer-assisted processing of SSA-SEM images from each cell type enabled three-dimensional (3D) modeling of the distribution of virus particles within an ACE-II, a M/Mϕ, and a Neu.IMPORTANCE Generally, it is difficult to observe IAV particles in postmortem samples from patients with seasonal influenza. In fact, only a few viral antigens are detected in bronchial epithelial cells from autopsied lung sections. Previously, we detected many viral antigens in AEC-IIs from the lung. This was because the majority of A/H1N1/pdm09 in the lung tissue harbored an aspartic acid-to-glycine substitution at position 222 (D222G) of the hemagglutinin protein. A/H1N1/pdm09 harboring the D222G substitution has a receptor-binding preference for α-2,3-linked sialic acids expressed on human AECs and infects them in the same way as H5N1 and H7N9 avian IAVs. Here, we report the first successful observation of virus particles, not only in AEC-IIs, but also in Ms/Mϕs and Neus, using electron microscopy. The finding of a M/Mϕ harboring numerous virus particles within vesicles and at the cell surface suggests that Ms/Mϕs are involved in the pathogenesis of IAV primary pneumonia. Influenza A viruses (IAVs) are enveloped viruses that contain a segmented negative-strand RNA genome. IAVs express the spike glycoproteins hemagglutinin (HA) and neuraminidase (NA) on the viral surface , 2. HA p5–5–Previously, we reported a fatal case of A/H1N1/pdm09 infection . The resHere, we analyzed the same lung specimens using a novel scanning electron microscopy (SEM) method in addition to TEM . Whereas16–Most of the AEC-IIs observed in the lung tissue specimen prepared for electron microscopy (EM) observation were desquamated from the basement membrane; 50 TEM images of AEC-IIs were analyzed. Virus particles were found in the alveolar space and around AEC-IIs  and c. T16–Ms/Mϕs were less common than AEC-IIs; therefore, 30 TEM images of Ms/Mϕs were analyzed. In four of them, several filamentous and spherical virus particles were observed within cytoplasmic vesicles . The meaNeus were present in numbers similar to those of Ms/Mϕs; therefore, 30 TEM images of Neus were analyzed. In three of them, Neus were closely associated with desquamated and degenerated AEC-IIs, suggesting the occurrence of phagocytosis . Six imaAn entire alveolus is too large  to be presented in cross-section in a single TEM or SEM image. Therefore, we built a set of contiguous SEM images and digitally stitched them together to generate a cross-section of an entire alveolus at high resolution. Briefly, a single 200-nm-thick section was mounted on a glass slide and stained with toluidine blue to facilitate selection of an appropriate region by light microscopy. The section was then stained with heavy metals, thereby enabling a 10-by-14 array of contiguous SEM images to be taken and digitally stitched together . In the SSA-SEM analysis revealed the distribution of specific structure throughout a single cell. Briefly, 100 serial sections (each 200 nm thick) were mounted on a platinum-palladium-coated glass slide and stained with toluidine blue. Then, sections containing the object cells were marked in yellow. Next, each marked serial section was captured by SEM after heavy metal staining . Six AECSix randomly selected AEC-IIs were analyzed by SSA-SEM. All were desquamated from the basement membrane. Virus particles were observed around the surfaces of three of six AEC-IIs . IntraveSix randomly selected Ms/Mϕs were analyzed by SSA-SEM  and 3. MAmong the 100 serial sections used for SSA-SEM imaging, Neus were less common than Ms/Mϕs. Thus, three randomly selected Neus were analyzed by SSA-SEM  and 3. VSpecific structures, including cytoplasm (light blue), nucleus (purple), virus particles within the cell (green), and virus particles outside the cell (red), in each SSA-SEM image from serial sections were traced manually . All SSAEM enables visualization and identification of the morphology of virus particles, although it has a limited observable range and can provide only partial observation of large-scale structures. Most EM images of influenza virus particles have been captured using infected cultured cells –25. Seve–26–In this study, we focused on AEC-IIs, Ms/Mϕs, and Neus and identified influenza virus particles and intranuclear dense tubules  in each cell type –20. The –In vitro IAV infection of human Ms/Mϕs results in productive infection and release of infectious particles . Unfortunately, we have no remaining samples available for immuno-EM studies. Since the specimens used for EM had been fixed with glutaraldehyde and osmium, their antigenicity has been destroyed. However, we point out that this is a common limitation of previous studies reporting EM images of influenza virus particles in human tissue , 26–29. 26–Despite being based on a single human case, these viruses were observed in postmortem influenza virus-infected human tissues. Conducting studies using postmortem biopsied lung tissue that is appropriate for image analysis of viral particles is extremely difficult. Thus, previous EM analyses of influenza viruses were conducted mainly in animal models or cultured cells. Despite the difficulties, we succeeded in obtaining clear images of the virus in human lung tissue.Additionally, the study used novel experimental methods that are unprecedented in the field of influenza research. First, we successfully obtained EM images of an entire alveolus, which can be viewed at low and high magnification, by stitching together SEM images. This technique enabled visualization of both virus particles and intranuclear dense tubules (M1 proteins) in a single EM image. Second, 3D reconstruction of SSA-SEM images of an AEC-II, a M/Mϕ, and a Neu revealed virus distribution within a single whole cell. Thus, the analysis provides insight that could not be obtained by conventional 2D imaging.This is the first report of the use of novel SEM methods to examine the distribution of IAV particles throughout a single whole cell in sections from an autopsy specimen. The differences between AEC-IIs, Ms/Mϕs, and Neus in terms of distribution of virus particles and virus-related structures suggest that each cell plays a different role in A/H1N1/pdm09 pneumonia.The subject of the study was a 33-year-old man who died of respiratory failure resulting from A/H1N1/pdm09 pneumonia complicated by development of ARDS on the 7th day after onset of disease. An autopsy was carried out 3 h after death. The detailed pathological and molecular-biological findings have been reported –11. This–To prepare the specimens for EM, small parts of the lung tissue were excised and prefixed with 2% glutaraldehyde and 2.5% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 2 h at 4°C and then postfixed in 1% osmium tetroxide, dehydrated through a graded series of alcohols and propylene oxide, and embedded in epoxy resin. Other lung tissue specimens were routinely fixed in 10% buffered formalin and embedded in paraffin to prepare them for optical microscopy. Hematoxylin-eosin staining was performed for histological analysis, and immunohistochemistry was performed to evaluate the IAV nucleoprotein antigen, as previously described , 10, 11.After trimming the epoxy resin-embedded tissue, ultrathin sections 70 nm thick were cut using an ultramicrotome , mounted on grids, and stained with 4% uranyl acetate and lead citrate for TEM using an H-7650 or an HT7700 . For SEM, ultrathin sections (200 nm) were cut using an ultramicrotome fitted with a histo-Jumbo diamond knife . Serial sections were placed on platinum-palladium-coated  glass slides and allowed to adhere at 55°C for 30 min. The sections were stained with 1% toluidine blue for light microscopy and scanned with a NanoZoomer 2.0-RS digital slide scanner . The observation regions were selected using NDP.view2 viewing software . The sections were stained with 1% uranyl acetate and lead citrate and observed by ultra-high-resolution field emission SEM . Regions of the ultrathin sections were imaged using backscattered electrons with an accelerating voltage of 1.5 kV. Images were taken at magnifications of ×7,000 to ×9,000, with a working distance of 3 mm. The workflows are shown in https://imagej.net/Image_Stitching) and viewed with the NDP.view2 viewing software. The workflows are shown in To observe a whole cross-section of one alveolus, a 10-by-14 array of images was taken under the following conditions: accelerating voltage, 1.5 kV; working distance, 3 mm; magnification, ×5,000. After acquisition, the images were automatically aligned using the image stitching plugin in Fiji/Image J software (https://imagej.net/StackReg). The aligned images were carefully checked visually and corrected manually if necessary. Subsequently, they were imported into Photoshop software , and the cytoplasm, nucleus, and viruses were manually selected by tracing their boundaries. After identification of the target structures in the serial images, 3D surface-rendering reconstruction was performed in 3D Slicer (https://www.slicer.org/).Serial section images were aligned automatically using the StackReg plugin in Fiji/Image J (