Planetary boundaries and Veterinary Services.

National Veterinary Services (NVS) play a crucial role in animal health, production and welfare. They are also intimately involved with safeguarding global health security and the health of the planet. Climate change is just one of the nine planetary boundaries (PBs), i.e. Earth system processes, that can be used to monitor the vital signs of our living planet. In this paper, the authors identify the positive and negative impacts of humaninduced management of aquatic and terrestrial animals in relation to these PBs. In the context of NVS, the authors provide an overview of the real and potential impacts of NVS policies on Earth systems and offer suggestions as to how new sustainability paradigms may assist with reviewing and revising NVS mandates and facilitating stakeholder engagement. Opportunities are proposed for the World Organisation for Animal Health to contribute to the global debate on the role of aquatic and terrestrial animal agriculture and wildlife in sustainable development. In addition, the paper suggests that a wider debate is required in relation to recent significant increases in domestic animal populations and PBs. Intersectoral and interdisciplinary collaboration are required to achieve the transformation of the framework in which NVS operate. While such transformations cannot be driven by the veterinary profession alone, veterinarians have proven very effective operators in the One Health arena. By building on these intersectoral linkages, it will be possible for our profession and NVS to actively contribute to the crucial discussions and transformations required to pull Earth system metrics back within safe boundaries.

Les Services vétérinaires nationaux jouent un rôle crucial dans les domaines de la santé animale, de la production animale et du bien-être des animaux. Mais ils sont aussi fortement engagés dans la protection de la sécurité sanitaire mondiale et de la santé de la planète. Le changement climatique est l'une des neuf frontières planétaires existantes, c'est-à-dire les neuf processus du système terrestre qui peuvent nous aider à surveiller les signes vitaux de notre planète vivante. Les auteurs font le point sur les impacts positifs et négatifs de la gestion par l'homme des animaux aquatiques et terrestres dans la perspective de ces frontières planétaires. S'agissant des Services vétérinaires nationaux, les auteurs donnent un aperçu de l'impact réel des politiques des Services vétérinaires sur les systèmes terrestres ainsi que des possibilités en la matière et font quelques propositions pour que les mandats de ces Services soient repensés et modifiés et la participation des parties prenantes facilitée à la lumière des nouveaux paradigmes de durabilité. Ils évoquent plusieurs occasions au cours desquelles l'Organisation mondiale de la santé animale (OIE) pourrait contribuer aux discussions à l'échelle mondiale sur le rôle des animaux terrestres et aquatiques, tant d'élevage que sauvages, dans le développement durable. En outre, les auteurs estiment qu'un débat plus général est nécessaire concernant l'augmentation récente et significative des populations d'animaux domestiques et les frontières planétaires. Une collaboration intersectorielle et interdisciplinaire sera nécessaire pour transformer le cadre dans lequel interviennent les Services vétérinaires nationaux. Certes, une transformation de cette nature ne peut être menée par la profession vétérinaire seule, mais les vétérinaires ont déjà fait preuve de l'efficacité de leurs capacités opérationnelles dans l'arène Une seule santé. En s'appuyant sur ces liens intersectoriels, la profession vétérinaire et les Services vétérinaires nationaux seront à même de contribuer activement aux discussions cruciales ainsi qu'aux transformations indispensables pour ramener les variables mesurables de la Terre à l'intérieur de frontières sûres.

La función de los Servicios Veterinarios nacionales, además de ser crucial para la sanidad, la producción y el bienestar animales, guarda estrecha relación con la salvaguarda de la seguridad sanitaria mundial y la salud del planeta. El cambio climático es solo uno de los nueve límites planetarios, esto es, procesos del sistema terrestre, que cabe utilizar para seguir de cerca los signos vitales del ser vivo que es nuestro planeta. Los autores exponen los efectos tanto positivos como negativos que en relación con estos límites planetarios trae consigo la gestión antrópica de animales acuáticos y terrestres. Por lo que respecta a los Servicios Veterinarios nacionales, los autores presentan a grandes líneas las repercusiones que las políticas de estos Servicios Veterinarios tienen o pueden tener en los sistemas terrestres y proponen fórmulas para que los nuevos paradigmas de la sostenibilidad ayuden a examinar y revisar el mandato de los Servicios Veterinarios nacionales y a facilitar la participación de los demás interlocutores. También señalan las oportunidades que tiene ante sí la Organización Mundial de Sanidad Animal para contribuir a las deliberaciones mundiales sobre la función de la producción de animales acuáticos y terrestres y de la gestión de la fauna silvestre con vistas al desarrollo sostenible. Los autores, además, consideran necesario un debate más amplio en relación con el reciente y considerable crecimiento de las poblaciones de animales domésticos y los límites planetarios. Para lograr una transformación de las coordenadas en las que operan los Servicios Veterinarios de los países se requiere una colaboración tanto intersectorial como interdisciplinar. Aunque estos cambios no pueden venir únicamente de la mano de la profesión veterinaria, la labor de los veterinarios en el ruedo de Una sola salud ha demostrado que son agentes muy eficaces. Profundizando en estos vínculos intersectoriales, será posible que nuestra profesión y los Servicios Veterinarios nacionales contribuyan activamente a las cruciales deliberaciones y transformaciones que se necesitan para que el valor de los parámetros del sistema terrestre vuelva a situarse dentro de los márgenes de seguridad.

Incidence of contamination of live bird markets in Bangladesh with influenza A virus and subtypes H5, H7 and H9.

In the absence of robust active surveillance of avian influenza viruses (AIV) affecting poultry in South Asian countries, monitoring of live bird markets (LBMs) can be an alternative. In a longitudinal study of 32 LBM, five environments were sampled as follows: market floor, stall floor, slaughter area, poultry holding cage and water used for meat processing. Samples were taken monthly for 5 months, September 2013-January 2014. Incidence rates (IRs) of LBM contamination with AIV and its subtypes H5, H7 and H9 were assessed. In 10 of the LBM selected, biosecurity measures had been implemented through FAO interventions: the other 22 were non-intervened. Standard procedures were applied to detect AIV and three subtypes in pooled samples (1:5). An LBM was considered positive for AIV or a subtype if at least one of the pooled samples tested positive. The incidence rates of LBM contamination with AIV, H5, H7 and H9 were 0.194 (95% confidence interval (CI) 0.136-0.276), 0.031 (95% CI 0.013-0.075), 0 and 0.175 (95% CI 0.12-0.253) per LBM-month at risk, respectively. The log IR ratio between the FAO-intervened and non-intervened LBM for contamination with AIV was -0.329 (95% CI -1.052 to -0.394, p = .372), 0.598 (95% CI -1.593 to 2.789, p = .593) with subtype H5 and -0.500 (95% CI -1.249 to 0.248, p = .190) with subtype H9, indicating no significant difference. The results obtained suggest that both H5 and H9 were circulating in LBM in Bangladesh in the second half of 2013. The incidence of contamination with H9 was much higher than with H5.

Biosecurity and Circulation of Influenza A (H5N1) Virus in Live-Bird Markets in Bangladesh, 2012.

Bangladesh has been considered as one of the five countries endemic with highly pathogenic avian influenza A subtype H5N1 (HPAI H5N1). Live-bird markets (LBMs) in south Asian countries are believed to play important roles in the transmission of HPAI H5N1 and others due to its central location as a hub of the poultry trading. Food and Agriculture Organization (FAO) of the United Nations has been promoting improved biosecurity in LBMs in Bangladesh. In 2012, by enrolling 32 large LBMs: 10 with FAO interventions and 22 without assistance, we assessed the virus circulation in the selected LBMs by applying standard procedures to investigate market floors, poultry stall floors, poultry-holding cases and slaughter areas and the overall biosecurity using a questionnaire-based survey. Relative risk (RR) was examined to compare the prevalence of HPAI H5N1 in the intervened and non-intervened LBMs. The measures practised in significantly more of the FAO-intervened LBMs included keeping of slaughter remnants in a closed container; decontamination of poultry vehicles at market place; prevention of crows' access to LBM, market/floor cleaning by market committee; wet cleaning; disinfection of floor/poultry stall after cleaning; and good supply of clean water at market (P < 0.05). Conversely, disposal of slaughter remnants elsewhere at market and dry cleaning were in operation in more of the FAO non-intervened LBMs (P < 0.05). The RR for HPAI H5N1 in the intervened and non-intervened LBMs was 1.1 (95% confidence interval 0.44-2.76), suggesting that the proportion positive of the virus in the two kinds of LBM did not vary significantly (P = 0.413). These observations suggest that the viruses are still maintained at the level of production in farms and circulating in LBMs in Bangladesh regardless of interventions, albeit at lower levels than in other endemic countries.

Factors limiting the health of semi-scavenging ducks in Bangladesh.

Duck rearing is well suited to coastal and lowland areas in Bangladesh. It is an important component of sustainable livelihood strategies for poor rural communities as an additional source of household income. An epidemiological study was conducted during January 2005-June 2006 on 379 households in Chatkhil of the Noakhali District, Bangladesh which were using the recently devised "Bangladesh duck model". The overall objective of the study was to identify factors that significantly contributed to mortality and constrained productivity and to generate sufficient knowledge to enable establishment of a disease surveillance system for household ducks. The overall mortality was 15.0% in Chatkhil, with predation causing a significantly higher mortality compared with diseases (p < 0.001). Common diseases were duck plague and duck cholera. Morbid ducks frequently displayed signs associated with diseases affecting the nervous and digestive systems. Haemorrhagic lesions in various organs and white multiple foci on the liver were frequently observed in dead ducks. Epidemiological analysis with a shared frailty model that accounted for clustering of data by farm was used to estimate the association between survival time and risk factors. The overall mortality rate due to disease was significantly lower in vaccinated than in non-vaccinated ducks in all zones except zone 2 (p < 0.001). Only vaccinated ducks survived in zone 1. In conclusion, duck mortality and untimely sale of ducks appeared to be important constraints for household duck production in Chatkhil. Vaccination against duck plague appears to be an effective preventive strategy in reducing the level of associated duck mortality. A successful network was established amongst farmers and the surveillance team through which dead ducks, with accompanying information, were readily obtained for analysis. Therefore, there is an opportunity for establishing a long-term disease surveillance programme for rural ducks in Chatkhil of the Noakhali District of Bangladesh.

Monitoring the health and production of household Jinding ducks on Hatia Island of Bangladesh.

Duck rearing is an important component of sustainable living in poor rural communities, especially as a source of subsistence. A study was conducted on 118 households (N = 1,373 Jinding ducks, Anas platyrhynchus) from December 2002 to February 2004 on Hatia Island in Bangladesh with the aim of identifying the factors that limit the health and production of Jinding ducks. Overall duck mortality was 29.3%, with disease (19.7%) being a more significant factor than predation (9.6%; p = 0.001). Duck mortality also varied significantly among study zones (p < 0.001). Common diseases were duck plague (21.1%) and duck cholera (32.1%). Helminth infection was prevalent, with endemic trematode (Prosthogonimus spp., Trichobilharzia spp., Echinostoma spp.) and nematode (Cyathostoma bronchialis, Amidostomum anseris, Heterakis gallinarum, Capillaria spp., and Echinuria spp.) infections and epidemic cestode infections due to Hymenolepsis setigera. The median egg production rate per duck per household was 93 for a 6-month laying period. The odds of diminished egg production (average ≤ 93 eggs per duck per household for a 6-month laying period) was 25.4 times higher in ducks that were kept in traditional duck houses (p < 0.001) and 14.2 times higher in ducks that experienced delays in the onset of sexual maturity (days 191 and 280; p < 0.001). Ducks that were provided snails for a shorter period of time over the laying period were 18.2 times more likely to produce fewer eggs than their longer fed peers (p = 0.002). In conclusion, duck mortalities due to diseases and predation and parasitic infections appear to be common constraints on household duck production on Hatia Island. Additionally, improving duck housing and providing longer nutritional supplementation with snails increased the production capabilities of household-raised Jinding ducks on Hatia Island.

Mortality rate and clinical features of highly pathogenic avian influenza in naturally infected chickens in Bangladesh.

A retrospective study was performed to assess the mortality rates and clinical signs in commercial and backyard chickens naturally infected with the highly pathogenic avian influenza (HPAI) H5N1 virus in Bangladesh. All of the 33 commercial and 25 backyard farms, recorded during the first wave of HPAI outbreaks (January to November 2007) in Bangladesh, were enrolled in this study. The farm profile data, stock information and major clinical signs recognised by the farmers and/or farm attendants, were collected through a prototype questionnaire. The investigating veterinarians' retrospective appraisals of the clinical signs were recorded by interview. After the clinical onset, the incidence rates of mortality in breeder, commercial broiler, commercial layer and backyard chickens were, respectively: 0.0215, 0.0341, 0.0179 and 0.0703 per chicken-day at risk. The four major clinical signs observed were cyanotic combs and wattles, oedema of the head and face, drowsiness and huddling and ecchymotic discoloration of the leg shanks. The first sign (cyanotic combs and wattles) was equally observed in chickens from commercial and backyard farms (p = 0.44) but the second (oedema of the head and face) was more common in backyard farms (p = 0.03).

Factors limiting traditional household duck production in Bangladesh.

A cross sectional survey of duck production was carried out in 2002 on 771 traditional, semiscavenging household duck farms on the coastal Island of Hatia. We determined the socioeconomic characteristics of duck farmers and their management systems, identified the factors associated with egg production, and measured the level of selected duck diseases and current preventive strategies. Household family size varied from 1 to 14 individuals and women were the main caretakers of ducks. Around 34% of keepers were illiterate. Most duck products (eggs and meat; 85%) were sold at the local market. Duck houses were poorly ventilated and a variety of bedding materials were used. Feed was available in nearby scavenging areas; however, additional feed was frequently supplied by farmers. Almost all farmers (96%) ranked the rainy season as the best time for rearing ducks due to greater feed availability. The annual egg production was 79 eggs per layer with a weight of 48 g and a hatchability rate of 87%. Egg production varied by zone (p < 0.05). The odds of suboptimal egg production was 0.5 times lower in educated farmers (p = 0.001). The odds of suboptimal egg production was 2.5 times more likely in ducks that attained sexual maturity at >22 weeks (p<0.001). Most farmers ranked duck plague as the most important disease, followed by duck cholera, botulism, and duck viral hepatitis. Preventive vaccination was sporadic and used by few farmers (28%). There are significant opportunities for improved duck production on the Island of Hatia and in Bangladesh generally.

Risk factors for infection with highly pathogenic influenza A virus (H5N1) in commercial chickens in Bangladesh.

A matched case-control study was carried out to identify risk factors for highly pathogenic avian influenza A virus (subtype H5N1) infection in commercial chickens in Bangladesh. A total of 33 commercial farms diagnosed with H5N1 before September 9, 2007, were enrolled as cases, and 99 geographically matched unaffected farms were enrolled as control farms. Farm data were collected using a pretested questionnaire, and analysed by matched-pair analysis and multivariate conditional logistic regression. Two factors independently and positively associated with H5N1 infection remained in the final model. They were 'farm accessible to feral and wild animals' (odds ratio [OR] 5.71, 95 per cent confidence interval [CI] 1.81 to 18.0, P=0.003) and 'footbath at entry to farm/shed' (OR 4.93, 95 per cent CI 1.61 to 15.1, P=0.005). The use of a designated vehicle for sending eggs to a vendor or market appeared to be a protective factor (OR 0.14, 95 per cent CI 0.02 to 0.88, P=0.036).

Serosurvey of five viruses in chickens on smallholdings in Bangladesh.

A serologic survey was undertaken in chickens in smallholdings in Bangladesh for avian influenza A virus (AIV), egg drop syndrome '76 virus (EDS'76V), infectious bronchitis virus (IBV), Newcastle disease virus (NDV) and reovirus (RV) in three phases: January 2002-May 2003, September 2003-August 2004, and August 2005-March 2006. Four hundred thirty-six sera collected in the 2nd phase, 295 in the first phase, 755 in the 1st plus 2nd phases and 295 in the 1st phase were investigated for AIV, EDS'76V, IBV and RV, respectively, using enzyme linked immunosorbent assays. All 854 sera collected in the three phases were screened for NDV using hemagglutination inhibition test. In chickens 20% were seropositive to AIV, 3% to EDS'76V, 74% to IBV, 88% to NDV, and 47% to RV. The seroprevalence in flocks was 23% to AIV, 6% to EDS'76V, 79% to IBV, 89% to NDV and 56% to RV. Twenty-five percent chickens had > or = 10log(2)HI titers to NDV.

Survivability and causes of loss of broody-hen chicks on smallholder households in Bangladesh.

We determined the flock sizes and rates of loss caused by different factors in broody-hen chicks (BHC) up to 60 days of age on 600 randomly selected smallholdings in Bangladesh. The smallholders were beneficiaries of a village poultry production chain called 'Smallholder Livestock Development Project-2' (SLDP-2) which was undertaken with the financial assistance of the Danish International Development Agency (DANIDA). For estimating survival time of BHC, we observed chicks in 80 smallholdings. SLDP-2 aims at ameliorating poverty among women by poultry rearing at village level; in total, 104,000 key rearers, constituting 96% of all of the beneficiaries of the SLDP-2 area, were enrolled in 26 upazilas (a lower administrative unit of Bangladesh). A key rearer is a smallholder who rears at least five 'Sonali' (RIR x Fayoumi) and some indigenous (desi) chickens in a semi-scavenging system. Sonali chickens are supplied from the development project, and have higher egg production while the broodiness of the desi hens is exploited to get chicks hatched for future stocks; thus, the chicks hatched and reared to 60 days old at key rearers' households are called BHC. In this study 32% of the smallholders had BHC each month. At the beginning of a month, the median number of chicks in a flock was 8, and the mean survival time was 50.5 days. Incidence rates of loss of BHC from disease, predation, selling and slaughtering were 0.102, 0.086, 0.009 and 0.002 per chick-month at risk, respectively. The major predators were crows, mongooses and eagles with incidence rates of loss being 0.018, 0.016 and 0.010 per chick-month at risk, respectively. Colibacillosis (both single and mixed infections) contributed to the death of 21% of dead BHC collected; Newcastle disease and salmonellosis contributed to the next highest (14 and 12%) proportional mortalities.

Causes of loss of Sonali chickens on smallholder households in Bangladesh.

In a 1-year-long prospective longitudinal study, we determined the causes of loss of 'Sonali' (male symbol Rhode Island Red x female symbol Fayoumi) chickens at key-rearers' households of the smallholder livestock development project-2 (SLDP-2) area in Bangladesh. A key rearer is a smallholder of chickens in the 'village poultry-production chain' (undertaken by SLDP-2 under the financial assistance of the DANIDA) who rears at least five Sonali and some 'Deshi' (non-descriptive and indigenous) chickens in their homesteads based on semi-scavenging system. The aim of this program is to ameliorate poverty, especially among women. Two co-ordination centers (set at the Potuakhali and Noakhali districts) supervised the development activities. We selected two upazilas (lower administration units) randomly from each of the two districts and in every selected upazila, we selected at random 125 key-rearer households. Incidence rates of loss of Sonali chickens from disease, predation, selling and slaughtering were 0.025, 0.023, 0.081 and 0.039 per bird-month at risk, respectively. The major predators of Sonali chickens in the study area were foxes, a kind of wild cat (Felis chaus), mongooses and human thieves. Colibacillosis (both single and mixed infections) had a contributory role in the death of 28% of dead Sonali birds collected for diagnosis; salmonellosis, Newcastle disease and internal parasites contributed to the next highest (14, 11and 10%) proportional mortalities.

A longitudinal study of the incidence of major endemic and epidemic diseases affecting semi-scavenging chickens reared under the Participatory Livestock Development Project areas in Bangladesh.

A 17-month (from January 2002 to May 2003) longitudinal study was undertaken to elucidate the epidemiology of important endemic and epidemic diseases affecting semi-scavenging chickens reared in the Participatory Livestock Development Project area in Bangladesh. This project was implemented in 17 northern and north-central districts of Bangladesh, under which 361 839 rural poor people were assisted to undertake poultry-rearing activity as a tool of poverty reduction. Of the total beneficiaries 93% were "key rearers". A key rearer is defined as a beneficiary who generally rears 10 to 13 hens in a semi-scavenging system with little additional feed supply. Households of 650 key rearers and some chick rearers were observed. During the study period 1227 birds, which belonged to different age, breed and sex categories, were found dead as a result of disease occurrence. From every dead bird organ samples such as the liver, heart, spleen, brain lung, trachea and bursa of Fabricius were collected. The incidence rate of mortality was 0.01976 per bird-months at risk. Of the total deaths 58.44% had single or mixed type of infections. Newcastle disease had the highest proportional mortality rate (15.81%). The proportional mortality caused by fowlpox, fowl cholera, salmonellosis, colibacillosis, aspergillosis, infectious bursal disease, mixed infections and undiagnosed cases were 8.96%, 6.76%, 7.09%, 6.93%, 0.33%, 2.04%, 10.51% and 41.56%, respectively. Newcastle disease affected a significant higher proportion (18.81%) of birds older than 60 days of age (P=0.00). Younger birds (age < or = 60 days) had a higher proportional mortality due to fowlpox and infectious bursal disease than older birds (P=0.00). Sonali (male Rhode Island Red x female Fayoumi) birds reared under the semi-scavenging system had a higher infection rate with Newcastle disease virus compared with indigenous and Fayoumi birds (P=0.00). Fowlpox was more prevalent in Fayoumi birds compared with Sonali. Surprisingly, Newcastle disease was more common in the vaccinated birds rather than the unvaccinated birds.

cDNA cloning and gene expression of lebocin, a novel member of antibacterial peptides from the silkworm, Bombyx mori.

A cDNA encoding lebocin, a novel member of insect antibacterial peptides, was isolated from the fat body cDNA library of Bombyx mori larvae immunized with Escherichia coli. The cDNA was 844 bp long and had an open reading frame (ORF) containing a probable signal peptide (16 amino acids), a putative prosegment (104 amino acids) and a mature peptide (32 amino acids) followed by 27 additional amino acids at its carboxyl-terminus. Northern blot analysis showed that lebocin gene expression was inducible by bacterial injection, occurred tissue-specifically in the fat bodies and continued at least for 48 h post-infection. These results suggest that lebocin has a unique precursor structure and shows typical gene expression pattern as insect antibacterial peptide.

Occurrence of novel types of nitric oxide synthase in the silkworm, Bombyx mori.

Nitric oxide (NO) synthase activity was detected in fat body and the Malpighian tubles of the silkworm, Bombyx mori. Main NO synthase activity in the fat body was Ca(2+)/calmodulin-dependent, inducible by bacterial lipopolysaccharide (LPS) and required NADPH, FAD, FMN, dithiothreitol (DTT) and tetrahydrobiopterin (BH4) as cofactors for the full expression of the activity. The Malpighian tubles contained two types of NO synthase. One was Ca(2+)-independent, calmodulin-dependent and constitutive and the other was Ca(2+)-dependent and constitutive. The former NO synthase required the same cofactors as fat body NO synthase. The activity of Malpighian tuble NO synthases increased dramatically at the end of the last instar period, just prior to spinning. These results indicate that B. mori contains new types of NO synthase, suggesting the wide distribution and different characteristics of this enzyme among vertebrates and invertebrates.

Immunogenicity of wild-type and vaccine strains of Japanese encephalitis virus and the effect of haplotype restriction on murine immune responses.

The Chinese live attenuated Japanese encephalitis (JE) virus vaccine clone SA14-14-2 produced in primary hamster kidney (PHK) cells has been adapted to primary dog kidney cells (PDK) for use as a live attenuated human vaccine. In this study we have compared the immunogenicity in mice of SA14-14-2 (PDK) and SA14-14-2 (PHK); also included was the wild-type parent to the vaccine clones, SA14, and another wild-type JE virus strain Nakayama (original). It was found that Balb/c (H-2d) mice given a single dose of 10(3) or 10(6) p.f.u. of live SA14-14-2 (PHK) virus elicited a superior neutralizing (N) antibody response as compared to the same dosages of live SA14-14-2 (PDK) virus. However, if the vaccine clones were inactivated and administered in a two-dose regime the N antibody response elicited was similar for the two viruses. This observation may be explained by differences in the replication efficiency in vivo of the respective vaccine clones. The humoral immune response to all the virus strains in this study elicited by different inbred mouse strains each carrying a discrete haplotype (Balb/c (H-2d), C3H (H-2k), C57BL/6 (H-2b)) were also assessed using haemagglutination inhibition (HAI) and N assays. Viruses were shown to elicit patterns of high and low N-antibody response depending on the major histocompatibility complex (MHC) make-up of the mouse strains. However, the patterns did not necessarily coincide when HAI and N titre reactivity patterns were compared for the same virus strain.

In vitro homotypic and heterotypic interference by defective interfering particles of West Nile virus.

Defective interfering (DI) particles of the flavivirus West Nile (WN) were generated after as few as two high multiplicity serial passages in Vero and LLC-MK2 cells. Six cell lines (Vero, LLC-MK2, L929, HeLa, BHK-21 and SW13) were used to assay interference by DI particles in a yield reduction assay. Interference was found to vary depending on the cell type used. The highest levels of interference were obtained in LLC-MK2 cells, whereas no detectable effect was observed in BHK-21 and SW13 cells. The ability of DI virus to be propagated varied depending on the cell line used; no detectable propagation of DI virus was observed in SW13 cells. Optimum interference was obtained following co-infection of cells with DI virus and standard virus at a multiplicity of 5. Interference between DI and standard viruses occurred only when they were co-infected or when cells were infected with DI virus 1 h before standard virus. Investigation of heterotypic interference by DI particles of WN virus strains from Sarawak, India and Egypt revealed that interference was dependent on the strain of WN virus or flavivirus used as standard virus. A measure of the similarity between five strains of WN virus and other flaviviruses was made on the basis of interference by DI viruses, and was found to be similar to that based on haemagglutination inhibition tests using a panel of monoclonal antibodies.