Mary J. Pantin-Jackwood

Pathogenicity of Influenza Viruses with Genes from the 1918 Pandemic Virus: Functional Roles of Alveolar Macrophages and Neutrophils in Limiting Virus Replication and Mortality in Mice

ABSTRACT The Spanish influenza pandemic of 1918 to 1919 swept the globe and resulted in the deaths of at least 20 million people. The basis of the pulmonary damage and high lethality caused by the 1918 H1N1 influenza virus remains largely unknown. Recombinant influenza viruses bearing the 1918 influenza virus hemagglutinin (HA) and neuraminidase (NA) glycoproteins were rescued in the genetic background of the human A/Texas/36/91 (H1N1) (1918 HA/NA:Tx/91) virus. Pathogenesis experiments revealed that the 1918 HA/NA:Tx/91 virus was lethal for BALB/c mice without the prior adaptation that is usually required for human influenza A H1N1 viruses. The increased mortality of 1918 HA/NA:Tx/91-infected mice was accompanied by (i) increased (>200-fold) viral replication, (ii) greater influx of neutrophils into the lung, (iii) increased numbers of alveolar macrophages (AMs), and (iv) increased protein expression of cytokines and chemokines in lung tissues compared with the levels seen for control Tx/91 virus-infected mice. Because pathological changes in AMs and neutrophil migration correlated with lung inflammation, we assessed the role of these cells in the pathogenesis associated with 1918 HA/NA:Tx/91 virus infection. Neutrophil and/or AM depletion initiated 3 or 5 days after infection did not have a significant effect on the disease outcome following a lethal 1918 HA/NA:Tx/91 virus infection. By contrast, depletion of these cells before a sublethal infection with 1918 HA/NA:Tx/91 virus resulted in uncontrolled virus growth and mortality in mice. In addition, neutrophil and/or AM depletion was associated with decreased expression of cytokines and chemokines. These results indicate that a human influenza H1N1 virus possessing the 1918 HA and NA glycoproteins can induce severe lung inflammation consisting of AMs and neutrophils, which play a role in controlling the replication and spread of 1918 HA/NA:Tx/91 virus after intranasal infection of mice.

Enteric Viruses Detected by Molecular Methods in Commercial Chicken and Turkey Flocks in the United States Between 2005 and 2006

Abstract Intestinal samples collected from 43 commercial broiler and 33 commercial turkey flocks from all regions of the United States during 2005 and 2006 were examined for the presence of astrovirus, rotavirus, reovirus, and coronavirus by reverse transcription-polymerase chain reaction (PCR), and for the presence of groups 1 and 2 adenovirus by PCR. Phylogenetic analysis was performed to further characterize the viruses and to evaluate species association and geographic patterns. Astroviruses were identified in samples from 86% of the chicken flocks and from 100% of the turkey flocks. Both chicken astrovirus and avian nephritis virus (ANV) were identified in chicken samples, and often both viruses were detected in the same flock. Turkey astrovirus type-2 and turkey astrovirus type-1 were found in 100% and 15.4% of the turkey flocks, respectively. In addition, 12.5% of turkey flocks were positive for ANV. Rotaviruses were present in 46.5% of the chicken flocks tested and in 69.7% of the turkey flocks tested. Based upon the rotavirus NSP4 gene sequence, the chicken and turkey origin rotaviruses assorted in a species-specific manner. The turkey origin rotaviruses also assorted based upon geographical location. Reoviruses were identified in 62.8% and 45.5% of chicken and turkey flocks, respectively. Based on the reovirus S4 gene segment, the chicken and turkey origin viruses assorted separately, and they were distinct from all previously reported avian reoviruses. Coronaviruses were detected in the intestinal contents of chickens, but not turkeys. Adenoviruses were not detected in any chicken or turkeys flocks. Of the 76 total chicken and turkey flocks tested, only three chicken flocks were negative for all viruses targeted by this study. Most flocks were positive for two or more of the viruses, and overall no clear pattern of virus geographic distribution was evident. This study provides updated enteric virus prevalence data for the United States using molecular methods, and it reinforces that enteric viruses are widespread in poultry throughout the United States, although the clinical importance of most of these viruses remains unclear.

Pathobiology of Asian Highly Pathogenic Avian Influenza H5N1 Virus Infections in Ducks

Abstract Ducks and other wild aquatic birds are the natural reservoir of type A influenza viruses, which normally are nonpathogenic in these birds. However, the Asian highly pathogenic avian influenza (HPAI) viruses have evolved from producing no disease or mild respiratory infections in ducks to some strains producing severe systemic disease and mortality. To further understand the pathogenicity of these strains in ducks, we studied the gross and histologic lesions and tissue distribution of viral antigen in 2- and 5-wk-old white Pekin ducks infected with different Asian-origin H5N1 AI viruses. Seven of eight 2-wk-old ducks inoculated with A/Egret/HK/757.2/02 developed acute disease, including severe neurological dysfunction and death. However, this virus killed only two of eight 5-wk-old ducks. Two additional viruses, A/Vietnam/1203/04 and A/Crow/Thailand/04, also produced high mortality in 2-wk-old ducks. Microscopic lesions and AI viral antigen were observed most frequently in the nasal cavity, brain, heart, adrenal glands, and pancreas. Another virus, A/Thailand PB/6231/04, killed three of eight 2-wk-old ducks but did not induce neurological signs. Furthermore, older ducks infected with this virus did not present clinical signs or gross lesions, and their tissues showed very few microscopic lesions. All the viruses studied established systemic infections in both younger and older ducks, with viral replication in tissues correlating with the severity of the clinical signs. The differences in mortality induced by HPAI H5N1 viruses in ducks are reflected in the pathological findings and antigen distribution in tissues. However, the observed differences in pathology between ducks infected at different ages is unclear and may be associated with a variety of factors including the virus strain, host immune response, host cell maturation, and capacity to support viral replication.

Pathogenesis of pandemic influenza A (H1N1) and triple-reassortant swine influenza A (H1) viruses in mice.

ABSTRACT The pandemic H1N1 virus of 2009 (2009 H1N1) continues to cause illness worldwide, primarily in younger age groups. To better understand the pathogenesis of these viruses in mammals, we used a mouse model to evaluate the relative virulence of selected 2009 H1N1 viruses and compared them to a representative human triple-reassortant swine influenza virus that has circulated in pigs in the United States for over a decade preceding the current pandemic. Additional comparisons were made with the reconstructed 1918 virus, a 1976 H1N1 swine influenza virus, and a highly pathogenic H5N1 virus. Mice were inoculated intranasally with each virus and monitored for morbidity, mortality, viral replication, hemostatic parameters, cytokine production, and lung histology. All 2009 H1N1 viruses replicated efficiently in the lungs of mice and possessed a high degree of infectivity but did not cause lethal disease or exhibit extrapulmonary virus spread. Transient weight loss, lymphopenia, and proinflammatory cytokine and chemokine production were present following 2009 H1N1 virus infection, but these levels were generally muted compared with a triple-reassortant swine virus and the 1918 virus. 2009 H1N1 viruses isolated from fatal cases did not demonstrate enhanced virulence in this model compared with isolates from mild human cases. Histologically, infection with the 2009 viruses resulted in lesions in the lung varying from mild to moderate bronchiolitis with occasional necrosis of bronchiolar epithelium and mild to moderate peribronchiolar alveolitis. Taken together, these studies demonstrate that the 2009 H1N1 viruses exhibited mild to moderate virulence in mice compared with highly pathogenic viruses.

PERIODIC MONITORING OF COMMERCIAL TURKEYS FOR ENTERIC VIRUSES INDICATES CONTINUOUS PRESENCE OF ASTROVIRUS AND ROTAVIRUS ON THE FARMS

Abstract A longitudinal survey to detect enteric viruses in intestinal contents collected from turkeys in eight commercial operations and one research facility was performed using molecular detection methods. Intestinal contents were collected from turkeys prior to placement, with each flock resampled at 2, 4, 6, 8, 10, and 12 wk of age. The samples were screened for astrovirus, rotavirus, reovirus, and turkey coronavirus (TCoV) by a reverse transcriptase and polymerase chain reaction (RT-PCR), and for groups 1 and 2 adenovirus by PCR. Rotavirus was the only virus detected prior to placement (7 of 16 samples examined). All of the commercial flocks were positive for rotavirus and astrovirus from 2 until 6 wk of age, and most were intermittently positive until 12 wk of age, when the birds were processed. Of the 96 samples collected from birds on the farms, 89.5% were positive for astrovirus, and 67.7% were positive for rotavirus. All flocks were negative for TCoV, reovirus, and group 1 adenovirus at all time points, and positive for group 2 adenovirus (hemorrhagic enteritis virus) at 6 wk of age. All the flocks monitored were considered healthy or normal by field personnel. Turkeys placed on research facilities that had been empty for months and thoroughly cleaned had higher body weights and lower feed conversion rates at 5 wk of age when compared to turkeys placed on commercial farms. Intestinal samples collected at 1, 2, and 3 wk of age from these turkeys were free of enteric viruses. This report demonstrates that astroviruses and rotaviruses may be present within a turkey flock through the life of the flock. Comparison of infected birds with one group of turkeys that were negative for enteric viruses by the methods used here suggests that astrovirus and/or rotavirus may affect production. The full impact on flock performance needs to be further determined.

Role of Poultry in the Spread of Novel H7N9 Influenza Virus in China

ABSTRACT The recent outbreak of H7N9 influenza in China has resulted in many human cases with a high fatality rate. Poultry are the likely source of infection for humans on the basis of sequence analysis and virus isolations from live bird markets, but it is not clear which species of birds are most likely to be infected and shedding levels of virus sufficient to infect humans. Intranasal inoculation of chickens, Japanese quail, pigeons, Pekin ducks, Mallard ducks, Muscovy ducks, and Embden geese with 106 50% egg infective doses of the A/Anhui/1/2013 virus resulted in infection but no clinical disease signs. Virus shedding was much higher and prolonged in quail and chickens than in the other species. Quail effectively transmitted the virus to direct contacts, but pigeons and Pekin ducks did not. In all species, virus was detected at much higher titers from oropharyngeal swabs than cloacal swabs. The hemagglutinin gene from samples collected from selected experimentally infected birds was sequenced, and three amino acid differences were commonly observed when the sequence was compared to the sequence of A/Anhui/1/2013: N123D, N149D, and L217Q. Leucine at position 217 is highly conserved for human isolates and is associated with α2,6-sialic acid binding. Different amino acid combinations were observed, suggesting that the inoculum had viral subpopulations that were selected after passage in birds. These experimental studies corroborate the finding that certain poultry species are reservoirs of the H7N9 influenza virus and that the virus is highly tropic for the upper respiratory tract, so testing of bird species should preferentially be conducted with oropharyngeal swabs for the best sensitivity. IMPORTANCE The recent outbreak of H7N9 influenza in China has resulted in a number of human infections with a high case fatality rate. The source of the viral outbreak is suspected to be poultry, but definitive data on the source of the infection are not available. This study provides experimental data to show that quail and chickens are susceptible to infection, shed large amounts of virus, and are likely important in the spread of the virus to humans. Other poultry species can be infected and shed virus but are less likely to play a role of transmitting the virus to humans. Pigeons were previously suggested to be a possible source of the virus because of isolation of the virus from several pigeons in poultry markets in China, but experimental studies show that they are generally resistant to infection and are unlikely to play a role in the spread of the virus.

Removal of Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) Inhibitors Associated with Cloacal Swab Samples and Tissues for Improved Diagnosis of Avian Influenza Virus by RT-PCR

Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) is routinely used for the rapid detection of Avian influenza virus (AIV) in clinical samples, but inhibitory substances present in some clinical specimens can reduce or block PCR amplification. Most commercial RNA extraction kits have limited capacity to remove inhibitors from clinical samples, but using a modified commercial protocol (Ambion® MagMAX™, Applied Biosystems, Foster City, CA) with an added high-salt wash of 2 M NaCl and 2 mM ethylenediamine tetra-acetic acid was shown to improve the ability of the kit to remove inhibitors from cloacal swabs and some tissues. Real-time RT-PCR was carried out in the presence of an internal positive control to detect inhibitors present in the purified RNA. Cloacal swabs from wild birds were analyzed by real-time RT-PCR comparing RNA extracted with the standard (MagMAX-S) and modified (MagMAX-M) protocols. Using the standard protocol on 2,668 samples, 18.4% of the samples had evidence of inhibitor(s) in the samples, but the modified protocol removed inhibitors from all but 21 (4.8%) of the problem samples. The modified protocol was also tested for RNA extraction from tissues using a TRIzol-MagMAX-M hybrid protocol. Tissues from chickens and ducks experimentally infected with high-pathogenicity Asian H5N1 AIV were analyzed by real-time RT-PCR, and the limit of detection of the virus was improved by 0.5–3.0 threshold cycle units with the RNA extracted by the MagMAX-M protocol. The MagMAX-M protocol reported in the present study can be useful in extracting high-quality RNA for accurate detection of AIV from cloacal swabs and tissues by real-time RT-PCR.

Lethal dissemination of H5N1 influenza virus is associated with dysregulation of inflammation and lipoxin signaling in a mouse model of infection

ABSTRACT Periodic outbreaks of highly pathogenic avian H5N1 influenza viruses and the current H1N1 pandemic highlight the need for a more detailed understanding of influenza virus pathogenesis. To investigate the host transcriptional response induced by pathogenic influenza viruses, we used a functional-genomics approach to compare gene expression profiles in lungs from 129S6/SvEv mice infected with either the fully reconstructed H1N1 1918 pandemic virus (1918) or the highly pathogenic avian H5N1 virus Vietnam/1203/04 (VN/1203). Although the viruses reached similar titers in the lung and caused lethal infections, the mean time of death was 6 days for VN/1203-infected animals and 9 days for mice infected with the 1918 virus. VN/1203-infected animals also exhibited an earlier and more potent inflammatory response. This response included induction of genes encoding components of the inflammasome. VN/1203 was also able to disseminate to multiple organs, including the brain, which correlated with changes in the expression of genes associated with hematological functions and lipoxin biogenesis and signaling. Both viruses elicited expression of type I interferon (IFN)-regulated genes in wild-type mice and to a lesser extent in mice lacking the type I IFN receptor, suggesting alternative or redundant pathways for IFN signaling. Our findings suggest that VN/1203 is more pathogenic in mice as a consequence of several factors, including the early and sustained induction of the inflammatory response, the additive or synergistic effects of upregulated components of the immune response, and inhibition of lipoxin-mediated anti-inflammatory responses, which correlated with the ability of VN/1203 to disseminate to extrapulmonary organs.

A Multiplex RT-PCR Test for the Differential Identification of Turkey Astrovirus Type 1, Turkey Astrovirus Type 2, Chicken Astrovirus, Avian Nephritis Virus, and Avian Rotavirus

Abstract Recent studies have revealed the presence of astroviruses and rotavirus in numerous poorly performing and healthy chicken and turkey flocks in the United States. The phylogenetic analysis of the sequence data produced during these studies has identified four groups of avian astroviruses circulating in the United States: turkey astrovirus types 1 and 2 (TAstV-1 and TAstV-2), avian nephritis virus (ANV), and a chicken-origin astrovirus (CAstV). As the molecular epidemiology of poultry enteric disease is poorly understood, the development of updated diagnostic assays is crucial to the continued surveillance and management of enteric disease in affected as well as healthy flocks. This report details the development of a multiplex reverse transcriptase–polymerase chain reaction (RT-PCR) assay specific for astroviruses and avian rotavirus in turkey-origin and chicken-origin samples. The assay consists of two multiplex tests, one for turkey-origin samples and one for chicken-origin samples. The turkey sample test differentially identifies TAstV-1, TAstV-2, ANV, and avian rotavirus. The test for chicken-origin samples differentially identifies CAstV, ANV, and avian rotavirus. Assay sensitivity varied by target sequence between approximately 10 copies for avian rotavirus alone and approximately 2 × 106 copies for TAstV-2 in the presence of a heterologous competitor RNA sequence. Each test was shown to be specific for the intended target by testing for cross-reaction with other common avian enteric viruses. The specificity was further shown by testing 109 chicken specimens and 32 turkey specimens from commercial flocks with the appropriate test and sequencing the RT-PCR amplicons to confirm amplification of the correct target.

Molecular Characterization and Typing of Chicken and Turkey Astroviruses Circulating in the United States: Implications for Diagnostics

Abstract Avian astroviruses were detected by reverse transcriptase and polymerase chain reaction in intestinal contents collected from commercial chickens and turkeys from throughout the United States from 2003 through 2005. Astroviruses were detected in birds from both healthy and poorly performing flocks with or without enteric disease. Phylogenetic analysis was performed with sequence data from the polymerase (ORF-1b) genes of 41 turkey-origin astroviruses and 23 chicken-origin astroviruses. All currently available avian astrovirus sequence data and selected mammalian astrovirus sequence data were included in the analysis. Four groups of avian astroviruses were observed by phylogenetic analysis: turkey astrovirus type 1 (TAstV-1)-like viruses, turkey astrovirus type 2 (TAstV-2)-like viruses, both detected in turkeys; avian nephritis virus (ANV)-like viruses, detected in both chickens and turkeys; and a novel group of chicken-origin astroviruses (CAstV). Among these four groups, amino acid identity was between 50.1% and 73.8%, and was a maximum of 49.4% for all avian isolates when compared with the mammalian astroviruses. There were multiple phylogenetic subgroups within the TAstV-2, ANV, and CAstV groups based on 9% nucleotide sequence divergence. Phylogenetic analysis revealed no clear assortment by geographic region or isolation date. Furthermore, no correlation was observed between the detection of a particular astrovirus and the presence of enteric disease or poor performance. Based on these data, a revision of the present taxonomic classification for avian astroviruses within the genus Avastrovirus is warranted.