Transboundary and Emerging Diseases
ORIGINAL ARTICLE
Virus Excretion and Antibody Dynamics in Goats Inoculated with a Field Isolate of peste des petits ruminants virus W. Liu1,2, X. Wu1, Z. Wang1, J. Bao1, L. Li1, Y. Zhao1 and J. Li1 1 2
National Diagnostic Center for Exotic Animal Diseases, China Animal Health and Epidemiology Center, Qingdao, China College of Animal Science and Technology, Qingdao Agricultural University, Qingdao, China
Keywords: Peste des petits ruminants virus (PPRV); qRT-PCR; ELISA; virus neutralization test Correspondence: Z. Wang. National Diagnostic Center for Exotic Animal Diseases, China Animal Health and Epidemiology Center, 369 Nanjing Road, Qingdao, Shandong 266032, China. Tel.: +86 532 87839188; Fax: +86 532 87839922; E-mail: [email protected] The first two authors contributed equally to this manuscript. Received for publication November 15, 2012
Summary A field isolate of peste des petits ruminants virus (PPRV) from an outbreak in Tibet, China, was inoculated into goats to investigate the dynamics of virus excretion and antibody production. Further, animals received PPRV vaccine strain Nigeria 75/1. Ocular, nasal and oral samples were tested for the presence of virus antigen by one-step real-time qualitative RT-PCR (qRT-PCR); competitive ELISA (c-ELISA) was used for the measurement of specific antibodies against PPRV. Virus particles could be detected as early as day 3 post-inoculation (pi) and virus excretion lasted for up to day 26 pi. All four goats inoculated with the PPRV field isolate were seropositive as early as day 10 pi. In animals inoculated with the vaccine strain, antibody was detected at day 14 pi, and levels of neutralizing antibodies remained above the protection threshold level (1 : 8) for 8 months. Both virus particles and neutralizing antibodies were detected earlier in goats challenged with the field isolate than in those receiving the vaccine strain.
doi:10.1111/tbed.12136
Introduction Peste des petits ruminants (PPR) also known as ovine rinderpest (or pseudo-rinderpest), is an important viral disease of goats, sheep and some small wild ruminants. It is usually characterized by fever, respiratory distress, ocular, nasal and oral discharges, pneumonia, stomatitis and gastroenteritis leading to severe diarrhoea followed by death or recovery from the disease (Gibbs et al., 1979; Hussain et al., 2008), although these signs are not always present at the same time. Morbidity and mortality vary but can be as high as 100% (Ndamukong et al., 1989; Abubakar et al., 2008; Al-Dubaib, 2009). Severity of the disease depends on virus virulence (Couacy-Hymann et al., 2007a), host breed (Anderson and McKay, 1994), seasonal variations (Singh et al., 2004) and geographical conditions (Lefevre and Diallo, 1990; Taylor et al., 2002; Abubakar et al., 2009). The etiological agent, peste des petits ruminants virus (PPRV), has only one known serotype with different geographical distributions, but at least four distinct genetic lineages (I–IV) have been described based on alignments of
N gene or F gene nucleotide sequences (Shaila et al., 1996; Kwiatek et al., 2007). Virus transmission is predominantly via the ocular and respiratory tracts, and virus particles are excreted in oculonasal discharges, saliva and faeces. There were no records of PPRV in China until the pathogen first emerged in the Ngari region of south-western Tibet, China, in 2007 (Zhiliang Wang et al., 2007). From this outbreak, we isolated a PPRV strain, named China/Tib/07. Phylogenetic analysis identified this strain as a member of lineage IV and most closely related to viruses currently circulating in neighbouring countries of southern Asia. Because PPR is a newly emerging viral disease in China, no studies are yet available regarding the incubation period, virus excretion, clinical course and pathogenesis of infection. Some earlier experimental studies on PPRV infection in goats employed different virus strains and addressed the immediate postinfection period (Bundza et al., 1988; Couacy-Hymann et al., 2007b, 2009; Ezeibe et al., 2008; Hammouchi et al., 2012). The objective of the present study was to evaluate the time course of excretion of this new PPRV field isolate
© 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 60 (Suppl. 2) (2013) 63–68
63
Antibody Dynamics of PPRV in Goats
W. Liu et al.
in China and the dynamics of antibody production following challenge of a local breed of Laoshan milch goats. Materials and Methods Cells, media and virus The Vero cell line maintained in Dulbecco’s modified Eagle medium (DMEM; Gibco, Grand Island, NY, USA) containing 10% inactivated calf serum was used for virus propagation and virus neutralization tests. The PPRV field isolate China/Tib/07 was initially isolated from the rectal tissues of an infected goat from Tibet, China. The fifth passage of the virus on Vero cells was used in this study; the vaccine strain used was the Africa vaccine strain Nigeria 75/1. Viruses were propagated and titreed on Vero cells; titres were expressed as the median tissue culture infective dose (TCID50). Animals Twelve Laoshan milch goats 1–2 years of age were employed. Before the experiments, the animals were confirmed to be seronegative for anti-PPRV antibodies using a monoclonal antibody-based c-ELISA for the presence of antibodies against the nucleoprotein of the virus. All animal experiments were conducted in a high-security containment facility at the Class III Biosafety animal lab of the China Animal Health and Epidemiology Center. Different groups of animals were maintained in separate rooms with separate feeding and drinking tanks; animals were always kept indoors. Each animal was treated with ivermectin (0.025 ml/kg), an anti-parasite drug, during the acclimatization period (15 day) before PPRV inoculation. Experimental designs Virus challenge The twelve goats were divided randomly into three groups, A–C. Group A animals (numbered 1# to 4#) were inoculated with the field isolate of China/Tib/07, group B animals (numbered 5# to 8#) with the vaccine strain of Nigeria 75/ 1; group C animals (numbered 9# to 12#) acted as a control. A single subcutaneous inoculation of 1 ml of the virus suspension at a concentration of 103 TCID50/ml was inoculated into group A animals, a dose which from our previous studies on the virulence of China/Tib/07 generally gave rise to productive infection (X. Wu, Z. Wang, J. Li, and X. Han, unpublished data). The same dose of Nigeria 75/1 was administered to group B. All animals were managed under similar conditions; a different attendant was assigned to each room to feed and water the infected and control animals. Animals were examined daily, and rectal temperature and other physical changes were recorded daily. 64
Sample collection Nasal, ocular and oral swabs were collected from all challenged animals from day 1 to day 40 pi. Animals of group C were sampled at 5 day intervals. All swabs were stored at 40°C until examined. Blood samples without anticoagulant were collected from the jugular vein at regular intervals. Serum samples were separated by centrifugation at 5000 g for 10 min and stored at 40°C until testing. Detection of viral excretion by qRT-PCR RNA from samples of nasal, ocular and oral swabs of day 1 to day 40 pi was extracted using the High Pure Viral RNA Kit (Roche) according to the manufacturer’s protocol. A specific TaqMan-based qRT-PCR amplification and detection reaction were performed as described previously, and samples with a cycle threshold (Ct) ≤40 for detection were considered to be nucleic acid positive (Bao et al., 2008). In all experiments, RNase-free water and total RNA extracted from Nigeria 75/1-infected Vero cells culture served as negative and positive controls, respectively. Confirmation of virus excretion by sequencing Primer pairs NP3/NP4 were used to amplify a 350-bp fragment from the PPRV N gene using a one-step RT-PCR technique (Invitrogen, Grand Island, NY, USA) (CouacyHymann et al., 2002); negative and positive controls were as described in the previous section. PCR products were analysed by 1.5% agarose gel electrophoresis. Three separately amplified fragments from animals of each challenge group were randomly selected for sequencing and PPRV strain analysis. Serological testing Peste des petits ruminants virus antibodies in the serum from challenged and vaccinated goats were measured using an approved c-ELISA Kit (CIRAD, Montpellier, France) following recommended protocols, and the samples with PI >50 were considered to be seropositive (Libeau et al., 1995; Swai et al., 2009). Virus neutralization tests Sera from days 10, 14, 16, 30, 60 and 240 pi were used for the detection of neutralizing antibody. Aliquots (50 ll) of heat-inactivated sera were subjected to serial twofold dilutions. Serum dilutions were mixed in multiple replicates with an equal volume of medium containing 100 TCID50 of the homologous PPRV strain in 96-well plates and incubated for 1 h at 37°C. To each well was added 100 ll of Vero cell suspension (2–5 9 105 cells/ml), and incubation was continued for 7 days. Cytopathic effects (CPE) were assessed by light microscopy. The antibody titre in each case was taken as the highest serum dilution that neutralized virus CPE in more than 50% of replicates. Controls
© 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 60 (Suppl. 2) (2013) 63–68
W. Liu et al.
Antibody Dynamics of PPRV in Goats
included cell control (no serum, no virus) and virus control (no serum). Titres of
ORIGINAL ARTICLE
Virus Excretion and Antibody Dynamics in Goats Inoculated with a Field Isolate of peste des petits ruminants virus W. Liu1,2, X. Wu1, Z. Wang1, J. Bao1, L. Li1, Y. Zhao1 and J. Li1 1 2
National Diagnostic Center for Exotic Animal Diseases, China Animal Health and Epidemiology Center, Qingdao, China College of Animal Science and Technology, Qingdao Agricultural University, Qingdao, China
Keywords: Peste des petits ruminants virus (PPRV); qRT-PCR; ELISA; virus neutralization test Correspondence: Z. Wang. National Diagnostic Center for Exotic Animal Diseases, China Animal Health and Epidemiology Center, 369 Nanjing Road, Qingdao, Shandong 266032, China. Tel.: +86 532 87839188; Fax: +86 532 87839922; E-mail: [email protected] The first two authors contributed equally to this manuscript. Received for publication November 15, 2012
Summary A field isolate of peste des petits ruminants virus (PPRV) from an outbreak in Tibet, China, was inoculated into goats to investigate the dynamics of virus excretion and antibody production. Further, animals received PPRV vaccine strain Nigeria 75/1. Ocular, nasal and oral samples were tested for the presence of virus antigen by one-step real-time qualitative RT-PCR (qRT-PCR); competitive ELISA (c-ELISA) was used for the measurement of specific antibodies against PPRV. Virus particles could be detected as early as day 3 post-inoculation (pi) and virus excretion lasted for up to day 26 pi. All four goats inoculated with the PPRV field isolate were seropositive as early as day 10 pi. In animals inoculated with the vaccine strain, antibody was detected at day 14 pi, and levels of neutralizing antibodies remained above the protection threshold level (1 : 8) for 8 months. Both virus particles and neutralizing antibodies were detected earlier in goats challenged with the field isolate than in those receiving the vaccine strain.
doi:10.1111/tbed.12136
Introduction Peste des petits ruminants (PPR) also known as ovine rinderpest (or pseudo-rinderpest), is an important viral disease of goats, sheep and some small wild ruminants. It is usually characterized by fever, respiratory distress, ocular, nasal and oral discharges, pneumonia, stomatitis and gastroenteritis leading to severe diarrhoea followed by death or recovery from the disease (Gibbs et al., 1979; Hussain et al., 2008), although these signs are not always present at the same time. Morbidity and mortality vary but can be as high as 100% (Ndamukong et al., 1989; Abubakar et al., 2008; Al-Dubaib, 2009). Severity of the disease depends on virus virulence (Couacy-Hymann et al., 2007a), host breed (Anderson and McKay, 1994), seasonal variations (Singh et al., 2004) and geographical conditions (Lefevre and Diallo, 1990; Taylor et al., 2002; Abubakar et al., 2009). The etiological agent, peste des petits ruminants virus (PPRV), has only one known serotype with different geographical distributions, but at least four distinct genetic lineages (I–IV) have been described based on alignments of
N gene or F gene nucleotide sequences (Shaila et al., 1996; Kwiatek et al., 2007). Virus transmission is predominantly via the ocular and respiratory tracts, and virus particles are excreted in oculonasal discharges, saliva and faeces. There were no records of PPRV in China until the pathogen first emerged in the Ngari region of south-western Tibet, China, in 2007 (Zhiliang Wang et al., 2007). From this outbreak, we isolated a PPRV strain, named China/Tib/07. Phylogenetic analysis identified this strain as a member of lineage IV and most closely related to viruses currently circulating in neighbouring countries of southern Asia. Because PPR is a newly emerging viral disease in China, no studies are yet available regarding the incubation period, virus excretion, clinical course and pathogenesis of infection. Some earlier experimental studies on PPRV infection in goats employed different virus strains and addressed the immediate postinfection period (Bundza et al., 1988; Couacy-Hymann et al., 2007b, 2009; Ezeibe et al., 2008; Hammouchi et al., 2012). The objective of the present study was to evaluate the time course of excretion of this new PPRV field isolate
© 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 60 (Suppl. 2) (2013) 63–68
63
Antibody Dynamics of PPRV in Goats
W. Liu et al.
in China and the dynamics of antibody production following challenge of a local breed of Laoshan milch goats. Materials and Methods Cells, media and virus The Vero cell line maintained in Dulbecco’s modified Eagle medium (DMEM; Gibco, Grand Island, NY, USA) containing 10% inactivated calf serum was used for virus propagation and virus neutralization tests. The PPRV field isolate China/Tib/07 was initially isolated from the rectal tissues of an infected goat from Tibet, China. The fifth passage of the virus on Vero cells was used in this study; the vaccine strain used was the Africa vaccine strain Nigeria 75/1. Viruses were propagated and titreed on Vero cells; titres were expressed as the median tissue culture infective dose (TCID50). Animals Twelve Laoshan milch goats 1–2 years of age were employed. Before the experiments, the animals were confirmed to be seronegative for anti-PPRV antibodies using a monoclonal antibody-based c-ELISA for the presence of antibodies against the nucleoprotein of the virus. All animal experiments were conducted in a high-security containment facility at the Class III Biosafety animal lab of the China Animal Health and Epidemiology Center. Different groups of animals were maintained in separate rooms with separate feeding and drinking tanks; animals were always kept indoors. Each animal was treated with ivermectin (0.025 ml/kg), an anti-parasite drug, during the acclimatization period (15 day) before PPRV inoculation. Experimental designs Virus challenge The twelve goats were divided randomly into three groups, A–C. Group A animals (numbered 1# to 4#) were inoculated with the field isolate of China/Tib/07, group B animals (numbered 5# to 8#) with the vaccine strain of Nigeria 75/ 1; group C animals (numbered 9# to 12#) acted as a control. A single subcutaneous inoculation of 1 ml of the virus suspension at a concentration of 103 TCID50/ml was inoculated into group A animals, a dose which from our previous studies on the virulence of China/Tib/07 generally gave rise to productive infection (X. Wu, Z. Wang, J. Li, and X. Han, unpublished data). The same dose of Nigeria 75/1 was administered to group B. All animals were managed under similar conditions; a different attendant was assigned to each room to feed and water the infected and control animals. Animals were examined daily, and rectal temperature and other physical changes were recorded daily. 64
Sample collection Nasal, ocular and oral swabs were collected from all challenged animals from day 1 to day 40 pi. Animals of group C were sampled at 5 day intervals. All swabs were stored at 40°C until examined. Blood samples without anticoagulant were collected from the jugular vein at regular intervals. Serum samples were separated by centrifugation at 5000 g for 10 min and stored at 40°C until testing. Detection of viral excretion by qRT-PCR RNA from samples of nasal, ocular and oral swabs of day 1 to day 40 pi was extracted using the High Pure Viral RNA Kit (Roche) according to the manufacturer’s protocol. A specific TaqMan-based qRT-PCR amplification and detection reaction were performed as described previously, and samples with a cycle threshold (Ct) ≤40 for detection were considered to be nucleic acid positive (Bao et al., 2008). In all experiments, RNase-free water and total RNA extracted from Nigeria 75/1-infected Vero cells culture served as negative and positive controls, respectively. Confirmation of virus excretion by sequencing Primer pairs NP3/NP4 were used to amplify a 350-bp fragment from the PPRV N gene using a one-step RT-PCR technique (Invitrogen, Grand Island, NY, USA) (CouacyHymann et al., 2002); negative and positive controls were as described in the previous section. PCR products were analysed by 1.5% agarose gel electrophoresis. Three separately amplified fragments from animals of each challenge group were randomly selected for sequencing and PPRV strain analysis. Serological testing Peste des petits ruminants virus antibodies in the serum from challenged and vaccinated goats were measured using an approved c-ELISA Kit (CIRAD, Montpellier, France) following recommended protocols, and the samples with PI >50 were considered to be seropositive (Libeau et al., 1995; Swai et al., 2009). Virus neutralization tests Sera from days 10, 14, 16, 30, 60 and 240 pi were used for the detection of neutralizing antibody. Aliquots (50 ll) of heat-inactivated sera were subjected to serial twofold dilutions. Serum dilutions were mixed in multiple replicates with an equal volume of medium containing 100 TCID50 of the homologous PPRV strain in 96-well plates and incubated for 1 h at 37°C. To each well was added 100 ll of Vero cell suspension (2–5 9 105 cells/ml), and incubation was continued for 7 days. Cytopathic effects (CPE) were assessed by light microscopy. The antibody titre in each case was taken as the highest serum dilution that neutralized virus CPE in more than 50% of replicates. Controls
© 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 60 (Suppl. 2) (2013) 63–68
W. Liu et al.
Antibody Dynamics of PPRV in Goats
included cell control (no serum, no virus) and virus control (no serum). Titres of
