ATP1A1-mediated Src signaling inhibits coronavirus entry into host cells.

UNLABELLED: In addition to transporting ions, the multisubunit Na(+),K(+)-ATPase also functions by relaying cardiotonic steroid (CTS)-binding-induced signals into cells. In this study, we analyzed the role of Na(+),K(+)-ATPase and, in particular, of its ATP1A1 α subunit during coronavirus (CoV) infection. As controls, the vesicular stomatitis virus (VSV) and influenza A virus (IAV) were included. Using gene silencing, the ATP1A1 protein was shown to be critical for infection of cells with murine hepatitis virus (MHV), feline infectious peritonitis virus (FIPV), and VSV but not with IAV. Lack of ATP1A1 did not affect virus binding to host cells but resulted in inhibited entry of MHV and VSV. Consistently, nanomolar concentrations of the cardiotonic steroids ouabain and bufalin, which are known not to affect the transport function of Na(+),K(+)-ATPase, inhibited infection of cells with MHV, FIPV, Middle East respiratory syndrome (MERS)-CoV, and VSV, but not IAV, when the compounds were present during virus inoculation. Cardiotonic steroids were shown to inhibit entry of MHV at an early stage, resulting in accumulation of virions close to the cell surface and, as a consequence, in reduced fusion. In agreement with an early block in infection, the inhibition of VSV by CTSs could be bypassed by low-pH shock. Viral RNA replication was not affected when these compounds were added after virus entry. The antiviral effect of ouabain could be relieved by the addition of different Src kinase inhibitors, indicating that Src signaling mediated via ATP1A1 plays a crucial role in the inhibition of CoV and VSV infections. IMPORTANCE: Coronaviruses (CoVs) are important pathogens of animals and humans, as demonstrated by the recent emergence of new human CoVs of zoonotic origin. Antiviral drugs targeting CoV infections are lacking. In the present study, we show that the ATP1A1 subunit of Na(+),K(+)-ATPase, an ion transporter and signaling transducer, supports CoV infection. Targeting ATP1A1 either by gene silencing or by low concentrations of the ATP1A1-binding cardiotonic steroids ouabain and bufalin resulted in inhibition of infection with murine, feline, and MERS-CoVs at an early entry stage. Infection with the control virus VSV was also inhibited. Src signaling mediated by ATP1A1 was shown to play a crucial role in the inhibition of virus entry by ouabain and bufalin. These results suggest that targeting the Na(+),K(+)-ATPase using cardiotonic steroids, several of which are FDA-approved compounds, may be an attractive therapeutic approach against CoV and VSV infections.

The effects of coronavirus on human nasal ciliated respiratory epithelium.

Human coronavirus (HCoV) accounts for 15-30% of common colds, but only one case report has described the effect of a coronavirus infection, that was asymptomatic, on human respiratory epithelium. The authors examined the effects of infection with HCoV on ciliary structure and function in healthy volunteers infected by intranasal inoculation with HCoV 229E. A further four volunteers were sham infected with ultraviolet-inactivated virus. Immediately before inoculation (day 0) and 3 days later (day 3), ciliated epithelium was obtained by brushing the inferior nasal turbinate. Ciliary beat frequency was determined and beat pattern analysed for evidence of dyskinesia (0=normal, 3=severely dyskinetic) using digital high-speed video photography. Ciliary ultrastructure was examined by transmission electron microscopy. Symptom diaries were kept for the duration of the study. All subjects inoculated with HCoV, including the three who did not develop symptoms of an upper respiratory tract infection, had disruption of their respiratory epithelium on day 3. Although there was no difference in the mean ciliary beat frequency between day 0 (11.3 Hz (95% confidence interval (CI): 8.6-14.0) and day 3 (9.4 Hz (95% CI 7.2-11.6)), there was a significant increase (p<0.05) in the ciliary dyskinesia score between day 0 (0.2 (95% CI 0-0.5)) and day 3 (1.1 (95% CI 0.5-1.7). In sham-infected subjects, no differences in epithelial integrity, or ciliary structure and function were found between day 0 and day 3. Inoculation of healthy volunteers with human coronavirus caused disruption of the ciliated epithelium and ciliary dyskinesia. This is likely to impair mucociliary clearance. Damage to the respiratory epithelium, due to human coronavirus infection, may occur without overt clinical symptoms.

[Mating activity of male SHR rats infected with sialodacryoadenitis virus].

The male SHR rats infected with sialodacryoadenitis (SDA) virus did not show any abnormal mating activity. However, high mortality was seen in embryos of diseased dams after cage-mating with infected males on Day 4 to Day 11 postinoculation.

An experimental model for dilated cardiomyopathy after rabbit coronavirus infection.

A rabbit model for coronavirus-induced dilated cardiomyopathy is described. Acute rabbit coronavirus infection results in virus-induced myocarditis and congestive heart failure. Of the survivors of rabbit coronavirus infection, 41% had increased heart weight and heart weight-to-body weight ratios, biventricular dilation, myocyte hypertrophy, myocardial fibrosis, and myocarditis consistent with the development of dilated cardiomyopathy. These changes were also seen in the remaining 59% of the survivors, except that the degree of myocyte hypertrophy was reduced and only right ventricular dilation was present. In most survivors, myocarditis was usually mild (1-5 foci/transverse section), but in some cases it was severe (> 20 foci/transverse section). Interstitial and replacement fibrosis was more pronounced in the papillary muscles. These data suggest that rabbit coronavirus infection may progress to dilated cardiomyopathy.

Reproductive disorders in female rats infected with sialodacryoadenitis virus.

Effects of sialodacryoadenitis virus infection on the reproduction of Wistar and Sprague-Dawley (SD) rats were studied. Estrous cycle was considerably out of order in about 40% of infected rats of both strains, starting on days 4 to 20 postinfection and persisting for 2 to 12 days. Of Wistar and SD dams infected on day 0 of gestation about 30% and 10% of the fetuses were found dead, while only 4 and 3% of non-infected dams. In severely diseased Wistar and SD dams infected on day 15 of gestation, the offspring showed death rates of 57% and 13%, respectively, because of inadequate nursing.

IL-6 activity in feline infectious peritonitis.

Involvement of IL-6 in the development of vasculitis and polyclonal gammopathy in feline infectious peritonitis (FIP) was investigated, by using the proliferative responses of two IL-6-dependent murine hybridoma cell clones, B3B1 and MH60.BSF-2 cells. A significant IL-6 activity was found in sera and ascitic fluids of cats with FIP, whereas no IL-6 activity was detected in sera from healthy cats. In these FIP cats, IL-6 activity in ascitic fluids was significantly higher than that in sera. Peritoneal exudate cells from FIP cats were also found to release a high level of IL-6 to the culture supernatant. The ascitic IL-6 activity was eluted into the fractions corresponding to the m.w. of 30,000 to 40,000 in gel filtration, and into the fractions at the salt concentration from 0.2 to 0.3 M NaCl in anion exchange chromatography. The level of ascitic IL-6 activity was inversely correlated to serum albumin/globulin ratio in these FIP cats. These findings indicate that IL-6 accumulated in the ascites might leaked into the systemic circulation, and be linked to systemic alterations such as enhanced synthesis of Ig and acute phase proteins.

Selection of variants of avian infectious bronchitis virus showing tropism for different organs.

Avian infectious bronchitis virus strain Kagoshima-34 isolated from the kidneys of a chicken that died of nephrosis/nephritis lost its nephropathogenicity during intratracheal passage in SPF chickens. The resultant virus acquired stronger respirotropism but reduced tropism for kidneys. On the other hand strain Tottori-2 isolated from the trachea of a chicken suffering from severe respiratory disease did not lose its respirotropism after serial intravenous passage in SPF chickens. The serological properties of the passaged virus were investigated by virus neutralisation test. The antibody titres of both strains of virus fluctuated with progressive passage. The serological properties of the virus isolated from respiratory organs were not necessarily the same as those of the isolates made from the kidneys.

Reproductive disorders in female SHR rats infected with sialodacryoadenitis virus.

Effects of sialodacryoadenitis virus infection on the reproduction of female SHR rats were studied. The oestrous cycle was considerably perturbed in most infected rats, the perturbation was observed initially between Days 0 to 10 post infection and the effect persisted for 6 to 18 days. About half of the foetuses of dams infected on Day 0 of gestation were found dead while only 4% of the foetuses from non-infected dams were found dead. Five or six days after infection on Day 0 of gestation, some infected dams were shown to have metritis, and virus antigen was detectable within the endometrium as well as exudate cells. In dams infected on Day 5 or later of gestation and severely diseased, the offspring showed a low survival rate possibly because of inadequate nursing.

Influence of viral infections on body weight, survival, and tumor prevalence in Fischer 344/NCr rats on two-year studies.

Sendai virus (SV), pneumonia virus of mice (PVM), and rat coronavirus/sialodacryoadenitis virus (RCV/SDAV) were common viral infections of rats in the National Cancer Institute-National Toxicology Program (NCI-NTP) studies from 1977 to 1983. Influence of these viral infections on body weight, survival, and prevalences of spontaneous tumors in the F344/NCr rats of 28 diet control groups at five different laboratories were evaluated. Tumor prevalences evaluated in this investigation included the following: leukemia and tumors of the anterior pituitary, lungs, salivary glands and Harderian glands in both sexes; adrenal pheochromocytomas in male rats; and mammary tumors in female rats. SV and PVM but not RCV/SDAV infections were associated with significant (P less than 0.05) decreases in body weights of male and female rats. Male rat groups with PVM infection had a lower prevalence of leukemia and male rat groups with RCV/SDAV infection had a higher prevalence of anterior pituitary tumors than the corresponding uninfected groups. Female rat groups with SV infection had greater survival and a higher prevalence of lung tumors than groups without SV infection. However, none of the tumor prevalence and survival differences were statistically significant when interlaboratory variability and time-related effects were taken into account.

Neutral endopeptidase and neurogenic inflammation in rats with respiratory infections.

Neuropeptides such as substance P are implicated in inflammation mediated by sensory nerves (neurogenic inflammation), but the roles in disease of these peptides and the peptidases that degrade them are not understood. It is well established that inflammation is a prominent feature of several airway diseases, including viral infections, asthma, bronchitis, and cystic fibrosis. These diseases are characterized by cough, airway edema, and abnormal secretory and bronchoconstrictor responses, all of which can be elicited by substance P. The effects of substance P and other peptides that may be involved in inflammation are decreased by endogenous neutral endopeptidase (NEP; also called enkephalinase, EC 3.4.24.11), which is a peptidase that degrades substance P and other peptides. In the present study, we report that rats with histories of infections caused by common respiratory tract pathogens (parainfluenza virus type 1, rat corona-virus, and Mycoplasma pulmonis) not only have greater susceptibility to neurogenic inflammatory responses than do pathogen-free rats but also have a lower activity of NEP in the trachea. This reduction in NEP activity may cause the increased susceptibility to neurogenic inflammation by allowing higher concentrations of substance P to reach tachykinin receptors in the trachea. Thus decreased NEP activity may exacerbate some of the pathological responses in animals with respiratory tract infections.

Changes in human nasal mucosa during experimental coronavirus common colds.

Twenty-four adult volunteers were inoculated with nasal drops containing a coronavirus of 229E serotype to determine the differences in the clinical and physiological reactions which occur between clinically infected, sub-clinically infected and non-infected individuals. Thirteen volunteers were clinically infected, 8 had sub-clinical infections and 3 were uninfected. Nasal airway resistance and the temperature of the nasal mucosa increased in all infected subjects both with and without symptoms: the core temperature increased also but to a lesser extent. Mucosal blood flow and nasal secretion increased only in those with symptoms. The albumin content of the nasal secretion increased in the clinically infected, suggesting that it was derived, partially at least, from the circulation. The nasal cycle of variation in airway resistance between the two sides of the nose was observed in all three groups but increased only in those clinically infected.

Changes in epithelial secretory cells and potentiation of neurogenic inflammation in the trachea of rats with respiratory tract infections.

In rats respiratory tract infections due to Sendai virus and coronavirus usually are transient, but they can have long-lasting consequences when accompanied by Mycoplasma pulmonis infections. Morphological alterations in the tracheal epithelium and a potentiation of the inflammatory response evoked by sensory nerve stimulation ("neurogenic inflammation") are evident nine weeks after the infections begin, but the extent to which these changes are present at earlier times is not known. In the present study we characterized these abnormalities in the epithelium and determined the extent to which they are present 3 and 6 weeks after the infections begin. We also determined the magnitude of the potentiation of neurogenic inflammation at these times, whether the potentiation can be reversed by glucocorticoids, and whether a proliferation of blood vessels contributes to the abnormally large amount of plasma extravasation associated with this potentiation. To this end, we studied Long-Evans rats that acquired these viral and mycoplasmal infections from other rats. We found that the tracheal epithelium of the infected rats had ten times as many Alcian blue-PAS positive mucous cells as did that of pathogen-free rats; but it contained none of the serous cells typical of pathogen-free rats, so the total number of secretory cells was not increased. In addition, the epithelium of the infected rats had three times the number of ciliated cells and had only a third of the number of globule leukocytes. In response to an injection of capsaicin (150 micrograms/kg i.v.), the tracheas of the infected rats developed an abnormally large amount of extravasation of two tracers, Evans blue dye and Monastral blue pigment, and had an abnormally large number of Monastral blue-labeled venules, particularly in regions of mucosa overlying the cartilaginous rings. This abnormally large amount of extravasation was blocked by dexamethasone (1 mg/day i.p. for 5 days). We conclude that M. pulmonis infections, exacerbated at the outset by viral infections, result within three weeks in the transformation of epithelial serous cells into mucous cells, the proliferation of ciliated cells, and the depletion of globule leukocytes. They also cause a proliferation of mediator-sensitive blood vessels in the airway mucosa, which is likely to contribute to the potentiation of neurogenic inflammation that accompanies these infections.

Effect of Gray strain infectious bronchitis virus and high dietary calcium on renal function of Single Comb White Leghorn pullets at 6, 10, and 18 weeks of age.

Experiments were designed to evaluate the effects of Gray strain infectious bronchitis virus (IBV) and high dietary calcium (Ca), alone and in combination, on renal function in pullets. Eight hundred female Single Comb White Leghorn chicks were raised on starter ration. Five hundred chicks were inoculated intravenously with Gray strain IBV at 4 wk of age; the remaining chicks were not exposed to IBV. At 6 wk of age, IBV-inoculated and uninoculated chicks were randomly divided into two diet treatment groups. Half the chicks were fed commercial grower ration (approximately 1.0% Ca, .6% available P) and half were fed commercial layer ration (approximately 3.25% Ca, .5% available P). Birds remained on their respective diets until 18 wk of age. Kidney function studies were conducted on anesthetized birds at 6 wk of age prior to initiation of the diet treatments, at 10 wk of age, and at 18 wk of age. The layer ration increased Ca excretion, decreased inorganic phosphate excretion, and decreased urine hydrogen ion concentration in 10-wk-old pullets in comparison with the grower ration. These diet effects on kidney function were attenuated when the pullets reached 18 wk of age. The layer ration also caused an 11.5% incidence of urolithiasis, and significantly increased kidney asymmetry in 18-wk-old pullets relative to the effects of the grower ration. Gray strain IBV exposure significantly increased kidney asymmetry in 18-wk-old pullets, but had no gross effect on kidney function clearly related to the etiology of urolithiasis. Gray strain IBV did not enhance the incidence of urolithiasis in any of the age groups.

Developmental toxicity of alprostadil in rats after subcutaneous administration or intravenous infusion.

Timed-pregnant Upj:TUC(SD)spf (Sprague-Dawley) rats were dosed with alprostadil (prostaglandin E1), either subcutaneously on Days 6-15 of gestation at 0.0, 0.5, 1.0, or 2.0 mg/kg/day or by iv infusion into the jugular vein (24 hr/day) on Days 7-15 at 0.0, 0.5, 1.0, 2.0, 4.0, or 6.0 mg/kg/day. Maternal toxicity was observed in all dams receiving alprostadil subcutaneously, the severity of which increased in a dose-related manner. Toxicity also was evident in the offspring in the 2.0 mg/kg/day group as evidenced by a significant increase in the percentage of resorptions and a significant decrease in the percentage of live fetuses. Mean fetal weight was significantly depressed in all three alprostadil-treated groups and several skeletal and visceral variations were significantly higher in the 1.0 and 2.0 mg/kg/day groups than in the vehicle control group; in addition, there were two instances of significantly increased frequencies of skeletal variations in the 0.5 mg/kg/day group. Gross, visceral, and skeletal malformations were significantly increased in the high-dose group. During iv infusion of alprostadil more than 50% of the dams in the 6.0 mg/kg/day group died and there was one death in the 4.0 mg/kg/day group. Significant decreases in maternal weight gain between Days 7 and 11 of gestation were observed at doses of 1.0 mg/kg/day and above. However, continuous iv infusion of this prostaglandin, at dosages which were not severely toxic to the dams, was judged not to be teratogenic or otherwise embryotoxic in rats. The increase in uterine contractions, observed at 1.0 and 2.0 mg/kg after sc administration to rats implanted with chronic uterine microballoons, was consistent with the hypothesis that the developmental toxicity observed after bolus sc administration was the consequence of decreased blood flow in the uterus and/or placenta and/or embryos.

Effects of infectious bronchitis virus (Arkansas strain) on laying chickens.

Seventy-seven-week-old white leghorn layers were inoculated intraocularly with the Arkansas strain of infectious bronchitis virus (AIBV) to study the effects of the virus on egg production and on antibody response of the birds. Infected hens laid fewer eggs than the controls, and those eggs weighed less than eggs laid by controls. Further, the shell quality and internal quality of eggs laid by infected birds were inferior. The serum hemagglutination-inhibition (HI) titers of infected birds increased continuously through 4 weeks postinfection; serum HI titers of the controls were negligible.

Coronavirus IBV: removal of spike glycopolypeptide S1 by urea abolishes infectivity and haemagglutination but not attachment to cells.

Urea has been used to remove the S1 spike glycopolypeptide from avian infectious bronchitis virus (IBV) strains M41 and Beaudette, without removing the S2 spike-anchoring glycopolypeptide. Reduction of the pH to 2.9 did not cause release of S1 although some S1 was released spontaneously from IBV Beaudette at pH 7.4. Virus that lacked S1 was no longer infectious or able to cause haemagglutination (HA). However, radiolabelled IBV that lacked S1 attached to erythrocytes and chick kidney cells to the same or similar extent as did intact virus. Treatment of IBV with a phospholipase C preparation, required to make IBV cause HA, did not increase binding of IBV to erythrocytes. The results indicate that while the attachment to cells of virus that lacks S1 is qualitatively different from that of intact virus, the decline in infectivity is the consequence of the loss of some other spike function.

Efficacy of experimental inactivated mycoplasma gallisepticum oil-emulsion bacterin in egg-layer chickens.

Six groups of white leghorn pullets were studied to determine the ability of beta-propiolactone-inactivated Mycoplasma gallisepticum (MG) oil-emulsion bacterins to counteract reductions in egg production caused by MG infection. The pullets were inoculated with 0.5 ml of MG bacterin subcutaneously in the neck at about 20 weeks of age and were challenged with MG near 28 weeks of age, when they were in peak egg production. Various challenge schemes with infectious bronchitis virus were used at the time of MG challenge to increase the reduction in egg production. MG bacterins afforded protection against moderate drops in egg production in at least three of the studies, where the unvaccinated challenged control hens exhibited reduced egg production.