Figura 1. Normal lung tissue in Rats Non infected by RCOV. The monoclonal IgG to RCOV was negative i...

Figura 2. Intense inflammation interstice, with epithelial detachment in Rat with COV infection 10 X...

Figura 3. Immunohistochemical of lung of rats, with IgG monoclonal anti RCOV, demonstrated with red ...

Figura 4. HE technique x 10. Thrombosis in lumen of lung venous marked with T. Micro Thrombus in lun...

Figura 5. The serum C3 (complement) levels in randomized RCOV rats, compared with non - infected RCO...

Figura 6. The phagocytic levels in randomized infected RCOV rats compared with non – infected RCOV...

Figura 7. Spontaneous hyper-phagocytosis and auto-phagocytosis of red blood in Rats infected by RCOV...

Figura 8. TNFα levels in RCOV infected and non-infected rats, demonstrated by ELISA p= 0.0027.

Figura 9. IgM levels determined by radial immune- diffusion in infected and in non-infected rats wit...

Figura 10. IgG levels determined by radial immunodiffusion technique in Infected and non-infected rat...

Figura 11. IgA levels determined by radial immunodiffusion in infected a non-infected rats with RCOV,...

Figura 12. Comparative Lymphocytes proliferative levels with concanavalin A and phytohemagglutinin st...

Introduction

Viruses that infect the lung are a significant cause of morbidity and mortality in animals and humans worldwide. Coronaviruses are being increasingly associated with severe diseases in the lower respiratory tract [1-3].

The coronavirus of humans and animals are increasingly being recognized as significant pathogens in the lower respiratory tract [3-5]. These viruses which infect not only man but cattle, pigs, rodents, cats, dogs and birds as well some are serious veterinary pathogens, specially chickens and rats in experimental animals [6].

2003 SARS-COV emerged from wildlife to cause a high level of human epidemic fatalities [1-5]. In animals, it appears to be restricted to rats; rats of all ages are susceptible.

The primary route of transmission appears to be respiratory. The virus spread quickly by aerosol or contact exposure. The virus is excreted from salivary glands for about 7 days after primary infection. It has not been detected in urine or faces as well as in intrauterine transmission. The residual chronic respiratory disease

due to corona rat virus infection remains an important disease of laboratory rats [6-11].

The association among these newly identified coronaviruses with a wide range of respiratory diseases, compromised from mild upper respiratory tract infections to severe pneumonia [3-12]. Acute lesions are present in serous and mixed salivary glands, lachrymal glands and the respiratory tract [9]. Infection begins with mild to severe necrotizing rhinotracheitis, which may be followed by focal interstitial pneumonitis [6-11].

Primary epithelial cell cultures derived from conducting airways have been studied as targets for several respiratory viruses including SARS-COV [12-22]. It is also important to understand the role of alveolar epithelial cells in initiating a regulating local immune response to viral infection in the alveoli through the expression of cytokines and chemokines [12-15,17-18,21-22].

Corona virus infection is often associated with the following clinical symptoms: inappetence, weight loss, sneezing, dyspnoea, and fatalities [1-5,23-24]. Viral antigen can be demonstrated in affected tissues by immunochemical methods during the first week of infection. Virus isolations are feasible, but not usually necessary [5,23-26].

Serum antibody to rat coronavirus can be detected one week post infection [6-11].

It is generally accepted that coronavirus infection precedes bacterial infections, with a synergism action in respiratory manifestations [6]. The immune spectrum of severe acute respiratory syndrome in rats is poorly understood [25-26].

The immunological response of animals presented several different steps from innate to acquired specific humoral and cellular reaction against RCOV.

This progression was naturally associated with respiratory symptoms and presence of specific IgG to Co V [8-10].

The other part of immune response was not highly investigated in order to explain the rest of pathological findings in the respiratory system [8-11,26].

The purpose of this work was to study the dynamics of the immune spectrum from innate to specific humoral and cellular profile in spontaneous respiratory corona virus infection trough nasal cavity in Wistar rats.

Material and methods

We had studied 80, four month–old female, Wistar rats with spontaneous coronavirus infections and respiratory manifestations and compared to 14 healthy Wistar rats, matching in age and sex. The work was performed in the period from 1998 to 2004.

All animals were controlled in respiratory symptoms as well as in clinical evolution. All animals with respiratory manifestations and controls were finally studied by autopsy. All of the animals were collected for the light microscopic evaluation after autopsy. Tissues were routinely fixed by immersion in 10% neutral buffered formalin. The lung and trachea were removed immediately at necropsy and fixed by intratracheal perfusion of formalin prior to immersing the lung in this fixative.

Tissues for light microscopy were embedded in paraffin, sectionized at 4-6 µm and stained with haematoxylin eosin [26].. All tissue sections were examined as unknowns and the tissues were graded on a scale of 0= no lesions; 1= minimal lesions; 2= moderate lesions; 3 = marked lesions as previously described [26].

These rats with and without respiratory symptoms, were studied in: Specific IgG to rat coronavirus by ELISA Xpress Bio – USA according with manufacturers instruction in all animals with respiratory disease (n: 80) and controls (n: 14). We separated by randomization the following studies: Lung histopathological study by monoclonal anti Co V (C7901-26 corona virus Mab Mox, purified 100 ug US Biological – USA) and biotin – streptavidin alkaline phosphatase label – fast red (Biogenex - USA) according with the laboratory instructions [26-29].

This procedure was performed by randomization in animals #: 1, 4,10, 15, 21, 25, 34, 37, 45, 56, 63, 68, 72, 77, and compared with control rats (n: 10) from # 1 to 10. All studied animals were examined as unknowns and tissues positive foci indicating viral antigen were recorded.

Serum complement C3 fraction determination (Biocientífica – Argentina) were performed by single radial immune diffusion, following the laboratory instructions in selected animals by randomization (n: 14): # 3, 6,12,15, 24, 27, 30, 34, 42, 45, 56, 60, 62 and compared with normal healthy control rats (n: 10, from rats # 1 to 10.

Phagocyte function was studied following the Björktein / Quie procedure [30] and Campbell et al. [31]. Briefly, the heparinized blood of rats was sedimented 1 hour in 1% dextran in saline. The next step was slow centrifugation and the leucocytes obtained was washed and counted, 5 x 106 PMN, next drop 0,1 ml of bacterial suspension (Staphylococcus aureus) and 0,4 ml of opsonin in 0,5 ml of PMN, next incubation 37°C and turntable at 20 rpm and finally the number of bacteria colonies was counted inside the PMN.

The S. aureus was intake by the CD 35 (CR1) complement receptor. Normal values for S. aureus are from 8 to 12 colonies per cell.

The phagocyte determinations were performed per triplicate by randomization in rats with RCOV infections (n: 14) #: 2, 4, 11, 16, 20, 35, 41, 50, 58, 61, 67, 72, 77, 80 and compared with controls (n: 10) from # 5 to 14 rats.

Dosage of rat TNF α ELISA kit (R&D) was performed following the laboratory instructions The results were expressed in pg/ml and the sensitivity of procedure was from 4,1 pg/ml to 1000 pg/ml. The determination serum TNF α was performed by randomization in (n: 10) animals with RCOV infection, # 3,6,9,12,15, 18, 42, 45, 47, 71 and control group (n: 10) from # 1 to 10.

IgM, IgG, IgA assays (Biocientífica – Argentina) were performed by single radial immune diffusion, following the manufacturer instructions, in selected animals by randomization (n: 14): # 3, 6,12,15, 24, 27, 30,34, 42, 45, 56, 60, 62, 71 and compared with normal healthy control rats (n: 10), from rats # 1 to 10.

Lymphocyte culture was performed as previously described [32], without and within stimulation with Con A and PHA (Sigma) in randomized selection of rats with RCOV infection (n:11), animal # 1, 5, 13, 17, 21, 25, 37, 41, 64, 70, 74 and compared with control Group (n: 10) from # 1 to10 animals. Assays to DNA synthesis was measured as incorporation of {3H} Thymidine (40 Ci/mM) and assayed at 48 hs of culture. The isotope was added 2 hr before harvest in 50 μl RPMI 1640 to a final concentration of 2 μCi/ml. Incorporation of isotope was measured as number of cpm on stimulated and non-stimulated culture.

Statistical analysis

All the results are expressed as mean and standard deviation (SD). Because of the small population, nonparametric tests were used. Statistical comparisons were performed using unpaired T test and F tests to compare variances, ANOVA with Burlett´s correction and Tukey multiple comparison (for 3 or more groups of comparisons) (Grafpad), Prisma 5, CA – USA). We considered p< 0.05 statistically significant.

Results

All animals with RCOV (n: 80) presented clinical manifestations of RCOV infection. This group presented at onset sialo-dacryoadenitis, following symptoms of respiratory difficult defined shortness of breath, fatigue, inappetence, loss weight when compared with the control group (Table1).

The RCOV infection presented specific IgG to RCOV in all cases of infected rats and negative in control group, p< 0,0001 (Table 2). The IgG positive by ELISA was confirmed by out-side laboratory in USA, NIH. 1999.

All RCOV infected animals presented in autopsy pneumonitis and the histopathological features of the bronchi were epithelial detachment and exudate, as well as in 9/80 rat (11%) presented pulmonary thromboembolism. Walls of alveoli presented intense inflammation associated to collagen deposition.

Those alterations represented the lung interstitial tissue compromised in different grades, when compared with the normal non infected rat (Table 3; Figures 1, 2, 4).

The selected randomized group of rats with RCOV infection presented positive specific IgG to Co V in lung tissues in all cases (n:10) and negative in controls (n:10) (Table 4; Figures 1, 2).

The serum complement C3 level was 555±22,01 mg% in RCOV rats and 164.5±15,29 mg% in controls, p< 0,0001 (Figure 5).

The phagocytic function was dramatically increased in RCOV infected rats when compared with controls, 36,50±4,8464 vs12,50±2,173 S. aureus/PMN, p< 0.0001 (Figures 6 y 7).

The serum TNF α levels was 308,9±40,94 pg/ml in RCOV infected group and 6,93±1,708 pg/ml in controls, p< 0,0001, and < 0,0001 with F test (Figure 8).

The total serum level of IgM was 154,5±6,308 mg% in RCOV infected rats and 128,6±3,086 mg% in controls, p=0,0001 (Mann Whitney test) (Figure 9).

The total serum level of IgG was 1442±64,71 mg% on RCOV infected rats and 1442±44,16 mg% on controls, NS (Figure 10).

The total serum IgA level was 14,71±0,673 mg% in RCOV animals and 121±11,37 mg% in controls, p< 0,0001 (Figure 11).

The lymphocyte cultures proliferation was different in these two groups, on RCOV infected rat group was similar from the basal to stimulated with PHA and Con A and in control group the stimulation induced intense mitogenic responses to PHA and Con A, p< 0,0001 (Figure 12).

Discussion

The study indicates that all RCOV infected rats presented predominately clinical respiratory symptoms expressed as: dyspnoea and fatigue as well as associated sialo-dacryo-adenitis. These findings were different from the normal control animals. The Rat COV was initially isolated from the salivary glands of with sialo-dacryoadenitis and porphyries [9].

Commonly the most frequent in adults form of RCOV infection was localized as a lesion of the upper respiratory tract [9].

However, in our study the lesions were present in the lung with different grades of intensity from mild to severe level of inflammatory responses.

This type of RCOV in our study was predominantly pneumotropic and produced interstitial pneumonia. Our finding was in agreement with several observations [7-8,10-11] and in special with Miura et al [12]. These last authors who describes the infection in suckling rats infected by RCOV presented more severe respiratory disease. RCOV was initially isolated from the lungs of asymptomatic rats. In contrast the infection RCOV are different from neonates to adults, while the group of young rat results in lethal interstitial pneumonia [10,12].

Viral infections in the lung are an important cause of morbidity and fatalities in human worldwide [1-5,23-24,27-28]. In order to design strategies for vaccination and therapy, we need to understand the molecular mechanism that onset and regulate effective antiviral immune responses in the lung [11,18,21-22,28,33].

In concordance with previous studies of animal models which in vitro mechanistic researches can be correlated with infections in vivo by the study performed with RCOV in rat alveolar epithelial cells as a model for the early events in respiratory coronavirus pathogenesis [12-20,33-34].

Intratracheal inoculation in adult rats with RCOV resulted in infection of the lung, primarily in alveolar type I cells [12,17-22,24,26,33-36].

Alveolar type I cells, which make up 95% of this surface area, are terminally differentiated in two functions: in gas exchange and fluid homeostasis [17]. Alveolar type II cells are dividing cells that trans-differentiate in vivo into the type I cell phenotype during the repair of damaged alveolar epithelium. Type II cells also produce surfactant proteins and lipids, which keep the alveoli expanded and function in innate defense of the lung [13-14].

Epithelial cells that line the respiratory tract initiate pulmonary inflammation in response to pollutants, allergens, or infectious agents [15].

Bronchial epithelial cells and alveolar type II cells express and secrete pro-inflammatory cytokines and chemokines in response to infection with respiratory viruses including respiratory syncytial virus, influenza A virus, and the SARS-associated coronavirus (SARS-COV) [12,19,22,33-35].

The RCOV infection caused transient weight loss and focal lesions with recruitment of neutrophils in the lung, followed by clearance of virus and probably resolution of inflammation [18-22]. Thus adult rats mounted an effective innate immune response to RCOV infection in the lung. In the cultures of differentiated rat alveolar type I cells, RCOV replicated and induced expression of neutrophil chemotactic chemokines (CXC) [12]. However, to now the role of alveolar type I cells in immune responses to viral infection is largely unknown [12]. Several strains of rat coronavirus (RCOV) have been isolated from the lungs of rats with clinical or subclinical manifestations [7-8,10-12].

Primary rat alveolar type II cells were transdifferentiated into the type I cell phenotype. Type I cells were productively infected with SDAV and RCOV-P, and both live virus and UV-inactivated virus induced mRNA and protein expression of three CXC chemokines: CINC-2, CINC-3, and LIX, which are neutrophil chemo-attractants [12,17-22,33-34]. Dual immunolabeling of type I cells for viral antigen and CXC chemokines showed that chemokines were expressed primarily by uninfected cells. Virus-induced chemokine expression was reduced by the IL-1 receptor antagonist, suggesting that IL-1 produced by infected cells induces uninfected cells to express chemokines. Primary cultures of alveolar epithelial cells are an important model for the early events in viral infection that lead to pulmonary inflammation [12,17-22,34].

In adult rats inoculated intra-nasally with RCOV-P, the virus replicates in the upper and lower respiratory tracts. Neutrophils are observed in the nasal cavity, trachea, and alveoli on days 2–5, followed by infiltration of mononuclear cells in the lung on day 5 after infection [12,33-38]. RCOV-P infection of adult rats causes interstitial pneumonia and focal edema in the alveoli, which resolves in day 8 after infection [21-22]. Virus-induced chemokine expression was reduced by IL-1 receptor antagonist, but not soluble TNF α receptor, indicating that IL-1 produced by infected cells may induce chemokine expression from the uninfected type I cells. These data suggest that alveolar type I cells can secrete chemokines in response to viral infection, which may lead to infiltration of neutrophils into the lung [12,16,18,20-22,33-38].

The RCOV depresses the IL 1 production as demonstrated by authors [12,35].

Our features of increment on the TNFα serum level were correlated with the acute pulmonary attack by neutrophils [16,37-38]. This last result was in agreement with different publications. These authors presented similar results in patients within and without SARS-COV infection when compared with our study [23-25].

The importance of the alveolar epithelium integrity is remarkable in the pathogenesis of lung fibrosis further implicated by the observation that epithelial cell growth factors protect against scarring in animal models. Our results were in agreement with these observations [39]. The core of the epithelial-mesenchymal hypothesis of pulmonary fibrosis is the contention that type II alveolar epithelial cells fail to repair the damaged epithelium as a result of ineffectual proliferation, migration, and/or differentiation, and this leads to interstitial scarring [39].

This ineffectual reconstitution of the epithelium is postulated to drive fibrosis by inducing the proliferation and differentiation of fibroblasts and the deposition of TypeII collagen [39].

On the other hand, our features presented intense hyper-phagocytic responses when compared the infected vs control rats. This finding is probably explained by the previous description of neutrophil chemotactic CXC chemokines expression by epithelial alveolar cells as well as the increase of production of the C 3 fraction of complement and the TNFα. These two proteins probably represented the acute phase proteins and the interactions between RCOV and alveolar epithelial cells that initiate and regulate the innate immune antiviral immunological responses in the lung [12,22,26,34].

The group of rats studied with immunohistochemical procedures presented, in all cases, positive specific IgG anti RCOV attached to alveolar epithelial cells and represented the link with RCOV to angiotensin Converting Enzyme Receptor 2 (ACE R2) has recently identified as functional cellular receptor for SARS-COV [40]. The RCOV is linked with this ACE R2 and probably regulates the inflammatory responses.

Studies of the tissue and cellular distribution of SARS-COV, and ACE2 protein expression, reveal new insights into the pathogenesis of this deadly disease [40].

ACE2 is expressed at high levels in the primary target cells of SARS-COV, pneumocytes and surface enterocytes of the small intestine. Despite the fact that SARS-COV can infect the lung and intestine, the tissue responses in these two organs are different. Notably, injection of SARS-COV Spike into mice worsens acute lung failure in vivo that can be attenuated by blocking the renin – angiotensin pathway [40].

These results provide a molecular explanation why SARS-COV infections cause severe and often lethal lung failure [40].

All other tissues and cell types expressing ACE2 may be potential targets of SARS-COV infection. Remarkably, endothelial cells, which express ACE2 to a high level, have not been shown to be infected by SARS-COV. There is also evidence that cell types without detectable ACE2 expression may also be infected Furthermore, studies in a new human cell culture model have indicated that the presence of ACE2 alone is not sufficient for maintaining viral infection. Therefore, other virus receptors or co-receptors may be required in different tissues [40].

Moreover, the interaction between SARS-COV and the immunological or lymphoid system remains to be defined.

Our study developed inside an immunology system presented in which the total IgM serum level was slightly increased in RCOV infected rats when compared with noninfected rats, this finding represented the first part of the onset of the specific immune viral response of B lymphocytes by IgM [41].

The IgG total serum level was similar in 2 groups, but the serum level of IgA was significantly different in these two groups. The RCOV infected rats decreased dramatically the IgA serum level when compared with the control group.This finding was probably explained by the viral action at the epithelial cell surface level on small bowels mucosa followed by subsequent lymphocyte T and B depression and the serum IgA fall [41].

Our lymphocyte culture study presented intense proliferative suppression in animals with RCOV infection when compared to rats without RCOV infection. These results are in accordance with previous studies with certain viruses including rat coronavirus have shown that inhibition or activation of mitogen-activated protein kinase (MAPK) was involved in induction or suppression of cytokines after virus infection [42].

The studies of this group showed that CD14 – positive monocytes were the leukocytes in SARS patients showing an increase in phospho-p38. The CD4-positive T cells from SARS patients appeared to have a suppressed intracellular phospho-p38; CD8 - positive T cells from SARS patients have similar findings, however both cells CD 4 /CD 8, have a significantly lower intracellular phospho-p38 level in this early stage [42].

The phospho-p38 expression in CD8 cells remained significantly suppressed in 2–3 weeks after admission, while those in CD14 and CD4 cells no longer had significant increase or decrease of phospho-p38 expression [42].

Results from this study, however, suggest that altered leukocyte p38 activation may contribute to abnormal blood cytokine profile in SARS patients. Study showed that an augmented p38 and activation in CD14 cells was associated with elevated IL-8 levels in SARS patients and neutrophilia, as well as cytokines storm.. Also, in agreement with these features, it is possible to explain the hyper-phagocytic responses in our animals with R COV infection [42].

Thus, further studies are needed to explore whether increase of p38 activation in monocytes, but decrease of p38 activation in CD 4 / CD 8 lymphocytes from SARS patients is really related to increase of immunosuppressive mediators or virus replication in the lung tissues.

Whether there is directly viral pneumonitis(virus cytotropic tissue damage) or indirectly immune-mediated tissue damage in SARS infections remains dark [42-46].

Our findings in lymphocyte culture depression were in agreement with these related works. Little is known about CD 4 + CD 8+ T cell immunity to the RCOV infection [11-23,42-46].

In conclusion, the tropism of RCOV for alveolar epithelial cells makes it an ideal model in which to study the mechanisms by which alveolar epithelial cells influence antiviral immune responses in special neutrophil and macrophage migration into the lungs and hyper phagocytic activity. All of these features were inside of innate immune responses and the final consequences, the fibrosis (3-4,12-15,25,34,39,47).

The other prominent findings were associated with specific depression of IgA serum levels as well as the activity of lymphocytes T demonstrated by lymphocyte cultures. It is clear that we are only at the dawn of our understanding of the pathogenesis of SARS (17-18,21-22,41-42).

The pulmonary thromboembolism is present in our findings in near of 11 % of animals with Coronavirus infection. In these selected rats the alteration of coagulation and lung embolism is probably explained by the cytokines storm and activation of the kinin system by the coronavirus, via ACE 2 receptor. (40,50) The dysregulates renin angiotensin aldosterone system, probably converts angiotensin II in angiotensin 1 – 7, this is associated with down regulation of ACE 2 activity – augmentation of ACE 2 signal and induction of pro- thrombotic pathway via proinflammatory cytokines and the oxidative stress (31,37,40,50-51).

On the other aspect is the dysregulation of innate response. The RCOV is influenced by the innate immunity, it plays a critical roll, as an early defense mechanism against microbial including RCOV and other. However, uncontrolled innate immune system response elicited by overactivated neutrophils, subsequent cytokine storm and TNF α cause thrombosis by various mechanisms (51). Our results presented high levels of C3 fraction of complement, hyper-phagocytic response and high level of TNF α, all of these in agreement with several authors (18,22,26,37,47-51).

1. Vijayanand P, Wilkins E, Woodhead M. Severe acute respiratory syndrome (SARS): a review. Clin Med. 2004; 4: 152 – 60.

2. Christian MD, Poutamen SM, Loufty MR, Muller MP, Low DE. Severe acute respiratory syndrome. Clin Infect Dis 2004; 38: 1420 – 7.

3. S M Peiris, S T Lai, L L M Poon, Y Guan, L Y C Yam, W Lim, J Nicholls, W K S Yee, W W Yan, M T Cheung, V C CCheng,K H Chan, D N C Tsang, R W H Yung, T K Ng, K Y Yuen, Coronavirus as a possible cause of severe acute respiratory syndrome THE LANCET • Published online April 8, 2003.

4. Rota PA, Oberste St, Monroe St S , Allan Nix W A Campagnoli R, Icenogle JP, Peñaranda S, Bankam B, Maher K, Min-hsin Chen MH l, Tong S, Tamin A, Lowell L, Frace M, DeRisi JL, Chen Q, Wanq D, Erdman DD, Peret TC, Burns C. Rollin PE, Sanchez A, Liffick S, Holloway B, Limor J, McCaustland K, Olsen-Rasmussen M, Fouchier R, Günther S, Osterhaus AD, Drosten C, Pallansch MA, Anderson LJ, Bellini WJ. Characterization of a Novel Coronavirus Associated with Severe Acute Respiratory Syndrome. Science, 2003;300: 1394 – 8.

5. Weiss SR, Navas-Martin S. Coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. Microbiol Mol Biol Rev. 2005; 69:635–64.

6. Shunck MK, Percy DH, SØren R . Effect of time of Exposure to Rat Coronavirus and Mycoplasma pulmonis or Respiratory Tract lesions in Wistar Rats. Can J Vet Res 1995; 59: 60 – 6.

7. Parker JC, Cross SS, Rowe WP. Rat coronavirus (RCV): a prevalent, naturally occurring pneumotropic virus of rats. Arch Gesamte Virusforsch 1970;31:293–302.

8. Percy DH, Williams KL. Experimental Parker’s coronavirus infection in Wistar rats. Lab Anim Sci 1990; 40:603-7.

9. Weir EC, Jacoby RO, Paturzo FX, Johnson EA. Infection of SDAV-immune rats with SDAV and rat coronavirus. Lab Anim Sci 1990; 40:363-6.

10. Watts D.M,. Peters C.J, Newman, P. . Wang N, Yoshikawa N., Tseng C.K, and. Wyde P.R. Evaluation of Cotton Rats as a Model for Severe Acute Respiratory Syndrome. Vector Borne Zoonotic Dis. 2008; 8: 339–44.

11. Compton SR, Smith AL, Gaertner DJ. Comparison of the pathogenicity in rats of rat coronaviruses of different neutralization groups. Lab Anim Sci. 1999;49:514–8.

12. T A. Miura, J Wang, K Holmes, and R J. MasonRat coronaviruses infect rat alveolar type I epithelial cells and induce expression of CXC chemokines. Virology, 2007;369:288-298.

13. Mason RJ, Lewis MC, Edeen KE, McCormick-Shannon K, Nielsen LD, Shannon JM. Maintenance of surfactant protein A and D secretion by rat alveolar type II cells in vitro. Am J Physiol Lung Cell Mol Physiol. 2002;282:249–58.

14. Mason RJ. Biology of alveolar type II cells. Respirology. 2006; 11: S12–5.

15. Martin LD, Rochelle LG, Fischer BM, Krunkosky TM, Adler KB. Airway epithelium as an effector of inflammation: molecular regulation of secondary mediators. Eur Respir J. 1997;10: 2139–46.

16. Wang SZ, Xu H, Wraith A, Bowden JJ, Alpers JH, Forsyth KD. Neutrophils induce damage to respiratory epithelial cells infected with respiratory syncytial virus. Eur Respir J. 1998;12:612-8.

17. Williams MC. Alveolar type I cells: molecular phenotype and development. Annu Rev Physiol. 2003; 65: 669–95.

18. Yen YT, Liao F, Hsiao CH, Kao CL, Chen YC, Wu-Hsieh BA. Modeling the early events of severe acute respiratory syndrome coronavirus infection in vitro. J Virol. 2006;80:2684–93

19. Vanderbilt JN, Mager EM, Allen L, Sawa T, Wiener-Kronish J, Gonzalez R, Dobbs LG. CXC chemokines and their receptors are expressed in type II cells and upregulated following lung injury. Am J Respir Cell Mol Biol. 2003;29:661–8.

20. Gonzalez R, Yang YH, Griffin C, Allen L, Tigue Z, Dobbs L. Freshly isolated rat alveolar type I cells, type II cells, and cultured type II cells have distinct molecular phenotypes. Am J Physiol Lung Cell Mol Physiol. 2005;288:179–189.

21. Borok Z, Hami A, Danto SI, Zabski SM, Crandall ED. Rat serum inhibits progression of alveolar epithelial cells toward the type I cell phenotype in vitro. Am J Respir Cell Mol Biol. 1995;12:50-5.

22. Miura TA, HolmesK V. Host-pathogen interactions during coronavirus infection of primary alveolar epithelial cells. J. Leukoc. Biol. 2009; 86: 1145– 51.

23. Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, Emery S, Tong S, Urbani C, Comer JA, Lim W, Rollin PE, Dowell SF, Ling AE, Humphrey CD, Shieh WJ, Guarner J, Paddock CD, Rota P, Fields B, DeRisi J, Yang JY, Cox N, Hughes JM, LeDuc JW, Bellini WJ, Anderson LJ. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med. 2003;348:1953–66.

24. Todar K. Severe Acute Respiratory Syndrome (SARS) The Microbial World Lectures in Microbiology by Kenneth Todar PhD University of Wisconsin-Madison Department of Bacteriology 2007.

25. Hawkins R. Preparing the Biochemistry Laboratory for the Next Outbreak: Lessons from SARS in Singapore. Clin Biochem Rev. 2005; 26: 59 – 64.

26. Bihun CGD, Percy DH : Coronavirus Infection in the Laboratory Rat: Degree of Cross Protection Following Immunization with Heterologus Strain. Can J Vet Res. 1994: 58: 224 – 9.

27. Weiss SR, Leibowitz JLCoronavirus pathogenesis.Adv Virus Res. 2011;81:85-164.

28. Ng EK, Lo YM. Molecular diagnosis of severe acute respiratory syndrome. Methods Mol Biol. 2006; 336:163-75.

29. Lio P, Golman N. Phylogenomics and bioinformatics of SARS-CoV. Trend in Microbiology, 2004 12: 106 – 11 .

30. Björsten B, Quie PG. Abnormalities of circulating phagocyte function. Recent advances in clinical immunology. Thompson editor. Churchill Livingstone –Edinburgh, 1977 ; pp181 – 202 .

31. Campbell D E, Douglas St D, Phagocytic Cell Functions, Oxidation and Chemotaxis. Manual of Clinical Laboratory Immunology. N Rose, Conway de Macario E, Folds J D, Lane HC, Nakamura RM. ASM press Washington.1997; pp 320 – 8 .

32. Fletcher M. A, Urban R G, Asthana D, Wallin J, Friedlander A, Page J B. Lymphocyte Proliferation. Manual of Clinical Laboratory Immunology. N Rose, Conway de Macario E, Folds J D, Lane HC, Nakamura RM. ASM press Washington.1997; pp 313 – 19.

33. Roberts, A, Lamirande, EW, Vogel, L, Jackson, JP, Paddock, CD, Guarner, J, Zaki, SR, Sheahan, T, Baric, R, Subbarao, K. Animal models and vaccines for SARS CoV infection. Virus Res 2008; 133: 20–32.

34. Funk C. J, Manzer R,. Miura T A, Groshong S D, Ito Y, Travanty E A, Leete J, Holmes K V, . Mason R J Rat respiratory coronavirus infection: replication in airway and alveolar epithelial cells and the innate immune response. J General Virology. 2009; 90, 2956 – 64.

35. Law AH, Lee DC, Cheung BK, Yim HC, Lau AS. Role for nonstructural protein 1 of severe acute respiratory syndrome coronavirus in chemokine dysregulation. J Virol. 2007;81:416–22.

36. Roberts A, Deming D, Paddock CD, Cheng A, Yount B, Vogel L, Herman BD, Sheahan T, Heise M, Genrich GL, Zaki SR, Baric R, Subbarao K. A Mouse-Adapted SARS-Coronavirus Causes Disease and Mortality in BALB/c Mice. PLoS Pathog. 2007;3(1):e5-Published online 2007 January 12. doi: 10.1371/journal.ppat.0030005

37. Gagneten S, Scanga CA, Dveksler GS, Beauchemin N, Percy D, Holmes KV. Attachment glycoproteins and receptor specificity of rat coronaviruses. Lab Anim Sci. 1996;46:159–66.

38. Chang YJ, Liu CY, Chiang BL, Chao YC, Chen CC. Induction of IL-8 release in lung cells via activator protein-1 by recombinant baculovirus displaying severe acute respiratory syndrome-coronavirus spike proteins: identification of two functional regions. J Immunol. 2004;173:7602–14.

39. H. Sisson, M Mendez, K Choi, N Subbotina, A Courey, A Cunningham , A Dave, JF. Engelhardt, X Liu , E. S. White, V. J. Thannickal1, B. B. Moore, P. J. Christensen, R H. Simon. Targeted Injury of Type II Alveolar Epithelial Cells Induces Pulmonary Fibrosis. Am J Respir Crit Care Med. 2010; 181. 254 – 63.

40. Tseng CT, Huang C, Newman P, Wang N, Narayanan K, Watts DM, Makino S, Packard MM, Zaki SR, Chan TS, Peters CJ. Severe acute respiratory syndrome coronavirus infection of mice transgenic for the human Angiotensin-converting enzyme 2 virus receptor. J Virol. 2007; 81: 1162–73.

41. Giordano M, Inmunidad por linfocitos B en Introducción a la Inmunología. Faiboin,Geffner. Panamericana Buenos Aires. 2011, pp 275 – 303.

42. Chen-Hsiang Lee, Rong-Fu Chen, Jien-Wei Liu, Wen-Tien Yeh, Jen-Chieh Chang, Po-Mai Liu, Hock-Liew Eng, Meng-Chih Lin, and Kuender D. YangAltered p38 Mitogen-Activated Protein Kinase Expression in Different Leukocytes with Increment of Immunosuppressive Mediators in Patients with Severe Acute Respiratory Syndrome. Journal of Immunology, 2004, 172: 7841–7847.

43. De Diego M L, Nieto-Torres J L,. Jiménez-GuardenJM, Regla-Nava JA, Alvárez E, Oliveros JC, Zhao J, C Fett C, Perlman S, Enjuanes L. Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Regulates Cell Stress Response and Apoptosis PLoSPathog. 2011 October; 7(10): e1002315 (On Line).

44. Chen JY, Chen WN, Poon KM, Zheng BJ, Lin X, Wang YX, Wen YM. Interaction between SARS-CoV helicase and a multifunctional cellular protein (Ddx5) revealed by yeast and mammalian cell two-hybrid systems.Arch Virol. 2009; 154: 507-12.

45. Cheng M, Chan CW, Cheung RC, Bikkavilli RK, Zhao Q, Au SW, Chan PK, Lee SS, Cheng G, Ho WK, Cheung WT.Cross-reactivity of antibody against SARS-coronavirus nucleocapsid protein with IL-11.Biochem Biophys Res Commun. 2005; 338:1654-60.

46. Liu Y, Zhang X Expression of Cellular Oncogene Bcl-x L Prevents Coronavirus-InducedCell Death and Converts Acute Infection to Persistent Infection in Progenitor Rat Oligodendrocytes JOURNAL OF VIROLOGY. 2005; 79: 47–56.

47. Glocker MT, Cabral H, Muiño JC. Lung features of Infection with Corona Virus in Wistar Rats. J Allergy Clinical Immunology, 2006, 117,2, abstract 390, pp S100.

48. Glocker MT, Cabral H, Muiño JC. Carta al editor sobre SARS COV 19. ARCH ARG Alergia e Inmunología Clínica. 2020, 51: 53-56.

49. Muiño JC, Gómez M. Coronavirus COVID 19 Aspectos sobresalientes de este virus en la pandemia que ha provocado. ARCH ARG Alergia e Inmunología Clínica. 2020, 51: 57 – 62.

50. Cossy Isasi S, Muiño JC SARS COVID 19 y AC2. Cumbres borrascosas, una relación estrecha, Escondida o Devastadora pero siempre profunda. ARCH ARG Alergia e Inmunología Clínica. 2020, 51: 69 – 82.

51. Haijiao Jing, Xiaoming Wu, MengqiXiang, Langjiao Liu, Valerie A. Novakovic, and Jialan Shi. Pathophysiological mechanisms of thrombosis in acute and long COVID-19 Front Immunol. 2022; 13: 992384.Published online 2022 Nov 16. doi:10.3389/fimmu.

Para descargar el PDF del artículo
Lung and Immunological features of rat corona virus infection

Haga click aquí