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Prevalence of Aves Polyomavirus 1 and Beak and Feather Disease Virus From Exotic Captive Psittacine Birds in Chile.

Abstract: Avian polyomavirus disease and psittacine beak and feather disease (PBFD) are both contagious viral diseases in psittacine birds with similar clinical manifestations and characterized by abnormal feathers. To determine the prevalence of Aves polyomavirus 1 (APyV) and beak and feather disease virus (BFDV) in captive, exotic psittacine birds in Chile, feathers from 250 psittacine birds, representing 17 genera, were collected and stored during the period 2013-2016. Polymerase chain reaction testing was used to detect APyV and BFDV were detected in feather bulb samples. The results indicated that 1.6% (4/250) of the samples were positive for APyV, 23.2% (58/250) were positive to BFDV, and 0.8% (2/250) were positive to both APyV and BFDV. This is the first report, to our knowledge, of APyV and BFDV prevalence in captive, exotic psittacine birds in South America. Analysis of 2 Chilean partial sequences of the gene encoding agnoprotein la (APyV) and the replication-associated protein (BFDV) extends the knowledge of genomic variability for both APyV and BFDV isolates and their spectrum of hosts. No geographical marker was detected for the local isolates.

Key words: polyomavirus 1, beak and feather disease virus, polymerase chain reaction, avian, Psittaciformes, psittacine birds


Avian polyomavirus and beak and feather disease virus (BFDV) are the most common viral diseases in psittacine birds, affecting both the feathers and physical appearance. (1) Moreover, these viruses can also cause serious and often fatal disease in psittacine birds. (2)

Aves polyomavirus 1 (APyV), formerly known as budgerigar fledgling disease virus, is a double-stranded DNA virus and a member of the family Polyomaviridae (genus Gammapolyomcivirus). (3) The viral particle is icosahedral, nonenveloped, and 4050 nm in size. (4) The viral genome (approximately 5 kb) is organized in 3 regions: early coding, late coding, and noncoding. Large and small T-antigen open reading frames (ORFs) are included in the early coding region. The late coding region includes 2 ORFs: 1 encodes the viral capsid proteins (VPs) VP1, VP2, and VP3 and structural proteins, (5) whereas the other encodes for the designated VP4, also referred to as agnoprotein la. Its location corresponds to that of the agnoprotein-coding region in mammalian polyomavirus genomes. (6) The protein encoded by this avian polyomavirus ORF has no apparent sequence homology to simian virus 40 VP4 or the mammalian polyomavirus agnoprotein. Viral capsid protein 4 of avian polyomavirus is a component of the mature virion, which is thought to be involved in packaging the viral genome and inducing apoptosis in cell cultures. (7-9) Mutants of APyV that lack VP4 exhibit reduced virulence, suggesting that VP4 may also contribute to acute pathogenesis. (10)

Budgerigar fledgling disease virus, the etiologic agent of psittacine beak and feather disease (PBFD), is a member of the family Circoviridae. It is one of the smallest viruses known to exist based on physicochemical and genetic characteristics. The BFDV has a relatively simple, but compact, circular, single-stranded DNA genome of approximately 2000 nucleotides. The DNA of the BFDV genome has 2 major ORFs, which encode a replication-associated protein (rep) and a capsid protein, respectively. (11-13) Similar to other single-stranded DNA viruses, BFDV is prone to a high rate of genetic mutation, although the rep gene is relatively conserved. This facilitates diagnosis of the infection by polymerase chain reaction (PCR) testing. (14)

As shown in previous studies, the feather bulb is an appropriate sample for molecular detection of BFDV. (1,15,16) Usually the predilection sites of BFDV are tissues isolated from stratum germinativum at the feather follicle and epidermal tissue. (17) The feather follicle is also used for APyV detection because it has been described as the main location of the virus. (1,16,18)

Psittacine birds are among the most popular companion birds in Chile. Psittacine birds are bred predominantly in domestic farms, but some are imported from other countries. Reportedly, 1519 psittacine birds were imported between June 2010 and May 2014 (Servicio Agricola y Ganadero, Chile, written communication; July 2, 2014). However, the numbers of psittacine birds that live in captivity in households in Chile has not been established.

The percentage of APyV viral genomes detected in psittacine birds from different countries ranges from 0.8% in Italy to 15.2% in Taiwan. (19-21) For BFDV, these percentages range from 3.5% in the United States to 41.2% in Taiwan and 64.5% in Thailand. (16,21,22) At present, BFDV has been identified as the most important viral infection of psittacine birds in Japan and other countries. (2) Recently, BFDV was detected at a high prevalence (56.2%) in psittacine birds in Australia; most of these cases had no observed clinical signs of PBFD. In non-psittacine birds that were tested, the prevalence of BFDV was 20%. (23)

No previous studies, to our knowledge, have reported the prevalence of APyV or BFDV in endemic or introduced psittacine species in South American countries. Recently, the first case of concurrent psittacine BFDV and APyV infection in a psittacine bird in captivity in Chile, with fatal consequence, was described. (24) The purpose of this investigation was to determine the prevalence, by PCR testing, of APyV and BFDV infection in imported and domestically bred psittacine birds in Chile. The data obtained from several viral sequences of infected birds were used to group the polyomaviruses and circovirus according to their phylogeny.


Total DNA was obtained from feathers, as was performed in previous epidemiologic studies. (15,16) To determine the prevalence of APyV and BFDV, the formula described by Wayne (25) was applied. A prevalence of 20% was expected, considering a 5% sampling error and a 95% confidence level. (19,21,26) A sample size of 246 feathers was required. However, a total of 250 feather samples from psittacine birds (from 21 avian breeders in different regions of the country) were collected between 2013 and 2016 and analyzed in the present study. Feather samples were categorized according to normal or abnormal appearance (retention of the feather sheaths, hemorrhage within the pulp cavity, fractures of the proximal rachis, midshaft constrictions, and failure of the developing feather to exsheath). (27) The association of feather appearance (normal or abnormal) with the presence or absence of infection with APyV and BFDV was determined by the Fisher exact test or [chi square] test with an a level of .05.

Viral DNA extraction from the feather calamus was performed by using the APyV kit (Bioingentech, Conception, Chile). Purification of DNA from samples was conducted in accordance with the protocol described by the manufacturer. The DNA extracted from feathers was dissolved in 13 (iL of 10 mM Tris(hydroxymethyl)aminomethane, ImM ethylenediaminetetraacetic acid and stored at --20[degrees]C until use. A TC 4000 thermocycler (Techne, Burlington, NJ, USA) was used for initial denaturation at 94[degrees]C for 3 minutes, then 30 cycles with 30 seconds for denaturation at 94[degrees]C, 30 seconds annealing at 57[degrees]C, and 30 seconds extension at 72[degrees]C, followed by a final extension step of 5 minutes at 72[degrees]C.

The PCR reaction was based on the specific primers of the VP4 nucleotide sequence in the APyV kit (Bioingentech), which resulted in an amplification product of nearly 500 base pair (bp). The presence of BFDV infection was also detected with the BFDV kit (Bioingentech), which is based on the specific primers for the rep protein gene. The temperature conditions were 94[degrees]C for 3 minutes, 30 cycles of 94[degrees]C for 30 seconds, 57[degrees]C for 30 seconds, and 72[degrees]C for 30 seconds; the final extension was performed at 72[degrees]C for 5 minutes. The expected length of the specific, amplified fragment for BFDV and internal control were 395 bp and 140 bp, respectively. The PCR products obtained were subjected to 1% agarose gel electrophoresis. Positive, negative, and internal controls for both kits were used to validate the procedures. The DNA samples of 1 randomly chosen APV- and BFDV-positive sample were amplified, and the PCR products were sequenced. The PCR products obtained were purified with the Kit Wizard SV gel (Promega, Madison, WI, USA) and the PCR cleanup system (Promega). Finally, purified PCR products from both APyV- and BFDV-positive samples were subjected to sequence analysis to verify adequate amplification. The PCR products were automatically sequenced in both directions with both primers (Pontifical Catholic University of Chile, Santiago, Chile). Sequencing was done on an ABI PRISM 3130 (Applied Biosystems, Foster City, CA, USA).

For bioinformatics analysis of the sequences and alignments, the Basic Local Alignment Search Tool (BLAST, National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) and the Clustal W online platform (NCBI) were used and matched with the GenBank (NCBI) database (accession numbers of partial sequences: KY696654 and KY69 6 6 56). (28) Two partial sequences from the BFDV replication protein (rep) gene were obtained from Chilean blue and yellow macaw (Ara ararauna) and plum-headed parakeet (Psittacula cyanocephala) samples (accession numbers: KY696656 and KU500899, respectively). In addition, 2 partial sequences from the psittacine bird APyV agnoprotein la gene were obtained from Chilean rose-ringed parakeet (Psittacula krameri) and plum-headed parakeet samples (accession numbers: KY696654 and KU500898, respectively). Both sequences KU500899 and KU500898 were obtained from the first fatal case reported in Chile from a psittacine bird with concurrent BFDV and APyV infection. (24) These sequences were compared with previously reported BFDV and APyV sequences available in GenBank.

For BFDV comparison, an area of 303 nucleotides within the rep gene was chosen. For APyV comparison, the area of 324 nucleotides from the agnoprotein la (Agnola) gene was evaluated. Sequences were pairwise compared, and multiple alignments were performed with Clustal W. (28) Identity percentages were obtained, and phylogenetic trees were constructed with Molecular Evolutionary Genetics Analysis freeware version 7.0 (MEGA7; (29) Phylogenetic trees were constructed using the neighbor-joining method and the Tajima and Nei model, with bootstrapping of 1000 replications. (30-32) Neighbor-joining trees were construct ed with Chilean psittacine bird sequences and with international psittacine bird sequences from different countries and hosts for both BFDV and APyV. From South America, the Brazilian BFDV sequence (accession number: JQ649410) from blue-fronted parrot (Amazona aestiva) was also included. (33)


The 250 feather samples were collected from, and were represented in, 17 genera of psittacine birds from 2 families. A total of 4 APyV-positive birds (1.6%) were identified in 2 genera (Table 1). The BFDV was detected more frequently. A total of 58 BFDV-positive birds (23.2%) were found in 13 genera. Only 0.8% (2/250) of the birds (1 being of the Psittacula genus and another of the Trichoglossus genus) were positive for both APyV and BFDV.

Twenty-six of 250 feather samples (10.4%) were categorized as abnormal. Fourteen of 26 abnormal feathers were BFDV positive, and 3 of 26 abnormal feathers (11.5%) were APyV-positive (Table 2). The genera of psittacine birds and the feather appearance in APyV-positive and BFDV-positive birds appear in the descriptive section of Table 2. Of the 250 feathers, 224 (89.6%) were categorized as normal. Twelve of 192 negative BFDV samples (6.3%) had abnormal feathers, and 14 of 58 BFDV-positive samples (24.1%) had abnormal feathers (Table 2). In the case of APyV, 3 of 4 APyV-positive birds (75%) had abnormal feathers, and 23 of 246 APyV-negative samples (9.3%) had abnormal feathers (Table 2). The appearance of feathers differs in the presence of infection by BFDV and APyV infection ([chi square] = 15.3; P < .05, respectively).

Sequence analysis of APyV showed that Chilean sequences from rose-ringed parakeet and plum-headed parakeet were very similar and shared 99.7% identity between them. The identity percentages among the other sequences included in the analysis were high and ranged from 98% to 99.7%. The highest identity between Chilean sequences and internationally reported sequences was 99.7%, and it was found with several sequences with different geographical and host origins.

Sequence analysis of BFDV revealed that Chilean sequences from blue and yellow macaw and plum-headed parakeet share 97% identity between them (data not shown). The comparison with other available BFDV sequences showed identity percentages ranging from 84% to 97.3% and from 85% to 100% for the blue and yellow macaw and plum-headed parakeet sequences, respectively (data not shown). The sequence from plum-headed parakeet shared the greatest identity with 2 Australian sequences from different hosts, grouped as genotype K, according to the Julian et al classification. (34) However, the blue-and-yellow macaw sequence shared the greatest identity percentage (100%) with an Italian sequence with no previous classification, which was isolated from an African grey parrot (Psiltacus erithacus; data not shown) and with 2 Polish sequences belonging to genotype J (99.7%) isolated from Australian king parrot (Alisterus scapular is) and Sengal parrot (Poicephalus senegalus).

The phylogenetic analysis for APyV was not entirely clear because bootstrapping at branches was low (<50%), and the sequences were very similar. Despite that, Chilean APyV sequences were maintained together in a branch near several sequences from Japan, Germany, and other hosts (Fig 1).

The phylogenetic tree for BFDV was in agreement with the identity percentages and showed a grouping of the Chilean blue and yellow macaw sequence together with Polish, United Kingdom, Turkish, and Italian sequences proposed as subgroup J1 within genotype J. (34) In addition, on another branch, the Chilean plum-headed parakeet sequence, was located near 2 Australian sequences typified as genotype K (Fig 2).


Infection with APyV has been observed in psittacine birds in many countries, including Canada, Australia, Germany, the United States, Switzerland, Slovakia, Italy, Taiwan, China, Thailand, Costa Rica, and Turkey. (1-16,19,21,35-42) Very few reports exist concerning APyV infections in psittacine birds in South America. The first known analyses of APyV infections by antibody detection of specimens from South America were performed in the United States in sun conures (Aratinga solstitialis) from Guyana that were exposed to birds from other areas. In addition, wild dusky-headed parakeets (Aratinga weddellii) from Peru were analyzed by serosurveys (complement fixation and virus neutralization). (43) In Chile, in serum neutralization tests, 36 of 100 psittacine birds (36%) in captivity showed antibodies against ApyV. (44) Also in Chile, DNA for APyV was also detected by PCR testing in a dead Pionus species parrot. (45) More recently, a fatal case of coinfection with APyV and BFDV was described in a 6-week-old plum-headed parakeet from a private bird collection in the metropolitan region of Chile. The bird died after presenting with depression, ataxia, tremors of the head, subcutaneous hemorrhage, and delayed crop emptying. (24)

Infection with BFDV is found in many countries, including Indonesia and the Philippines, the United Kingdom, New Zealand, Germany, the United States, South Africa, Italy, Australia, Taiwan, Thailand, Costa Rica, Poland, Turkey, and Saudi Arabia. (1-16,21,22,19,42,46-52) In South America, viral nucleic acid of BFDV was detected by DNA in situ hybridization in Brazil, in a white cockatoo (Cacatua alba). (53) The prevalence of psittacine bird BFDV was also investigated in Brazilian native parrots with normal feathering arriving at rescue and triage centers for wild animals. The occurrence of BFDV detected by PCR was 6.3%. (33) Other researchers identified the infection by real-time PCR in 11 of 20 psittacine birds (55%) in a study conducted in Argentina. (54)

In this study, we report for the first time, to our knowledge, the prevalence of APyV (1.6%) and BFDV (23.2%) in psittacine birds in captivity in Chile, as detected by PCR. However, a 36% seropositivity for APyV in captive psittacine birds was previously reported in Chile (44) These differences between prevalence and seropositivity may be a result of the different techniques used or because of the higher probability of finding antibodies, rather antigens, in a population. The prevalence determined in the present study (1.6%) was similar to that reported in an investigation performed in Italy. (19) The APyV-positive birds were found in 1 of 21 aviaries (4.8%). It has been suggested that APyV prevalence can be underestimated by the type of sample used and that cloacal swab samples should be taken for PCR analysis to determine the presence of APyV in psittacine birds. (27) However, in this study, feather samples were used based on a report of 165 psittacine birds in Taiwan in which both APyV and BFDV detection rates in feather samples were higher than in fecal samples. (21)

Regarding BFDV, the rate of PBFD infection (23.2%) that we obtained was similar to that observed in studies of psittacine birds in Poland (25.3%>).26 The BFDV-positive birds in the present study were found in 11 of 21 aviaries (52.4%). One reason for the high prevalence determined in the present study may be that most of the aviaries in Chile do not have sanitary protocols and do not implement adequate disinfection measures in cages when new birds are introduced. The characteristics of the viral agent favor that situation. In addition, BFDV is highly contagious and highly stable in the environment. The main transmission is via feather dust, feces, and crop secretions. However, the virus can also be transmitted to unhatched chicks by their infected mothers. (55)

The percentages of APyV- and BFDV-positive samples obtained in this study are believed to reflect the current situation for captive, exotic psittacine birds in Chile. Based on these findings, our recommendations for preventing the spread of the viruses include inspection of aviaries, followed by molecular diagnostic testing to determine the prevalence of the viral causative agents BFDV and APyV. When considering the introduction of new psittacine birds into the aviary, knowing the infection status of the existing collection before introduction and testing new birds while they remain in quarantine are critical to prevent disease spread. Testing the nursery environment for viral contamination is also important. Given the findings of this study, we recommend that native Chilean psittacine birds (e.g., Patagonian conures [Cyanoliseus patagonus bloxami], slender-billed parakeets [Enicognathus leptorhynchus], and austral conures [Enicognathus ferrugineus]) should not be in proximity to exotic psittacine birds in zoos, rescue centers, or private collections. Both viruses could represent a substantial threat to the recovery of native psittacine species and, potentially, could threaten their survival in the wild. In addition, the international parrot trade could facilitate the spread of pathogenic BFDV and APyV variants that could further endanger wild parrot populations.

Confirming the definitive location of the Chilean psittacine BFDV and APyV sequences in the phylogenetic trees reported here is difficult because of a lack of whole genome information. However, the location is most likely accurate in the case of BFDV. Although partial BFDV sequences were obtained, the segments belong to the rep gene, which is one of the recommended areas for circovirus classification within the species. (56) Considering Julian et al BFDV classification, Chilean sequences would be localized into 2 different groups (J and K) that show no host or geographical association. (34) This is in agreement with findings in most reports that several circovirus strains can infect different psittacine hosts. (57-62) The genomic similarity of the tested psittacine bird circovirus sequences with reference to European and Australian sequences could indicate the exotic origin of the BFDV strains detected in the present study, suggesting their introduction in Chile by the legal or illegal trade of psittacine birds from those geographic regions. The findings of this study reinforce the recommendation that all imported birds should be tested for BFDV by PCR to prevent entry of this virus.

Regarding psittacine bird Chilean APyV sequences, their placement into discrete clades was not possible. The evaluated segments were useful for APyV detection purposes, but unfortunately, they were not suitable for phylogenetic analysis. Because of the limited size of the sequences obtained, only a small, conserved area was available for pairwise comparison. As a result, no differences among Chilean and international APyV sequences could be observed, and the tree was not statistically supported with less than 50% bootstrapping.

The Polyomaviridae Study Group of the International Committee on Taxonomy of Viruses recommends sequences from the later T antigen (LTAg) gene for phylogenetic purposes. (3) In our case, we used the Agnola-coding region, which is a conserved region within the Aves polyomavirus I, but apparently, it is not variable enough to support clade formation.

Finally, including more APyV and BFDV sequences from the southern hemisphere (tropical, subtropical, and temperate regions of South America) in NCBI databases is necessary because wild psittacine birds are naturally found predominantly in these regions.


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Gisela Gonzalez-Hein, DVM, PhD, Isabel Aguirre Gil, DVM, PhD, Rodolfo Sanchez, MS, and Bernardo Huaracan, MS

From Bioingentech, Paseo Bulnes 107 oficina 57, Santiago 8330243, Chile (Gonzalez-Hein): the Laboratorio de Biotecnologia y Patologia Acuatica, Departamento de Patologia Animal, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Isla Teja, Valdivia 5090000, Chile (Aguirre Gil); and Bioingentech. Bernardo O'Higgins 1186 oficina 1307, Concepcion 4070242, Chile (Sanchez, Huaracan).

Caption: Figure 1. Aves polyomavirus 1 (APyV) phylogenetie tree. Chilean APyV sequences are indicated with black diamonds. They are maintained together in a branch near sequences from Japan and Germany. Partial nucleotide sequences of Agno-la protein gene (324 nucleotides [nt]) were analyzed by the neighbor-joining method. (30) Evolutionary distances were computed by the Tajima and Nei method. (31) Evolutionary analyses were conducted in Molecular Evolutionary Genetics Analysis freeware version 7.0 (MEGA7). (29) Sequences from Chile and different countries available on GenBank were included. Accession numbers, host species, and country of origin codes are indicated in each branch. Bootstrapping of 1000 replicates was performed. Bootstrap percentages <70% are not shown. Country codes are noted as AUS indicating Australia; CHIN, China; CHL, Chile; JPN, Japan; GER, Germany; POL, Poland; and USA, United States.

Caption: Figure 2. Beak and feather disease virus (BFDV) phylogenetic tree. Chilean BFDV sequences are indicated with black diamonds. The Chilean blue-and-yellow macaw sequence is grouped together with Polish, United Kingdom, Italian, and Turkish sequences, proposed as genotype J1. (34) The Chilean plum-headed parakeet sequence is located near Australian sequences identified as genotype K. Country codes are noted as AUS indicating Australia; BRA. Brazil; CHL, Chile; CHN, China; GER, Germany; IRN, Iran; ITA. Italy; JPN, Japan; NCL, New Caledonia; NZL, New Zealand; POL, Poland; THA, Thailand; TUR. Turkey; UK, United Kingdom: USA, United States; and ZAF, South Africa.
Table 1. The genera of psittacine birds analyzed for aves
polyomavirus 1 (APyV) and beak and feather disease virus (BFDV)
infections. A total of 250 feather samples were collected to
determine APyV and BFDV prevalence in Chile. Four APyV-positive
birds (1.6%) and 58 BFDV-positive birds (23.2%) were found.


Family        Genus            no. positive/no. examined (%)

Cacatuidae    Nymphicus                   0/8 (0)
Psiltacidae   Agapornis                   0/2 (0)
              Alisterus                   0/1 (0)
              Amazona                     0/20 (0)
              Ara                         0/17 (0)
              Aratinga                    0/1 (0)
              Barnardius                  0/7 (0)
              Bolborhynchus               0/3 (0)
              Neopsepliotus               0/3 (0)
              Pionus                      0/3 (0)
              Platycercus                 0/69 (0)
              Polytelis                   0/11 (0)
              Psephotus                   0/1 (0)
              Psittacula                 3/79 (3.8)
              Psittacus                   0/6 (0)
              Pyrrhura                    0/5 (0)
              Trichoglossus              1/14(7.1)
Total                                   4/250 (1.6)


Family        Genus              no. positive/no. examined (%)

Cacatuidae    Nymphicus                 1/8 (12.5)
Psiltacidae   Agapornis                 2/2 (100)
              Alisterus                 1/1 (100)
              Amazona                    1/20 (5)
              Ara                      4/17 (23.5)
              Aratinga                   0/1 (0)
              Barnardius                5/7 (71.4)
              Bolborhynchus              0/3 (0)
              Neopsepliotus             2/3 (66.7)
              Pionus                     0/3 (0)
              Platycercus              20/69 (29.0)
              Polytelis                2/11 (18.2)
              Psephotus                  0/1 (0)
              Psittacula               12/79 (15.2)
              Psittacus                 2/6 (33.3)
              Pyrrhura                  3/5 (60.0)
              Trichoglossus            3/14 (21.4)
Total                                  58/250 (23.2)

Table 2. The genera of psittacine birds and feather
appearance in both beak and feather disease virus
(BFDV)- and aves polyomavirus 1 (APyV)-positive
birds; 14 of 58 BFDV-positive samples (24.1%) had
abnormal feathers and 3 of 4 APyV positive birds
(75.0%) had abnormal feathers.

                 BFDV positive.       APyV positive.
                 n = 58               n = 4

                 Abnormal   Normal    Abnormal   Normal
Genus            feather    feather   feather    feather

Agapomis            0          2         0          0
Alisterus           1          0         0          0
Am a: on a          1          0         0          0
Ara                 0          4         0          0
Barnardius          2          3         0          0
Neopsephotus        0          2         0          0
Nymphicus           1          0         0          0
Platycercus         2         18         0          0
Polytelis           0          2         0          0
Psittacula          3          9         2          1
Psittacus           2          0         0          0
Pvrrhura            0          3         0          0
Trichoglossus       2          1         1          0
Total               14        44         3          1
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Article Details
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Title Annotation:Research Brief
Author:Gonzalez-Hein, Gisela; Gil, Isabel Aguirre; Sanchez, Rodolfo; Huaracan, Bernardo
Publication:Journal of Avian Medicine and Surgery
Geographic Code:3CHIL
Date:Jun 1, 2019
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