Journal of General Virology (2015), 96, 24–29
Short Communication
DOI 10.1099/vir.0.068106-0
Isolation of multiple novel paramyxoviruses from pteropid bat urine Jennifer Barr,1 Craig Smith,2 Ina Smith,1 Carol de Jong,2 Shawn Todd,1 Debra Melville,2 Alice Broos,2 Sandra Crameri,1 Jessica Haining,1 Glenn Marsh,1 Gary Crameri,1 Hume Field2,3 and Lin-Fa Wang1,4
Correspondence
1
Lin-Fa Wang
2
[email protected]
CSIRO Australian Animal Health Laboratory, Geelong, Australia Queensland Department of Agriculture, Fisheries and Forestry, Brisbane, Australia
3
Ecohealth Alliance, New York, NY, USA
4
Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, Singapore
Received 20 May 2014 Accepted 11 September 2014
Bats have been found to harbour a number of new emerging viruses with zoonotic potential, and there has been a great deal of interest in identifying novel bat pathogens to determine the risk to human and animal health. Many groups have identified novel viruses in bats by detection of viral nucleic acid; however, virus isolation is still a challenge, and there are few reports of viral isolates from bats. In recent years, our group has developed optimized procedures for virus isolation from bat urine, including the use of primary bat cells. In previous reports, we have described the isolation of Hendra virus, Menangle virus and Cedar virus in Queensland, Australia. Here, we report the isolation of four additional novel bat paramyxoviruses from urine collected from beneath pteropid bat (flying fox) colonies in Queensland and New South Wales during 2009–2011.
In recent years, bats have been found to be the reservoir host of several significant groups of emerging zoonotic viruses including paramyxoviruses, coronaviruses and filoviruses (Calisher et al., 2006; Halpin et al., 2007; Smith & Wang, 2013; Wong et al., 2007). As more zoonotic viruses are linked to bats, identifying novel agents harboured by bats has become increasingly important (Baker et al., 2012, 2013b; Lau et al., 2010; Wang et al., 2011). Detection of viral nucleic acid by PCR to identify viruses in bats represents the most common method employed in published studies. Several groups have also used nextgeneration sequencing platforms to identify most, if not all, of the viruses in a target species of bat, termed the ‘bat virome’ (Anthony et al., 2013; Baker et al., 2013a; Donaldson et al., 2010; Ge et al., 2012; Li et al., 2010). However, determining the zoonotic and pathogenic potential of these agents is very difficult based on sequence information alone. Virus isolation and animal infection trials remain the best method to determine which of these may present a risk to human and/or animal health. At the present time, isolation of live virus from bats remains a challenge. In recent years, our group has focused on developing optimized procedures for isolation of live viruses; from urine collection protocols, sample transportation medium and storage conditions to the development of specialized bat primary cell lines (Crameri et al., The GenBank/EMBL/DDBJ accession numbers for the virus sequences determined in this study are KJ716812–KJ716815.
24
2009). In previous reports, we have described the isolation of Hendra virus (HeV) (Smith et al., 2011), Menangle virus (MenPV) (Barr et al., 2012) and the novel henipavirus, Cedar virus (CedPV) (Marsh et al., 2012), from beneath colonies of pteropid bats (commonly known as flying foxes) in Queensland (QLD), Australia, utilizing these optimized procedures. In this study, urine was collected from beneath various flying-fox colonies in QLD and New South Wales (NSW) in August and September 2009 and July and August 2011, respectively. The bat colonies sampled were targeted based on their close proximity to a known HeV spillover event into a horse(s). The Cedar Grove and Tolga Scrub colonies were the exception; these colonies were routinely sampled by members of our team as part of a longitudinal study (Field et al., 2011). No HeV spillover events occurred in these parts of QLD in 2009. Generally, once a horse had been confirmed to be infected with HeV, the team would identify the closest readily accessible bat colony to the location of the spillover event and sample urine from beneath the colony as described previously (Field et al., 2011). Briefly, urine was pooled off plastic sheets from underneath bat colonies and collected in a tube containing transport medium (either 500 ml sucrose-phosphate-glutamate-albumin plus antibiotic/antimycotic buffer, or 100 ml 10 % BSA in PBS plus double-strength antibiotic/ antimycotic). The tubes were subsequently transported cold to the Biosecurity Sciences Laboratory in QLD, frozen 068106 G 2015 The Authors
Printed in Great Britain
Virus isolation from bat urine
at -80 uC and later transported on dry ice to the CSIRO Australian Animal Health Laboratory (AAHL) in Geelong for virus isolation. The urine samples were screened for HeV by reverse transcription-PCR, and positive samples were targeted for virus isolation. The Cedar Grove 25 September 2009 collection had an unusually high rate of HeV PCR positives (12 % compared with ,5 % seen previously) and was of great interest, as this was the first time that such a high proportion of HeV positives was observed in bat pooled urine samples (Field et al., 2011). In addition, 2011 was an unprecedented year with regard to the number of HeV cases. There were 18 spillover events in that year alone, compared with 14 from 1994 to 2010 (Clayton et al., 2013; Field et al., 2012). The proportion of HeV PCR-positive pooled urine samples collected during the outbreak period in 2011 was extremely high (up to 67 % for some collections). These samples were therefore targeted for virus isolation. Virus isolation was carried out as described previously (Barr et al., 2012). Due to the relatively high rate of HeV PCR-positive samples, isolation was conducted initially in a Biosafety Level 3 laboratory at AAHL, and then transferred and continued in a BSL4 facility when syncytial cytopathic effect (CPE) became apparent. Briefly, the samples were thawed at room temperature and centrifuged to pellet debris. The urine was diluted 1 : 10 in cell culture medium (Dulbecco’s modified Eagle’s medium nutrient mixture F-12 Ham supplemented with double-strength antibiotic/antimycotic and 10 % FCS), centrifuged again to clarify it and then dispersed across both Vero and primary Pteropus alecto kidney (PaKi) (Crameri et al., 2009) cell monolayers. The flasks were rocked for 30 min at 37 uC, cell culture medium was added and the cells were incubated for 7 days at 37 uC. Supernatant and cells were passaged two more times if no CPE was observed. The cell monolayers were observed for toxicity, bacterial or fungal contamination, and viral CPE. The samples collected from Cedar Grove, Yeppoon and Tolga Scrub in August/September 2009 and from Hervey Bay, Nambucca Heads and Boonah in July/August 2011 all resulted in the isolation of live viruses (Tables 1 and 2). Some samples, such as those from Cedar Grove 2009 and the three locations sampled in 2011, produced a surprisingly high number of isolates. Virus isolation was
attempted on 32 pooled urine samples from Cedar Grove across two time points, which resulted in 10 isolations of five different viruses; 15 pooled urine samples from Boonah across three time points resulted in 37 isolations of seven different viruses, seven pooled urine samples from Nambucca Heads from one time point resulted in five isolations of two different viruses and 16 pooled urine samples from Hervey Bay across three time points resulted in eight isolations of five different viruses. In this study, syncytial CPE was observed in both Vero and PaKi cell monolayers as early as day 2 post-inoculation during the first passage or as late as the third blind passage (Table 2). Cultures displaying syncytial CPE were screened using subfamily- or genus-specific primer sets following protocols described elsewhere (Tong et al., 2008). PCR products were sequenced directly for preliminary characterization of the viral genomic sequences. When sequencing was ambiguous due to either poor PCR quality or mixing of more than one virus from the same cell culture isolation, PCR products were cloned and individual clones were sequenced. The consensus sequence from each virus isolate was deposited in GenBank and used for subsequent analysis. Routine sequence analysis was conducted using Clone Manager 9 (Sci-Ed Software), whereas phylogenetic analysis was performed using MEGA5.2 as described previously (Barr et al., 2012). Previously, our group has reported viruses isolated from this collection of flying-fox urine: four bat isolates of HeV (Smith et al., 2011), a bat isolate of MenPV (Barr et al., 2012) and the novel henipavirus, CedPV (Marsh et al., 2012). In addition to these viruses, we have now isolated four additional novel paramyxoviruses, all in the genus Rubulavirus. The number of rubulaviruses isolated has outnumbered all other viruses. This may be explained either by the fact that we may be inadvertently targeting rubulaviruses by our methods of isolation and the cell lines used, or, alternatively, the number of rubulaviruses present in the urine of these bat species is greater than any other virus type. We tried to address this by conducting a viral metagenome analysis using pooled samples from various urine collections. However, due to the pool sample qualities and some technical issues, we were unable to obtain conclusive data from this first attempt. This will be one of the focuses in our future surveillance studies.
Table 1. Summary of all paramyxoviruses isolated from this study Genus
(Proposed) species
Total no. isolations
Location (no. times isolated at that location)
Henipavirus
Cedar (CedPV) Hendra (HeV) Menangle (MenPV) Hervey (HerPV) Grove (GroPV) Teviot (TevPV) Yeppoon (YepPV)
1 8 11 9 10 17 8
Cedar Grove (1) Cedar Grove (1), Yeppoon (2), Tolga Scrub (1), Boonah (1), Hervey Bay (3) Cedar Grove (1), Boonah (9), Hervey Bay (1) Hervey Bay (1), Boonah (5), Nambucca Heads (3) Cedar Grove (4), Boonah (6) Cedar Grove (3), Boonah (12), Nambucca Heads (2) Yeppoon (1), Hervey Bay (3), Boonah (4)
Rubulavirus
http://vir.sgmjournals.org
25
J. Barr and others
Table 2. Isolation detail of the four novel bat rubulaviruses (Proposed) species
Hervey (HerPV)
Grove (GroPV)
Teviot (TevPV)
Yeppoon (YepPV)
Isolate no.
1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1 2 3 4 5 6 7 8
Location of isolation
Hervey Bay Boonah Boonah Boonah Boonah Boonah Nambucca Heads Nambucca Heads Nambucca Heads Cedar Grove Cedar Grove Cedar Grove Cedar Grove Boonah Boonah Boonah Boonah Boonah Boonah Cedar Grove Cedar Grove Cedar Grove Boonah Boonah Boonah Boonah Boonah Boonah Boonah Boonah Boonah Boonah Boonah Boonah Nambucca Heads Nambucca Heads Yeppoon Hervey Bay Hervey Bay Hervey Bay Boonah Boonah Boonah Boonah
We named these four novel rubulaviruses based on the name of locations close to where the bats were sampled. The isolate with closest sequence relatedness to the known bat rubulavirus Tioman paramyxovirus (TioPV) was designated Teviot paramyxovirus (TevPV). TevPV was isolated multiple times from Cedar Grove, Boonah and Nambucca Heads across the 2009–2011 time period and was more likely to be isolated from Vero cells than from PaKi cells (Table 2). TioPV was first identified during the 26
CPE first observed Cell type
Passage no.
PaKi PaKi PaKi Vero Vero PaKi Vero Vero PaKi Vero PaKi Vero PaKi Vero PaKi PaKi Vero Vero Vero PaKi Vero Vero Vero Vero Vero PaKi Vero PaKi Vero Vero PaKi Vero Vero Vero Vero Vero PaKi Vero PaKi PaKi Vero PaKi Vero PaKi
1 1 1 1 1 1 2 2 2 2 1 1 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 1 1 2 1 1 1 1
hunt for Nipah virus and was isolated from bat urine collected from Tioman Island (Chua et al., 2001). There is serological evidence that TioPV is able to infect humans, although it is not known whether the infection is associated with clinical manifestation (Yaiw et al., 2007). MenPV was isolated from the 2009 Cedar Grove collection and reported previously (Barr et al., 2012). MenPV is another known bat rubulavirus, closely related to TioPV, and was found to be the aetiological agent of a disease in pigs in Journal of General Virology 96
Virus isolation from bat urine
Australia in 1997 and responsible for flu-like sickness of two persons (Philbey et al., 1998). Since the initial isolation of MenPV from flying-fox urine, it has been isolated from the urine collected in 2011 from Hervey Bay and Boonah a further 10 times (J. Barr, unpublished results). A novel rubulavirus, designated Yeppoon paramyxovirus (YepPV), was isolated initially from the Yeppoon 2009 collection. A virus most related to YepPV was subsequently isolated from Cedar Grove and designated Grove paramyxovirus (GroPV). The fourth novel rubulavirus was isolated in PaKi cells and designated Hervey paramyxovirus (HerPV). HerPV was originally isolated from the Hervey Bay 20 July 2011 collection and then subsequently isolated from Boonah and Nambucca Heads samples collected in 2011. Phylogenetic analysis based on the 530 bp fragment of the large (L) gene obtained using pan-Paramyxovirinae primers (Tong et al., 2008), which is the most used sequence for paramyxovirus phylogeny studies (Drexler et al., 2012), indicated that three of the four newly isolated bat rubulaviruses clustered with the two previously identified Asian bat viruses, MenPV and TioPV. However, the fourth newly isolated virus, HerPV, was more closely related to the Achimota virus 2 isolated from bats in Ghana, Africa (Fig. 1). These data suggested that it is likely that these rubulaviruses have been associated with bats for a long time and that their geographical distribution can cover a wide range from Asia to Africa.
93 95 100
*Grove virus/Bat/2009/Cedar Grove (KJ716812) *Yeppoon virus/Bat/2009/Yeppoon (KJ716815) Menangle virus Australia/Bat/2009/Cedar Grove (AFY09794)
99
Tioman virus (NP665871) Achimota virus 2 (AFX75118)
25 86
In summary, this study illustrated that pteropid bat urine is a rich source of paramyxoviruses, with a potential bias towards rubulaviruses, and that, using optimized procedures and appropriate cell lines, it is possible to isolate live virus directly from urine samples. In many cases, multiple viruses were identified from a single pooled urine sample and in some cases in only one cell type. Further purification and characterization of these novel viruses is required. Much more in-depth studies are needed to assess the animal and public health risk of these novel bat rubulaviruses. In addition to MenPV and TioPV, both
*Teviot virus/Bat/2009/Cedar Grove (KJ716814)
41
0.1
As a first step in assessing the potential of these viruses to spill over into livestock (such as pigs) or human populations, a comparative growth study with two known bat rubulaviruses was conducted using four different cell lines, derived from human (HeLa), monkey (Vero), pig (PK15a) and bat (PaKi), respectively. As shown in Fig. 2, all viruses grew well in Vero cells as expected. MenPV, the only virus known to cause disease in pigs, grew best in PK15a cells. YepPV seemed to grow well in all three nonbat cell lines and reached similar growth at 48 h in all four cell lines. Based on this initial analysis, YepPV may be a worthwhile target to follow in future surveillance and infection studies for potential spillover into other host(s). However, these are very preliminary data and more followup study and in-field surveillance is required to assess their real potential for cross-species transmission.
64
*Hervey virus/Bat/2011/Hervey Bay (KJ716813) Sosuga virus (AHH02041)
100
Tuhoko virus 1 (ADI80715) Mumps virus (NP054714)
96 74
Parainfluenza virus 5 (YP138518) Newcastle disease virus (NP071471) Measles virus (NP056924) Bat paramyxovirus BatPV/Eid_hel/GH-M74a/GHA/2009 (AET43339)
100
Cedar virus/Bat/2009/Cedar Grove (AFP87280)
97
Hendra virus (NP047113)
96 100
Nipah virus (NP112028) Human metapneumovirus (YP012613)
100
Human respiratory syncytial virus (NP056866)
Fig. 1. Phylogenetic tree of paramyxoviruses. The tree was generated from 19 partial sequences of the L gene from selected members of the family Paramyxoviridae. Sequences derived from viruses isolated or detected in bats are in bold, with the four newly isolated viruses from this study indicated by an asterisk. GenBank accession numbers are within parentheses next to the virus names. Neighbour-joining trees were reconstructed using MEGA5.2 (www.megasoftware.net/) with bootstrapping at 1000 replicates. Bar, nucleotide substitutions per site. http://vir.sgmjournals.org
27
J. Barr and others
Vero PaKi HeLa PK15a
TCID50 ml–1
107 106 105 104 103
107 106 105 104 103
102
102
101 100
101 100 0
24
48
TioPV
108
TCID50 ml–1
MenPV
108
0
72
24
HerPV
107
107
106
106
105 104 103
102 101 100 48
0
72
24
TevPV
106
106
TCID50 ml–1
TCID50 ml–1
107
105 104 103
72
105 104 103
102
102
101 100
101 100 24
YepPV
108
107
0
48
Time (h)
Time (h) 108
72
104 103
101 100 24
48
105
102
0
72
GroPV
108
TCID50 ml–1
TCID50 ml–1
108
48 Time (h)
Time (h)
48
72
Time (h)
0
24 Time (h)
Fig. 2. Comparative growth analysis in cells of different species origin. HeLa (human), Vero (monkey), PK15a (pig) and PaKi (bat) cell monolayers were prepared overnight in six-well tissue culture plates. Cell culture medium was removed and the cells were infected at an m.o.i. of 0.01 with MenPV, TioPV, HerPV, GroPV, TevPV or YepPV for 1 h at 37 6C and then washed four times with PBS before adding back the cell culture medium. The culture supernatant was sampled at four time points (0, 24, 48 and 72 h) and stored at ”80 6C until titration in Vero cells to determine TCID50 ml”1.
known to infect humans, a novel rubulavirus, named Sosuga virus, was recently isolated from a US wildlife biologist who had travelled to South Sudan and Uganda for bat work before returning to the USA and becoming sick (Albarin˜o et al., 2014). Although the causative relationship was not conclusively established from this single human infection, it is quite possible that Sosuga virus and many other bat rubulaviruses have the potential to cause zoonotic human infections in many parts of the world. This is further demonstrated by the isolation of another bat rubulavirus, Achimota virus, from African bats (Eidolon helvum) and serological indication of human infection in our previous published study (Baker et al., 2013b). Finally, 28
it is interesting to note that our high success rate of virus isolation from the 2011 samples correlates with the unprecedented number of HeV spillover events that occurred in 2011. Whether this is a co-incident event or whether there is some inherent physiological or ecological link between these two observations is yet to be determined by more longitudinal studies currently being conducted by our group.
Acknowledgements This study was supported in part by the CSIRO OCE Science Leader Award (to L.-F. W.) and the State of Queensland through the Queensland Centre for Emerging Infectious Diseases. Journal of General Virology 96
Virus isolation from bat urine
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Short Communication
DOI 10.1099/vir.0.068106-0
Isolation of multiple novel paramyxoviruses from pteropid bat urine Jennifer Barr,1 Craig Smith,2 Ina Smith,1 Carol de Jong,2 Shawn Todd,1 Debra Melville,2 Alice Broos,2 Sandra Crameri,1 Jessica Haining,1 Glenn Marsh,1 Gary Crameri,1 Hume Field2,3 and Lin-Fa Wang1,4
Correspondence
1
Lin-Fa Wang
2
[email protected]
CSIRO Australian Animal Health Laboratory, Geelong, Australia Queensland Department of Agriculture, Fisheries and Forestry, Brisbane, Australia
3
Ecohealth Alliance, New York, NY, USA
4
Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, Singapore
Received 20 May 2014 Accepted 11 September 2014
Bats have been found to harbour a number of new emerging viruses with zoonotic potential, and there has been a great deal of interest in identifying novel bat pathogens to determine the risk to human and animal health. Many groups have identified novel viruses in bats by detection of viral nucleic acid; however, virus isolation is still a challenge, and there are few reports of viral isolates from bats. In recent years, our group has developed optimized procedures for virus isolation from bat urine, including the use of primary bat cells. In previous reports, we have described the isolation of Hendra virus, Menangle virus and Cedar virus in Queensland, Australia. Here, we report the isolation of four additional novel bat paramyxoviruses from urine collected from beneath pteropid bat (flying fox) colonies in Queensland and New South Wales during 2009–2011.
In recent years, bats have been found to be the reservoir host of several significant groups of emerging zoonotic viruses including paramyxoviruses, coronaviruses and filoviruses (Calisher et al., 2006; Halpin et al., 2007; Smith & Wang, 2013; Wong et al., 2007). As more zoonotic viruses are linked to bats, identifying novel agents harboured by bats has become increasingly important (Baker et al., 2012, 2013b; Lau et al., 2010; Wang et al., 2011). Detection of viral nucleic acid by PCR to identify viruses in bats represents the most common method employed in published studies. Several groups have also used nextgeneration sequencing platforms to identify most, if not all, of the viruses in a target species of bat, termed the ‘bat virome’ (Anthony et al., 2013; Baker et al., 2013a; Donaldson et al., 2010; Ge et al., 2012; Li et al., 2010). However, determining the zoonotic and pathogenic potential of these agents is very difficult based on sequence information alone. Virus isolation and animal infection trials remain the best method to determine which of these may present a risk to human and/or animal health. At the present time, isolation of live virus from bats remains a challenge. In recent years, our group has focused on developing optimized procedures for isolation of live viruses; from urine collection protocols, sample transportation medium and storage conditions to the development of specialized bat primary cell lines (Crameri et al., The GenBank/EMBL/DDBJ accession numbers for the virus sequences determined in this study are KJ716812–KJ716815.
24
2009). In previous reports, we have described the isolation of Hendra virus (HeV) (Smith et al., 2011), Menangle virus (MenPV) (Barr et al., 2012) and the novel henipavirus, Cedar virus (CedPV) (Marsh et al., 2012), from beneath colonies of pteropid bats (commonly known as flying foxes) in Queensland (QLD), Australia, utilizing these optimized procedures. In this study, urine was collected from beneath various flying-fox colonies in QLD and New South Wales (NSW) in August and September 2009 and July and August 2011, respectively. The bat colonies sampled were targeted based on their close proximity to a known HeV spillover event into a horse(s). The Cedar Grove and Tolga Scrub colonies were the exception; these colonies were routinely sampled by members of our team as part of a longitudinal study (Field et al., 2011). No HeV spillover events occurred in these parts of QLD in 2009. Generally, once a horse had been confirmed to be infected with HeV, the team would identify the closest readily accessible bat colony to the location of the spillover event and sample urine from beneath the colony as described previously (Field et al., 2011). Briefly, urine was pooled off plastic sheets from underneath bat colonies and collected in a tube containing transport medium (either 500 ml sucrose-phosphate-glutamate-albumin plus antibiotic/antimycotic buffer, or 100 ml 10 % BSA in PBS plus double-strength antibiotic/ antimycotic). The tubes were subsequently transported cold to the Biosecurity Sciences Laboratory in QLD, frozen 068106 G 2015 The Authors
Printed in Great Britain
Virus isolation from bat urine
at -80 uC and later transported on dry ice to the CSIRO Australian Animal Health Laboratory (AAHL) in Geelong for virus isolation. The urine samples were screened for HeV by reverse transcription-PCR, and positive samples were targeted for virus isolation. The Cedar Grove 25 September 2009 collection had an unusually high rate of HeV PCR positives (12 % compared with ,5 % seen previously) and was of great interest, as this was the first time that such a high proportion of HeV positives was observed in bat pooled urine samples (Field et al., 2011). In addition, 2011 was an unprecedented year with regard to the number of HeV cases. There were 18 spillover events in that year alone, compared with 14 from 1994 to 2010 (Clayton et al., 2013; Field et al., 2012). The proportion of HeV PCR-positive pooled urine samples collected during the outbreak period in 2011 was extremely high (up to 67 % for some collections). These samples were therefore targeted for virus isolation. Virus isolation was carried out as described previously (Barr et al., 2012). Due to the relatively high rate of HeV PCR-positive samples, isolation was conducted initially in a Biosafety Level 3 laboratory at AAHL, and then transferred and continued in a BSL4 facility when syncytial cytopathic effect (CPE) became apparent. Briefly, the samples were thawed at room temperature and centrifuged to pellet debris. The urine was diluted 1 : 10 in cell culture medium (Dulbecco’s modified Eagle’s medium nutrient mixture F-12 Ham supplemented with double-strength antibiotic/antimycotic and 10 % FCS), centrifuged again to clarify it and then dispersed across both Vero and primary Pteropus alecto kidney (PaKi) (Crameri et al., 2009) cell monolayers. The flasks were rocked for 30 min at 37 uC, cell culture medium was added and the cells were incubated for 7 days at 37 uC. Supernatant and cells were passaged two more times if no CPE was observed. The cell monolayers were observed for toxicity, bacterial or fungal contamination, and viral CPE. The samples collected from Cedar Grove, Yeppoon and Tolga Scrub in August/September 2009 and from Hervey Bay, Nambucca Heads and Boonah in July/August 2011 all resulted in the isolation of live viruses (Tables 1 and 2). Some samples, such as those from Cedar Grove 2009 and the three locations sampled in 2011, produced a surprisingly high number of isolates. Virus isolation was
attempted on 32 pooled urine samples from Cedar Grove across two time points, which resulted in 10 isolations of five different viruses; 15 pooled urine samples from Boonah across three time points resulted in 37 isolations of seven different viruses, seven pooled urine samples from Nambucca Heads from one time point resulted in five isolations of two different viruses and 16 pooled urine samples from Hervey Bay across three time points resulted in eight isolations of five different viruses. In this study, syncytial CPE was observed in both Vero and PaKi cell monolayers as early as day 2 post-inoculation during the first passage or as late as the third blind passage (Table 2). Cultures displaying syncytial CPE were screened using subfamily- or genus-specific primer sets following protocols described elsewhere (Tong et al., 2008). PCR products were sequenced directly for preliminary characterization of the viral genomic sequences. When sequencing was ambiguous due to either poor PCR quality or mixing of more than one virus from the same cell culture isolation, PCR products were cloned and individual clones were sequenced. The consensus sequence from each virus isolate was deposited in GenBank and used for subsequent analysis. Routine sequence analysis was conducted using Clone Manager 9 (Sci-Ed Software), whereas phylogenetic analysis was performed using MEGA5.2 as described previously (Barr et al., 2012). Previously, our group has reported viruses isolated from this collection of flying-fox urine: four bat isolates of HeV (Smith et al., 2011), a bat isolate of MenPV (Barr et al., 2012) and the novel henipavirus, CedPV (Marsh et al., 2012). In addition to these viruses, we have now isolated four additional novel paramyxoviruses, all in the genus Rubulavirus. The number of rubulaviruses isolated has outnumbered all other viruses. This may be explained either by the fact that we may be inadvertently targeting rubulaviruses by our methods of isolation and the cell lines used, or, alternatively, the number of rubulaviruses present in the urine of these bat species is greater than any other virus type. We tried to address this by conducting a viral metagenome analysis using pooled samples from various urine collections. However, due to the pool sample qualities and some technical issues, we were unable to obtain conclusive data from this first attempt. This will be one of the focuses in our future surveillance studies.
Table 1. Summary of all paramyxoviruses isolated from this study Genus
(Proposed) species
Total no. isolations
Location (no. times isolated at that location)
Henipavirus
Cedar (CedPV) Hendra (HeV) Menangle (MenPV) Hervey (HerPV) Grove (GroPV) Teviot (TevPV) Yeppoon (YepPV)
1 8 11 9 10 17 8
Cedar Grove (1) Cedar Grove (1), Yeppoon (2), Tolga Scrub (1), Boonah (1), Hervey Bay (3) Cedar Grove (1), Boonah (9), Hervey Bay (1) Hervey Bay (1), Boonah (5), Nambucca Heads (3) Cedar Grove (4), Boonah (6) Cedar Grove (3), Boonah (12), Nambucca Heads (2) Yeppoon (1), Hervey Bay (3), Boonah (4)
Rubulavirus
http://vir.sgmjournals.org
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J. Barr and others
Table 2. Isolation detail of the four novel bat rubulaviruses (Proposed) species
Hervey (HerPV)
Grove (GroPV)
Teviot (TevPV)
Yeppoon (YepPV)
Isolate no.
1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1 2 3 4 5 6 7 8
Location of isolation
Hervey Bay Boonah Boonah Boonah Boonah Boonah Nambucca Heads Nambucca Heads Nambucca Heads Cedar Grove Cedar Grove Cedar Grove Cedar Grove Boonah Boonah Boonah Boonah Boonah Boonah Cedar Grove Cedar Grove Cedar Grove Boonah Boonah Boonah Boonah Boonah Boonah Boonah Boonah Boonah Boonah Boonah Boonah Nambucca Heads Nambucca Heads Yeppoon Hervey Bay Hervey Bay Hervey Bay Boonah Boonah Boonah Boonah
We named these four novel rubulaviruses based on the name of locations close to where the bats were sampled. The isolate with closest sequence relatedness to the known bat rubulavirus Tioman paramyxovirus (TioPV) was designated Teviot paramyxovirus (TevPV). TevPV was isolated multiple times from Cedar Grove, Boonah and Nambucca Heads across the 2009–2011 time period and was more likely to be isolated from Vero cells than from PaKi cells (Table 2). TioPV was first identified during the 26
CPE first observed Cell type
Passage no.
PaKi PaKi PaKi Vero Vero PaKi Vero Vero PaKi Vero PaKi Vero PaKi Vero PaKi PaKi Vero Vero Vero PaKi Vero Vero Vero Vero Vero PaKi Vero PaKi Vero Vero PaKi Vero Vero Vero Vero Vero PaKi Vero PaKi PaKi Vero PaKi Vero PaKi
1 1 1 1 1 1 2 2 2 2 1 1 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 1 1 2 1 1 1 1
hunt for Nipah virus and was isolated from bat urine collected from Tioman Island (Chua et al., 2001). There is serological evidence that TioPV is able to infect humans, although it is not known whether the infection is associated with clinical manifestation (Yaiw et al., 2007). MenPV was isolated from the 2009 Cedar Grove collection and reported previously (Barr et al., 2012). MenPV is another known bat rubulavirus, closely related to TioPV, and was found to be the aetiological agent of a disease in pigs in Journal of General Virology 96
Virus isolation from bat urine
Australia in 1997 and responsible for flu-like sickness of two persons (Philbey et al., 1998). Since the initial isolation of MenPV from flying-fox urine, it has been isolated from the urine collected in 2011 from Hervey Bay and Boonah a further 10 times (J. Barr, unpublished results). A novel rubulavirus, designated Yeppoon paramyxovirus (YepPV), was isolated initially from the Yeppoon 2009 collection. A virus most related to YepPV was subsequently isolated from Cedar Grove and designated Grove paramyxovirus (GroPV). The fourth novel rubulavirus was isolated in PaKi cells and designated Hervey paramyxovirus (HerPV). HerPV was originally isolated from the Hervey Bay 20 July 2011 collection and then subsequently isolated from Boonah and Nambucca Heads samples collected in 2011. Phylogenetic analysis based on the 530 bp fragment of the large (L) gene obtained using pan-Paramyxovirinae primers (Tong et al., 2008), which is the most used sequence for paramyxovirus phylogeny studies (Drexler et al., 2012), indicated that three of the four newly isolated bat rubulaviruses clustered with the two previously identified Asian bat viruses, MenPV and TioPV. However, the fourth newly isolated virus, HerPV, was more closely related to the Achimota virus 2 isolated from bats in Ghana, Africa (Fig. 1). These data suggested that it is likely that these rubulaviruses have been associated with bats for a long time and that their geographical distribution can cover a wide range from Asia to Africa.
93 95 100
*Grove virus/Bat/2009/Cedar Grove (KJ716812) *Yeppoon virus/Bat/2009/Yeppoon (KJ716815) Menangle virus Australia/Bat/2009/Cedar Grove (AFY09794)
99
Tioman virus (NP665871) Achimota virus 2 (AFX75118)
25 86
In summary, this study illustrated that pteropid bat urine is a rich source of paramyxoviruses, with a potential bias towards rubulaviruses, and that, using optimized procedures and appropriate cell lines, it is possible to isolate live virus directly from urine samples. In many cases, multiple viruses were identified from a single pooled urine sample and in some cases in only one cell type. Further purification and characterization of these novel viruses is required. Much more in-depth studies are needed to assess the animal and public health risk of these novel bat rubulaviruses. In addition to MenPV and TioPV, both
*Teviot virus/Bat/2009/Cedar Grove (KJ716814)
41
0.1
As a first step in assessing the potential of these viruses to spill over into livestock (such as pigs) or human populations, a comparative growth study with two known bat rubulaviruses was conducted using four different cell lines, derived from human (HeLa), monkey (Vero), pig (PK15a) and bat (PaKi), respectively. As shown in Fig. 2, all viruses grew well in Vero cells as expected. MenPV, the only virus known to cause disease in pigs, grew best in PK15a cells. YepPV seemed to grow well in all three nonbat cell lines and reached similar growth at 48 h in all four cell lines. Based on this initial analysis, YepPV may be a worthwhile target to follow in future surveillance and infection studies for potential spillover into other host(s). However, these are very preliminary data and more followup study and in-field surveillance is required to assess their real potential for cross-species transmission.
64
*Hervey virus/Bat/2011/Hervey Bay (KJ716813) Sosuga virus (AHH02041)
100
Tuhoko virus 1 (ADI80715) Mumps virus (NP054714)
96 74
Parainfluenza virus 5 (YP138518) Newcastle disease virus (NP071471) Measles virus (NP056924) Bat paramyxovirus BatPV/Eid_hel/GH-M74a/GHA/2009 (AET43339)
100
Cedar virus/Bat/2009/Cedar Grove (AFP87280)
97
Hendra virus (NP047113)
96 100
Nipah virus (NP112028) Human metapneumovirus (YP012613)
100
Human respiratory syncytial virus (NP056866)
Fig. 1. Phylogenetic tree of paramyxoviruses. The tree was generated from 19 partial sequences of the L gene from selected members of the family Paramyxoviridae. Sequences derived from viruses isolated or detected in bats are in bold, with the four newly isolated viruses from this study indicated by an asterisk. GenBank accession numbers are within parentheses next to the virus names. Neighbour-joining trees were reconstructed using MEGA5.2 (www.megasoftware.net/) with bootstrapping at 1000 replicates. Bar, nucleotide substitutions per site. http://vir.sgmjournals.org
27
J. Barr and others
Vero PaKi HeLa PK15a
TCID50 ml–1
107 106 105 104 103
107 106 105 104 103
102
102
101 100
101 100 0
24
48
TioPV
108
TCID50 ml–1
MenPV
108
0
72
24
HerPV
107
107
106
106
105 104 103
102 101 100 48
0
72
24
TevPV
106
106
TCID50 ml–1
TCID50 ml–1
107
105 104 103
72
105 104 103
102
102
101 100
101 100 24
YepPV
108
107
0
48
Time (h)
Time (h) 108
72
104 103
101 100 24
48
105
102
0
72
GroPV
108
TCID50 ml–1
TCID50 ml–1
108
48 Time (h)
Time (h)
48
72
Time (h)
0
24 Time (h)
Fig. 2. Comparative growth analysis in cells of different species origin. HeLa (human), Vero (monkey), PK15a (pig) and PaKi (bat) cell monolayers were prepared overnight in six-well tissue culture plates. Cell culture medium was removed and the cells were infected at an m.o.i. of 0.01 with MenPV, TioPV, HerPV, GroPV, TevPV or YepPV for 1 h at 37 6C and then washed four times with PBS before adding back the cell culture medium. The culture supernatant was sampled at four time points (0, 24, 48 and 72 h) and stored at ”80 6C until titration in Vero cells to determine TCID50 ml”1.
known to infect humans, a novel rubulavirus, named Sosuga virus, was recently isolated from a US wildlife biologist who had travelled to South Sudan and Uganda for bat work before returning to the USA and becoming sick (Albarin˜o et al., 2014). Although the causative relationship was not conclusively established from this single human infection, it is quite possible that Sosuga virus and many other bat rubulaviruses have the potential to cause zoonotic human infections in many parts of the world. This is further demonstrated by the isolation of another bat rubulavirus, Achimota virus, from African bats (Eidolon helvum) and serological indication of human infection in our previous published study (Baker et al., 2013b). Finally, 28
it is interesting to note that our high success rate of virus isolation from the 2011 samples correlates with the unprecedented number of HeV spillover events that occurred in 2011. Whether this is a co-incident event or whether there is some inherent physiological or ecological link between these two observations is yet to be determined by more longitudinal studies currently being conducted by our group.
Acknowledgements This study was supported in part by the CSIRO OCE Science Leader Award (to L.-F. W.) and the State of Queensland through the Queensland Centre for Emerging Infectious Diseases. Journal of General Virology 96
Virus isolation from bat urine
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