The induction of immune responses by the delivery of inactivated pathogens has been a standard and successful vaccination approach for many years, and licenced, inactivated vaccines for diseases such as poliomyelitis 63 and rabies 64 are commercially available. The long history of this approach is underpinned by a well‐defined regulatory framework that can be readily applied to new disease targets 65. The major challenge for the inactivated virus approach is that infection is not established, and therefore a full adaptive immune response is generally not achieved. However, because of the absence of living pathogens, these types of vaccines are safe and a basic capability to prepare such vaccines, especially for emergency use, might be worthwhile as a stop‐gap while alternative longer‐term approaches are developed. In this regard, studies of virus inactivation with X‐ray radiation (as a simple and cheap alternative to gamma irradiation by the use of radioactive isotopes), which maintain the tertiary antigenic structures of virus particles while destroying infectivity, have shown useful promise for a range of applications including vaccination (B. Afrough, unpublished).

Synthetic peptide‐based epitope‐vaccines (EVs) make use of short antigen‐derived peptide fragments that can be presented either to T cells or B cells 58. EVs offer several advantages over other forms of vaccines, particularly with regard to safety, ease of production, storage and distribution, without cold chain issues. They also offer the opportunity to vaccinate against several pathogens or multiple epitopes from the same pathogen. However, drawbacks include poor immunogenicity and the restriction of the approach to patients of a given tissue type [human leucocyte antigen (HLA) haplotype] 59 and, as such, they need to be tailored to accommodate the natural variation in HLA genes. Although initially this was thought to be a major impediment, new technologies have made this personalized‐medicine approach feasible 60, 61. Recently, bioinformatics tools have been developed to identify putative CD4+ T cell epitopes, mapped to the surface glycoproteins of the emerging viruses LASV, NipV and Hendra 62. While these vaccine candidates still need to be experimentally tested, the approach represents an interesting and novel strategy that shows promise for vaccination and which could also address immunity in particular target populations.

1University of Minnesota, Saint Paul, MN, USA, 2University of Minnesota, Minneapolis, MN, USA :::

Emily Coffey

1, Kim Little1, Davis Seelig1, Aaron Rendahl2, Jennifer Granick1

Quantitative bacterial culture and sensitivity testing is the gold standard for diagnosing bacterial urinary tract infections. Samples are commonly transported to external reference laboratories prior to inoculation of culture media. The objective of this study was to compare the results of bacterial culture and sensitivity testing of canine and feline urine samples when streak plate inoculation is performed immediately after sample collection versus when streak plate inoculation is delayed until arrival at a reference laboratory.

This was a prospective, observational study that included urine samples from 194 canines and 45 felines submitted for routine urinalysis and urine culture and sensitivity testing. Streak plate inoculations of urine samples were performed immediately after sample collection, and again after receipt by an outside reference laboratory. Culture and sensitivity results were compared. Additional data collected included signalment, comorbidities, presence of lower urinary tract signs, urinalysis results, and antimicrobial history.

Overall agreement between immediate and delayed culture was 87%. While 16% of samples were collected via free‐catch, they represented 41% of discrepant culture results. However only 8% of free‐catch, versus 50% of cystocentesis, culture discrepancies were deemed to have a clinical impact. Pyuria was significantly associated with positive culture results.

Use of external reference laboratories for urine culture and sensitivity testing is generally reliable if samples are stored and transported according to pre‐established guidelines. Though discordant results of immediate versus delayed culture of cystocentesis samples were more likely to alter clinical decisions, these discrepancies were uncommon.

Lung samples from infected and control animals were used for the microdissection study. For each animal, 20 μm sections were cut from formalin-fixed paraffin-embedded (FFPE) lung tissue and mounted in PEN-membrane slides (two sections per slide). Prior to deparaffinization, slides were placed into an oven at 60 °C for 25 min. For each lung sample, sections were cut, deparaffinised, and rehydrated using standard protocols with RNase-free reagents, and stained with 1% cresyl violet acetate (SIGMA, C5042), and alcoholic 1% eosin (Alvarez, 10-3051). Stained slides were then dehydrated through a series of graded ethanol steps prepared with DEPC treated water (Ambion, P/N AM9915G) to 100% ethanol. The slides were then air-dried for 10 min, and individually frozen at −80 °C in 50 mL parafilm sealed falcons, before being transferred to the LCM microscope (LMD6500; Leica Microsystems) for simple microdissection.

Bronchiolar, vascular, and alveolar areas were separately selected for analysis using the Leica LMD6500 (Leica) system (20× magnification, Laser Microdissection 6000 software version 6.7.0.3754). Selected areas were chosen by a pathologist as observed in Figure 1. In infected animals, areas which exhibited pathological lesions were preferably selected. The total dissected area per selected lung compartment and animal rose approximately to 1.5 mm2. One cap was used per anatomic dissected compartment and animal.

The different dissected areas were then collected separately into RNAse-free 1.5 mL PCR tubes, per lung compartment.

Right lung lobe sections (cranial and caudal lobes), were taken for histological examination. The tissues were fixed for 24–48 h in neutral-buffered 10% formalin, and then embedded in paraffin wax in two different blocs containing one portion of the cranial and the caudal right lung lobes, consecutively taken. One of the paraffin blocks was sectioned at 3 µm, and stained with haematoxylin and eosin (HE) for examination under light microscopy; the second paraffin block was used for microdissection studies.

Cross sections of the cranial and caudal pulmonary lobes for each animal were histopathologically and separately evaluated. Semiquantitative assessment of IAV-associated microscopic lesions in the lungs was performed. The lesional scoring was graded on the basis of lesion severity as follows: grade 0 (no histopathological lesions observed), grade 1 (mild to moderate necrotizing bronchiolitis), grade 2 (bronchointerstitial pneumonia characterised by necrotizing bronchiolitis and alveolar damage in adjacent alveoli), and grade 3 (necrotizing bronchiolitis and diffuse alveolar damage in the majority of the pulmonary parenchyma). Microscopic lesional scores were assigned for each lobe, and the means of the two lobes were used for the final histopathological score for each animal.

Franziska Sonderegger, Thierry Francey, Ariane Schweighauser, Eliane Marti, Jelena Mirkovitch, Simone Schuller

Leptospiral pulmonary hemorrhage syndrome (LPHS) is a severe manifestation of leptospirosis, which affects both humans and animals, and is associated with high mortality. The pathogenic mechanisms of LPHS are poorly understood. Aim of this prospective observational clinical study was to examine whether soluble I‐CAM‐1 (sI‐CAM‐1), a serum marker of endothelial cell activation and dysfunction, is altered in dogs with leptospirosis and whether this marker is correlated with the occurrence of pulmonary hemorrhage and outcome.

Ethical approval was obtained for the study. Prospectively collected day 1 from healthy dogs and day 1 and 3 serum samples from dogs with leptospirosis with and without pulmonary hemorrhage and dogs with acute kidney injury (AKI) due to other causes were included. Dogs were deemed healthy based on an uneventful history and normal physical examination. Dogs with leptospirosis presented consistent clinical, laboratory and imaging findings and at least one microscopic agglutination test (MAT) titre ≥1:800 or Leptospira‐PCR positivity. Dogs with AKI due to other causes showed negative MAT on one or paired samples and a convincing alternative diagnosis. Serum sI‐CAM‐1 was measured using a commercial ELISA‐kit (SEA548Ca; Brunschwig) and results compared between groups using the Kruskal‐Wallis Test. Associations between sI‐CAM‐1, disease group and outcome were tested using logistic regression analysis. The diagnostic accuracy of day 1 sI‐CAM‐1 to predict the occurrence of LPHS was tested using ROC curve analysis.

Median sI‐CAM‐1 serum concentrations were significantly higher in dogs with AKI (43.6 ng/ml, IQR 18.9‐72.1; n = 18) and in dogs with LPHS (64.2 ng/ml, IQR 42.4‐107.1; n = 14) compared to heathy controls (15.8 ng/ml, IQR 8.8‐29.9; n = 31; p < 0.001). Median sI‐CAM‐1 was higher in dogs with leptospirosis without LPHS (37.6 ng/ml, IQR 20.9‐53.4, n = 10) compared controls, and lower than in the LPHS group, but these differences did not reach statistical significance. Few day 3 samples were available for analysis, but showed similar trends with highest sI‐CAM‐1 in dogs with LPHS (87.5 ng/ml, IQR 44.1‐101.7; n = 5) compared to healthy controls, dogs with AKI (21.9 ng/ml, IQR 10.5‐71.3; n = 5) and leptospirosis without LPHS (26.2 ng/ml, IQR 23.2‐29.3; n = 2). However, only the difference between healthy and LPHS group was statistically significant (p = 0.015). There was no significant association between sI‐CAM‐1, disease group and outcome. Day 1 sI‐CAM‐1 predicted the development of LPHS with reasonable accuracy (AUC 0.79; sensitivity 79%, CI 49‐95; specificity 73%, CI 60‐84).

These findings suggest, that in the context of leptospirosis, overexpression of endothelial ICAM‐1 may be associated with the development of pulmonary hemorrhage.

The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the National Animal Disease Center-USDA-Agricultural Research Service. Thirty-two 10-week-old cross-bred pigs were obtained from a U.S. high-health herd and were found to be free of PRRSV and influenza virus antibodies using commercially available enzyme-linked immunosorbent assay (ELISA) kits (HerdChek PRRS 2XR; IDEXX Laboratories, Westbrook, Maine) and NP ELISA (MultiS ELISA, IDEXX, Westbrook, Maine), respectively. Pigs were also confirmed negative for porcine circovirus type 2 by quantitative real-time PCR. One day prior to starting the experiment, pigs were bled, weighed and randomly assigned to one of four groups. Group 1 (N = 8) consisted of negative control pigs, which received an intranasal 2 ml sham inoculum of minimum essential media (MEM) on 0 dpi. Group 2 pigs (N = 12) were challenged intranasally with 2 ml of 1 × 106 50% tissue culture infective dose (TCID50)/ml of Chinese PRRSV strain rJXwn06 in Animal Biosafety Level-3-Agriculture (ABSL-3-Ag) housing, where they remained for the duration of the experiment. Group 3 consisted of naïve pigs (N = 4) that were placed in contact with Group 2 swine on 2 dpi. Group 4 pigs (N = 8) were challenged intranasally with 2 ml of 1 × 106 TCID50/ml of Type 2 prototype strain VR-2332. Groups 1 and 2 were housed in separate isolation rooms in an ABSL2 facility. Animal care and euthanasia were conducted in accordance with the Report of the AVMA Panel on Euthansia and under the supervision of IACUC of NADC. Serum and bronchoalveolar lung lavage fluid (BALF) were tested for infectious virus as described previously. Lungs were scored for gross lesions and sections fixed for histopathology. Swabs were collected from BALF, and various sites for bacterial isolation.

MARC-145 cells were cultured in minimum essential medium (EMEM, SAFC 56416C) with 10% fetal bovine serum at 37°C, 5% CO2. Wild-type (wt) Type 2 PRRSV strain VR-2332 (GenBank U87392), passage 6 on MARC-145 cells, was titrated and used for the swine study. Virus (rescued JXwn06; rJXwn06) was rescued from a cloned cDNA of Chinese highly pathogenic Type 2 PRRSV strain JXwn06 [pWSK-JXwn; GenBank EF641008,] and passaged 3 times on MARC-145 cells for use in the swine study.

The incubation period of Lassa fever ranges from 7 to 21 days. The clinical disease begins as a flu-like illness characterized by fever, general weakness, and malaise, which may be accompanied by cough, sore throat, and severe headache. Gastrointestinal manifestations such as nausea, vomiting, and diarrhea are also common (Table 2). The differential diagnosis of Lassa fever based on the presenting symptoms can be problematic due to the many other acute undifferentiated febrile illnesses circulating in West Africa. Although, hemorrhagic manifestations are not an important feature of Lassa fever, perturbation of vascular function is likely to be central to Lassa fever-associated pathobiology, since the signs of increased vascular permeability, such as facial edema and pleural and pericardial effusions, indicate a poor prognosis for the disease outcome. Recovery from Lassa fever generally begins within 8 to 10 days of disease onset. In severe cases, the condition of the patient deteriorates rapidly between the 6th and 10th day of illness with severe pulmonary edema, acute respiratory distress, clinical signs of encephalopathy, sometimes with coma and seizures, and terminal shock. Bleeding from mucosal surfaces is often observed; however, it is usually not of a magnitude to produce shock by itself. Sensorineural deafness is commonly observed in patients in the late stages of disease or in early convalescence in survivors.

The level of viremia is highly predictive of the disease outcome. In a study involving 137 patients with Lassa fever, patients that presented with viremia < 103 median tissue culture infectious dose (TCID50)/ml on the day of hospitalization had 3.7 times greater chance of survival than those admitted with higher levels of viremia. Similarly, the probability of fatal outcome in patients with serum titers > 103 TCID50/ml and serum levels of aspartate aminotransferase (AST) ≥ 150 international units (IU)/L was 21 times higher than that in patients not meeting either of these criteria. Virtually all patients with fatal Lassa fever whose sera were tested were viremic at the time of death with terminal viremia ranging from 103 to 108 TCID50/ml. Detailed studies have shown that viremia peaks between 4 and 9 days after the onset of symptomatic disease and is followed by pronounced clinical manifestations. Patients recovering from Lassa fever clear virus from blood circulation about 3 weeks after the beginning of illness.

The current knowledge of Lassa fever pathogenesis does not include the chain of events that take place during disease development and lead to death of severely ill patients. Apparently, failure to develop the cellular immune response that would control dissemination of LASV, which is indicated by high serum virus titers, combined with disseminated replication in tissues and absence of neutralizing antibodies, leads to the development of fatal Lassa fever. However, considering the high mortality and truly dramatic course of the disease, the pathological findings do not provide the basis that would explain the mechanism of disease progression and the cause of death from Lassa fever.

Physical examination of patients after the onset of fever often reveals purulent pharyngitis, bilateral conjunctival hemorrhages, facial edema, and generalized abdominal tenderness. Macroscopic pathological changes can include pleural effusions, pulmonary edema, ascites, and hemorrhagic manifestations in the gastrointestinal mucosa. Microscopic findings include hepatocellular necrosis and apoptosis, splenic necrosis, adrenocortical necrosis, mild mononuclear interstitial myocarditis without myocardial fiber necrosis, alveolar edema with capillary congestion and mild interstitial pneumonitis, lymph nodal sinus histiocytosis with mitoses, gastrointestinal mucosal petechiae, renal tubular injury, and interstitial nephritis. A comprehensive postmortem histopathological examination of 21 virologically confirmed community-acquired cases of Lassa fever in Sierra Leone revealed variable levels of hepatic necrosis involving from 1 to 40% of hepatocytes. The necrotic hepatocytes were randomly distributed often forming foci of contiguous cells. Mononuclear phagocytes were observed either contacting or phagocytosing necrotic hepatocytes. Interestingly, this phagocytic reaction, although highly variable from case to case and even from one necrotic focus to another in the same case, demonstrated a tendency towards homogeneity of the level of involvement within a particular patient. The predominant distribution of splenic necrosis was observed in the marginal zone of the periarteriolar lymphocytic sheath. Close examination of thin tissue sections revealed the presence of fibrin in addition to the debris of necrotic cells. Splenic venous subendothelium appeared to be infiltrated by lymphocytes and other mononuclear cells. Microscopic examination of adrenal glands showed prominent spherical, hyaline, acidophilic cytoplasmic inclusions in cells near the junction of zona reticularis and medulla. In most cases these cells appeared to be adrenocortical cells of the zona reticularis; however, some cells were of adrenal medulla origin. Additionally, multifocal adrenocortical cellular necrosis was detected that was most prominent in the zona fasciculata and was often associated with focal inflammatory reaction. However, in all examined cases adrenal necrosis was mild and ≥ 90% of the cells of adrenal cortex appeared viable.

The major and most common lesions of Lassa fever in humans occur in the liver. There are four principal features of LASV hepatitis can be derived: 1) focal cytoplasmic degeneration of hepatocytes suggestive of phagocytosed apoptotic fragments; 2) randomly distributed multifocal hepatocellular necrosis; 3) monocytic reaction to necrotic hepatocytes; 4) hepatocellular mitoses. These morphologic effects do not uniformly occur in all cases, but in some instances can be found simultaneously. Based on the degree of hepatic damage, three general nosopoetic phases have been proposed to divide patients with fatal Lassa fever into categories with respect to pathogenic events in fatal LASV hepatitis. The first phase, active hepatocellular injury, is defined by the presence of focal cytoplasmic degeneration with <20% of hepatocytes undergoing necrosis. This phase may represent the late stage of viremic spread and early cellular injury, which is, most likely, caused by direct viral action rather than mediated by a cellular immune response, since lymphocytic infiltration is not detected. The second phase, the peak of Lassa hepatitis, is characterized by 20-50% necrosis of hepatocytes, widespread focal cytoplasmic degeneration and limited phagocytic infiltration. This is suggested not only progressive hepatocellular damage, but, also, early liver recovery through phagocytic removal of necrotic hepatocytes and regeneration of new cells. The third phase, hepatic recovery, is defined by <10% of hepatocellular necrosis, absence of focal cytoplasmic degeneration and clear evidence of mitoses, which indicate liver regeneration. Interestingly, no correlation has been observed between the degree of hepatic necrosis and chemical indicators of liver damage, such as elevated levels of AST, alanine transaminase (ALT), and lactate dehydrogenase (LDH) in serum. Overall it is apparent that the level of liver damage can vary dramatically in patients that die from Lassa fever. Therefore, it can be concluded that liver disease is a necessary, but not sufficient, condition in the chain of pathological events that lead to fatal outcome.

Lassa fever is not considered to be associated with coagulation dysfunction, e.g., neither decrease in the coagulation factors nor disseminated intravascular coagulation (DIC) has been observed in infected patients. However, moderate thrombocytopenia with significantly impaired functionality of thrombocytes is detected in patients with severe Lassa fever.

One possible mechanism involved in Lassa fever pathogenesis could be infection-triggered induction of uncontrolled cytokine expression similar to what is seen in sepsis. This hypothesis is supported by experimental data obtained from a case of fatal Lassa fever imported into Germany in 2000. In this patient, who died from multi-organ failure and hemorrhagic shock, the proinflammatory cytokines, interferon γ (IFN- γ) and tumor necrosis factor α (TNF- α), rose to extremely high levels shortly before death. However, in another study no elevation of both cytokine levels was observed in the examined fatal cases of Lassa fever, which suggests that the levels of IFN- γ and TNF- α are either elevated only in a fraction of patients or during a very short period that would require frequent sampling for detection.

Another possibility is that virus-induced immunosuppression may be involved in the pathogenesis of severe Lassa fever. Thus, infection with LASV fails to activate monocyte-derived dendritic cells (DC) and macrophages (MP) of human origin. Infected DC fail to secrete proinflammatory cytokines, do not upregulate costimulatory molecules, such as CD40, CD80, and CD86, and poorly induce proliferation of T cells. Importantly, human DC infected with the naturally nonpathogenic Mopeia virus, a closely related arenavirus that shares 75% amino acid similarity with LASV and was isolated from the same rodent reservoir, induces stronger CD4 and CD8 T-cell responses than those infected with LASV. Downregulation of immune responses caused by LASV infection demonstrated in vitro is also in agreement with the results of clinical observations showing that fatal outcome of Lassa fever correlates with low levels or absence of interleukin (IL) 8 and IFN inducible protein 10 (IP-10) in circulation.

Mice and guinea pigs have been evaluated as models of LASV infection. However, normal adult mice are highly resistant to peripheral routes of inoculation. Mice expressing humanized MHC-I are LASV-susceptible and develop a severe illness. Genotype of a particular mouse strain has significant influence on the development of pathologic manifestations in infected mice. Thus, newborn mice develop an asymptomatic infection upon inoculation with LASV with high virus titers in the brain, lung, and muscle, while intracranial inoculation of adult mice results in a fatal convulsive disorder resembling classical murine LCM immunopathology. However, other investigators failed to reproduce these findings. Pathogenicity of LASV for guinea pigs largely depends on the host strain and the virus used for inoculation. For instance, Josiah strain of LASV causes a uniformly lethal disease in inbred strain 13 guinea pigs; however, the lethality varies from approximately 30% to more than 60% in outbred Hartley guinea pigs. Inbred animals have higher viral titers in all target tissues than outbred guinea pigs. LASV-infected guinea pigs destined to die develop respiratory insufficiency with pulmonary edema, alveolar hyaline membranes, myocarditis, and focal calcification of myocardial fibers and hepatocytes. Terminally ill animals are viremic and contain virus in nearly every organ tested. There are two major differences between Lassa fever pathogenesis in humans and guinea pigs. In humans LASV is particularly hepatotropic, and patients with severe Lassa fever develop hepatocellular necrosis; however, in guinea pigs only foci of calcified hepatocytes are observed. On the other hand, LASV is myocardiotropic and myocardiopathic in guinea pigs, findings that have not been reported in humans. Also, several isolates of LASV from clinical human cases are benign in guinea pigs, and some are lethal when tested in cynomolgus monkeys. Therefore, in order to reliably model the pathogenesis of Lassa fever in humans, studies in non-human primates are required.

Several non-human primate species have been evaluated as potential models for Lassa fever including squirrel monkeys, capuchin monkeys, marmosets, hamadryas baboons, African green monkeys, cynomolgus monkeys, and rhesus monkeys. Capuchin and squirrel monkeys seroconverted upon infection with LASV; however, the animals of both species uniformly survived low and high dose inoculations. Virus was detected in virtually all organs of infected squirrel monkeys, but the liver, lymph nodes, and kidneys were the key early targets. Later in infection, the spleen, heart, and brain also showed pathological alterations. Viremia persisted in all animals for up to 28 days after infection. Histopathologic changes were mild and included germinal center necrosis in the spleen and lymph nodes, myocarditis, acute arteritis, renal tubular necrosis and regeneration, chronic inflammation of the choroid plexus, ependyma, and meninges, and cerebral perivascular cuffing. Hepatocytic regeneration suggesting recovery from disease-mediated liver damage was also observed. Hamadryas baboons infected with LASV develop a disease that clinically resembled a severe form of human Lassa fever. The animals developed fever, characteristic pathologic and hemorrhagic manifestations, and high levels of viremia. Interestingly, African green monkeys and rhesus macaques challenged with low doses (10-15 pfu) of LASV uniformly succumbed to the infection, whereas a high challenge dose (106 pfu) was only partially lethal in these animals. The gross pathological changes in LASV-infected rhesus macaques were petechiae and, in some animals, mild-to-moderate pleural effusions. Viremia appeared between days 5 and 10 in all animals and increased progressively until the animals died or met the criteria for euthanasia. Virus was detected in all organs tested, namely adrenal glands, spleen, liver, duodenum, jejunum, colon, bone marrow, lymph nodes, thymus, heart, lungs, pleural fluid, kidneys, skeletal muscle, pancreas, salivary glands, ovaries, bladder, cerebrum, cerebellum, brain stem, cerebrospinal fluid, and aqueous humor of the eye. The largest quantities of virus were generally detected in adrenal glands, spleen, liver, bone marrow, and intestines. The microscopic lesions included mononuclear pulmonary arteritis with intraluminal mononuclear cell aggregates and mononuclear cell infiltration of the subendothelium and the pulmonary arterial wall accompanied by endothelial hypertrophy and hyperplasia, hepatocellular necrosis, interstitial pneumonia, adrenal gland necrosis, encephalitis and uveitis. Therefore, infected rhesus monkeys exhibited pathologic lesions similar to human Lassa fever, such as the amounts and organ distribution of virus, necrosis of hepatocytes, adrenal cortical cells, and splenic marginal zone of the periaortic lymphocytic sheath, and interstitial nephritis. However, meningoencephalomeningitis, pulmonary vasculitis, systemic arteritis, and skeletal myositis were significantly more prominent in the monkey model than in human Lassa fever. The clinical manifestations of LASV-infected cynomolgus macaques included fever, weight loss, depression, and acute respiratory syndrome. Other clinical features included thrombocytopenia, lymphopenia, lymphadenopathy, splenomegaly, infiltration of mononuclear cells, and pathologic alterations in the liver, lungs, and endothelium, which essentially mirrored observations from human cases. An additional feature of the disease observed in cynomolgus monkeys was multifocal to severe central nervous system lesions at terminal stages. Dysregulation of the host immune response characterized by increased circulating levels of proinflammatory cytokines/chemokines including IL-1β, IL-6, MCP-1, and eotaxin were detected in the infected animals. Histopathologic evaluation of tissues revealed a sequence of events in LASV infection in cynomolgus macaques, which is initiated with dendritic cells in the lymphoid tissues, then progresses to infection of Kupffer cells in liver and parenchymal cells in liver and adrenal glands, and endothelial cells in various organs including the central nervous system. Experimental infection of common marmosets results in systemic disease from clinical and pathologic standpoints highly similar to disease observed in fatal cases of Lassa fever in humans. Among the main clinical features are fever, weight loss, high viremia and viral RNA loads in tissues, elevated levels of liver enzymes, and severe morbidity between days 15 and 20. Histopathologic changes include multifocal hepatic necrosis associated with mild inflammation and hepatocyte proliferation, adrenal necrosis, lymphoid depletion, and interstitial nephritis. The necrotic hepatocellular foci observed in LASV-infected marmosets contained predominantly macrophages with the near absence of CD20-, CD8-, or CD3-positive lymphocytes, markedly decreased expression of MHC-II molecules, and hepatocyte proliferation. The levels of MHC-II expression were also significantly reduced in lymph nodes. Overall numbers of CD20- and CD3-positive lymphocytes in the spleen of infected animals were reduced, and the destruction of lymphoid follicular architecture was evident. These findings strongly suggest that the replication of LASV in target tissues may cause pathologic changes that directly impair the adaptive immune response to LASV infection.

Coordinates and structure factors have been deposited in the Protein Data Bank with accession number 6JGY for crystal structure of fusion core of LASV GP2 protein.

Lassa fever is an acute viral hemorrhagic illness occurring in West Africa, having posed a serious public health threat in many countries (Sogoba et al., 2012; Shaffer et al., 2014). Its case-fatality rate is 1% for overall infection and 15∼20% for severe cases among patients hospitalized. This mortality rate will increase sharply during epidemics or in pregnant women (Asogun et al., 2012). The etiologic agent of Lassa fever is Lassa virus (LASV), belonging to the arenavirus family. Arenavirus has more than 30 members divided into two groups: the New World viruses (or Tacaribe complex) and the Old World viruses (or LCM-Lassa complex). The New World family mainly contains Venezuelan hemorrhagic fever (VHF), Junín virus (JUNV), Machupo virus (MACV), and Bolivian hemorrhagic fever (BHF), while the Old World viruses includes, for example Lujo virus (LUJV), Lymphocytic choriomeningitis virus (LCMV), Morogoro virus (MORV), and LASV. These arenaviruses have both geographical and genetic differences.

Lassa virus is an enveloped, single-stranded RNA virus. The two RNA segments in its genome encode four viral proteins, including zinc-binding protein (Z), RNA polymerase (L), nucleoprotein (NP), and the surface glycoprotein precursor (GP, or spike protein). GP is cleaved into envelope glycoproteins GP1 and GP2 (Cao et al., 1998). GP1 that is responsible for receptor binding (including α-dystroglycan, heparin sulfate, DC-SIGN, etc.) and GP2 that mediates membrane fusion interact with each other to form a stable trimer complex on the LASV viral envelope (Li et al., 2016; Hastie et al., 2017). Upon receptor binding, LASV enters the target cell via clathrin- and dynamin-independent endocytosis with subsequent transport to late endosomal compartments, where fusion occurs at low pH (Vela et al., 2007; Rojek et al., 2008). A recent study has also found that after GP1 binds to cell receptor α-dystroglycan, LASV enters into the target cell through the unusual micropinocytosis pathway and membrane fusion under low pH condition (Oppliger et al., 2016).

So far, no approved vaccines and specific treatment modalities against LASV are available. Given that the first peptide-based antiviral drug enfuvirtide (T20) inhibits human immunodeficiency virus (HIV) fusion with and entry into the target cell by targeting the heptad repeat domain in the viral envelope glycoprotein gp41 (Qi et al., 2017; Su et al., 2017), the heptad repeat domain in GP2 of LASV may also serve as a target for the design of LASV fusion inhibitors. We have previously demonstrated that the N-terminal heptad repeat 1 (HR1) domain binds to C-terminal heptad repeat 2 (HR2) domain to form a stable six-helix bundle (6-HB) to mediate viral entry; therefore, peptides derived from HR2 should specifically bind to the homotrimeric HR1, interfering with the formation of 6-HB and, hence, blocking viral entry (Zhu et al., 2015, 2016, Xia et al., 2019). Therefore, it is essential to determine the 6-HB core structure and characterize the interaction sites in the HR1 and HR2 domains, in order to design the HR2-derived peptides against LASV infection. Here, we solved the crystal structure of the post-fusion 6-HB formed by LASV HR1 and HR2 domains. Based on the structure, we designed several peptides spanning the HR2 domain and tested their antiviral activities. Concurrent with the preparation of the present manuscript, another group reported a post-fusion structure of LASV using the insect baculovirus expression system (Shulman et al., 2019), which allows us to compare the structural differences of 6-HB cores formed by the HR1 and HR2 domains in their truncated version of LASV GP2 spanning residues 306–421 with those in our LASV HR1-T-loop-HR2 construct. These comparisons provide more comprehensive knowledge for better understanding the entry mechanism of LASV and designing of peptide-based LASV fusion inhibitors.

Development of an effective asexual blood stage malarial vaccine candidate has been difficult and fraught with problems. Apparently, to generate the most effective candidate new antigen choices as well as new delivery technologies need to be developed. Here we demonstrated the addition of two new promising B-cell epitopes P27-NC and P27A from the Tex1 antigen to the SAPN delivery system. In particular, the P27A antigen is currently being evaluated in clinical trials as a synthetic peptide (ClinicalTrials.gov; PACTR201310000683408), thus improving its immunogenicity by coupling to a suitable carrier seems to be a highly promising approach.

The humoral immune response to the two segments of P27-NC clearly shows the relationship of immunodominant epitopes versus less immunogenic regions: The most prominently exposed portions of P27-NC, the C-terminal portion of the trimeric coiled coil (i.e. P27-C) induces a stronger immune response compared to the antibody production against the N-terminal region P27-N (Table 2), which is more buried in the core of the SAPNs (see Fig. 2d). Remarkably, for the corresponding shorter fragment Pept-P27-C 62% is recognized by 62% of donor’s sera from Burkina Faso (Table 1). Since the P27-C is also highly conserved among many different Plasmodium species (Fig. 5), this is a welcome feature of our SAPN design as it may induce cross-protection among different Plasmodium species, including the most prominent parasites species P. falciparum and P. vivax.

One of the major advantages of our SAPN technology is that it enables the addition of two B cell antigens, one on each terminus of the monomer to be displayed on the surface of the SAPNs. In addition, T-cell epitopes can be engineered into the SAPN core to be delivered to the antigen processing machinery of the cells. This unique design therefore allows for the inclusion of multiple T and B cell epitopes. In this case we included both P27-NC and P27A B-cell epitopes, which both have been established to be immunogenic in mice (P27), rabbits and humans (P27A, manuscript in preparation) in combination with a variety of CD4 and/or CD8 epitopes. Human purified antibodies specific for P27 and P27A have been shown to be active in ADCI [10, 14] and are associated with protection in people in endemic areas (unpublished results). By including both P27-NC and P27A antigens it is likely to have a higher probability of inducing protective antibodies in a population at large. The present design has the particular advantage of being very immunogenic without the need of any adjuvant.

In summary, these new SAPNs represent a new vaccine strategy. The epitopes P27 and P27A are expressed primarily on the asexual blood stage of the parasite’s life cycle, while CSP is expressed on the pre-erythrocytic stage. A CSP based vaccine will only prevent infection before clinical symptoms develop. An asexual blood stage vaccine will actually help deal with preventing parasitemia from further developing and reducing clinical symptoms. As both approaches continue to be developed they eventually can be admixed to generate a vaccine candidate that prevents the initial infection, and can eventually prevent any breakthrough infection.

Listeria were grown overnight in BHI at 37°C and cultures were centrifuged at 8000 g for 5 min. Bacterial pellets were resuspended in 75 µg/mL lysozyme and incubated at 37°C for 1 h. DNA was then extracted using the DNeasy blood and tissue kit (Qiagen) and quantified by spectrophotometry (Nanodrop). For transfection assays, THP-1 macrophages were transfected with 200 ng/mL DNA with 2% lipofectamine 2000 (Invitrogen) and incubated for 24 h. Following incubation, supernatants were collected for IFN-β analysis. For pretreatment of DNA with DNase, DNase was added at final concentration of 100 µg/mL for 45 min at 37°C.

For cytokine analysis, macrophages were infected with Listeria strains at MOI 10∶1, centrifuged at 300 g for 2 min and incubated at 37°C for 15 min. Following phagocytosis, monolayers were washed twice followed by incubation in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and gentamicin (20 µg/mL). Supernatants were collected at various time points, for detection of IFN-β by ELISA. For transcript analysis, macrophages were infected with Listeria strains at MOI 20∶1 and incubated at 37°C for 1 h to allow phagocytosis. Monolayers were washed and incubated in DMEM supplemented with 10% FCS and gentamicin (5 µg/mL). After 2 h, medium was changed to DMEM supplemented with 10% FCS and gentamicin (1 µg/mL). Cells were lysed at various time points and RNA collected for qPCR analysis.

Malaria, the mosquito-borne parasitic disease caused by members of the Plasmodium genus, resulted in at least 438,000 deaths worldwide in 2014. Currently, there is an attempt to eliminate the disease from the human population. Attempts at vector control, diagnosis, and development of pharmaceuticals have all been successful in the reducing the number of cases, however, a major missing piece in the elimination campaign is an effective vaccine candidate. The current most successful vaccine candidate RTS,S is only about 30% protective in children in Sub-Saharan Africa, the most vulnerable population [2, 3]. The goal of malaria elimination and the weaknesses of RTS,S necessitate the development of new more successful vaccine candidates.

One of the most appealing, yet elusive, malaria vaccine candidates is one targeting the asexual blood stage, the clinical stage of the disease. Issues such as antigen polymorphism, lack of MHC I molecules on erythrocytes, and speed of erythrocyte infection all hamper the development of an asexual blood stage vaccine [4–9]. Many different antigen candidates and technical approaches have been attempted with little to no success. These problems have led to the search for new potential blood stage antigens using different screening mechanisms including bioinformatic approaches.

In one such bioinformatic approach coiled-coil domains present in the asexual blood stage were targeted. Coiled-coil domains are a common oligomerization domain in proteins known for their characteristic heptad repeat and stability, making them excellent choices for vaccine development. One of the most interesting targets identified in this screen was P27, which showed complete conservation in field isolates. P27 is a 27 amino acid sequence contained on the 1103 amino acid trophozoite export protein (Tex1). However, P27 was only able to activate 30% of peripheral blood mononuclear cells (PBMC) from semi-immune individuals, leading to fears that it would not offer broad protection in a vaccinated diverse population. This lead to further screening of the Tex1 protein and the identification of P27A in the N-terminal portion of the protein, a region that was predicted to be intrinsically unstructured. Initial screenings demonstrated that people living in endemic areas had antibodies for P27A, it was immunogenic in small mammal models, and antibodies raised against P27A were capable of inhibiting P. falciparum in antibody-dependent cellular inhibition parasite-growth assay. Currently, good immunogenicity results were obtained in phase Ia and Ib clinical trials of P27A combined with the adjuvants GLA-SE or Alhydrogel (ClinicalTrials.gov; PACTR201310000683408; manuscript in preparation).

Subunit vaccines based solely on recombinant proteins are generally weakly immunogenic and will most likely not be successful for vaccination in humans. To be an effective vaccine an important consideration is the delivery system. Several recent effective vaccine candidates are based on particulate delivery systems that are able to repetitively express antigens. One promising delivery system is the self-assembling protein nanoparticle (SAPN). Each SAPN monomer, the subunit that oligomerizes to form the nanoparticle, contains two coiled-coil domains held together by a linker. Antigenic B-cell epitopes are engineered on either the N or C terminal ends of the gene encoding the monomer. In the same way T-cell epitopes can be added to the core leading to a vaccine candidate that is able to activate both the humoral and cellular branches of the immune system. SAPNs show great promise for vaccine design [15, 17–19] and many SAPN-based malaria vaccines have previously been developed in our laboratories [20–23].

Normally, the sequence of amino acids that form the pentameric and trimeric oligomerization domains are de novo designed sequences that do not have homology to any human proteins to minimize the possibility of inducing an immune response that could be detrimental to the host receiving the vaccine. Here we detail the unusual step in the development of a SAPN vaccine candidate by using a parasite native coiled-coil sequence, the P27 epitope from the Tex1 protein, to form part of the core oligomerization domains. In combination with the intrinsically unstructured epitope P27A, this vaccine candidate is immunogenic in a murine model and induces antibodies that are recognizing the same antigens as sera from seropositive individuals in Burkina Faso, a malaria endemic region.

This study was undertaken on three groups of cats. Group 1.1 comprised 16 cats with FIP, further subdivided into natural infection (12 pet cats; age: 5 months to 2 years; Group 1.1a) and experimental infection (four female specific pathogen free (SPF) cats; age: 14–16 weeks; Group 1.1b), see Table 1. All 1.1a cats were submitted for diagnostic post mortem examination with full owner consent. The 1.1b cats had been euthanased with FIP after experimental intra-peritoneal infection with the serotype I FCoV strain FIPV-UCD at the University of Utrecht, The Netherlands. Approval for this experiment was obtained from the Ethical Committee of Utrecht University (approval number: 0502.0802). All Group 1.1b cats showed clinical signs of FIP which necessitated euthanasia of two cats at 3.5 and 4 weeks post infection (p.i.) whilst the remaining two were euthanased at the end of the experiment (11 weeks p.i.).

The diagnosis of FIP was confirmed in all cases by gross, histological, and immunohistological examination. Six of the 16 cats with FIP had effusions, including three of the four experimental cases (data was unavailable for three animals).

Group 1.2 consisted of 14 clinically healthy, male SPF cats that had been per-orally infected at an age of 8.5 to 27 weeks with previously isolated serotype I FCoV field strains of enteric pathotype (FCoVZu1, 2, 3, and 5 -feline enteric coronavirus; FECV) and had been euthanased between 2 and 12 weeks p.i.. This experiment was performed under the Swiss regional legislation (project license number TVB 66/2000). All cats had tested positive for FCoV shedding and those euthanased more than 2 weeks p.i. seroconverted. All were confirmed to be systemically FCoV infected by the presence of a FCoV viraemia. These cats were used as a comparison group to provide relatively uniform baseline cytokine level as pet cats without FIP would be subject to wide variations in terms of pathogen exposure, FCoV infection status, and concurrent disease. This also allowed evaluation of the effect of FIP on the animal rather than FCoV infection per se.

All group 1.1 and 1.2 animals were necropsied within 1 h of death. Liver samples from grossly normal regions (i.e., without FIP lesions) were collected and immediately frozen at −80 °C for RNA extraction, whilst normal and lesion samples were fixed in 10% buffered formalin for 24–48 h and routinely paraffin wax embedded for histological and immunohistological examination.

The third group (Group 1.3) comprised six healthy untreated SPF cats, aged 36–38 months, that had been euthanased at the University of Glasgow, UK as part of a study performed under UK Home Office Project Licence PPL 60/3735. From these cats, formalin fixed, paraffin embedded liver samples were kindly provided by Prof M Hosie.

The immunolabelling of cell markers was done using the avidin-biotin complex method (ABC Vector Elite, Vector laboratories, USA) as described previously. Briefly, 4 μm thick sections were dewaxed and rehydrated, followed by endogenous peroxidase inhibition with 3% H2O2 in methanol for 30 min. Depending on the epitope of interest, antigen retrieval in the tissue sections was performed by enzymatic trypsin/alpha-chymotrypsin (for CD3 and MAC-387) or by microwaving the sections in citric acid pH6.0 (for CD79a) or pH6.0 citrate buffer (for PRRSV nucleocapsid N protein). The slides were mounted in a Sequenza Immunostaining Centre (Shandon Scientific, UK) and washed with Tris buffered saline (TBS; pH 7.6, 0.005 M; Sigma–Aldrich, UK) and incubated for 30 min at room temperature with 100 μL per slide of blocking solution. The primary antibodies used were monoclonal anti-human CD3 (1:1000; Dako, UK), monoclonal anti-human CD79a (1:400; Dako, UK), and monoclonal anti-human MAC-387 (1:100; AbDSerotec, UK). Each antibody was applied for 1 h at room temperature. In each case, the corresponding biotinylated secondary antibody (Vector Laboratories, UK) was then incubated for 30 min at room temperature. Slides were then incubated for 30 min with avidin-biotin complex and labelling performed using 3,30-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich, UK). Sections were counterstained with Mayer’s haematoxylin, dehydrated and mounted. Positive and negative controls, as well as isotype controls, were included in each IHC run.

The immunolabelled Med-LN sections were examined by light microscopy, and immunolabelling measurements recorded using a score ranking from −3 to 0 (cellular depletion) and from 0 to 3 (cellular increment) compared to the control group. Positive scale: 0 = absence (<1 positive cell/structure); 1 = scarce (1–10 positive cells/structure); 2 mild-moderate (11–30 positive cells/structure); 3 abundant (>31 positive cells/structure). Negative scale: 0 = absence (no different to control group); −1 = scarce (5% less positive cells compares with control group); −2 mild-moderate (6-10% less positive cells compares with control group); −3 abundant (more than 10% less positive cells compares with control group).

Pigs were monitored daily throughout the study, and clinical signs, including rectal temperatures, were scored and recorded as previously described. The Med-LN is the main draining lymph node for the apical and medial lobes of the lungs. Since PRRSV is most frequently detected in these lung lobes, Med-LN was selected for this study and the gross pathology evaluated during post-mortem examination. For the analysis by RT-qPCR, a piece of 10×10×3 mm of Med-LN was embedded and cryopreserved in optimal cutting temperature (OCT) compound (Sakura Finetek Europe B.V., The Nederland) as previously described. For histopathology examination, the remaining Med-LN samples were fixed in 10% buffered formalin, routinely processed, embedded in paraffin-wax, and 4 μm tissue sections stained with haematoxylin and eosin. The histopathological lesions were evaluated in these slides using a light microscope.

This study was undertaken on three additional groups of cats. Group 2.1 comprised 18 pet cats (age: 2 months to 3 years; mean age: 14 months) that had died or were euthanased with FIP; the diagnosis was confirmed as described above. See Table 2.

Group 2.2 comprised 10 cats that had been euthanased due to non-inflammatory diseases not expected to have any systemic impact (24 months to 19 years; mean age: 9 years) which were further grouped by age (Group 2.2a (n = 4), (two each); Group 2.2b (n = 6), aged 9–19 years, mean age: 13.4 years) to acknowledge the fact that age has an effect on constitutive cytokine expression in the myocardium. See Table 2.

Group 2.3 comprised three cats with systemic inflammatory diseases other than FIP. See Table 2.

All cats had been euthanased and submitted for diagnostic post mortem examination with full owner consent. They were necropsied within 1 h of death. Pleuritis involving the outer pericardium was observed in one of the FIP cats, however, neither this cat nor any of the others exhibited any gross changes in the heart. 14 of the 18 cats with FIP had effusions (data was unavailable for one animal). Hearts were removed and samples collected from both atria, both ventricular free walls, and the interventricular septum into RNAlaterTM Stabilization Solution (Thermo Fisher Scientific, Ilkirch Cedex, France) and stored at −80 °C until RNA extraction. The remaining heart was fixed in 10% buffered formalin for 24–48 h, trimmed, routinely paraffin wax embedded and subjected to a histological examination. This did not reveal pathological changes in the myocardium of any of the cats.

A promising route in the fight against major disease, such as malaria, SARS, influenza, HIV and toxoplasmosis, is a novel family of nanoparticle-based vaccines. They rely on a special class of self-assembling protein nanoparticles (called SAPNs) that form from multiple copies of a purpose-designed protein chain, functionalized to present epitope antigens on the particle surface. Other approaches to design protein-based nanoparticulate systems have been published by various research groups. The architecture of such designs have been described with high accuracy. A major challenge in the rational design of such SAPNs lies in the control of their surface structures, as building blocks can self-assemble into a spectrum of different particle morphologies. Starting with the work of Raman et al., several SAPN species have been synthesized, but their structures have not been completely determined in most cases, and nanoparticle populations are usually characterized in terms of the diameter of the particles only. In some studies, the numbers of the protein chains composing the particle have been identified. For example, Kaba et al. and Raman et al. report particles corresponding to assemblies of 60 chains; Pimentel et al. describe SAPNs with 120 chains; Yang et al. discuss species made of 180 and 300 chains; and finally, Indelicato et al. report assemblies of 240, 300, 360 chains. Also smaller assemblies, so-called LCM units containing 15 protein chains have been discussed and reported. However, an exhaustive enumeration of all possible nanoparticle morphologies that can arise from multiple copies of a given type of building block is currently lacking. This presents a bottleneck in the prediction of the display of B-cell epitopes on the surface of the SAPNs to render them optimal repetitive antigen display systems.

The challenge of enumerating all possible SAPN geometries is reminiscent of the one faced in the classification of virus structures. Similar to SAPNs, viruses assemble the protein containers that encapsulate their genomes (viral capsids) from multiple copies of a small number of different capsid proteins, in many cases a single type of capsid protein. These proteins typically group together in clusters of two, three, five or six in the capsid surface, akin to the clusters seen in SAPN architectures. Caspar & Klug’s seminal classification scheme of viral architectures relies on a geometric approach, predicting the spectrum of possible virus architectures in terms of the numbers and relative positions of these protein clusters (capsomeres) with reference to spherical surface lattices. This classification has revolutionized our understanding of virus structure, and plays a key role in the interpretation of experimental data in virology. This classification of virus architectures has been developed for particles with icosahedral symmetry and, as such, can be used also for synthetic vaccines based on virus-like particles, but is not suitable to model SAPNs.

We develop here a classification scheme for SAPN morphologies in terms of surface tessellations and associated graphs that pinpoint the positions of the protein building blocks in the particle surfaces. Our approach exploits the geometric relation of SAPN morphologies with fullerene architecture, and further develops tools that have been introduced for fullerene classification. As a result, we present a procedure to classify SAPN morphologies both symmetric and asymmetric, and we deliver a classification for high and low symmetry particles seen in the experiments. In particular, we explicitly determine particle morphologies for symmetric particles formed from up to 360 protein building blocks, as there is experimental evidence that spherical particles up to this size should exist, and these are relevant for vaccine design. Defective nanoparticles are not considered in this work as they require a different mathematical model, and will be the object of future investigation.

The classification presented here provides, to our knowledge, the first complete atlas of SAPN geometries of D3 symmetry or higher, and provides a construction method for all particles, including low symmetry and asymmetric ones. We have demonstrated previously that a combinatorial analysis of SAPN structures can be an invaluable tool in the interpretation of experimental data. In particular, biophysical methods such as analytical ultracentrifugation can provide information on the numbers of chains N in the particles that occur in the self-assembly process. Combinatorics does then narrow down the spectrum of options to a limited ensemble of particle geometries compatible with this range of chain numbers, and identifies the precise surface structures of the particles in terms of the placements of all protein chains and threefold and fivefold coiled coils. It also offers a glimpse at the complexity of the assembly process in terms of the numbers of different particles that can occur in a given range of chain numbers. In previous work, a full classification had not yet been available. It was therefore only possible to identify possible candidates for the particles seen in experiment, but an exhaustive enumeration was not possible.

The construction method with reference to fullerene architecture introduced here provides a step change. It offers for the first time, to our knowledge, insights into the full spectrum of particles of arbitrary size and morphology occurring in an experiment. This exhaustive approach therefore opens up opportunities for the analysis of experimental data that had not been possible before. For example, it is now possible to apply statistical mechanics approaches and construct partition functions describing the outcome of the assembly experiments. These can be used to better understand the assembly process itself in terms of the most likely, dominant assembly pathways. This, in turn, will provide pointers for experimentalists on how to optimize the assembly procedure, e.g. in terms of the yield of desired particle types. The detailed insights into the connectivity of each chain in the nanoparticle surface moreover enable computer reconstructions of the nanoparticles, as in the example in figure 1c. These can then be used to engineer specific architectures by controlling the rigidity of the links and the angle between the coiled coils (an issue not addressed here).

Most importantly, however, the results obtained here enable the identification of SAPN morphologies that have not yet been synthesized, and thus enable the rational design of desired particle morphologies. In particular, our approach links SAPN morphologies with epitope positions, and therefore provides a tool for the identification of SAPN morphologies with optimal properties for vaccine design. However, if the SAPNs are co-assembled from different chains, i.e. if the SAPNs are composed of epitope-decorated units and protein chains lacking epitopes, then the assembly forms will be much more difficult to predict. Depending on the B-cell epitope, chains with epitope may cluster together if there are attracting forces between the B-cell epitopes. Also, we do not exclude the possibility that SAPNs may be formed that have an irregular assembly form of protein chains owing to imperfect propagation of the lattice in all directions. If so, this would lead to chimeric forms of SAPNs with respect to their architecture as described here.

Figure 1 shows a representative placental specimen following immunolabeling with anti-cytokeratin antibody and laser capture microdissection of trophoblasts. Cytokeratin labeling was specific for trophoblasts.

Statistical evaluation of fetal viability from early-and late-term gestation was previously reported. Statistical analyses of immunomodulator expression for early-and late-term control and FIV-B-2542-infected trophoblasts were done using single-factor ANOVA and the two independent sample Wilcoxon rank sum test (SOCR Analysis, University of California, Los Angeles, CA).

Blood samples from the selected animals were collected at the market of source (at 0 hour) and at the market of destination (immediately after arrival and then again 24 hours after arrival at the destination market).

Blood samples, 6 ml per animal, were collected from the jugular veins of the animals and placed in two separate vials: one containing EDTA for haematological evaluation and one having no EDTA for biochemical and hormonal evaluation. Each vial contained 3 ml of the blood sample. The blood samples obtained were transferred through insulated ice eskies to the laboratory at Chittagong Veterinary and Animal Sciences University between 2 and 16 hours from the destination and source market, respectively. The blood samples without anticoagulant were centrifuged at 1500 rpm for 10 minutes to separate the serum samples. The serum samples were transferred to the Eppendorf tube (2 ml) and then stored in −18°C for further analysis. The blood samples with anticoagulants were stored at 4°C. All laboratory analyses were performed within 24 hours.