Search

Highly pathogenic avian influenza viruses (HPAIVs) cause severe hemorrhagic disease in terrestrial poultry and are a threat to the poultry industry, wild life, and human health. HPAIVs arise from low pathogenic avian influenza viruses (LPAIVs), which circulate in wild aquatic birds. HPAIV emergence is thought to occur in poultry and not wild aquatic birds, but the reason for this species-restriction is not known. We hypothesized that, due to species-specific tropism and replication, intrahost HPAIV selection is favored in poultry and disfavored in wild aquatic birds. We tested this hypothesis by co-inoculating chickens, representative of poultry, and ducks, representative of wild aquatic birds, with a mixture of H7N7 HPAIV and LPAIV, mimicking HPAIV emergence in an experimental setting. Virus selection was monitored in swabs and tissues by RT-qPCR and immunostaining of differential N-terminal epitope tags that were added to the hemagglutinin protein. HPAIV was selected in four of six co-inoculated chickens, whereas LPAIV remained the major population in co-inoculated ducks on the long-term, despite detection of infectious HPAIV in tissues at early time points. Collectively, our data support the hypothesis that HPAIVs are more likely to be selected at the intrahost level in poultry than in wild aquatic birds and point towards species-specific differences in HPAIV and LPAIV tropism and replication levels as possible explanations., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 de Bruin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The emergence of several bat coronavirus-related disease outbreaks in human and domestic animals has fueled surveillance of coronaviruses in bats worldwide. However, little is known about how these viruses interact with their natural hosts. We demonstrate a Betacoronavirus (subgenus Merbecovirus ), PN-βCoV, in the intestine of its natural host, Nathusius's Pipistrelle Bat ( Pipistrellus nathusii) , by combining molecular and microscopy techniques. Eighty-eight P. nathusii bat carcasses were tested for PN-βCoV RNA by RT-qPCR, of which 25 bats (28%) tested positive. PN-βCoV RNA was more often detected in samples of the intestinal tract than in other sample types. In addition, viral RNA loads were higher in intestinal samples compared to other sample types, both on average and in each individual bat. In one bat, we demonstrated Merbecovirus antigen and PN-βCoV RNA expression in intestinal epithelium and the underlying connective tissue using immunohistochemistry and in situ hybridization, respectively. These results indicate that PN-βCoV has a tropism for the intestinal epithelium of its natural host, Nathusius's Pipistrelle Bat, and imply that the fecal-oral route is a possible route of transmission. IMPORTANCE Virtually all mammal species circulate coronaviruses. Most of these viruses will infect one host species; however, coronaviruses are known to include species that can infect multiple hosts, for example the well-known virus that caused a pandemic, SARS-CoV-2. Chiroptera (bats) include over 1,400 different species, which are expected to harbor a great variety of coronaviruses. However, we know very little about how any of these coronaviruses interact with their bat hosts; for example, we do not know their modes of transmissions, or which cells they infect. Thus, we have a limited understanding of coronavirus infections in this important host group. The significance of our study is that we learned that a bat coronavirus that occurs in a common bat species in Europe has a tropism for the intestines. This implies the fecal-oral route is a likely transmission route.

Central nervous system (CNS) disease is one of the most common extrarespiratory tract complications of influenza A virus infections. Remarkably, zoonotic H5N1 virus infections are more frequently associated with CNS disease than seasonal or pandemic influenza viruses. Little is known about the interaction between influenza A viruses and cells of the CNS; therefore, it is currently unknown which viral factors are important for efficient replication. Here, we determined the replication kinetics of a seasonal, pandemic, zoonotic, and lab-adapted influenza A virus in human neuron-like (SK-N-SH) and astrocyte-like (U87-MG) cells and primary mouse cortex neurons. In general, highly pathogenic avian influenza (HPAI) H5N1 virus replicated most efficiently in all cells, which was associated with efficient attachment and infection. Seasonal H3N2 and to a lesser extent pandemic H1N1 virus replicated in a trypsin-dependent manner in SK-N-SH but not in U87-MG cells. In the absence of trypsin, only HPAI H5N1 and WSN viruses replicated. Removal of the multibasic cleavage site (MBCS) from HPAI H5N1 virus attenuated, but did not abrogate, replication. Taken together, our results showed that the MBCS and, to a lesser extent, the ability to attach are important determinants for efficient replication of HPAI H5N1 virus in cells of the CNS. This suggests that both an alternative hemagglutinin (HA) cleavage mechanism and preference for α-2,3-linked sialic acids allowing efficient attachment contribute to the ability of influenza A viruses to replicate efficiently in cells of the CNS. This study further improves our knowledge on potential viral factors important for the neurotropic potential of influenza A viruses. IMPORTANCE Central nervous system (CNS) disease is one of the most common extrarespiratory tract complications of influenza A virus infections, and the frequency and severity differ between seasonal, pandemic, and zoonotic influenza viruses. However, little is known about the interaction of these viruses with cells of the CNS. Differences among seasonal, pandemic, and zoonotic influenza viruses in replication efficacy in CNS cells, in vitro , suggest that the presence of an alternative HA cleavage mechanism and ability to attach are important viral factors. Identifying these viral factors and detailed knowledge of the interaction between influenza virus and CNS cells are important to prevent and treat this potentially lethal CNS disease., (Copyright © 2019 Siegers et al.)

Background: The 1918 Spanish H1N1 influenza pandemic was the most severe recorded influenza pandemic with an estimated 20-50 million deaths worldwide. Even though it is known that influenza viruses can cause extrarespiratory tract complications-which are often severe or even fatal-the potential contribution of extrarespiratory tissues to the pathogenesis of 1918 H1N1 virus infection has not been studied comprehensively., Methods: Here, we performed a time-course study in ferrets inoculated intranasally with 1918 H1N1 influenza virus, with special emphasis on the involvement of extrarespiratory tissues. Respiratory and extrarespiratory tissues were collected after inoculation for virological, histological, and immunological analysis., Results: Infectious virus was detected at high titers in respiratory tissues and, at lower titers in most extrarespiratory tissues. Evidence for active virus replication, as indicated by the detection of nucleoprotein by immunohistochemistry, was observed in the respiratory tract, peripheral and central nervous system, and liver. Proinflammatory cytokines were up-regulated in respiratory tissues, olfactory bulb, spinal cord, liver, heart, and pancreas., Conclusions: 1918 H1N1 virus spread to and induced cytokine responses in tissues outside the respiratory tract, which likely contributed to the severity of infection. Moreover, our data support the suggested link between 1918 H1N1 infection and central nervous system disease.

Severe influenza is often associated with disease manifestations outside the respiratory tract. While proinflammatory cytokines can be detected in the lungs and blood of infected patients, the role of extra-respiratory organs in the production of proinflammatory cytokines is unknown. Here, we show that both 2009 pandemic H1N1 influenza A (H1N1) virus and highly pathogenic avian influenza A (H5N1) virus induce expression of tumor necrosis factor α, interleukin-6, and interleukin-8 in the respiratory tract and central nervous system. In addition, H5N1 virus induced cytokines in the heart, pancreas, spleen, liver, and jejunum. Together, these data suggest that extra-respiratory tissues contribute to systemic cytokine responses, which may increase the severity of influenza., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Influenza A viruses can replicate in the olfactory mucosa and subsequently use the olfactory nerve to enter the central nervous system (CNS). It is currently unknown whether intervention strategies are able to reduce or prevent influenza virus replication within the olfactory mucosa and subsequent spread to the CNS. Therefore, we tested the efficacy of homologous vaccination and prophylactic oseltamivir to prevent H5N1 virus CNS invasion via the olfactory nerve in our ferret model., Methods: Ferrets were vaccinated intramuscularly or received oseltamivir (5 mg/kg twice daily) prophylactically before intranasal inoculation of highly pathogenic H5N1 virus (A/Indonesia/05/2005) and were examined using virology and pathology., Results: Homologous vaccination reduced H5N1 virus replication in the olfactory mucosa and prevented subsequent virus spread to the CNS. However, prophylactic oseltamivir did not prevent H5N1 virus replication in the olfactory mucosa sufficiently, resulting in CNS invasion via the olfactory nerve causing a severe meningoencephalitis., Conclusions: Within our ferret model, vaccination is more effective than prophylactic oseltamivir in preventing CNS invasion by H5N1 virus via the olfactory nerve. This study highlights the importance of including the olfactory mucosa, olfactory nerve, and CNS tissues in future vaccine and antiviral studies, especially for viruses with a known neurotropic potential., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Hepatitis A virus (HAV) is an ancient and ubiquitous human pathogen recovered previously only from primates. The sole species of the genus Hepatovirus, existing in both enveloped and nonenveloped forms, and with a capsid structure intermediate between that of insect viruses and mammalian picornaviruses, HAV is enigmatic in its origins. We conducted a targeted search for hepatoviruses in 15,987 specimens collected from 209 small mammal species globally and discovered highly diversified viruses in bats, rodents, hedgehogs, and shrews, which by pairwise sequence distance comprise 13 novel Hepatovirus species. Near-complete genomes from nine of these species show conservation of unique hepatovirus features, including predicted internal ribosome entry site structure, a truncated VP4 capsid protein lacking N-terminal myristoylation, a carboxyl-terminal pX extension of VP1, VP2 late domains involved in membrane envelopment, and a cis-acting replication element within the 3D(pol) sequence. Antibodies in some bat sera immunoprecipitated and neutralized human HAV, suggesting conservation of critical antigenic determinants. Limited phylogenetic cosegregation among hepatoviruses and their hosts and recombination patterns are indicative of major hepatovirus host shifts in the past. Ancestral state reconstructions suggest a Hepatovirus origin in small insectivorous mammals and a rodent origin of human HAV. Patterns of infection in small mammals mimicked those of human HAV in hepatotropism, fecal shedding, acute nature, and extinction of the virus in a closed host population. The evolutionary conservation of hepatovirus structure and pathogenesis provide novel insight into the origins of HAV and highlight the utility of analyzing animal reservoirs for risk assessment of emerging viruses.

Central nervous system (CNS) disease is the most common extrarespiratory complication of influenza in humans. However, the pathogenesis, including the route of virus entry, is largely unknown. Here we present, for the first time, evidence of influenza virus entry into the CNS via the olfactory route in an immune-compromised infant. Since the nasal cavity is a primary site of influenza virus replication and is directly connected to the CNS via the olfactory nerve, these results imply that influenza virus invasion of the CNS may occur more often than previously believed., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

We determined the pattern of attachment of the avian-origin H7N9 influenza viruses A/Anhui/1/2013 and A/Shanghai/1/2013 to the respiratory tract in ferrets, macaques, mice, pigs, and guinea pigs and compared it to that in humans. The H7N9 attachment pattern in macaques, mice, and to a lesser extent pigs and guinea pigs resembled that in humans more closely than the attachment pattern in ferrets. This information contributes to our knowledge of the different animal models for influenza.

Highly pathogenic avian influenza H5N1 virus causes a severe, often fatal, pneumonia in humans. The tropism and pathogenesis of highly pathogenic avian influenza H5N1 virus can partly be explained by the presence of H5N1 virus receptors in the human alveoli, which are the site of inflammation during pneumonia. Although studies on the distribution of influenza virus receptors in normal respiratory tract tissues have provided significant insights into the cell tropism and pathogenesis of influenza viruses, the distribution of influenza virus receptors have not been studied during influenza virus infection. Therefore, we studied the distribution of H5N1 virus receptors, by virus and lectin histochemistry, during highly pathogenic avian influenza H5N1 virus infection in alveolar tissues of humans, macaques, ferrets, and cats. In all species, we observed a decrease of H5N1 virus receptors in influenza virus-infected and neighboring cells. The observed decrease of H5N1 virus receptors was associated with the presence of MxA, a known marker for interferon activity. Taken together, our data suggest that the decrease of H5N1 virus receptors might be part of a defense mechanism that limits viral replication in the lower respiratory tract., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The hepatitis B virus (HBV), family Hepadnaviridae, is one of most relevant human pathogens. HBV origins are enigmatic, and no zoonotic reservoirs are known. Here, we screened 3,080 specimens from 54 bat species representing 11 bat families for hepadnaviral DNA. Ten specimens (0.3%) from Panama and Gabon yielded unique hepadnaviruses in coancestral relation to HBV. Full genome sequencing allowed classification as three putative orthohepadnavirus species based on genome lengths (3,149-3,377 nt), presence of middle HBV surface and X-protein genes, and sequence distance criteria. Hepatic tropism in bats was shown by quantitative PCR and in situ hybridization. Infected livers showed histopathologic changes compatible with hepatitis. Human hepatocytes transfected with all three bat viruses cross-reacted with sera against the HBV core protein, concordant with the phylogenetic relatedness of these hepadnaviruses and HBV. One virus from Uroderma bilobatum, the tent-making bat, cross-reacted with monoclonal antibodies against the HBV antigenicity determining S domain. Up to 18.4% of bat sera contained antibodies against bat hepadnaviruses. Infectious clones were generated to study all three viruses in detail. Hepatitis D virus particles pseudotyped with surface proteins of U. bilobatum HBV, but neither of the other two viruses could infect primary human and Tupaia belangeri hepatocytes. Hepatocyte infection occurred through the human HBV receptor sodium taurocholate cotransporting polypeptide but could not be neutralized by sera from vaccinated humans. Antihepadnaviral treatment using an approved reverse transcriptase inhibitor blocked replication of all bat hepadnaviruses. Our data suggest that bats may have been ancestral sources of primate hepadnaviruses. The observed zoonotic potential might affect concepts aimed at eradicating HBV.

Hepatitis C virus (HCV) is among the most relevant causes of liver cirrhosis and hepatocellular carcinoma. Research is complicated by a lack of accessible small animal models. The systematic investigation of viruses of small mammals could guide efforts to establish such models, while providing insight into viral evolutionary biology. We have assembled the so-far largest collection of small-mammal samples from around the world, qualified to be screened for bloodborne viruses, including sera and organs from 4,770 rodents (41 species); and sera from 2,939 bats (51 species). Three highly divergent rodent hepacivirus clades were detected in 27 (1.8%) of 1,465 European bank voles (Myodes glareolus) and 10 (1.9%) of 518 South African four-striped mice (Rhabdomys pumilio). Bats showed anti-HCV immunoblot reactivities but no virus detection, although the genetic relatedness suggested by the serologic results should have enabled RNA detection using the broadly reactive PCR assays developed for this study. 210 horses and 858 cats and dogs were tested, yielding further horse-associated hepaciviruses but none in dogs or cats. The rodent viruses were equidistant to HCV, exceeding by far the diversity of HCV and the canine/equine hepaciviruses taken together. Five full genomes were sequenced, representing all viral lineages. Salient genome features and distance criteria supported classification of all viruses as hepaciviruses. Quantitative RT-PCR, RNA in-situ hybridisation, and histopathology suggested hepatic tropism with liver inflammation resembling hepatitis C. Recombinant serology for two distinct hepacivirus lineages in 97 bank voles identified seroprevalence rates of 8.3 and 12.4%, respectively. Antibodies in bank vole sera neither cross-reacted with HCV, nor the heterologous bank vole hepacivirus. Co-occurrence of RNA and antibodies was found in 3 of 57 PCR-positive bank vole sera (5.3%). Our data enable new hypotheses regarding HCV evolution and encourage efforts to develop rodent surrogate models for HCV.

Black-headed gulls (Chroicocephalus ridibundus) are a suitable host species to study the epidemiology of low-pathogenic avian influenza virus (LPAIV) infection in wild waterbirds because they are a common colony-breeding species in which LPAIV infection is detected frequently, limited mainly to the H13 and H16 subtypes. However, the sites of virus replication and associated lesions are poorly understood. We therefore performed virological and pathological analyses on tissues of black-headed gulls naturally infected with LPAIV. We found that 24 of 111 black-headed gulls collected from breeding colonies were infected with LPAIV (10 birds with H16N3, one bird with H13N8, 13 birds undetermined), based on virus and viral genome detection in pharyngeal and cloacal swabs. Of these 24 gulls, 15 expressed virus antigen in their tissues. Virus antigen expression was limited to epithelial cells of intestine and cloacal bursa. No histological lesions were detected in association with virus antigen expression. Our findings show that LPAIV replication in the intestinal tract of black-headed gulls is mainly a superficial infection in absence of detectable lesions, as determined recently for natural LPAIV infection in free-living mallards (Anas platyrhynchos). These findings imply that LPAIV in black-headed gulls has adapted to minimal pathogenicity to its host and that potentially the primary transmission route is faecal-oral.

The primary complication of seasonal influenza in humans is viral pneumonia. A conventional animal model--intranasal inoculation of ferrets with 10(6) median tissue culture infectious dose of virus--results in disease that is neither consistent nor comparable with severe viral pneumonia in humans. Therefore, the authors modified the experimental procedures by increasing the median tissue culture infectious dose to 10(9) and by inoculating via the intratracheal route, testing these procedures with H1N1 strains (A/Bilthoven/3075/1978 and A/Netherlands/26/2007) and H3N2 strains (A/Bilthoven/16190/1968 and A/Netherlands/177/2008) of seasonal influenza virus. The ferrets of all groups (n = 3 per virus strain) had clinical signs, increased body temperature, virus excretion from day 1, loss of body weight, and increased relative lung weight at 4 days postinoculation. All ferrets had severe pulmonary consolidation, and histologic examination revealed moderate to severe necrotizing bronchointerstitial pneumonia with severe edema, necrosis of alveolar epithelium, inflammatory infiltrates in alveolar septa and lumina, epithelial regeneration, and perivascular and peribronchiolar inflammatory infiltrates. The lesions were associated with the presence of influenza virus antigen in respiratory epithelium by immunohistochemistry. Although all 4 virus strains caused pulmonary lesions of comparable severity, virus isolation in the lungs, trachea, nasal concha, and tonsils showed higher mean virus titers in the H1/07 and H3/68 groups than in the H1/78 and H3/08 groups. In conclusion, the above H1N1 and H3N2 strains cause severe pneumonia in ferrets by use of the modified experimental procedures and provide a good model for pneumonia caused by seasonal influenza A virus infection in humans.

The route by which highly pathogenic avian influenza (HPAI) H5N1 virus spreads systemically, including the central nervous system (CNS), is largely unknown in mammals. Especially, the olfactory route, which could be a route of entry into the CNS, has not been studied in detail. Although the multibasic cleavage site (MBCS) in the hemagglutinin (HA) of HPAI H5N1 viruses is a major determinant of systemic spread in poultry, the association between the MBCS and systemic spread in mammals is less clear. Here we determined the virus distribution of HPAI H5N1 virus in ferrets in time and space-including along the olfactory route-and the role of the MBCS in systemic replication. Intranasal inoculation with wild-type H5N1 virus revealed extensive replication in the olfactory mucosa, from which it spread to the olfactory bulb and the rest of the CNS, including the cerebrospinal fluid (CSF). Virus spread to the heart, liver, pancreas, and colon was also detected, indicating hematogenous spread. Ferrets inoculated intranasally with H5N1 virus lacking an MBCS demonstrated respiratory tract infection only. In conclusion, HPAI H5N1 virus can spread systemically via two different routes, olfactory and hematogenous, in ferrets. This systemic spread was dependent on the presence of the MBCS in HA.

Highly pathogenic avian influenza virus (HPAIV) H5N1 can infect mammals via the intestine; this is unusual since influenza viruses typically infect mammals via the respiratory tract. The dissemination of HPAIV H5N1 following intestinal entry and associated pathogenesis are largely unknown. To assess the route of spread of HPAIV H5N1 to other organs and to determine its associated pathogenesis, we inoculated infected chicken liver homogenate directly into the intestine of cats by use of enteric-coated capsules. Intestinal inoculation of HPAIV H5N1 resulted in fatal systemic disease. The spread of HPAIV H5N1 from the lumen of the intestine to other organs took place via the blood and lymphatic vascular systems but not via neuronal transmission. Remarkably, the systemic spread of the virus via the vascular system was associated with massive infection of endothelial and lymphendothelial cells, resulting in widespread hemorrhages. This is unique for influenza in mammals and resembles the pathogenesis of HPAIV infection in terrestrial poultry. It contrasts with the pathogenesis of systemic disease from the same virus following entry via the respiratory tract, where lesions are characterized mainly by necrosis and inflammation and are associated with the presence of influenza virus antigen in parenchymal, not endothelial cells. The marked endotheliotropism of the virus following intestinal inoculation indicates that the pathogenesis of systemic influenza virus infection in mammals may differ according to the portal of entry.

Only two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that influenza virus becomes resistant to these antiviral drugs and spreads in the human population. The 2009 pandemic A/H1N1 influenza virus is naturally resistant to adamantanes. Recently a novel neuraminidase I223R mutation was identified in an A/H1N1 virus showing cross-resistance to the neuraminidase inhibitors oseltamivir, zanamivir and peramivir. However, the ability of this virus to cause disease and spread in the human population is unknown. Therefore, this clinical isolate (NL/2631-R223) was compared with a well-characterized reference virus (NL/602). In vitro experiments showed that NL/2631-I223R replicated as well as NL/602 in MDCK cells. In a ferret pathogenesis model, body weight loss was similar in animals inoculated with NL/2631-R223 or NL/602. In addition, pulmonary lesions were similar at day 4 post inoculation. However, at day 7 post inoculation, NL/2631-R223 caused milder pulmonary lesions and degree of alveolitis than NL/602. This indicated that the mutant virus was less pathogenic. Both NL/2631-R223 and a recombinant virus with a single I223R change (recNL/602-I223R), transmitted among ferrets by aerosols, despite observed attenuation of recNL/602-I223R in vitro. In conclusion, the I223R mutated virus isolate has comparable replicative ability and transmissibility, but lower pathogenicity than the reference virus based on these in vivo studies. This implies that the 2009 pandemic influenza A/H1N1 virus subtype with an isoleucine to arginine change at position 223 in the neuraminidase has the potential to spread in the human population. It is important to be vigilant for this mutation in influenza surveillance and to continue efforts to increase the arsenal of antiviral drugs to combat influenza.

Highly pathogenic avian influenza virus (HPAIV) of the subtype H5N1 causes severe, often fatal pneumonia in humans. The pathogenesis of HPAIV H5N1 infection is not completely understood, although the alveolar macrophage (AM) is thought to play an important role. HPAIV H5N1 infection of macrophages cultured from monocytes leads to high percentages of infection accompanied by virus production and an excessive pro-inflammatory immune response. However, macrophages cultured from monocytes are different from AM, both in phenotype and in response to seasonal influenza virus infection. Consequently, it remains unclear whether the results of studies with macrophages cultured from monocytes are valid for AM. Therefore we infected AM and for comparison macrophages cultured from monocytes with seasonal H3N2 virus, HPAIV H5N1 or pandemic H1N1 virus, and determined the percentage of cells infected, virus production and induction of TNF-alpha, a pro-inflammatory cytokine. In vitro HPAIV H5N1 infection of AM compared to that of macrophages cultured from monocytes resulted in a lower percentage of infected cells (up to 25% vs up to 84%), lower virus production and lower TNF-alpha induction. In vitro infection of AM with H3N2 or H1N1 virus resulted in even lower percentages of infected cells (up to 7%) than with HPAIV H5N1, while virus production and TNF-alpha induction were comparable. In conclusion, this study reveals that macrophages cultured from monocytes are not a good model to study the interaction between AM and these influenza virus strains. Furthermore, the interaction between HPAIV H5N1 and AM could contribute to the pathogenicity of this virus in humans, due to the relative high percentage of infected cells rather than virus production or an excessive TNF-alpha induction.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), caused by influenza A virus H5N1 and severe acute respiratory syndrome coronavirus (SARS-CoV), supposedly depend on activation of the oxidative-stress machinery that is coupled with innate immunity, resulting in a strong proinflammatory host response. Inflammatory cytokines, such as interleukin 1β (IL-1β), IL-8, and IL-6, play a major role in mediating and amplifying ALI/ARDS by stimulating chemotaxis and activation of neutrophils. To obtain further insight into the pathogenesis of SARS-CoV-associated ALI, we compared SARS-CoV infections in two different nonhuman primate species, cynomolgus macaques and African green monkeys. Viral titers in the upper and lower respiratory tract were not significantly different in SARS-CoV-infected macaques and African green monkeys. Inflammatory cytokines that play a major role in mediating and amplifying ALI/ARDS or have neutrophil chemoattractant activity, such as IL-6, IL-8, CXCL1, and CXCL2, were, however, induced only in macaques. In contrast, other proinflammatory cytokines and chemokines, including osteopontin and CCL3, were upregulated in the lungs of African green monkeys to a significantly greater extent than in macaques. Because African green monkeys developed more severe ALI than macaques, with hyaline membrane formation, some of these differentially expressed proinflammatory genes may be critically involved in development of the observed pathological changes. Induction of distinct proinflammatory genes after SARS-CoV infection in different nonhuman primate species needs to be taken into account when analyzing outcomes of intervention strategies in these species.

To demonstrate that pandemic (H1N1) 2009 virus may cause respiratory disease in cats, we intratracheally infected cats. Diffuse alveolar damage developed. Seroconversion of sentinel cats indicated cat-to-cat virus transmission. Unlike in cats infected with highly pathogenic avian influenza virus (H5N1), extrarespiratory lesions did not develop in cats infected with pandemic (H1N1) 2009 virus.

Influenza viruses vary markedly in their efficiency of human-to-human transmission. This variation has been speculated to be determined in part by the tropism of influenza virus for the human upper respiratory tract. To study this tropism, we determined the pattern of virus attachment by virus histochemistry of three human and three avian influenza viruses in human nasal septum, conchae, nasopharynx, paranasal sinuses, and larynx. We found that the human influenza viruses-two seasonal influenza viruses and pandemic H1N1 virus-attached abundantly to ciliated epithelial cells and goblet cells throughout the upper respiratory tract. In contrast, the avian influenza viruses, including the highly pathogenic H5N1 virus, attached only rarely to epithelial cells or goblet cells. Both human and avian viruses attached occasionally to cells of the submucosal glands. The pattern of virus attachment was similar among the different sites of the human upper respiratory tract for each virus tested. We conclude that influenza viruses that are transmitted efficiently among humans attach abundantly to human upper respiratory tract, whereas inefficiently transmitted influenza viruses attach rarely. These results suggest that the ability of an influenza virus to attach to human upper respiratory tract is a critical factor for efficient transmission in the human population.

The emergence of viral respiratory pathogens with pandemic potential, such as severe acute respiratory syndrome coronavirus (SARS-CoV) and influenza A H5N1, urges the need for deciphering their pathogenesis to develop new intervention strategies. SARS-CoV infection causes acute lung injury (ALI) that may develop into life-threatening acute respiratory distress syndrome (ARDS) with advanced age correlating positively with adverse disease outcome. The molecular pathways, however, that cause virus-induced ALI/ARDS in aged individuals are ill-defined. Here, we show that SARS-CoV-infected aged macaques develop more severe pathology than young adult animals, even though viral replication levels are similar. Comprehensive genomic analyses indicate that aged macaques have a stronger host response to virus infection than young adult macaques, with an increase in differential expression of genes associated with inflammation, with NF-kappaB as central player, whereas expression of type I interferon (IFN)-beta is reduced. Therapeutic treatment of SARS-CoV-infected aged macaques with type I IFN reduces pathology and diminishes pro-inflammatory gene expression, including interleukin-8 (IL-8) levels, without affecting virus replication in the lungs. Thus, ALI in SARS-CoV-infected aged macaques developed as a result of an exacerbated innate host response. The anti-inflammatory action of type I IFN reveals a potential intervention strategy for virus-induced ALI.

Pentraxin 3 (PTX3) is an acute-phase response protein that initiates innate immunity against diverse microorganisms. It is produced in response to proinflammatory stimuli by many cell types including myeloid cells. Increased serum levels of PTX3 are found in pregnancy, a condition characterized by increased serum levels of the pregnancy hormone human chorionic gonadotropin (hCG). As myeloid cells bear the receptor for hCG, we hypothesized that hCG can promote innate immunity by affecting the PTX3 production by myeloid cells. In this paper, we demonstrate that hCG increases PTX3 expression by human monocytes, mouse dendritic cells, and mouse macrophages in vitro. This increased PTX3 expression by hCG is mediated via the protein kinase A signaling pathway. hCG injection in mice also increases the PTX3 serum levels. This serum PTX3 is produced mainly by blood monocytes, which from pregnant women, express more PTX3 compared with nonpregnant controls. The hCG-induced hormones progesterone and estrogen also increase the PTX3 production by human monocytes. In conclusion, hCG increases innate immunity via induction of PTX3 in myeloid cells.

The pregnancy hormone human chorionic gonadotropin (hCG) has been suggested to play an immunoregulatory role in addition to its endocrine function, thus contributing to the prevention of fetal rejection. We hypothesized that hCG is involved in the maternal-fetal immune tolerance by the regulation of dendritic cell (DC) function. Therefore, we studied the effect of hCG on DC maturation. Upon hCG treatment in combination with LPS, mouse bone marrow-derived DC (BMDC) increased the ratio of IL-10:IL-12p70, down-regulated TNF-alpha, and decreased antigen-specific T cell proliferation. Addition of hCG together with LPS and IFN-gamma blocked MHC class II up-regulation, increased IL-10 production, and decreased the antigen-specific T cell proliferation by DC. Splenic DC showed similar results. Upon hCG treatment, IDO mRNA expression and its metabolite kynurenine were increased by LPS- and IFN-gamma-stimulated DC, suggesting its involvement in the decreased T cell proliferation. To study the effect of hCG on DC differentiation from precursors, BMDC were generated in the continuous presence of hCG. Under this condition, hCG decreased cytokine production and the induction of T cell proliferation. These data are suggestive for a contribution of hCG to the maternal-fetal tolerance during pregnancy by modifying DC toward a tolerogenic phenotype.

The pathogenesis of severe acute respiratory syndrome coronavirus (SARS-CoV) is likely mediated by disproportional immune responses and the ability of the virus to circumvent innate immunity. Using functional genomics, we analyzed early host responses to SARS-CoV infection in the lungs of adolescent cynomolgus macaques (Macaca fascicularis) that show lung pathology similar to that observed in human adults with SARS. Analysis of gene signatures revealed induction of a strong innate immune response characterized by the stimulation of various cytokine and chemokine genes, including interleukin (IL)-6, IL-8, and IP-10, which corresponds to the host response seen in acute respiratory distress syndrome. As opposed to many in vitro experiments, SARS-CoV induced a wide range of type I interferons (IFNs) and nuclear translocation of phosphorylated signal transducer and activator of transcription 1 in the lungs of macaques. Using immunohistochemistry, we revealed that these antiviral signaling pathways were differentially regulated in distinctive subsets of cells. Our studies emphasize that the induction of early IFN signaling may be critical to confer protection against SARS-CoV infection and highlight the strength of combining functional genomics with immunohistochemistry to further unravel the pathogenesis of SARS.