Macrovascular and microvascular type 2 diabetes complications are interrelated in a mouse model.

AIM: Evaluate the development of multiple complications, their interactions, and common mechanisms in the same individual with T2D. MATERIAL AND METHODS: 4-week-old male C57BL/6J mice were divided into: control (n = 6) and T2D (n = 6). T2D was induced through a high-carbohydrate-diet and low doses of streptozotocin. T2D was validated by metabolic parameters. Diabetic neuropathy was evaluated by mechanical and thermal sensitivity tests. We performed a histopathological analysis of the heart, kidney, liver, and parotid salivary glands and changes in bone microarchitecture by µCT. We calculated the relative risk (RR), odd ratios (OR) and Pearson correlation coefficients between the different complications and metabolic features. RESULTS: T2D mice have cardiomyopathy, neuropathy, nephropathy, liver steatosis and fibrosis, structural damage in parotid salivary glands, and bone porosity. RR analysis shows that all complications are interrelated by hyperglycaemia, insulin resistance, obesity, and systemic inflammation. CONCLUSIONS: T2D mice develop multiple complications simultaneously, which are related to each other, and this is associated with metabolic alterations. Our findings open up new approaches for the study and new therapeutic approaches of the pathophysiology of T2D and its complications.

CD4+ T-cell activation of bone marrow causes bone fragility and insulin resistance in type 2 diabetes.

Type 2 diabetes mellitus (T2DM) causes an increased risk of bone fractures. However, the pathophysiology of diabetic bone fragility is not completely understood. It has been proposed that an inflammatory microenvironment in bone could be a major mechanism by inducing uncontrolled bone resorption, inadequate bone formation and consequently more porous bones. We propose that activated T-cells in the bone marrow cause a pro-inflammatory microenvironment in bone, and cause bone fragility in T2DM. We induced T2DM in C57BL/6 male mice through a hypercaloric diet rich in carbohydrates and low doses of streptozocin. In T2DM mice we inhibited systemic activation of T-cells with a fusion protein between the extracellular domain of Cytotoxic T-Lymphocyte Antigen 4 and the Fc domain of human immunoglobulin G (CTLA4-Ig). We analysed the effects of T2DM or CTLA4-Ig in lymphocyte cell subsets and antigen-presenting cells in peripheral blood and femoral bone marrow; and their effect on the metabolic phenotype, blood and bone cytokine concentration, femoral bone microarchitecture and biomechanical properties, and the number of osteoblast-like cells in the femoral endosteum. We performed a Pearson multiple correlation analysis between all variables in order to understand the global mechanism. Results demonstrated that CTLA4-Ig decreased the number of activated CD4+ T-cells in the femoral bone marrow and consequently decreased TNF-α and RANK-L concentration in bone, notably improved femoral bone microarchitecture and biomechanical properties, increased the number of osteoblast-like cells, and reduces osteoclastic activity compared to T2DM mice that did not receive the inhibitor. Interestingly, we observed that blood glucose levels and insulin resistance may be related to the increase in activated CD4+ T-cells in the bone marrow. We conclude that bone marrow activated CD4+ T-cells cause poor bone quality and insulin resistance in T2DM.

Single-Nucleotide Polymorphism Analysis to Select Conserved Regions for an Improved Real-Time Reverse Transcription-PCR Test Specific for Newcastle Disease Virus.

A bioinformatics approach using single-nucleotide polymorphism (SNP) analysis was performed to improve the current real-time reverse transcription-PCR (RRT-PCR) tests for the rapid detection of Newcastle disease virus (NDV). In total, 422 NDV complete genomes were analyzed using the Virus Pathogen Resource to compare the conservation of the primer and probe sequences and to select regions to develop new RRT-PCR tests. The sensitivity and specificity of the three new RRT-PCR tests targeting the nucleoprotein (NP) and polymerase (L) genes were optimized and were compared with established tests for NDV detection. The SNP analysis was also used to identify the number of mismatches between selected primers/probes and the NDV complete genome sequences. The SNP analysis, averaged over the entire primer or probe, showed the primer/probe sequences of three new tests were more conserved than the primer/probe sequences of the commonly used test targeting the matrix (M) gene. The M RRT-PCR test was compared with the new tests on a panel of 46 viruses, comprising 31 NDV isolates. Limit of detection (LOD) varied from 1.3 to 3.7 log 50% egg-infective doses using five isolates from different genotypes by all tests. The two RRT-PCR tests targeting the L and M genes detected three out of five isolates with the lowest LOD. The NP and M RRT-PCR tests had the lowest and highest rates of genetic variants, respectively, among all probes. Because currently used tests are likely to miss some isolates, the availability of validated alternative tests provides alternatives for detection of viral variants that can be rapidly deployed to diagnostic laboratories.

Análisis del polimorfismo de un nucleótido simple para seleccionar regiones conservadas para establecer prueba mejoradas de transcripción reversa y PCR en tiempo real específicas para el virus de la enfermedad de Newcastle. Se llevó a cabo un enfoque bioinformático utilizando el análisis del polimorfismo de un nucleótido simple (SNP) para mejorar las pruebas actuales de transcripción reversa y PCR en tiempo real (RRT-PCR) para la detección rápida del virus de la enfermedad de Newcastle (NDV). Se analizaron un total de 422 genomas completos del virus de Newcastle utilizando la base de datos llamada Recursos en Patógenos Virales (Virus Pathogen Resource) para comparar la conservación de las secuencias de iniciadores y de sondas y para seleccionar regiones adecuadas para desarrollar nuevas pruebas de transcripción reversa y PCR en tiempo real. La sensibilidad y especificidad de las tres nuevas pruebas dirigidas a los genes de la nucleoproteína (NP) y de la polimerasa (L) se optimizaron y se compararon con las pruebas establecidas para la detección del virus de Newcastle. El análisis del polimorfismo de un nucleótido simple también se usó para identificar el número de discrepancias entre los iniciadores y sondas seleccionados con las secuencias completas del genoma del virus de Newcastle. El análisis del polimorfismo de un nucleótido simple, promediado sobre el iniciador o sonda completos, mostró que las secuencias de iniciadores y sondas de las tres pruebas nuevas estaban más conservadas que las secuencias de iniciadores y sondas de la prueba comúnmente utilizada dirigida al gene de la matriz (M). La prueba dirigida para la matriz se comparó con las pruebas nuevas mediante un panel de 46 virus, que comprendió 31 virus de Newcastle. El límite de detección (LOD) mostró un rango de 1.3 a 3.7 logaritmos de dosis infecciosas de embrión de pollo 50% usando cinco aislamientos de diferentes genotipos en todas las pruebas. Las dos pruebas dirigidas a los genes L y M detectaron tres de cinco aislamientos con los límites de detección más bajos. Las pruebas dirigidas a los genes NP y M mostraron las tasas más bajas y las más altas de variantes genéticas, respectivamente, de entre todas las sondas. Debido a que es probable que las pruebas utilizadas actualmente pueden omitir algunos aislamientos, la disponibilidad de pruebas alternativas validadas proporciona alternativas para la detección de variantes virales que se pueden establecer rápidamente en los laboratorios de diagnóstico.

Armoured exogenous internal control for real-time PCR diagnosis of avian influenza.

An exogenous "armoured" PCR internal control (IC) short RNA was analyzed in conjunction with real-time RT-PCR method for diagnosis of avian influenza. The resistance to nucleases and increased physical stability of the IC was ensured using branched polyethyleneimine (PEI) which was in complex with IC-RNA. The option to add the IC directly to pathological material suspensions allows measurement of the nucleic acids extraction efficiency. Stability of armoured RNA-IC during storage and tissue suspension preparation was shown. The advantage of exogenous "armoured" IC was demonstrated in the experiment with AIV genome detection by qPCR in samples from different species of wild birds. The exogenous IC gave reproducible homogeneous Ct values in all tests.

Presence of Newcastle disease viruses of sub-genotypes Vc and VIn in backyard chickens and in apparently healthy wild birds from Mexico in 2017.

Virulent Newcastle disease viruses (NDV) have been present in Mexico since 1946, and recently, multiple outbreaks have been reported in the country. Here, we characterized eleven NDV isolated from apparently healthy wild birds and backyard chickens in three different locations of Jalisco, Mexico in 2017. Total RNA from NDV was reverse-transcribed, and 1285 nucleotides, which includes 3/4 of the fusion gene, was amplified and sequenced using a long-read MinION sequencing method. The sequences were 99.99-100% identical to the corresponding region obtained using the Illumina MiSeq. Phylogenetic analysis using MinION sequences demonstrated that nine virulent NDV from wild birds belonged to sub-genotypes Vc and VIn, and two backyard chicken isolates were of sub-genotype Vc. The sub-genotype Vc viruses had nucleotide sequence identity that ranged from 97.7 to 98% to a virus of the same sub-genotype isolated from a chicken in Mexico in 2010. Three viruses from pigeons had 96.3-98.7% nucleotide identity to sub-genotype VIn pigeon viruses, commonly referred to as pigeon paramyxovirus, isolated in the USA during 2000-2016. This study demonstrates that viruses of sub-genotype Vc are still present in Mexico, and the detection of this sub-genotype in both chickens and wild birds suggests that transmission among these species may represent a biosecurity risk. This is the first detection and complete genome sequencing of genotype VI NDV from Mexico. In addition, the utilization of an optimized long-read sequencing method for rapid virulence and genotype identification using the Oxford nanopore MinION system is demonstrated.

Age-dependent pathogenesis of clade 2.3.4.4A H5N2 HPAIV in experimentally infected Broad Breasted White turkeys.

Highly pathogenic avian influenza (HPAI) is a viral disease with devastating consequences to the poultry industry as it results in high morbidity, mortality and international trade restrictions. In the present study, we characterized age-related differences in terms of pathology in commercial white broad breasted turkeys inoculated with A/turkey/Minnesota/12582/2015 (H5N2) HPAIV clade 2.3.4.4A, a virus from the largest HPAI poultry outbreak that affected the Unites States in 2014-2015. Turkeys infected at 6-weeks of age showed inapparent to little clinical signs with rapid disease progression, reaching 100% mortality at 3 days post infection (dpi). In contrast, turkeys infected at 16-weeks of age developed ataxia and lethargy and reached 100% mortality by 5 dpi. Infection in the 6-weeks old turkeys resulted in peracute lesions consistent of extensive hemorrhages, edema and necrosis, but inflammation was not prominent. In the 16-weeks old turkeys, necrosis and hemorrhages in tissues were accompanied by a more prominent subacute inflammatory infiltrate. Both age groups showed presence of avian influenza virus (AIV) nucleoprotein (NP) in multiple cell types including neurons, glial cells, ependymal cells, respiratory epithelial cells, air capillary epithelium and pulmonary macrophages, cardiac myocytes, smooth muscle fibers, pancreatic acini and ductal cells. Cells of the vascular walls stained strongly positive for viral antigens, but no positivity was found in the endothelial cells of any organs. These findings indicate that age is a determinant factor in the progression of the disease and delay of mortality during infection with the H5N2 clade 2.3.4.4A HPAI virus in naïve white broad breasted turkeys.

Impact of treated wastewater for irrigation on soil microbial communities.

The use of treated wastewater (TWW) for irrigation has been suggested as an alternative to use of fresh water because of the increasing scarcity of fresh water in arid and semiarid regions of the world. However, significant barriers exist to widespread adoption due to some potential contaminants that may have adverse effects on soil quality and or public health. In this study, we investigated the abundance and diversity of bacterial communities and the presence of potential pathogenic bacterial sequences in TWW in comparison to synthetic fresh water (SFW) using pyrosequencing. The results were analyzed using UniFrac coupled with principal coordinate analysis (PCoA) to compare diversity and abundance of different bacterial groups in TWW irrigated soils to soils treated with SFW. Shannon diversity index values (H') suggest that microbial diversity was not significantly different (P<0.086) between soils with TWW and SFW. Pyrosequencing detected sequences of 17 bacterial phyla with Proteobacteria (32.1%) followed by Firmicutes (26.5%) and Actinobacteria (14.3%). Most of the sequences associated with nitrifying bacteria, nitrogen-fixing bacteria, carbon degraders, denitrifying bacteria, potential pathogens, and fecal indicator bacteria were more abundant in TWW than in SFW. Therefore, TWW effluent may contain bacterial that may be very active in many soil functions as well as some potential pathogens.

Characteristics of pigeon paramyxovirus serotype-1 isolates (PPMV-1) from the Russian Federation from 2001 to 2009.

Monitoring programs for highly dangerous avian diseases in the Russian Federation from 2001 to 2009 detected 77 samples that were PCR positive for avian paramyxovirus serotype-1 (APMV-1) from sick or dead feral and domestic pigeons. Nucleotide sequences of the fusion (F) gene, including a nucleotide sequence encoding the F protein cleavage site, were determined for these isolates. All of the studied isolates possessed virulent F0 protein cleavage sites (112KRKKRF117, 112RRQKRF117, or 112KRQKRF117). Intracerebral pathogenicity index (ICPI) values determined for seven of the isolates exceeded the value of 0.7 (the range from 0.8 to 1.41). Based on partial genome sequencing and phylogenetic analysis, the isolates were assigned to two individual sublineages within class II genotype VIb. It was determined that most of these Newcastle disease virus isolates (70/77) recovered from the pigeons belonged to a relatively poorly studied sublineage VIb/2. The complete nucleotide sequence of the genome for the Pigeon/Russia/Vladimir/687/05 isolate of sublineage VIb/2 was determined.

Pathogenicity of two Egyptian H5N1 highly pathogenic avian influenza viruses in domestic ducks.

Domestic ducks have been implicated in the dissemination and evolution of H5N1 highly pathogenic avian influenza (HPAI) viruses. In this study, two H5N1 HPAI viruses belonging to clade 2.2.1 isolated in Egypt in 2007 and 2008 were analyzed for their pathogenicity in domestic Pekin ducks. Both viruses produced clinical signs and mortality, but the 2008 virus was more virulent, inducing early onset of neurological signs and killing all ducks with a mean death time (MDT) of 4.1 days. The 2007 virus killed 3/8 ducks with a MDT of 7 days. Full-genome sequencing and phylogenetic analysis were used to examine differences in the virus genes that might explain the differences observed in pathogenicity. The genomes differed in 49 amino acids, with most of the differences found in the hemagglutinin protein. This increase in pathogenicity in ducks observed with certain H5N1 HPAI viruses has implications for the control of the disease, since vaccinated ducks infected with highly virulent strains shed viruses for longer periods of time, perpetuating the virus in the environment and increasing the possibility of transmission to susceptible birds.

Phylogenetic analysis of hemagglutinin and neuraminidase genes of highly pathogenic avian influenza H5N1 Egyptian strains isolated from 2006 to 2008 indicates heterogeneity with multiple distinct sublineages.

The Eurasian-lineage H5N1 highly pathogenic avian influenza (HPAI) virus caused widespread outbreaks in Egypt in 2006 and eventually become enzootic in poultry. Although outbreaks have a seasonal pattern, with most occurring during the cooler winter months, it remains unclear whether this seasonality reflects virus maintenance within Egypt or yearly introductions of the virus into the country. To evaluate the epidemiology of H5N1 HPAI in Egypt, sequence analysis of the hemagglutinin (HA) and neuraminidase (NA) genes of selected Egyptian isolates from early 2006 to 2008 was conducted. The data from this study identifies distinct genetic markers in both HA and NA genes and suggests grouping Egyptian isolates into two major HA isolate sublineages from 2006 to 2008 and into three smaller, emergent subgroups. The NA phylogenetic and sequence analysis showed a similar pattern, except that two of the emergent groups from the HA phylogenetic tree clustered together, evidence of likely reassortment. The different subgroups did not appear to segregate by relation to the date of isolation, to the species of origin, nor to the geographic location of the viruses. The conclusion is that H5N1 is continuing to mutate with multiple heterogenic strains persisting in Egypt.

Evolution of highly pathogenic avian influenza H5N1 virus in natural ecosystems of northern Eurasia (2005-08).

Fifty-four strains of H5N1 highly pathogenic avian influenza (HPAI) virus were isolated from wild birds in the ecosystems of northern Eurasia and from poultry in the south of western Siberia (July 2005), at the mouth of Volga River (November 2005), at Uvs-Nur Lake on the boundary of the Great Lakes Depression in western Mongolia and the Tyva Republic of Russia (June 2006), in the vicinity of Moscow (February 2007), in the southeastern part of the Russian Plain (September 2007 and December 2007), and in the far east (April 2008) of the Russian Federation and were phenotypically characterized and deposited into the Russian state collection of viruses. Complete genome nucleotide sequences for 24 strains were obtained and deposited into GenBank. In all cases when strains were isolated from both wild birds and poultry in the same outbreak these strains were genetically closely related to each other. Until 2008 all HPAI H5N1 strains isolated in northern Eurasia clustered genetically with the viruses from Kukunor Lake (Qinghai Province, China), known as genotype 2.2 or the "Qinghai-Siberian" genotype. The viruses from the Qinghai-Siberian genotype have continued to evolve from those initially introduced into western Siberia in 2005 into two genetic groups: "Iran-North Caucasian" and "Tyva-Siberian." In vitro replication potential (50% tissue-culture infectious dose in porcine embryo kidney) of Qinghai-Siberian strains decreased over time, which could reflect decreasing virulence. Comparison of genome sequences with biological characteristics of the respective strains permitted us to identify point mutations in PB2, PB1, PA, HA, NP, NA, M2, NS1, and NS2 that possibly influenced the level of replication potential. The HPAI H5N1 virus, which penetrated into the south of the Russian Far East in spring 2008, belonged to genotype 2.3.2.

Development and evaluation of an avian influenza, neuraminidase subtype 1, indirect enzyme-linked immunosorbent assay for poultry using the differentiation of infected from vaccinated animals control strategy.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed using baculovirus, purified, recombinant N1 protein from A/chicken/Indonesia/PA7/2003 (H5N1) virus. The N1-ELISA showed high selectivity for detection of N1 antibodies, with no cross-reactivity with other neuraminidase subtypes, and broad reactivity with sera to N1 subtype isolates from North American and Eurasian lineages. Sensitivity of the N1-ELISA to detect N1 antibodies in turkey sera, collected 3 wk after H1N1 vaccination, was comparable to detection of avian influenza antibodies by the commercial, indirect ELISAs ProFLOK AIV Plus ELISA Kit (Synbiotics, Kansas City, MO) and Avian Influenza Virus Antibody Test Kit (IDEXX, Westbrook, ME). However, 6 wk after vaccination, the Synbiotics ELISA kit performed better than the N1-ELISA and the IDEXX ELISA kit. An evaluation was made of the ability of the N1-ELISA to discriminate vaccinated chickens from subsequently challenged chickens. Two experiments were conducted, chickens were vaccinated with inactivated H5N2 and H5N9 viruses and challenged with highly pathogenic H5N1 virus, and chickens were vaccinated with recombinant poxvirus vaccine encoding H7 and challenged with highly pathogenic H7N1 virus. Serum samples were collected at 14 days postchallenge and tested by hemagglutination inhibition (HI), quantitative neuraminidase inhibition (NI), and N1-ELISA. At 2 days postchallenge, oropharyngeal swabs were collected for virus isolation (VI) to confirm infection. The N1-ELISA was in fair agreement with VI and HI results. Although the N1-ELISA showed a lower sensitivity than the NI assay, it was demonstrated that detection of N1 antibodies by ELISA was an effective and rapid assay to identify exposure to the challenge virus in vaccinated chickens. Therefore, N1-ELISA can facilitate a vaccination strategy with differentiation of infected from vaccinated animals using a neuraminidase heterologous approach.

Pathobiological characterization of low-pathogenicity H5 avian influenza viruses of diverse origins in chickens, ducks and turkeys.

We undertook one of the most comprehensive studies on the replication and intraspecies transmission characteristics of low-pathogenicity avian influenza viruses in ducks, chickens and turkeys. Our results indicated that most of these isolates could replicate and be transmitted in poultry without inducing clinical disease. However, differences in transmission to contact control birds were noted, emphasizing the importance of having contact control cage mates in biological characterization experiments. Ducks supported the replication of viruses of wild aquatic bird origin in their respiratory and digestive tracts equally well. The viruses from wild aquatic birds were not effectively transmitted among chickens. In contrast, the wild-bird isolates and viruses of domestic bird origin from live-bird markets and commercial poultry operations replicated and were transmitted more efficiently in turkeys than in chickens or ducks. We also found a lower minimal infectious dose requirement for infection of turkeys compared to chickens and ducks. Our data support an important role of turkeys as being more susceptible hosts for avian influenza viruses than domestic ducks and chickens. These results highlight the role of turkeys as intermediate or bridging hosts in the transmission of influenza viruses from wild birds to land-based domestic poultry or among different land-based bird species.

Phylogenetic and biological characterization of highly pathogenic H5N1 avian influenza viruses (Vietnam 2005) in chickens and ducks.

Analysis of Asian H5N1 avian influenza (AI) virus hemagglutinin (HA) genes shows a common origin, but the virus has evolved into at least three major clades (clades 0, 1, and 2) over the last 13 years. Previous reports of Vietnam viruses have documented predominantly clade 1 viruses. Unexpectedly, 19 viruses from northern Vietnam isolated in December 2005 fell into clade 2. These viruses further clustered into two distinct sublineages. Representative viruses from each sublineage were chosen for antigenic and pathogenic evaluation. Two distinct antigenic groups correlating with the genetic information were present when comparing hemagglutination inhibition (HI) titers. All viruses were highly virulent not only in chickens, killing them within 2 days of experimental inoculation, but also in 2- and 5-week-old Pekin ducks, causing 100% mortality within 4 days of challenge. The information gained about these viruses provides insight with regards to implementing control programs, including vaccine seed strain selection.

Pathobiology of triple reassortant H3N2 influenza viruses in breeder turkeys and its potential implication for vaccine studies in turkeys.

Triple reassortant (TR) H3N2 influenza viruses have been isolated from turkeys in the United States since 2003. These TR H3N2 virus infections have been associated with drastic declines in egg production in breeder turkeys although co-infection with multiple agents could have been responsible for exacerbating the clinical signs. In this study, we experimentally confirmed that TR H3N2 influenza virus alone can cause drastic reduction/complete cessation of egg production and pathology of the reproductive tract in 26-week-old breeder turkeys. We confirmed high levels of virus replication and abundant distribution of avian specific alpha2,3 sialic acid-galactose receptors in the oviduct of these turkeys. Although 2-6-week-old turkeys are routinely used for pathogenicity and vaccine protection studies, the low levels of viral shedding and asymptomatic infections in this age group often pose difficulty in interpretation of results. Our study shows that breeder turkeys should be used to assess the potential pathogenicity of TR H3N2 viruses and the viral titers and pathology of the oviduct as well as egg production data can be good measures of protection following in vivo challenge in vaccine efficacy studies.

Evaluation of chicken-origin (DF-1) and quail-origin (QT-6) fibroblast cell lines for replication of avian influenza viruses.

Avian influenza viruses (AIVs) are isolated routinely and propagated in specific pathogen free embryonated chicken eggs (ECE) and mammalian origin Madin-Darby Canine Kidney (MDCK) cell line. Continuous avian cell lines offer advantages for propagation of AIVs over MDCK cells because they maintain species specificity, and lower recurring costs compared to ECE. In this study, the characteristics of two avian fibroblast cell lines were evaluated, DF-1 (chicken-origin) and QT-6 (quail-origin), and their ability to support the growth of AIVs (n=19) belonging to nine different hemagglutinin subtypes from a variety of avian species. The replication efficiency of the AIVs in QT-6 and DF-1 cells was comparable to those in primary chicken embryo fibroblast (CEF) and MDCK cells. Receptor distribution analysis demonstrated high prevalence of SA alpha2,3-gal linked receptors in QT-6 and DF-1 cells which support a high growth of AIVs in these cell lines. Furthermore, the QT-6 and DF-1 cells supported high plaque-forming ability of representative highly pathogenic Eurasian H5N1 and H7N1 subtype AIVs. These two avian cell lines, especially QT-6 cells, also showed high transfection efficiency and could be useful for reverse genetics based rescue of AIVs. This study indicates that the DF-1 and QT-6 cell lines may be useful as a substitute for primary CEF and MDCK cells for AIV research in the areas of in vitro host range, molecular pathobiology and molecular genetics.

Characterization of influenza virus variants with different sizes of the non-structural (NS) genes and their potential as a live influenza vaccine in poultry.

From a stock of A/turkey/Oregon/71-delNS1 (H7N3) virus, which has a 10 nucleotide deletion in the coding region of the NS1 gene, we found that several variants with different sizes of NS genes could be produced by passaging the virus in 10- and 14-day-old embryonating chicken eggs (ECE), but not in 7-day-old ECE or Vero cells. We were able to rescue the reassortant virus that has different sizes of the NS genes and confirmed that those NS genes are genetically stable. By conducting in vivo studies in 2-week-old chickens, we found two plaque purified variants (D-del pc3 and pc4) which can be used as a potential live-attenuated vaccine. The variants were highly attenuated in chickens and did not transmit the virus from infected chickens to uninoculated cage mates. At the same time, the variants induced relatively high antibody titers which conferred good protection against a high dose heterologous virus challenge. Our study indicates that naturally selected NS1 deletion variants might be useful in the development of live-attenuated influenza vaccines in poultry. Furthermore, deletion in the NS1 protein can be potentially useful as a negative marker for a differentiating infected from vaccinated animals (DIVA) approach.

Genetic and antigenic relatedness of H3 subtype influenza A viruses isolated from avian and mammalian species.

In 2004, we isolated triple reassortant H3N2 influenza viruses from turkey breeder hens in Ohio and Illinois. The Illinois flock was vaccinated twice with an inactivated H3N2 vaccine containing a swine origin virus before the outbreak. Additionally, a commercial inactivated vaccine containing an H3N4 virus of duck origin is being used in some turkey breeders. This prompted us to initiate a comparative study on the antigenic and genetic relatedness of various H3 subtype influenza viruses isolated from turkeys, ducks, pigs and humans. The antigenic relatedness between the different viruses was evaluated with the Archetti and Horsfall formula, while nucleotide genetic similarities were calculated using pairwise alignments. Results obtained indicated a high degree of antigenic (>90%) and genetic (>99%) similarities among the turkey-origin H3N2 viruses. However, the turkey viruses were antigenically distantly related to the swine-origin vaccine virus (<30%), although they had approximately 95% genetic similarity in the HA1 gene. Additionally, major genetic and antigenic changes were observed between the turkey viruses and the H3N4 duck vaccine virus as well as the H3N2 human virus. Such genetic and antigenic differences between the turkey-origin viruses and other H3 subtype viruses including vaccine strains could be the reason for the failure in protection in the Illinois turkey breeders vaccinated with swine origin virus. This also emphasizes the importance of using viruses for vaccines that are antigenically similar to the field strains.

Pathogenicity and transmission studies of H5N2 parrot avian influenza virus of Mexican lineage in different poultry species.

In 2004, a low pathogenic H5N2 influenza virus (A/parrot/CA/6032/04) was identified in a psittacine bird for the first time in the United States. Sequence and phylogenetic analysis of the hemagglutinin gene grouped the parrot isolate under the Mexican lineage H5N2 viruses (subgroup B) with highest similarity to recent chicken-origin isolates from Guatemala. Antigenic analysis further confirmed the close relatedness of the parrot isolate to Mexican lineage viruses, the highest cross-reactivity being demonstrated to Guatemala isolates. In vivo studies of the parrot isolate in chickens, ducks and turkeys showed that the virus, though did not cause any clinical signs, could replicate to high titers in these birds and efficiently transmit to contact control cage mates. The possibility that the parrot harboring the virus was introduced into the United States as a result of illegal trade across the border provides additional concern for the movement of foreign animal diseases from neighboring countries. Considering the potential threat of the virus to domestic poultry, efforts should be continued to prevent the entry and spread of influenza viruses by imposing effective surveillance and monitoring measures.

Age at infection affects the pathogenicity of Asian highly pathogenic avian influenza H5N1 viruses in ducks.

The Asian highly pathogenic avian influenza (HPAI) H5N1 viruses have changed from producing no disease or mild respiratory infections in ducks to some strains causing systemic disease and death. Differences in pathogenicity between four of these viruses as well as the effect of host age on the outcome of infection were studied in ducks. Three of the viruses were highly lethal in 2-week-old ducks and induced severe neurological dysfunction. Neurological signs were also observed in 5-week-old ducks inoculated with one of these viruses; however mortality was low. The fourth virus studied did not induce neurological signs in 2-week-old ducks, but did produce moderate mortality. This virus caused no clinical signs or death in 5-week-old ducks. All viruses studied were isolated from oropharyngeal and cloacal swabs, and also from brain, heart, lung and muscle tissues, demonstrating systemic infection. All viruses evaluated transmitted efficiently to contact ducks. Phylogenetic analysis of the viruses studied and other Asian H5N1 HPAI viruses with diverse pathogenicity in ducks, showed changes in several genes, but none clearly associated with pathogenicity. In conclusion, the pathogenicity of circulating H5N1 HPAI viruses in ducks varies depending on the virus strain and the age of the duck and correlates with the level of viral replication in tissues. High titers of virus in organs, high viral shedding, and variable mortality enable ducks to circulate H5N1 HPAI viruses.