Alteration of blood coagulation and complement system in neotropical primates infected with Junin virus

The neotropical primate Callithrix jacchus infected with Junin virus presented an acute disease with hematological and neurological manifestations and died 17 to 24 days after infection. This picture is similar to that of human Argentine hemorrhagic fever (AHF). Blood coagulation and complement studies were performed in ten C jacchus animals inoculated with 10³ TCID₅₀ of Junin virus, the prototype pathogenic XJ strain. Four monkeys were used as normal controls. Infected monkeys and normal controls were bled to death on days 7, 14, 17, and 21. A progressive decrease in the number of platelets was found after day 7 of infection. On day 21, the last monkey had a value of 24,000/μl. The levels of blood clotting factors did not change until day 17, when a shortened partial thromboplastin time activated with Kaolin (PTTK) (36 sec) and increased factors VIII (192.2%) and VII-X (266.6%) were found. On day 21, the PTTK was prolonged (50.7 sec) and factors II, V, and VIII, were decreased. Thrombin time was found prolonged from day 14 onward. Fibrinogen and fibrin degradation products (FDPs) were increased on days 17 (754 mg/dl and 9.2 μg/ml) and 21 (457 mg/dl and 29.4 μg/ml). No changes in the levels of α₂ macroglobulin were observed. Complement hemolytic levels were found to be low on day 7 (58.3 UCH₅₀, increased on day 14 (165.1), and within normal range at the end of infection (107.2). C3 levels showed a similar pattern. The bone marrow was active and hypercellular, and the number and morphology of megakaryocytes were normal in all but one of infected animals. The results of blood clotting suggest a limited activation. The complement system presented a profile of activation followed by a rebound phenomenon. The activation of complement appeared ten days before the alteration of the clotting system was evident.     

Viremia in respiratory syncytial virus infection]

Viremia was demonstrated to occur in experimental respiratory syncytial (RS) virus infection in suckling cotton rats and in natural infection in children. RS virus was isolated from the whole blood of the animals in 3 out of 6 experiments at 2, 5, 6, 7 and 15 days after inoculation, the maximum infectious titer being more than 10(4) TCPD50/0.1 ml. RS virus was also isolated from the blood of 7 out of 15 examined children presenting the typical clinical picture of RS virus disease during the epidemic season of RS virus infection. In 6 patients RS virus was isolated from one blood specimen at 1, 6 and 7 days after the onset, in one patient from 3 blood specimens at 6, 9 and 22 days after the onset. The demonstrated long-term persistence of virus in the blood suggests the possibility of existence of chronic RS virus infection.     

Dynamics of complement hemolytic activity in experimental Ebola infection

The dynamic hemolytic activity of complements (HAC) was investigated in blood of guinea pigs in lethal and non-lethal Ebola infection. The increasing HAC dynamic activity in the animal blood was found to correlate with the infection lethal course. HAC as observed in animals with lethal infection was sweepingly increasing after they, were infected with Ebola virus, and yet after 15 hours from the infection time the complement activity parameters topped 2-fold the basic values in 100% of guinea pigs. They began to be dropping by the end of day 1, their decrease reached, when the incubation time was over (days 3-4 after infection) the basic value, after which they continued to go down to the zero value in 2-3 days before the lethal outcome. The described phenomenon, like the phenomenon of accelerated death, was even more pronounced, when the animals were infected after a single immunization by activated Ebola virus. In case, guinea pigs were infected by a non-lethal Ebola virus strain, the compliment synthesis was observed to be activated only at the end of the incubation period; the process was accompanied with a gradual raise and with a plateau-type or wave-type increase of the complement during the treatment time--it was equally accompanied with normalizing activity parameters during recovery. The detected specificity could be important in prognosticating a disease outcome. A reliable correlation was demonstrated between the complement hemolytic activity and the level of circulating immune complexes in blood of experimental animals, which can be traced both in lethal and non-lethal infection.     

Studies on the virus of hepatitis contagiosa canis (HCC)

Summary Guinea pigs, inoculated subcutaneously with large doses of HCC virus, displayed obvious symptoms of disease: apathy, inappetence, ruffled fur. Up to 25% of the animals died. Perihepatitis and perisplenitis were observed in the majority of the animals. A rise in the body temperature occurred with a maximum at 48 to 72 hours after inoculation. Lymphocytosis, culminating after about 7 days, was recorded. Virus was recovered from several organs 6 and 12 hours after inoculation; no virus was demonstrable after 24 hours; at 36 hours active virus reappeared and maximum concentrations were recorded between 60 and 96 hours after inoculation. At this stage small amounts of virus were found in the blood. Neutralizing antibodies appeared, after a single dose of even minute amounts of live virus. It is concluded that guinea pigs are uniformly and highly susceptible to experimental infection with HCC virus.     

Coxsackievirus B infection in pregnant mice and transplacental infection of the fetus.

Direct instillation of coxsackievirus B1 into the gastrointestinal tracts of albino mice caused viremia in more than 85% of the animals within 1 day. In pregnant mice infected early in gestation (7 days), the geometric mean titer of virus in the blood was lower (P = 0.02) and the duration of viremia was shorter (P = 0.07) than in nonpregnant female mice, but infection of the heart, liver, and uterus did not differ on each of 5 days after infection. Although transplacental infection of the placenta or fetus or both occurred, the high spontaneous abortion rate (48%) obviated comparison of transplacental infection in these mice with mice infected later in gestation. Pregnant mice infected in the third trimester had significantly greater geometric mean titers of virus in the blood, heart, liver, and uterus, and infection persisted longer than in nonpregnant mice (P = 0.04). A very high geometric mean titer of virus was recovered from the uteri of these mice for 3 days after infection, whereas simultaneous geometric mean titers of virus in the placentas and fetuses were lower. In the majority of third trimester pregnant mice, virus was found in low titers in the fetuses at 2 and 3 days after maternal infection, and virus was not detected after day 3. We conclude that coxsackievirus B1 infection in late gestational pregnant mice is more severe than in mice at earlier gestational stages and in nonpregnant mice and that transplacental infection of the fetus occurs transiently during maternal infection. This model will prove useful in the study of perinatal enterovirus infection and in examination of the numerous factors that may influence outcome of infection of perinatally infected newborn infants.     

Real-time RT-PCR detection of Bovine Viral Diarrhoea virus in whole blood using an external RNA reference

A novel two-step real-time RT-PCR assay using SYBR Green I was developed for the detection of acute Bovine Viral Diarrhoea virus (BVDV) infection in whole blood from cattle. During infection animals experience a characteristic transient leucopenia and the number of cells per volume of blood changes over time; so quantitation of viral load by reference to a cellular housekeeping gene is not ideal as this may hide significant animal to animal variation. Therefore, to facilitate comparison of different samples, an external RNA reference was used for normalisation whereby each sample was spiked with the RNA virus, Canine Enteric Coronavirus (CECov), prior to RNA extraction, for comparative purposes. Real-time RT-PCR was carried out with two primer sets designed to amplify either a 156 bp region of the BVDV 5'-UTR or a 280 bp region of the CECov nucleocapsid protein gene. Linearity and efficiency of the assay was established and the method assessed using samples from BVDV-challenged calves. Viral RNA was quantified on days 6 and 14 post-challenge by real-time RT-PCR. Infectious virus isolation by traditional cell culture was negative after day 7. This study demonstrates encouraging results for rapid, sensitive and reliable detection of acute BVDV infection and provides an alternative real-time RT-PCR method for use on whole blood samples or samples where suitable housekeeping genes are not available.     

Experimental reovirus infection in chickens: observations on early viraemia and virus distribution in bone marrow, liver and enteric tissues

The nature of viraemia and tissue distribution of reovirus were studied in the early phase after oral infection of 1-day-old specific-pathogen-free (SPF) White Leghorn chicks with the R2 strain of avian reovirus. A range of tissues collected up to 3 weeks after infection was titrated for their viral content. Virus was present in the plasma, erythrocyte and mononuclear fractions of the blood within 30 hours post-inoculation (p.i.) and was widely distributed in tissues, including the bone marrow by 3 to 5 days p.i. A greater part of the viraemia was associated with plasma, virus in the blood mononuclear fraction being detected only occasionally. There was more infectious virus in the duodenum than the liver and the highest virus titres were found in cloacal swabs taken 1 to 5 days p.i. It was also evident that virus reached the liver within a very short time after infection (<6 hours p.i.) although the source of this early hepatic virus was considered to be residual inoculum absorbed directly into the portal blood. Viraemic virus titres could not be correlated either with duodenal or hepatic virus titre alone.     

Studies of experimental Jembrana disease in Bali cattle. I. Transmission and persistence of the infectious agent in ruminants and pigs, and resistance of recovered cattle to re-infection

The agent causing Jembrana disease, an enzootic disease of Bos javanicus (Bali cattle) occurring in Bali, Indonesia, was shown to occur at high titres in the blood of animals during the febrile period of the disease and to persist in cattle for 25 months after clinical recovery. During the febrile period of the disease, most of the infectious agent appeared to be associated with the plasma fraction of whole blood. There was a linear relationship between the number of organisms inoculated into susceptible Bali cattle and the incubation period, which varied from 4.5 to 12 days. Seventeen of 18 animals in which Jembrana disease had been experimentally induced up to 22 months previously did not develop clinical signs when re-challenged with the infectious agent. Ongole cattle (Bos indicus), Friesian cattle (Bos taurus), buffaloes (Bubalus bubalis) and pigs, but not sheep or goats, developed a mild febrile response, but no other overt clinical signs of the disease after inoculation with the Jembrana disease agent. Ongole and Friesian cattle, buffaloes, and sheep developed a persistent infection after inoculation; the infectious agent persisted in blood or spleen for at least 9 months in buffaloes and for shorter periods in the other species.     

Potentiation of coxsackievirus B3 infection in adult mice pretreated with a gold salt.

In mice treated with sodium aurothiomalate (myocrisin), prior to infection with Coxsackievirus B3, 90% of the animals died by the 11th day postinfection (p.i.). A mortality of 10% was noted in mice receiving myocrisin only, and no deaths occurred in animals infected with virus alone. The highest amount of virus was recovered from the pancreas of myocrisin-treated mice on day 3 p.i. This was over 500-fold higher than the virus titer found in the pancreas of mice infected with virus only. Generally the titer of virus present in different organs was higher at every point in drug-treated animals as compared to intact mice infected with the virus. A high and persistent viremia was present in myocrisin-treated mice; in contrast a low viremia followed by virus clearance from the blood was observed in intact mice infected with the virus. The antibody response was studied in intact and myocrisin-treated mice infected with the virus. In both groups, no neutralizing antibodies were detected on days 1, 2, and 3 p.i. On day 7 after infection, the titers of antibodies were 1:16 and 1:12 in intact and myocrisin-treated mice, respectively. Administration of hyperimmune anti-Coxsackievirus B3 serum 6 hours after infection protrected in myocrisin-treated group of mice against lethal disease. The results of these studies suggest that (1) antibodies alone may not be sufficient to limit the spread and persistence of virus in natural infections and (2) in the absence of any apparent histopathological differences the increased multiplication of Coxsackievirus B3 could be the cause of death in myocrisin-treated mice.     

Passive serum antibody causes temporary recovery from influenza virus infection of the nose, trachea and lung of nude mice.

BALB/c normal and nude mice were infected with a non-lethal mouse-passaged A/PC/1/73 (H3N2) influenza virus in order to assess the role of T cells on the course of disease of the nose, trachea and lung. The tracheal epithelium of both mouse strains was desquamated by 3 days after infection. Although normal regeneration began, nude mice never completed that regeneration whereas normal mice had fully regenerated tracheas by Day 14. This failure to complete the recovery was also evident from the continued virus shedding by the nude mouse. In order to assess the role of serum antibody on recovery from infection, ferret, goat or mouse antibody to H3N2 influenza virus was passively administered to nude mice after infection. It resulted in a transient decrease in virus shedding from the nose, trachea and lung, and complete but temporary regeneration of the tracheal epithelium. However, later in the course of the infection, when serum antibody levels were no longer detectable, the tracheal epithelium of these animals redesquamated and large amounts of virus were again shed from nose, trachea and lungs. We conclude that: (i) desquamation of the ciliated epithelium of the trachea is not T-cell dependent; and (ii) serum antibody can contribute to temporary recovery from infection, but by itself is insufficient for permanent recovery of the nose, trachea or lung.     

Pathogenesis of Herpesvirus sylvilagus infection in cottontail rabbits.

Experimental infection with Herpesvirus sylvilagus produces clinical and histopathologic changes in its natural host, the cottontail rabbit (Sylvilagus floridanus), similar to those observed in humans acutely infected with Epstein-Barr virus (EBV). Twenty-seven seronegative cottontail rabbits were infected with Herpesvirus sylvilagus and all developed antibodies within 10 days. Neutralizing antibody was detected as early as 7 days after infection. Virus was isolated from blood mononuclear cells, spleen, bone marrow, thymus, lymph nodes, kidneys, lung, and liver as early as 3 days after infection. Infected animals showed leucocytosis, monocytosis, and lymphocytosis with the appearance of atypical lymphocytes. Peripheral blood abnormalities peaked at 10-14 days after infection, and returned to normal by 28 days after infection, with the exception of atypical lymphocytosis that persisted in some animals for more than 2 years after experimental infection. More severe histopathologic changes were seen in virus-infected juvenile rabbits than adult rabbits; these changes included viral myocarditis, interstitial pneumonia, and lymphocytic myositis. Reactive hyperplasia and subsequent lymphocytic depletion of spleen and lymph nodes were reminiscent of that seen in virus-associated hemophagocytosis syndrome. Prominent lymphoid hyperplasia of many nonlymphoid organs, most notably the kidney and lungs, was observed. The development of these lymphoproliferative lesions and other lymphoid changes during H. sylvilagus infection suggest that this system may be a model to study similar lesions induced by EBV infection in humans.     

Capsaicin-sensitive peptidergic neurons are involved in the zosteriform spread of herpes simplex virus infection.

The intraneuronal transport of herpes simplex virus (HSV) is an essential component in disease pathogenesis. Capsaicin, a neuropharmacologic agent lacking direct antiviral activity, has been shown to protect animals against HSV-induced disease. It has been hypothesized that capsaicin acts by interfering with the intraneuronal transport of virus. Since animal models have been useful in studying the spread of virus, we used two guinea pig models of zosteriform herpes to examine the effect of capsaicin on HSV spread. Capsaicin was subcutaneously administered to Hartley guinea pigs prior to intravaginal or cutaneous HSV-2 inoculation. Treatment did not prevent the development of herpetic vesicles at the site of inoculation but significantly reduced the zosteriform spread of lesions in male and female animals. Further, after recovery from primary infection, capsaicin-treated male guinea pigs were observed to have fewer days with recurrent herpetic lesions. These results suggest that capsaicin-sensitive nerve fibers play a role in the pathogenesis of primary and recurrent HSV infections. Capsaicin appears to reduce the severity of cutaneous HSV infections by interfering with the spread of virus.     

Lymphocyte Reactivity Contributes to Protection Conferred by Specific Antibody Passively Transferred to Herpes Simplex Virus-Infected Mice

Passively acquired immunity to herpes simplex virus (HSV) was studied in antithymocyte serum (ATS)-treated mice and athymic nude mice to determine whether immunocompetent lymphocytes contribute to the protection observed after transfer of HSV-specific antibody to infected animals. Mice were given three intraperitoneal injections of 0.1 ml of ATS at 24-h intervals. This treatment reduced concanavalin A and lipopolysaccharide stimulation of lymphocytes harvested from these animals by 90% when compared with the stimulation of lymphocytes harvested from untreated animals. It was found that intraperitoneal injection of 0.5 ml of specific antibody 8 h after corneal HSV type 1 infection or subcutaneous HSV type 2 infection did not protect ATS-treated animals from virus infection. Specific antibody passively transferred to ATS-treated animals 8 and 120 h postinfection also failed to protect lymphocyte-depleted animals from HSV. However, ATS-treated animals were protected from HSV infection by passively acquired antibody when lymphocytes harvested from these animals regained 80% of their ability to be stimulated with concanavalin A and lipopolysaccharide. It was also found that specific antibody conferred protection to nude mice infected with HSV only if they were first reconstituted with syngeneic thymus cells 48 h before infection. The results suggest that both antiviral antibody and thymus-derived lymphocytes contribute to the recovery of HSV-infected hosts after passive immunization.     

Experimental respiratory Marburg virus haemorrhagic fever infection in the common marmoset (Callithrix jacchus)

Marburg virus causes a highly infectious and lethal haemorrhagic fever in primates and may be exploited as a potential biothreat pathogen. To combat the infection and threat of Marburg haemorrhagic fever, there is a need to develop and license appropriate medical countermeasures. To determine whether the common marmoset (Callithrix jacchus) would be an appropriate model to assess therapies against Marburg haemorrhagic fever, initial susceptibility, lethality and pathogenesis studies were performed. Low doses of virus, between 4 and 28 TCID₅₀, were sufficient to cause a lethal, reproducible infection. Animals became febrile between days 5 and 6, maintaining a high fever before succumbing to disease between 8 and 11 days postchallenge. Typical signs of Marburg virus infection were observed including haemorrhaging and a transient rash. In pathogenesis studies, virus was isolated from the animals’ lungs from day 3 postchallenge and from the liver, spleen and blood from day 5 postchallenge. Early signs of histopathology were apparent in the kidney and liver from day 3. The most striking features were observed in animals exhibiting severe clinical signs, which included high viral titres in all organs, with the highest levels in the blood, increased levels in liver function enzymes and blood clotting times, decreased levels in platelets, multifocal moderate‐to‐severe hepatitis and perivascular oedema.     

Experimental pathogenesis of murine herpesvirus in newborn mice

Newborn white mice were susceptible to peroral (p.o.) infection with murine alphaherpesvirus isolated from free-living Clethrionomys glareolus. Death occurred within 6-8 days in animals infected with the higher virus dose of 4.8 log10 TCID50. Clinical symptoms also occurred in some animals infected with lower doses, while others developed inapparent infection as judged by presence of humoral antibodies at 60 days post-infection (p.i.). The virus was detected in the lungs, blood, liver, spleen, kidneys, heart muscle, brain and urinary bladder of sick animals. Necrotising pneumonia accompanied the replication of the virus in the epithelial cells of alveolar ducts and alveolar lining as confirmed by immunofluorescence and histological examination. Latent infection of Gasserian ganglia in the survivors was not necessarily related to the administered dose of infectious virus. Two of mother females, which had eaten their diseased offspring, became inapparently infected as proved by reisolation of the virus from trigeminal ganglion explants and by detection of specific antibodies at 60 days p.i.     

Variation Between Strains of Hamsters in the Lethality of Pichinde Virus Infections

Infection by Pichinde virus, a member of the arenavirus group, was studied in Golden Syrian hamsters (Mesocricetus auratus) with regard to possible mechanisms of resistance to virus infection in adult hamsters. Two hamster strains were found to differ in their susceptibility to lethal Pichinde virus infection. LVG/Lak randomly bred hamsters were found to be 100% susceptible to low doses of Pichinde virus during the first 6 days of life, but after 8 days of life, mortality was uncommon. Peak virus titers in the serum of animals infected at 3 days of life were 4 logs greater than in animals infected at 12 days. MHA/Lak inbred hamsters, in contrast, were found to be susceptible to lethal virus infection both as newborns and as adults. Peak virus titers of greater than 10(8) plaque-forming units/ml were observed in serum 8 days after infection of adult MHA hamsters as compared with less than 10(3) plaque-forming units/ml in the serum of adult LVG hamsters. Cultured primary kidney cells and peritoneal macrophages from either hamster strain supported Pichinde virus replication equally well in vitro. Antibodies to the complement-fixing antigens and to antigens at the surface of virus-infected cells were produced by both strains of hamsters. Cyclophosphamide immunosuppression rendered adult LVG animals susceptible to lethal infections, and virus grew to high titers in the treated animals. These findings suggest that immunological factors that appear early in life in LVG hamsters and are deficient in MHA hamsters limit Pichinde virus infection. Unlike previously reported arenavirus diseases, the observations suggest that death is produced by a direct viral effect and not through immunopathological mechanisms.     

Persistence of Adenovirus 5 in Guinea Pigs

One intracardiac inoculation of adenovirus 5 in guinea pigs leads to virus persistence in different organs, viz., 5 days in lungs and liver, 14 days in blood and lymph nodes, and 56 days or more in the spleen. After cultivation of tissue cells for 1 week, virus was recovered from blood, lymph nodes, or spleen lymphocytes, but virus could be detected directly in cells only when organs were removed within 48 h of inoculation. To determine how the virus persisted in low concentrations and as a latent infection, spleens were primarily selected for study by three techniques: homogenization of spleens, suspended Maitland fragment cultures, and in vitro cultivation of spleen cells. The last procedure showed virus in fibroblast-like cells (probably macrophages or reticuloendothelial cells) for 56 days after infection of guinea pigs. With other methods, the virus was found only within the first 2 days after inoculation.     

Does ovarian stimulation for in-vitro fertilization induce a hypercoagulable state?

Effects on blood coagulation and fibrinolytic activity during ovarian stimulation for in-vitro fertilization (IVF) were examined in 12 women. Blood samples were taken prior to hormonal stimulation (days 2-3 of the menstrual cycle, mean serum oestradiol concentration 0.16 nmol/l) and the day after ovulation induction with human chorionic gonadotrophin (HCG) (days 10-12, mean serum oestradiol concentration 5.35 nmol/l). We measured whole blood clotting time, whole blood clot lysis time, plasma fibrinogen, factor VII and antithrombin III. The whole blood clotting time was slightly, but not significantly shortened after ovarian stimulation. A significant rise in plasma fibrinogen (P less than 0.001) and reduction in antithrombin III (P less than 0.001) were observed, whereas no change in factor VII was found. The blood fibrinolytic activity was significantly reduced as evaluated by an increase in the clot lysis time (P less than 0.02). These results indicate that ovarian stimulation for IVF may create a state of hypercoagulability.     

Influenza infection in ferrets: role of serum antibody in protection and recovery.

The passive administration of ferret antiserum to Ao (H0N1) influenza virus failed to protect the recipient ferrets from subsequent infection with homologous virus. This susceptibility to infection was observed even when the passively acquired serum hemagglutination inhibition (HI) titer was similar to peak convalescent titers. It is therefore concluded that serum antibody alone is probably not a major factor in the prevention of influenza infection. This does not rule out a possible role for serum antibody in prevention of illness. Subsequent to infection, ferrets that had received passive antisera failed to develop high levels of serum HI antibody. In fact, many had no detectable serum antibody (less than 1:8). These animals shed virus for periods of time quite similar to those of infected control animals, which did develope serum antibody. From these data it was concluded that detectable serum HI antibody does not play a significant role in the recovery of ferrets from influenza infection. Interferon was present in high concentrations in the secretions a few days prior to cessation of virus shedding, but it is not clear whether this was the cause of the recovery or merely a concomitant event. Twenty-one days after initial infection two-thirds of the ferrets that had received passive antibody and all control animals were immune to reinfection with the homologous influenza virus. Since the former group had little or no detectable serum HI antibody but most members were immune, there must be some other host mechanism to account for the immunity.     

Cell-mediated immunity in experimental parainfluenza type 3 virus infection

Local respiratory-tract infection was produced experimentally in guinea-pigs by intranasal instillation of a suspension of parainfluenza virus type 3. Histologically, interstitial pneumonitis developed within 10 days and persisted for at least 70 days. Cell-mediated immunity was measured at intervals for 70 days after infection. Dermal reactivity could not be elicited. Leucocyte-migration inhibition and macrophage-migration inhibition were increased. Macrophage aggregation was present. Increased cell-mediated immunity could be transferred from infected donor animals to normal recipient animals by adoptive spleen-cell transfer even 60 and 70 days after infection.