Novel Gyrovirus genomes recovered from free-living pigeons in Southern Brazil.

Wild birds carry a number of infectious agents, some of which may have pathogenic potential for the host and others species, including humans. Domestic pigeons (Columba livia) are important targets of study since these increasingly cohabit urban spaces, being possible spillover sources of pathogens to humans. In the present study, two genomes (PiGyV_Tq/RS/Br and PiGyV_RG/RS/Br), representative of Gyrovirus genus, family Anelloviridae, were detected in sera of free-living pigeons collected in Southern Brazil. The genomes exhibit less than 50% identity to previously described members of Gyrovirus genus, suggesting that they constitute a new viral species circulating in pigeons, to which the name "pigeon gyrovirus (PiGyV)" is proposed. The current study characterizes these two PiGyV genomes which, to date, are the first gyrovirus species identified in domestic pigeons.

Isolation and genetic characterization of virulent strains of avian paramyxovirus-1 from multiple avian species in Azad Jammu and Kashmir 2017-2018.

Despite intensive vaccination, endemicity of Avian paramyxoviruses-1 (APMV-1) is a significant problem in developing countries in Africa, Middle East, and Asia. Given the importance of APMV-1 in poultry and multiple non-poultry avian species, it is important to continue surveillance programs, routine monitoring and characterization of field isolates in the region where viruses are endemic. The purpose of this study was to pathotyped and genetically characterized 21 APMV-1s isolated from multiple avian species reared in different regions of Azad Jammu and Kashmir (AJK). Phylogenetic analysis based on complete fusion (F) gene sequences showed that 17 APMV-1 isolates obtained from commercial poultry and backyard birds belonged to sub-genotype VIIi. Though, one pigeon-origin APMV-1 isolate was clustered in sub-genotype VIg and three in recently designated new sub-genotype VIm of genotype VI. The pigeon-origin isolates had the following two motifs 113-RKKR↓F-117 and 113-RQRR↓F-117, while all other isolates had the polybasic amino acid sequence 113-RQKR↓F-117 at the F-cleavage site, which is characteristic of virulent APMV-1 strains. These results are consistent with the five viruses that had intracerebral pathogenicity indices (ICPIs) of between 1.50 and 1.73, corresponding to a velogenic pathotype. The APMV-1s isolated from commercial poultry and backyard birds in this study showed low nucleotide distance (0.3-0.9%) and genetically closely related (> 97%) to viruses repeatedly isolated (2011-2017) from multiple avian species in other states of Pakistan. Strengthened surveillance programs in both commercial poultry and backyard flocks are needed to better assess the commercial-backyard bird interface and form a basis for evidence-based measures to limit and prevent APMV-1 transmission.

Virulent Newcastle disease viruses from chicken origin are more pathogenic and transmissible to chickens than viruses normally maintained in wild birds.

Five, class II, virulent Newcastle disease virus (vNDV) isolates of different genotypes from different host species were evaluated for their ability to infect, cause disease, and transmit to naïve chickens. Groups of five birds received a low, medium, or high dose, by the oculonasal route, of one of the following vNDV: three chicken-origin, one cormorant-origin, and one pigeon-origin. Three naïve birds were added to each group at two days post-inoculation (DPI) to evaluate transmission. Virus shedding was quantified from swabs (2/4/7 DPI), and seroconversion was evaluated at 14 DPI. All inoculated and contact birds in the chicken-origin vNDV groups succumbed to infection, displaying clinical signs typical of Newcastle disease and shed virus titers above 6 log10 EID50/ml. Birds receiving a high and medium dose of the cormorant virus showed primarily neurological clinical signs with 80% and 60% mortality, respectively. The chickens showing clinical disease shed virus at titers below 4 log10 EID50/ml, and the remaining bird in the high dose group seroconverted with a high HI titer. For the pigeon-origin virus, no clinical signs were observed in any of the birds, but all 5 chickens in the high challenge dose and one bird in the medium challenge group shed virus at mean titers of 3.1 and 2.2 log10 EID50/ml, respectively. Overall, the chicken-origin viruses infected chickens and efficiently transmitted to naïve birds, while the cormorant- and pigeon-origin viruses infected chickens only at the higher doses and did not transmit to other birds.

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.

Columbid circoviruses detected in free ranging pigeons from Southern Brazil: insights on PiCV evolution.

Pigeon circovirus (PiCV) is taxonomically classified as a member of the Circovirus genus, family Circoviridae. The virus contains a single stranded DNA genome of approximately 2 kb, with minor length variations among different isolates. The occurrence of PiCV infections in pigeons (Columba livia) has been documented worldwide over the past 20 years; however, in Brazil there were still no reports on PiCV detection. This study identifies seven PiCV genomes recovered from domestic pigeons of South Brazil through high-throughput sequencing and shows a high frequency of PiCV infection, through quantitative real-time PCR. Phylogenetic classification was performed by maximum likelihood analysis of the full genomes, ORF V1 (Rep) and ORF C1 (Cap). The results show that either full genome or Cap based analysis allowed PiCV classification into five major clades (groups A to E), where Brazilian sequences were classified as A, C or D. Recombination analyses were carried out with Simplot and RDP4 and the results show that both Rep and Cap ORFs contain several recombination hotspots, pointing to an important role for such events in PiCV evolution.

Evaluation of Argentinean Bird Species as Amplifying Hosts for St. Louis Encephalitis Virus (Flavivirus, Flaviviridae).

St.Louis encephalitis virus (SLEV) is an emerging human pathogen flavivirus in Argentina. Recently, it has reemerged in the United States. We evaluated the role as amplifying host of six resident bird species and analyzed their capacity as host during the 2005 encephalitis outbreak of SLEV in Córdoba. Eared Dove, Picui Ground Dove, and House Sparrow were the three species with highest host competence index. At a city level, Eared Dove and Picui Ground Dove were the most important amplifying hosts during the 2005 SLEV human outbreak in Córdoba city. This finding highlighted important differences in the SLEV ecology between Argentina and the United States. Characterizing and evaluating the SLEV hosts contribute to our knowledge about its ecology and could help us to understand the causes that promote its emergence as a human pathogen in South America.

Avian coronavirus isolated from a pigeon sample induced clinical disease, tracheal ciliostasis, and a high humoral response in day-old chicks.

The detection of avian coronaviruses (AvCoV) in wild birds and the emergence of new AvCoV have increased in the past few years. In the present study, the pathogenicity of three AvCoV isolates was investigated in day-old chicks. One AvCoV isolated from a pigeon, which clustered with the Massachusetts vaccine serotype, and two AvCoV isolated from chickens, which grouped with a Brazilian genotype lineage, were used. Clinical signs, gross lesions, histopathological changes, ciliary activity, viral RNA detection, and serology were evaluated during 42 days post infection. All AvCoV isolates induced clinical signs, gross lesions in the trachea, moderate histopathological changes in the respiratory tract, and mild changes in other tissues. AvCoV isolated from the pigeon sample caused complete tracheal ciliostasis over a longer time span. Specific viral RNA was detected in all tissues, but the highest RNA loads were detected in the digestive tract (cloacal swabs and ileum). The highest antibody levels were also detected in the group infected with an isolate from the pigeon. These results confirm the pathogenicity of Brazilian variants, which can cause disease and induce gross lesions and histopathological changes in chickens. Our results suggest that non-Galliformes birds can also play a role in the ecology of AvCoV.

Phylogenetic and pathotypic characterization of a Newcastle disease virus strain isolated from ducks and pigeons in Hubei, China

Newcastle disease is a highly contagious disease responsible for major outbreaks and considerable economic losses in the poultry industry in China. There is still little information available regarding gene characterization of the NDV, especially in ducks and pigeons. Therefore, the aim of this study was to investigate NDV isolated from ducks and pigeons in Hubei, China. In this study, three NDVs from ducks and pigeons were isolated between 2013 and 2015.The fusion protein (F) gene of the NDV isolates was sequenced and phylogenetically analyzed. The clinical signs and gross histopathological lesions were examined. Phylogenetic analysis of these strains indicated that all the sequences are classified as genotype II. The isolates shared a 112 G-R-Q-G-R-L 117motif at the F protein cleavage site, indicating that these three isolates strains are lentogenic. Necropsy and histopathology showed the typical pathological changes. It was concluded that commercial ducks and pigeons in Hubei province carry lentogenic NDV strains with regular genetic divergence, indicating that these species may act as the main reservoirs of NDV in poultry. Therefore, strategies and surveillance should be undertaken to reduce the risk of ND outbreaks.

Phylogenetic and pathotypic characterization of a Newcastle disease virus strain isolated from ducks and pigeons in Hubei, China

Newcastle disease is a highly contagious disease responsible for major outbreaks and considerable economic losses in the poultry industry in China. There is still little information available regarding gene characterization of the NDV, especially in ducks and pigeons. Therefore, the aim of this study was to investigate NDV isolated from ducks and pigeons in Hubei, China. In this study, three NDVs from ducks and pigeons were isolated between 2013 and 2015.The fusion protein (F) gene of the NDV isolates was sequenced and phylogenetically analyzed. The clinical signs and gross histopathological lesions were examined. Phylogenetic analysis of these strains indicated that all the sequences are classified as genotype II. The isolates shared a 112 G-R-Q-G-R-L 117motif at the F protein cleavage site, indicating that these three isolates strains are lentogenic. Necropsy and histopathology showed the typical pathological changes. It was concluded that commercial ducks and pigeons in Hubei province carry lentogenic NDV strains with regular genetic divergence, indicating that these species may act as the main reservoirs of NDV in poultry. Therefore, strategies and surveillance should be undertaken to reduce the risk of ND outbreaks.(AU)

Clinical and serological tests for arboviruses in free-living domestic pigeons (Columba livia).

BACKGROUND: In this study, we evaluated the role of free-living domestic pigeons (Columba livia) as a reservoir of arboviruses in the city of Belém, state of Pará, Brazil. We investigated the presence of antibodies against the most prevalent arboviruses. OBJECTIVES: This study was aimed at evaluating some clinical and physical parameters of domestic pigeons, including the presence of antibodies to Amazon-endemic arboviruses. METHODS: Eighty-five healthy pigeons were captured in Mangal das Garças Park, in Belém, and were bled. Upon capture, the birds were subjected to a clinical examination in search of alterations that could indicate the presence of arboviruses. Blood samples were converted to serum and tested using the haemagglutination inhibition (HI) technique with a panel of 19 antigens of arboviruses circulating in the Amazon. The confirmation assay for the positive reactions to the viral species tested by HI was a neutralisation test in new-born Swiss albino mice (Mus musculus) [mouse neutralisation test (MNT)]. FINDINGS: A total of 10 (11.8%) serum samples tested positive for antiflavivirus antibodies by HI. All the samples positive for the HI test were subjected to MNT for detection of viruses and yielded negative results (logarithmic neutralisation index < 1.7). MAIN CONCLUSION: The results represent the first serological detection of antiarbovirus antibodies in domestic pigeons as potential hosts of arboviruses in Brazil. The detection of haemagglutination-inhibiting antibodies against genus Flavivirus indicated that there was recent contact between the analysed domestic pigeons and these arboviruses. Further studies are needed to evaluate the role of free-living pigeons in the maintenance cycle and spread of arboviruses in the Amazon.

Clinical and serological tests for arboviruses in free-living domestic pigeons (Columba livia)

BACKGROUND In this study, we evaluated the role of free-living domestic pigeons (Columba livia) as a reservoir of arboviruses in the city of Belém, state of Pará, Brazil. We investigated the presence of antibodies against the most prevalent arboviruses. OBJECTIVES This study was aimed at evaluating some clinical and physical parameters of domestic pigeons, including the presence of antibodies to Amazon-endemic arboviruses. METHODS Eighty-five healthy pigeons were captured in Mangal das Garças Park, in Belém, and were bled. Upon capture, the birds were subjected to a clinical examination in search of alterations that could indicate the presence of arboviruses. Blood samples were converted to serum and tested using the haemagglutination inhibition (HI) technique with a panel of 19 antigens of arboviruses circulating in the Amazon. The confirmation assay for the positive reactions to the viral species tested by HI was a neutralisation test in new-born Swiss albino mice (Mus musculus) [mouse neutralisation test (MNT)]. FINDINGS A total of 10 (11.8%) serum samples tested positive for antiflavivirus antibodies by HI. All the samples positive for the HI test were subjected to MNT for detection of viruses and yielded negative results (logarithmic neutralisation index < 1.7). MAIN CONCLUSION The results represent the first serological detection of antiarbovirus antibodies in domestic pigeons as potential hosts of arboviruses in Brazil. The detection of haemagglutination-inhibiting antibodies against genus Flavivirus indicated that there was recent contact between the analysed domestic pigeons and these arboviruses. Further studies are needed to evaluate the role of free-living pigeons in the maintenance cycle and spread of arboviruses in the Amazon.

Detección molecular de virus de encefalitis de Saint Louis en mosquitos de Buenos Aires, Argentina

Durante el mes de marzo de 2013 una población de palomas torcazas (Zenaida auriculata) se instaló en una zona céntrica de la ciudad de Buenos Aires. Conociendo el rol que poseen estas aves como hospedadores competentes del virus de la encefalitis de Saint Louis (SLEV), fue colocada en el lugar una trampa de luz tipo CDC, a fin de realizar una vigilancia entomológica. Durante ese mes,fueron capturados 5 grupos de mosquitos (n = 48), 3 correspondieron a la especie Culex pipiens (n = 10) y 2 a Culex spp.(n = 38), no pudiéndose determinar en estos últimos con precisión la especie por encontrarse dañados. En un grupo de mosquitos Culex spp. se detectó el SLEV por técnicas moleculares. Posteriormente fue secuenciado y clasificado como perteneciente al genotipo III.

During March 2013 a population of eared doves (Zenaida auriculata) was established in the center of City of Buenos Aires. Considering the role of these birds as host competent for Saint Louis encephalitis virus (SLEV), a CDC light trap was put in place to perform entomologic surveillance. During this month 5 pools of mosquitoes (n = 48) were collected and taxonomically determined. Three of them were classified as Culex pipiens (n = 10) and the other two were Culex spp. (n = 38). In this case, the mosquitoes species could not be determined due to that individuals were damaged. One of the Culex spp. pool was found to be positive for Saint Louis encephalitis virus by molecular techniques. This was then sequenced and classified as genotype III.

[Molecular detection of Saint Louis encephalitis virus in mosquitoes in Buenos Aires]. / Detección molecular de virus de encefalitis de Saint Louis en mosquitos de Buenos Aires, Argentina.

During March 2013 a population of eared doves (Zenaida auriculata) was established in the center of City of Buenos Aires. Considering the role of these birds as host competent for Saint Louis encephalitis virus (SLEV), a CDC light trap was put in place to perform entomologic surveillance. During this month 5 pools of mosquitoes (n = 48) were collected and taxonomically determined. Three of them were classified as Culex pipiens (n = 10) and the other two were Culex spp. (n = 38). In this case, the mosquitoes species could not be determined due to that individuals were damaged. One of the Culex spp. pool was found to be positive for Saint Louis encephalitis virus by molecular techniques. This was then sequenced and classified as genotype III.

A liquid-phase-blocking concanavalin A enzyme-linked immunosorbent assay for the detection of antibodies against Newcastle disease virus in serum of free-ranging pigeons.

A competitive liquid-phase-blocking concanavalin A enzyme-linked immunosorbent assay (LPB-ConA-ELISA) was developed in the current study. The assay used ConA as a capture reagent, and the sera of specific pathogen-free chickens immunized with nonpurified Newcastle disease virus (NDV) suspension as detector antibodies, to detect and quantify specific antiviral antibodies in serum samples from free-ranging pigeons. The comparison between the LPB-ConA-ELISA and the hemagglutination inhibition (HI) test for the detection of antibodies in serum samples from 107 pigeons showed significant correlation between the assays (r = 0.875), a high sensitivity (100%), specificity (95.8%), accuracy (96.3%) for the ELISA, and good agreement (κ = 0.83) between the 2 assays. The results of this study suggest that the LPB-ConA-ELISA could be a useful alternative to HI test in the serodiagnosis of NDV in pigeons, or other species of birds.

Detection of and phylogenetic studies with avian metapneumovirus recovered from feral pigeons and wild birds in Brazil.

The aim of the present study was to determine whether avian metapneumovirus (aMPV)-related viruses were present in wild and synanthropic birds in Brazil. Therefore, we analysed samples from wild birds, feral pigeons and domestic chickens in order to perform a phylogenetic comparison. To detect the presence of aMPV, a nested reverse transcriptase-polymerase chain reaction was performed with the aim of amplifying a fragment of 270 bases for subtype A and 330 bases for subtype B, comprising the gene coding the G glycoprotein. Positive samples for aMPV subtypes A and B were found in seven (13.2%) different asymptomatic wild birds and pigeons (50%) that had been received at the Bosque dos Jequitibás Zoo Triage Center, Brazil. Also analysed were positive samples from 15 (12.9%) domestic chickens with swollen head syndrome from several regions of Brazil. The positive samples from wild birds, pigeons and domestic chickens clustered in two major phylogenetic groups: some with aMPV subtype A and others with subtype B. The similarity of the G fragment nucleotide sequence of aMPV isolated from chickens and synanthropic and wild avian species ranged from 100 to 97.5% (from 100 to 92.5% for the amino acids). Some positive aMPV samples, which were obtained from wild birds classified in the Orders Psittaciformes, Anseriformes and Craciformes, clustered with subtype A, and others from the Anas and Dendrocygma genera (Anseriformes Order) with subtype B. The understanding of the epizootiology of aMPV is very important, especially if this involves the participation of non-domestic bird species, which would add complexity to their control on farms and to implementation of vaccination programmes for aMPV.

Characterization of Newcastle disease viruses isolated from chicken, gamefowl, pigeon and quail in Mexico.

Velogenic Newcastle disease has threatened the Mexican poultry industry since 1946. Seven strains of velogenic Newcastle disease virus were isolated from poultry and other avian species in central and northern Mexico from 1998 to 2006 and subjected to phylogenetic analysis and biological characterization using standard pathogenicity tests and challenge studies. Phylogenetic analysis showed that all velogenic strains belonged to genetic group V and are clearly divided in two lineages, since phylogenetic similarities between groups are of only 93-94%. Isolates from 1998 to 2001 are closely related to the strain responsible for the 2000 year outbreak raised in La Laguna region (Torreon strain), and are phylogenetically distinct from viruses isolated between 2004 and 2006 that are genetically related to the Chimalhuacan strain isolated in 1973. All the viruses of both, the Chimalhuacan and the Torreon groups, contained a virulent fusion protein cleavage site represented by the motif "GGRRQKRF", revealing that evolutionary changes occurred at a different site. Chicken embryo mean death time value was shorter for the Chimalhuacan-like viruses (43.9 hours), when compared with the 1998-2001 average (54.3 hours). ICPI average value was higher (1.92) for viruses isolated during 2004-2006 than that for viruses isolated before 2001 (1.74). Microscopic evaluation of bursa of Fabricius and thymus of 5w-o broiler chickens challenged with 106 LD50/0.2 ml showed that Chimalhuacan-like isolate caused more severe lesions at 48 hpi in bursa and 72 and 96 hpi in thymus than Torreon-like isolate. Along with the MDT, ICPI and microscopic results, our findings suggest that some distinct selective pressure on the very virulent Chimalhuacan strain isolated in early 1970's may have led to the appearance of the still velogenic but less virulent new group (Torreon-like) in the middle of 1990's.

Eared dove (Zenaida auriculata, Columbidae) as host for St. Louis encephalitis virus (Flaviviridae, Flavivirus).

St. Louis encephalitis virus (SLEV) is an emerging Flavivirus in South American countries. Its ecology and biological transmission cycles are scarcely known. Eared doves (Zenaida auriculata) have frequently been found infected by SLEV, and therefore, could be suspected as SLEV hosts. Thirty post-hatch-year eared doves were subcutaneously inoculated with the genotype V SLEV 78V-6507 viral strain and subsequently bled. No deaths or clinical signs of illness were observed in the inoculated doves. The viremia titers ranged from 2 to 5.5 log(10) plaque-forming units (PFU)/mL during 1-7 days postinoculation (dpi), the highest being observed on the 4th dpi. Mosquitoes were collected using can traps baited with chicken and eared doves for comparison. A total of 2792 mosquitoes belonging to 5 species were collected. Ninety percent of the mosquitoes collected in eared dove-baited can traps were Culex quinquefasciatus. Statistical differences were not observed in either Cx. quinquefasciatus (Chi(2) = 0.86; df = 1; p = 0.354) or in Cx. interfor (Chi(2) = 0.63; df = 1; p = 0.426) mosquitoes collected in both chicken- and eared dove-baited can traps. Considering that eared doves were frequently found naturally infected by SLEV, that they developed viremia titers higher than the minimum infection threshold needed to infect Cx. quinquefasciatus, and that these mosquitoes also fed on eared doves, they could be considered competent hosts for SLEV.

Matrix protein gene sequence analysis of avian paramyxovirus 1 isolates obtained from pigeons.

The matrix protein gene was cloned and sequenced for several recent isolates of avian paramyxovirus type 1 (APMV-1). Specifically, isolates from pigeons and doves, members of the Columbidae family were examined. APMV-1 is the causative agent of Newcastle disease and the virus is associated with disease among a diverse number of avian species. Newcastle disease virus (NDV) isolates from pigeons have also been classified as pigeon paramyxovirus type 1 (PPMV-1). Matrix protein gene sequences for PPMV-1 isolates clustered together as a group relative to isolates from other species phylogenetically. However, there were also isolates from pigeons or doves that grouped with APMV-1 isolates from other species. This indicates that PPMV-1 may be circulating among Columbidae members as a distinct lineage, but that these avian species may also harbor other NDV strains as well. Of particular interest was a dove isolate from Europe that had an aberrant fusion protein cleavage site and was an outlying member phylogenetically between the two major groups of APMV-1 isolates.

Biological and molecular characterization of a pigeon paramyxovirus type-1 isolate found in Argentina.

In this report, we describe the biological and molecular characterization of a paramyxovirus type-1 (PPMV-1) isolate found in wild pigeons in an urban habitat in Buenos Aires, Argentina. Of the nine pigeons captured, three were moribund, and the other six showed diarrhea, ataxia, tremor, torticolis, and wing paralysis. The intracerebral pathogenicity index was 1.29, and the amino acid (aa) sequence at the fusion protein cleavage site was 112GRQ KRF117. These characteristics correspond to a virulent Newcastle disease virus isolate. Nevertheless, it was not possible to reproduce the disease in chickens experimentally although the chickens exhibited seroconversion after inoculation. On the other hand, pigeons inoculated with the isolate became sick. These results provide further evidence about the unusual pathogenicity of PPMV-1 for chickens and show once more the need for more biological determinations in these cases to arrive at a final conclusion.