Development and application of a higher throughput RSV plaque assay by immunofluorescent imaging.

Viral plaque assays are important tools in the development and evaluation of new antiviral drugs or vaccines in both preclinical and clinical research. While plaque assays are the standard tools to measure infectious virus, the methodology is time-consuming and requires experience in recognizing plaques. The assays are also prone to variation among analysts due to plaque recognition and manual counting errors. Here we describe the development of two simplified plaque assays for measuring RSV virus titers and anti-RSV antibody neutralization titers using 96 well plate formats. First, we evaluated multiple parameters to build up a quantitative plaque assay to measure infectious RSV. We then optimized the assay conditions to assess the fundamental changes from the traditional plaque assay, which were elimination of overnight pre-seeding host cells and addition of a centrifugation step after viral infection of the cells. We designed DoE to refine four key parameters within one experiment for host cell density, host cell volume, viral inoculum volume, host cell and viral mixture incubation time to make this assay more robust. We have also adapted these conditions into a second assay, which was an automated plaque reduction neutralization assay (PRNT) to determine neutralization titers of anti-RSV antibodies. Both assays utilize immune fluorescence staining to detect viral plaques. The images of the immuno-stained wells are captured by the PerkinElmer EnSight instrument and show clear visualization of plaques harvesting on day 3. Software algorithm was specifically designed for automatic counting of these fluorescent "objects". The quantitative plaque assay provided titers of RSV similar to those obtained from the traditional plaque assay. The method has been successfully utilized to screen multiple vaccine candidates in viral shedding efficacy studies. The automated PRNT assay provided antibody neutralizing titers that matched with published data. This automated 96 well plaque assay has made it possible to screen RSV samples in a higher throughput manner, and can be extended to other infectious organisms that form plaques for vaccine or drug evaluation.

A novel polyomavirus in sigmodontine rodents from São Paulo State, Brazil.

The nearly complete genome sequence of a novel polyomavirus from blood samples of Akodon montensis and Calomys tener collected in Brazil was determined by high-throughput sequencing. This virus showed a typical polyomaviruses genome organization, and it was classified as a member of the genus Betapolyomavirus. Our results expand the host range and viral diversity of the family Polyomaviridae.

Experimental infection of Rio Mamore hantavirus in Sigmodontinae rodents

This study shows an experimental spillover infection of Sigmodontinae rodents with Rio Mamore hantavirus (RIOMV). Necromys lasiurus and Akodon sp were infected with 103 RNA copies of RIOMV by intraperitoneal administration. The viral genome was detected in heart, lung, and kidney tissues 18 days after infection (ai), and viral excretion in urine and faeces began at four and six ai, respectively. These results reveal that urine and faeces of infected rodents contain the virus for at least 18 days. It is possible that inhaled aerosols of these excreta could transmit hantavirus to humans and other animals.

Intranasal nanoemulsion-based inactivated respiratory syncytial virus vaccines protect against viral challenge in cotton rats.

Respiratory Syncytial Virus is a leading cause of bronchiolitis and pneumonia in infants, the elderly and individuals with compromised immune systems. Despite decades of research, there is currently no available vaccine for RSV. Our group has previously demonstrated that intranasal immunization of mice with RSV inactivated by and adjuvanted with W805EC nanoemulsion elicits robust humoral and cellular immune responses, resulting in protection against RSV infection. This protection was achieved without the induction of airway hyper-reactivity or a Th2-skewed immune response. The cotton rat Sigmodon hispidus has been used for years as an excellent small animal model of RSV disease. Thus, we extended these rodent studies to the more permissive cotton rat model. Intranasal immunization of the nanoemulsion-adjuvanted RSV vaccines induced high antibody titers and a robust Th1-skewed cellular response. Importantly, vaccination provided sterilizing cross-protective immunity against a heterologous RSV challenge and did not induce marked or severe histological effects or eosinophilia in the lung after viral challenge. Overall, these data demonstrate that nanoemulsion-formulated whole RSV vaccines are both safe and effective for immunization in multiple animal models.

Pinhal virus, a new arenavirus isolated from calomys tener in Brazil

Arenavirus Sabiá was originally isolated from a fatal human infection in Brazil, and after the occurrence of the second fatal human case in São Paulo state, epidemiologic and virologic studies were performed in the area where the patient lived, aiming at the identification of the Sabiá natural rodent reservoir. A broadly cross-reactive enzyme-linked immunosorbent assay (ELISA) was used to screen for antibody-positive samples. Antibodies to arenavirus were detected in two of the 55 samples of Calomys tener, and from these results, samples of rodents were analyzed by a broad RT-PCR assay. RT-PCR amplification detected arenavirus sequences in five of the 55 C. tener samples, and sequencing showed that this virus is a distinct form of Sabiá virus. Thus, we describe here the evidence for the circulation of a new arenavirus in Brazil (proposed name Pinhal virus) and its genetic characterization compared to other arenaviruses. This study also suggests C. tener as a probable rodent reservoir for this virus and associates this new virus with the lineage C of New World arenaviruses. Although we have defined some characteristics of this virus, so far, there is no evidence of its involvement in human disease.

Condiciones para la transmision del hantavirus en zona andina de Río negro, Argentina

El Síndrome Pulmonar por Hantavirus (SPH) es una enfermedad de etiología viral que causa en el hombre un cuadro respiratorio grave. En Patagonia, la enfermedad es causada por el virus Andes Sur (AND), transmitido por el roedor Oligoryzomys longicaudatus. El objetivo del presente trabajo fue identificar las actividades del hombre que favorecen su exposición a roedores, denominados escenarios de contagio. Se realizó un estudio retrospectivo a partir de información recolectada en investigaciones de casos ocurridos en Río Negro, mediante Fichas Clínico-Epidemiológicas e informes de evaluación ecológico/ambiental. Se definieron como variables a ser consideradas: edad, sexo, época del año, grado de urbanización, localización geográfica, integración del hombre al hábitat de roedores, fuente probable de exposición, actividad humana y nivel de saneamiento. Se estudiaron 32 casos. La exposición rural se verificó en 18 (56.2%) de los casos y 10 (31.3%) en paraje rural (grupo de viviendas en zona rural). En relación al ambiente antropogénico 24 (75%) resultaron en ambientes modificados por el hombre y 8 (25%) en áreas poco modificadas. El sitio de exposición de mayor importancia en El Bolsón fue el interior de edificaciones en 8 de los 18 casos allí registrados (44.5%), mientras que en Bariloche fueron ambientes de exterior con 8/14 (57.1%) casos. La actividad de riesgo fue laboral en 23 (71.9%) de los casos y recreacional en 7 (28.1%). Determinar los escenarios de contagio a nivel local ha aportado información para aplicar todos los recursos disponibles en materia de prevención y educación sanitaria.

Hantavirus Pulmonary Syndrome (HPS) is a disease of viral etiology that affects humans causing severe acute respiratory symptoms. In Patagonia the disease is caused by the Andes Virus (AND) and transmitted by the rodent Oligoryzomys longicaudatus. The aim of this study was to identify those human activities that increase the risk of exposure to rodents, what we call "contagious scenarios". A retrospective study was performed with data obtained from cases in Rio Negro, which included clinic-epidemiological records and ecological/environmental assessment reports. The following variables were considered: age, sex, season, percentage of urbanization, geographic location, human settlements in rodent infested areas, probable source of exposure, type of activity and level of sanitary development. In total 32 cases were studied. Exposure was verified in 18 (56.2 %) cases in rural areas and 10 cases (31.3%) in small rural towns. In relation to anthropogenic environment, 24 (75%) cases were reported in developed settlements and 8 cases (25%) were related to slightly modified areas. Major exposition in El Bolson identified 8 cases of indoor activities of the total 18 reported in the area (44.5%), while in Bariloche 8 (57.1%) cases out of 14 were reported in outdoor surroundings. In general, activities that generated greater risk were work-related, accounting for 23 (71.9%) cases while 7 were related to recreational activities (28.1%). The identification of "contagious scenarios" at local level provided information for an effective application of available resources in terms of prevention and sanitary education.

Endothelial cell permeability and adherens junction disruption induced by junín virus infection.

Junín virus (JUNV) is endemic to the fertile Pampas of Argentina, maintained in nature by the rodent host Calomys musculinus, and the causative agent of Argentine hemorrhagic fever (AHF), which is characterized by vascular dysfunction and fluid distribution abnormalities. Clinical as well as experimental studies implicate involvement of the endothelium in the pathogenesis of AHF, although little is known of its role. JUNV has been shown to result in productive infection of endothelial cells (ECs) in vitro with no visible cytopathic effects. In this study, we show that direct JUNV infection of primary human ECs results in increased vascular permeability as measured by electric cell substrate impedance sensing and transwell permeability assays. We also show that EC adherens junctions are disrupted during virus infection, which may provide insight into the role of the endothelium in the pathogenesis of AHF and possibly, other viral hemorrhagic fevers.

Identification of rodent homologs of hepatitis C virus and pegiviruses.

UNLABELLED: Hepatitis C virus (HCV) and human pegivirus (HPgV or GB virus C) are globally distributed and infect 2 to 5% of the human population. The lack of tractable-animal models for these viruses, in particular for HCV, has hampered the study of infection, transmission, virulence, immunity, and pathogenesis. To address this challenge, we searched for homologous viruses in small mammals, including wild rodents. Here we report the discovery of several new hepaciviruses (HCV-like viruses) and pegiviruses (GB virus-like viruses) that infect wild rodents. Complete genome sequences were acquired for a rodent hepacivirus (RHV) found in Peromyscus maniculatus and a rodent pegivirus (RPgV) found in Neotoma albigula. Unique genomic features and phylogenetic analyses confirmed that these RHV and RPgV variants represent several novel virus species in the Hepacivirus and Pegivirus genera within the family Flaviviridae. The genetic diversity of the rodent hepaciviruses exceeded that observed for hepaciviruses infecting either humans or non-primates, leading to new insights into the origin, evolution, and host range of hepaciviruses. The presence of genes, encoded proteins, and translation elements homologous to those found in human hepaciviruses and pegiviruses suggests the potential for the development of new animal systems with which to model HCV pathogenesis, vaccine design, and treatment. IMPORTANCE: The genetic and biological characterization of animal homologs of human viruses provides insights into the origins of human infections and enhances our ability to study their pathogenesis and explore preventive and therapeutic interventions. Horses are the only reported host of nonprimate homologs of hepatitis C virus (HCV). Here, we report the discovery of HCV-like viruses in wild rodents. The majority of HCV-like viruses were found in deer mice (Peromyscus maniculatus), a small rodent used in laboratories to study viruses, including hantaviruses. We also identified pegiviruses in rodents that are distinct from the pegiviruses found in primates, bats, and horses. These novel viruses may enable the development of small-animal models for HCV, the most common infectious cause of liver failure and hepatocellular carcinoma after hepatitis B virus, and help to explore the health relevance of the highly prevalent human pegiviruses.

Respiratory syncytial virus fusion glycoprotein expressed in insect cells form protein nanoparticles that induce protective immunity in cotton rats.

Respiratory Syncytial Virus (RSV) is an important viral agent causing severe respiratory tract disease in infants and children as well as in the elderly and immunocompromised individuals. The lack of a safe and effective RSV vaccine represents a major unmet medical need. RSV fusion (F) surface glycoprotein was modified and cloned into a baculovirus vector for efficient expression in Sf9 insect cells. Recombinant RSV F was glycosylated and cleaved into covalently linked F2 and F1 polypeptides that formed homotrimers. RSV F extracted and purified from insect cell membranes assembled into 40 nm protein nanoparticles composed of multiple RSV F oligomers arranged in the form of rosettes. The immunogenicity and protective efficacy of purified RSV F nanoparticles was compared to live and formalin inactivated RSV in cotton rats. Immunized animals induced neutralizing serum antibodies, inhibited virus replication in the lungs, and had no signs of disease enhancement in the respiratory track of challenged animals. RSV F nanoparticles also induced IgG competitive for binding of palivizumab neutralizing monoclonal antibody to RSV F antigenic site II. Antibodies to this epitope are known to protect against RSV when passively administered in high risk infants. Together these data provide a rational for continued development a recombinant RSV F nanoparticle vaccine candidate.

Evidencia serológica de infección por hantavirus (Bunyaviridae: Hantavirus) en roedores del Departamento de Sucre, Colombia / Serological evidence of hanta virus infection (Bunyaviridae: Hantavirus) in rodents from the Sucre Department in Colombia

Objetivo Determinar la frecuencia de anticuerpos específicos a hantavirus en roedores del municipio de San Marcos, departamento de Sucre. Métodos Se capturaron 144 roedores con trampas Sherman® en áreas urbanas y rurales del municipio de San Marcos, desde diciembre de 2007 hasta julio de 2009. Los anticuerpos Ig G específicos contra el Virus Sin Nombre (VSN) fueron detectados en muestras de plasma mediante ELISA indirecto. Resultados La seroprevalencia de anticuerpos contra hantavirus fue del 8,3 % (12/144 capturas). Los porcentajes de seropositividad específicos por especie variaron entre 6,8 % (3/44, Zygodontomys brevicauda) y 50 % (1/2, Neacomys spinosus). No se encontró diferencia estadística en la seroprevalencia con respecto al área de muestreo, sexo y etapa reproductiva (p>0,05); sin embargo, hubo un mayor número de machos adultos seropositivos. Conclusiones Se evidenció por primera vez seropositividad a hantavirus en roedores de la subfamilia Murinae en Colombia. La detección de anticuerpos contra el virus refuerza la hipótesis que sugiere la circulación de al menos un hantavirus en roedores del norte colombiano.

Objective The main goal of this research was to determine the frequency of hantavirus-specific antibodies in rodents from the municipality of San Marcos in the Sucre department of Colombia. Methods 144 rodents were captured in San Marcos' urban and rural areas using Sherman traps between December 2007 and July 2009. "Virus sin Nombre" (SNV)-specific antibodies were detected in plasma samples by an indirect ELISA immunoassay. Results An 8.3 % (12/144) seroprevalence rate was found. Specific seropositivity rates ranged from 6.8 % (3/44, Zygodontomysbrevicauda) to 50 % (1/2, Neacomysspinosus). No significant differences were found in seroprevalence according to capture area, gender and/or reproductive stage (p>0.05); however, there were more seropositive adult males. Conclusion This is the first evidence of hanta virus seropositivity in rodents from the Murinae subfamily in Colombia. The presence of SNV antibodies in rodents in San Marcos supported the hypothesis that at least one hantavirusis circulating in rodents from northern Colombia.

Detection of the first incidence of Akodon paranaensis naturally infected with the Jabora virus strain (Hantavirus) in Brazil

We characterised hantaviruses circulating in different Akodon rodent species collected in midwestern Santa Catarina (SC), southern Brazil, where the Jabora hantavirus (JABV) strain was first identified in Akodon montensis. Genetic and phylogenetic analyses based on a partial S segment indicated that, in SC, Akodon paranaensis and A. montensis carried the same type of hantavirus. Additionally, we conducted the first genomic characterisation of the complete S segment from the Brazilian JABV strain. This is the first report of A. paranaensis infected with the JABV.

Geographic distribution of hantaviruses associated with neotomine and sigmodontine rodents, Mexico.

To increase our knowledge of the geographic distribution of hantaviruses associated with neotomine or sigmodontine rodents in Mexico, we tested 876 cricetid rodents captured in 18 Mexican states (representing at least 44 species in the subfamily Neotominae and 10 species in the subfamily Sigmodontinae) for anti-hantavirus IgG. We found antibodies against hantavirus in 35 (4.0%) rodents. Nucleotide sequence data from 5 antibody-positive rodents indicated that Sin Nombre virus (the major cause of hantavirus pulmonary syndrome [HPS] in the United States) is enzootic in the Mexican states of Nuevo León, San Luis Potosí, Tamaulipas, and Veracruz. However, HPS has not been reported from these states, which suggests that in northeastern Mexico, HPS has been confused with other rapidly progressive, life-threatening respiratory diseases. Analyses of nucleotide sequence data from 19 other antibody-positive rodents indicated that El Moro Canyon virus and Limestone Canyon virus are geographically widely distributed in Mexico.

Características de Oligoryzomys longicaudatus asociadas a la presencia del virus Andes (Hantavirus) / Oligoryzomys longicaudatus characteristics' associated with the presence of Andes virus (Hantavirus)

Oligoryzomys longicaudatus is the main reservoir of Andes virus (AND), which causes hantavirus pulmonary syndrome in Patagonia. The factors associated with the presence of antibodies against AND in this species are unknown. This study used a logistic regression model to analyze which characteristics of O. longicaudatus, captured in northern Argentinean Patagonia, led to an increased probability of an animal having antibodies against AND and to relate these characteristics to possible mechanisms of transmission of the virus within the population. Sex, age, body mass, and wounds were important predictors regarding the presence of antibodies against AND within O. longicaudatus populations. The probability of a wounded male O. longicaudatus adult having AND antibodies increased in parallel with the body mass. The probability of having antibodies was more than 80% in individuals with body masses above 44 gram. However, the possible transmission mechanism of AND within O. longicaudatus population is still uncertain and further studies involving a larger number of individuals and prolonged monitoring including the process of seroconversion are needed.

Oligoryzomys longicaudatus es el principal reservorio del virus Andes Sur (AND) causante del síndrome pulmonar por hantavirus en la Patagonia. Aún se desconoce qué características individuales están asociadas a una mayor presencia de anticuerpos contra AND en esta especie. En este estudio, mediante un modelo de regresión logística evaluamos qué características de O. longicaudatus, capturados en la Patagonia norte de Argentina, incrementan la probabilidad de un individuo de presentar anticuerpos contra AND para relacionarlos con posibles mecanismos de transmisión del virus dentro de la población. El sexo, la edad, la masa corporal y las heridas resultaron factores importantes para la circulación y persistencia del virus dentro de la población de O. longicaudatus. La probabilidad de que un O. longicaudatus, macho, adulto con heridas presente anticuerpos contra AND aumentó con el incremento de la masa corporal, siendo esta probabilidad mayor al 80% en individuos con masas corporales mayores a 44 g. Sin embargo, el posible mecanismo de transmisión de AND dentro de la población de O. longicaudatus queda aún incierto, por lo que son necesarios estudios futuros que involucren un mayor número de individuos y un tiempo prolongado de seguimiento en su proceso de seroconversión.

Genetic characterization of hantaviruses associated with sigmodontine rodents in an endemic area for hantavirus pulmonary syndrome in southern Brazil.

An ecological assessment of reservoir species was conducted in a rural area (Jaborá) in the mid-west of the state of Santa Catarina in southern Brazil, where hantavirus pulmonary syndrome is endemic, to evaluate the prevalence of hantavirus infection in wild rodents. Blood and tissue samples were collected from 507 rodents during seven field trips from March 2004 to April 2006. Some of the animals were karyotyped to confirm morphological identification. Phylogenetic reconstructions of rodent specimens, based on the mitochondrial DNA cytochrome b gene sequences, were also obtained. Hantavirus antibody was found in 22 (4.3%) of the 507 rodents: 5 Akodon montensis, 2 Akodon paranaensis, 14 Oligoryzomys nigripes, and 1 Sooretamys angouya. Viral RNAs detected in O. nigripes and A. montensis were amplified and sequenced. O. nigripes virus genome was 97.5% (nt) and 98.4% (nt) identical to sequences published for Araucaria (Juquitiba-like) virus based on N and G2 fragment sequences. Viral sequences from A. montensis strain showed 89% and 88% nucleotide identities in a 905-nt fragment of the nucleocapsid (N) protein-coding region of the S segment when it was compared with two other Akodontine rodent-associated viruses from Paraguay, A. montensis and Akodon cursor, respectively. Phylogenetic analysis showed the cocirculation of two genetic hantavirus lineages in the state of Santa Catarina, one from O. nigripes and the other from A. montensis, previously characterized in Brazil and Paraguay, respectively. The hantavirus associated with A. montensis, designed Jaborá virus, represents a distinct phylogenetic lineage among the Brazilian hantaviruses.

Geographical range of Rio Mamoré virus (family Bunyaviridae, genus Hantavirus) in association with the small-eared pygmy rice rat (Oligoryzomys microtis).

Hantavirus HTN.007 was originally isolated from a small-eared pygmy rice rat (Oligoryzomys microtis) captured in northeastern Peru. The results of analyses of nucleotide and amino acid sequence data in this study indicated that HTN.007 is a strain of Rio Mamoré virus (RIOMV) which is enzootic in small-eared pygmy rice rat populations in Bolivia. As such, the results of this study extend our knowledge of the geographical range of RIOMV and support the notion that the small-eared pygmy rice rat is the principal host of RIOMV.

Genetic diversity between and within the arenavirus species indigenous to western Venezuela.

The results of analyses of Z, RNA-dependent RNA polymerase, glycoprotein precursor, and nucleocapsid protein gene sequence data suggested that Guanarito virus was the most common cause of Venezuelan hemorrhagic fever in a 7-year period in the 1990s and that the evolution of Pirital virus in association with Sigmodon alstoni (Alston's cotton rat) has occurred at a significantly higher rate than the evolution of Guanarito virus in association with Zygodontomys brevicauda (short-tailed cane mouse) on the plains of western Venezuela. The results of analyses of the primary structures of the glycoproteins of the 8 strains of Guanarito virus isolated from humans suggested that these strains would be highly cross-reactive in neutralization assays. Thus, passive antibody therapy may prove beneficial in the treatment of human disease caused by strains of Guanarito virus that are enzootic in the region in which Venezuelan hemorrhagic fever is endemic.

Hantavirus and arenavirus antibodies in persons with occupational rodent exposure.

Rodents are the principal hosts of Sin Nombre virus, 4 other hantaviruses known to cause hantavirus pulmonary syndrome in North America, and the 3 North American arenaviruses. Serum samples from 757 persons who had worked with rodents in North America and handled neotomine or sigmodontine rodents were tested for antibodies against Sin Nombre virus, Whitewater Arroyo virus, Guanarito virus, and lymphocytic choriomeningitis virus. Antibodies against Sin Nombre virus were found in 4 persons, against Whitewater Arroyo virus or Guanarito virus in 2 persons, and against lymphocytic choriomeningitis virus in none. These results suggest that risk for infection with hantaviruses or arenaviruses usually is low in persons whose occupations entail close physical contact with neotomine or sigmodontine rodents in North America.

Immunocompetent, semi-permissive cotton rat tumor model for the evaluation of oncolytic adenoviruses.

Oncolytic adenovirus (Ad) vectors belong to a new class of cancer therapy agents that destroy cancer cells as part of the virus's lytic infectious cycle. In this chapter we describe an immunocompetent, semi-permissive cotton rat tumor model to evaluate the safety and efficacy of oncolytic Ad vectors. With this model one can investigate the effect of the host immune system on the vector-tumor interaction as well as the vector's effect on normal host cells in vivo. This chapter describes procedures for analyzing the growth and cytolytic properties of oncolytic Ad vectors in cotton rat cells in vitro. We discuss handling and husbandry issues and techniques for subcutaneous, intratumoral, and intravenous injection of cotton rats. We present methods for generating subcutaneous tumors in cotton rats and assessing the efficacy of Ad vectors upon intratumoral injection. Also, we discuss procedures for determining the biodistribution of a replicating Ad in cotton rats.

Primera evidencia serológica de infección por Hantavirus en roedores, en Colombia / First serological evidence of Hantavirus infection in rodents in Colombia

OBJETIVO: Determinar la prevalencia de infección por hantavirus en roedores del Departamento de Córdoba, Colombia. METODOLOGIA: Captura de roedores con trampas tipo Sherman live-capture traps (8x9x23 cm; Sherman Traps, Inc., Tallahassee, FL) en áreas domésticas y peridomésticas en el departamento de Córdoba. Analisis de anticuerpos IgG por ELISA, empleando como antígeno una proteína recombinante de la nucleocapside del Sin Nombre Virus (SNV) (CDC, Atlanta, Georgia, USA). RESULTADOS: Durante los meses de enero de 2003 a noviembre de 2004, en 79 noches de trampeo fueron capturados 336 roedores en once municipios del departamento de Córdoba (Murinae: 249; Sigmodontinae: 68; Heteromyidae: 17; Echimyidae: 2) (éxito de captura del 8,5 por ciento). La seroprevalencia de anticuerpos contra hantavirus fue del 2,1 por ciento (7 de 336 capturas). Los porcentajes de seropositividad específicos por género oscilaron entre 5,9 por ciento (1 de 17, Heteromys) a 50 por ciento (1 de 2, Proechimys). CONCLUSIONES: La prevalencia de anticuerpos contra el SNV en roedores de Córdoba, Colombia; indica que al menos un hantavirus es endémico en roedores del norte colombiano y esta frecuentemente trasmitido a residentes rurales.

The cotton rat provides a useful small-animal model for the study of influenza virus pathogenesis.

Influenza A virus continues to cause annual epidemics. The emergence of avian viruses in the human population poses a pandemic threat, and has highlighted the need for more effective influenza vaccines and antivirals. Development of such therapeutics would be enhanced by the use of a small-animal model that is permissive for replication of human influenza virus, and for which reagents are available to dissect the host response. A model is presented of nasal and pulmonary infection in adult inbred cotton rats (Sigmodon hispidus) that does not require viral 'adaptation'. It was previously demonstrated that animals infected intranasally with 10(7) TCID50 of a recent H3N2 influenza, A/Wuhan/359/95, have increased breathing rates. In this report it is shown that this is accompanied by weight loss and decreased temperature. Virus replication peaked within 24 h in the lung, with peak titres proportional to the infecting dose, clearing by day 3. Replication was more permissive in nasal tissues, and persisted for 6 days. Pulmonary pathology included early bronchiolar epithelial cell damage, followed by extensive alveolar and interstitial pneumonia, which persisted for nearly 3 weeks. Interleukin 1 alpha (IL1alpha), alpha interferon (IFN-alpha), IL6, tumour necrosis factor alpha (TNF-alpha), GROalpha and MIP-1beta mRNA were elevated soon after infection, and expression coincided with virus replication. A biphasic response was observed for RANTES, IFN-gamma, IL4, IL10 and IL12-p40, with increased mRNA levels early during virus replication followed by a later increase that coincided with pulmonary inflammation. These results indicate that cotton rats will be useful for further studies of influenza pathogenesis and immunity.