The Molecular Basis for Antigenic Drift of Human A/H2N2 Influenza Viruses.

Influenza A/H2N2 viruses caused a pandemic in 1957 and continued to circulate in humans until 1968. The antigenic evolution of A/H2N2 viruses over time and the amino acid substitutions responsible for this antigenic evolution are not known. Here, the antigenic diversity of a representative set of human A/H2N2 viruses isolated between 1957 and 1968 was characterized. The antigenic change of influenza A/H2N2 viruses during the 12 years that this virus circulated was modest. Two amino acid substitutions, T128D and N139K, located in the head domain of the H2 hemagglutinin (HA) molecule, were identified as important determinants of antigenic change during A/H2N2 virus evolution. The rate of A/H2N2 virus antigenic evolution during the 12-year period after introduction in humans was half that of A/H3N2 viruses, despite similar rates of genetic change.IMPORTANCE While influenza A viruses of subtype H2N2 were at the origin of the Asian influenza pandemic, little is known about the antigenic changes that occurred during the twelve years of circulation in humans, the role of preexisting immunity, and the evolutionary rates of the virus. In this study, the antigenic map derived from hemagglutination inhibition (HI) titers of cell-cultured virus isolates and ferret postinfection sera displayed a directional evolution of viruses away from earlier isolates. Furthermore, individual mutations in close proximity to the receptor-binding site of the HA molecule determined the antigenic reactivity, confirming that individual amino acid substitutions in A/H2N2 viruses can confer major antigenic changes. This study adds to our understanding of virus evolution with respect to antigenic variability, rates of virus evolution, and potential escape mutants of A/H2N2.

Determinants of virulence of influenza A virus.

Influenza A viruses cause yearly seasonal epidemics and occasional global pandemics in humans. In the last century, four human influenza A virus pandemics have occurred. Occasionally, influenza A viruses that circulate in other species cross the species barrier and infect humans. Virus reassortment (i.e. mixing of gene segments of multiple viruses) and the accumulation of mutations contribute to the emergence of new influenza A virus variants. Fortunately, most of these variants do not have the ability to spread among humans and subsequently cause a pandemic. In this review, we focus on the threat of animal influenza A viruses which have shown the ability to infect humans. In addition, genetic factors which could alter the virulence of influenza A viruses are discussed. The identification and characterisation of these factors may provide insights into genetic traits which change virulence and help us to understand which genetic determinants are of importance for the pandemic potential of animal influenza A viruses.

Transmission of influenza A/H5N1 viruses in mammals.

Highly pathogenic avian H5N1 influenza A viruses occasionally infect humans and cause severe respiratory disease and fatalities. Currently, these viruses are not efficiently transmitted from person to person, although limited human-to-human transmission may have occurred. Nevertheless, further adaptation of avian H5N1 influenza A viruses to humans and/or reassortment with human influenza A viruses may result in aerosol transmissible viruses with pandemic potential. Although the full range of factors that modulate the transmission and replication of influenza A viruses in humans are not yet known, we are beginning to understand some of the molecular changes that may allow H5N1 influenza A viruses to transmit via aerosols or respiratory droplets among mammals. A better understanding of the biological basis and genetic determinants that confer transmissibility to H5N1 influenza A viruses in mammals is important to enhance our pandemic preparedness.

Predicting 'airborne' influenza viruses: (trans-) mission impossible?

Repeated transmission of animal influenza viruses to humans has prompted investigation of the viral, host, and environmental factors responsible for transmission via aerosols or respiratory droplets. How do we determine-out of thousands of influenza virus isolates collected in animal surveillance studies each year-which viruses have the potential to become 'airborne', and hence pose a pandemic threat? Here, using knowledge from pandemic, zoonotic and epidemic viruses, we postulate that the minimal requirements for efficient transmission of an animal influenza virus between humans are: efficient virus attachment to (upper) respiratory tissues, replication to high titers in these tissues, and release and aerosolization of single virus particles. Investigating 'airborne' transmission of influenza viruses is key to understand-and predict-influenza pandemics.

Pandemic 2009 H1N1 influenza virus causes diffuse alveolar damage in cynomolgus macaques.

The pathogenesis of lower respiratory tract disease from the pandemic 2009 H1N1 (H1N1v) influenza A virus is poorly understood. Therefore, either H1N1v virus or a seasonal human H1N1 influenza A virus was inoculated into cynomolgus macaques as a nonhuman primate model of influenza pneumonia, and virological, pathological, and microarray analyses were performed. Macaques in the H1N1v group had virus-associated diffuse alveolar damage involving both type I and type II alveolar epithelial cells and affecting an average of 16% of the lung area. In comparison, macaques in the seasonal H1N1 group had milder pulmonary lesions. H1N1v virus tended to be reisolated from more locations in the respiratory tract and at higher titers than seasonal H1N1 virus. In contrast, differential expression of messenger RNA transcripts between H1N1v and seasonal H1N1 groups did not show significant differences. The most upregulated genes in H1N1v lung samples with lesions belonged to the innate immune response and proinflammatory pathways and correlated with histopathological results. Our results demonstrate that the H1N1v virus infects alveolar epithelial cells and causes diffuse alveolar damage in a nonhuman primate model. Its higher pathogenicity compared with a seasonal H1N1 virus may be explained in part by higher replication in the lower respiratory tract.

Idiopathic immune-mediated hemolytic anemia: treatment outcome and prognostic factors in 149 dogs.

BACKGROUND: Canine idiopathic immune-mediated hemolytic anemia (IMHA) is associated with a high mortality, especially in the 1st 2 weeks after diagnosis despite treatment. OBJECTIVES: To determine treatment outcome and identify prognostic variables in order to define areas of future research. ANIMALS: One hundred forty-nine dogs with hematocrit <30% and either a positive Coombs' test or spherocytosis and with no evidence of disease that can trigger IMHA were included. METHODS: Retrospective cohort study. All dogs were treated with prednisolone and azathioprine according to a standard protocol. Survival analysis was performed by the Kaplan-Meier method. Variables recorded at the time of diagnosis were tested as possible prognostic variables in a univariate and multivariate Cox proportional hazard model. RESULTS: The main predictors for mortality in dogs with idiopathic IMHA are the presence of increased plasma urea concentration, bands, thrombocytopenia, and petechiae at the time of diagnosis. The estimated Kaplan-Meier half-year survival was 72.6% (95% confidence interval [CI]: 64.9-81.3%). Mortality occurred mostly within the 1st 2 weeks. Cox proportional hazards analysis indicated that increased plasma urea concentration, icterus, and petechiae were the major independent predictors of mortality in the 1st 2 weeks. In most dogs that survived IMHA, a 3-month protocol of azathioprine with prednisolone maintained clinical remission. The estimated half-year survival for dogs that survived the 1st 2 weeks was 92.5% (95% CI: 86-99.3%). CONCLUSIONS AND CLINICAL IMPORTANCE: If the dogs survived IMHA, a 3-month protocol of prednisolone and azathioprine was effective with regard to survival and clinical outcome. Future research should be directed at identifying whether thrombotic tendency in dogs with IMHA is the main contributor to the development of increased plasma urea concentration, icterus, thrombocytopenia, and petechiae.

Sensory neuropathy in two Border collie puppies.

A peripheral sensory neuropathy was diagnosed in two Border collie puppies. Neurological, electrophysiological and histopathological examinations suggested a purely sensory neuropathy with mainly distal involvement. Urinary incontinence was observed in one of the puppies and histological examination of the vagus nerve revealed degenerative changes. An inherited disorder was suspected.

Dyschezia in dogs with discrete erosive anal disease and histological lesions suggestive of mucocutaneous lupus erythematosus.

Eight dogs suffered chronic defecation problems characterised by severe dyschezia, manifested by signs of fear and vocalisation on defecation. One or more depigmented, discrete erosions, easily bleeding on palpation, were found in the perineal region. Histopathological changes included interface dermatitis, apoptotic keratinocytes, focal hydropic degeneration of basal epidermal cells, focal thickening of the basement membrane zone and dermal lichenoid infiltrations with mononuclear and plasma cells. A diagnosis of cutaneous or discoid lupus erythematosus was made on the basis of these clinical, physical and specific histopathological changes. The dogs were treated systemically with immunomodulating drugs and good results were obtained.

Mesenteric volvulus in the dog: a retrospective study of 12 cases.

Mesenteric volvulus was diagnosed in 12 dogs over a nine-year period. Each case was presented with abdominal distension and shock. Haematochezia, which is frequently reported in association with mesenteric volvulus, was present in only two of the dogs. The diagnosis, which in all cases was based on radiography, was followed by immediate treatment for shock and surgical intervention whenever possible. In five of the 12 cases, the volvulus was treated successfully and these patients survived. This indicates that the prognosis for mesenteric volvulus might be better than is currently believed, and immediate laparotomy is recommended if mesenteric volvulus is suspected.

Experimental induction of diarrhoea in newly-weaned piglets.

To induce diarrhoea and hypovolaemia, newly-weaned conventionally bred piglets (3- to 4-weeks-old), were either given secretagogues or were inoculated with enterotoxigenic Escherichia coli (ETEC). Choleratoxin (n = 2), E. coli heat-labile enterotoxin (n = 2) or castor oil (n = 2) were given intragastrically or piglets were intraperitoneally injected with DL 5-hydroxytryptophan (n = 3). These substances induced a transient diarrhoea without clinical symptoms of dehydration. Therefore, a combination of castor oil and DL 5-hydroxytryptophan was given two times a day during 3 consecutive days to 3 piglets. Although diarrhoea lasted for 5 days, still no hypovolaemia occurred. Probably the secretagogues have to be given continuously to mimic the continuous release of enterotoxins during secretory colibacillary diarrhoea. It was, therefore, tried to reproduce colibacillosis in the just-weaned piglets. Animals were inoculated with K88ac fimbriae producing ETEC strains (O149:K91:K88ac; LT, STa and STb positive) (n = 7), or pretreated with chloramphenicol followed by the ETEC inoculation (n = 8), or pretreated with the antibiotic, inoculated with an enteropathogenic coronavirus, transmissible gastroenteritis virus (TGEV), and subsequently inoculated with ETEC (n = 18). Only the last procedure induced a reproducible diarrhoea (93%) and dehydration resulting in a mortality of 80%. It was concluded that the latter experimental procedure could be used to study diarrhoea and hypovolaemia in newly-weaned piglets and to evaluate the effect of potentially antisecretory drugs on postweaning diarrhoea in piglets.

Escherichia coli sepsis and endotoxemia in conscious young pigs.

In conscious pigs the influence of intravenous infusion of live E. coli (7 x 10(8)/kg), of the equivalent amount of endotoxin (20 micrograms/kg) or of a high dose of endotoxin (2.5 mg/kg) on the hemodynamic, clinical and pathological parameters and on survival rate was studied. E. coli and endotoxin infusion resulted in pulmonary hypertension, systemic arterial hypotension, a decrease in cardiac output and an increase in heart rate. Clinical signs were characterized by respiratory and nervous disturbances, whereas necropsy revealed hemorrhages and edema in several organs. Although these findings were similar in the three groups, a marked difference in lethality was observed. Infusion of E. coli or of the high dose of endotoxin resulted in a significant mortality, whereas all pigs survived the infusion of the low dose of endotoxin. This suggests that the lethal pathophysiological mechanisms may only become activated when a sufficient amount of endotoxin is released into the circulation.

Endotoxic shock in the awake young pig: absence of beneficial effect of prednisolone sodium succinate treatment.

In nonanesthetized young pigs, the influence of prednisolone sodium succinate therapy on a 65% lethal dose of Escherichia coli endotoxin was studied by evaluating clinical signs, several hemodynamic variables, survival rate, and changes seen at necropsy. Endotoxin infusion induced reproducible clinical signs characterized by nausea, vomiting, dyspnea, cyanosis, and moderate excitement followed by severe CNS depression. Among the hemodynamic variables, there were decreases in arterial blood pressure and cardiac output and increases in pulmonary arterial pressure, heart rate, and total peripheral and pulmonary vascular resistances. Core temperature and arterial pH did not change significantly. Survival rate at 30 hours after the start of the endotoxin infusion was 35%. According to the necropsy, marked edema and hemorrhages were in several organs. Treating the experimental animals with prednisolone sodium succinate (3 injections of 10 mg/kg of body weight after the start of the endotoxin infusion) did not influence any of the monitored hemodynamic variables, except for arterial blood pressure, which was higher at the end of the hemodynamic recording period (270 minutes after the start of the endotoxin infusion). Clinical signs, survival rate, and changes at necropsy were similar in both treated and nontreated pigs. This lack of effect can be due to an inappropriate dosage of the steroid or failure of steroid treatment to alleviate endotoxin-mediated effects.

Hemodynamic evaluation of endotoxic shock in anesthetized piglets: antagonism of endogenous vasoactive substances.

In anesthetized piglets the influence of an LD100 of Escherichia coli endotoxin (0.5 mg/kg IV, bolus injection) on several hemodynamic parameters and on survival time was studied. Endotoxin provoked a pronounced decrease in arterial pressure and cardiac output and an increase in portal venous and pulmonary arterial pressure and heart rate. Total peripheral and mesenteric vascular resistances displayed an initial increase followed by a sustained decrease, whereas pulmonary vascular resistance revealed a pronounced biphasic increase. All pigs died within 210 min following endotoxin administration. Pretreatment with the 5-HT2-antagonists R 41468 and R 50970 and with the prostanoid-synthesis inhibitor flurbiprofen induced a significant attenuation of the increase in pulmonary vascular resistance and beneficial effects on survival. Best survival results were obtained with prednisolone sodium succinate, although these results can only partly be ascribed to beneficial hemodynamic effects. The experiments with the opiate antagonists, however, point to detrimental effects.