Emerging Infectious Diseases

Lecture 9 & Sample Questions

X.J. Meng

Emerging Viral Diseases of Swine

Lecture Notes: Nipah Virus (NiV)

The Disease. A new pig disease characterized by pronounced respiratory and neurologic syndrome and sometimes sudden death of sows and boars spread among pig farms in Peninsular Malaysia during late 1998 to 1999. The pig disease appeared to be associated with viral encephalitis epidemic in pig farm workers. The disease is referred to as Barking Pig Syndrome (BSP) because of the characteristic loud barking cough, which differs from other common swine respiratory diseases. Based upon the characteristic clinical symptoms, porcine respiratory and encephalitis syndrome is suggested as the technical name for the disease.

Etiology. The Japanese encephalitis virus (JEV) was originally thought to be the cause of the disease. However, measure to control JEV did not decrease the disease incidence, and JEV was soon ruled out to be the cause of the viral encephalitis epidemic in pig handlers. An apparently new virus, designated as “Nipah virus (NiV)”, was identified and confirmed to be the cause of both the human and the pig disease. The name “Nipah” came after the first isolation of the new virus from a human in the village “Sungai Nipah” in the State of Negeri Semilan. NiV is an enveloped, nonsegmented, single negative-stranded RNA virus in the family of the Paramyxoviridae. NiV is related to, but distinct from, the Hendra virus isolated in 1994 in Australia. NiV and Hendra virus share about 78% nucleotide sequence identity in the N gene. NiV can be easily cultivated in cell cultures.

Epidemiology. The epidemic is believed to begin in the State of Perak and spread south to the States of Negeri Sembilan and Selangor. Movement of pigs is believed to be the mode of transmission among these States. The mode of transmission between farms is not known but sharing boar semen and domestic animals (cats and dogs) may contribute the spread of the disease. Transmission between pigs is likely due to direct contact with infected pigs or with contaminated excretory and secretary fluid (urine, salia, pharyngeal and bronchial secretions). Pigs could be experimentally infected by oral route inoculation and virus was excreted oronasally. Under experimental conditions, the virus spread quickly to contact pigs. During the outbreaks in Peninsular Malaysia, pigs, dogs and humans were all infected with the virus. Other animals such as cats, horses and goats could also be infected. Fruit bats have neutralizing antibody to NiV and might be a reservoir for the virus. Human-to-human transmission of NiV has not been documented.

Clinical Presentations.

Pigs: Four weeks to 6-month-old pigs primarily manifest acute febrile respiratory illness such as rapid and labored respiration, loud baking cough. Mortality is low, <1 to 5%, but morbidity is up to 100%. Sows and boars primarily manifest neurological disease and may be accompanied by sudden death, labored breathing, increased salivation, nasal discharge, early first trimester abortion, etc. Piglets often show open-mouth breathing, leg weakness with muscle tremors and neurological twitches.

Humans. During March 1999, febrile encephalitic and respiratory illnesses associated with NiV infection among pig handlers were reported in Malaysia and Singapore. As of June, 1999, 265 cases of febrile encephalitis were reported in Malaysia with 105 deaths. The apparent source of infection in humans continues to be exposure to infected pigs. In Singapore, during March 13-19, 11 abattoirs workers developed febrile encephalitis and respiratory illnesses associated with NiV infection with 1 death. Febrile encephalitis continues to be reported in Malaysia but has decreased following the massive culling of pigs in outbreak areas. No new cases of disease associated with NiV infection were reported in Singapore since the closing of abattoirs on March 19, 1999.

Pathological findings. Most affected pigs had severe lung lesions with varying degree of lung consolidation, emphysema and hemorrhages. The bronchi and trachea may be filled with fluid with or without blood. Brain tissue may have generalized congestion and edema. Occasionally, kidney showed congestion on the surface and cortex. Other organs apparently are not affected. Microscopically, the characteristic and main lesion is a moderate to severe interstitial pneumonia with hemorrhages and syncytial cell formations in the endothelial cells of the lung blood vessels. Brain tissues had non-suppurative meningitis with gliosis.

Diagnosis. An ELISA has been developed to diagnose the disease. Other diagnostic assays such as PCR, virus isolation etc. can only be performed in BL-4 laboratories.

Prevention and Control. The culling of all pigs in the infected areas has successfully controlled the human epidemic in the affected States of Peninsular Malaysia. From Feb. 28 to April 26, 1999, an estimated total of 1 million pigs from about 900 pig farms were destroyed. With the availability of an ELISA test, pigs can now be screened and monitored for possible NiV infection. Pig farmers and swine veterinarians should be educated about the clinical signs of the disease and the risk of zoonotic infections. Pig handlers should avoid direct contacts with affected pigs. Disinfectants such as sodium hyperchlorite, Lysol etc, are recommended for use in pig farms.

References.

CDC (1999), Outbreaks of Hendra-like virus in Malaysia and Singapore, 1998-1999. MMWR 48:265-269.

CDC (1999). Update: outbreak of Nipah virus-Malaysia and Singapore, 1999. MMWR 48:335-337.

Chua KB, Goh KJ, Wong KT, Kamarulzaman A, Tan PS, Ksiazek TG, Zaki SR, Paul G, Lam SK, Tan CT (1999). Fatal encephalitis due to Nipah virus among pig-farmers in Malaysia. Lancet 1999 Oct. 9;354 (9186):1257-1259.

Chua KB, Bellini WJ, Rota PA, Harcourt BH, Tamin A, Lam SK, Ksiazek TG, Rollin PE, Zaki SR, et al (2000). Nipah virus: a recently emergent deadly paramyxovirus. Science. 288:1432-1435.

Daniels, P (1999). Experimental infection of pigs and cats at CSIRO-AAHL: preliminary observations. A working paper for WHO meeting on zoonotic paramyxoviruses, Kuala Lumpur, Malaysia, July 19-21.

Field H, Yob J, Rashdi A, Morrissy C (1999). Nipah virus: the search for a natural reservoir. A working paper for WHO meeting on zoonotic paramyxoviruses, Kuala Lumpur, Malaysia, July 19-21.

Kerr JR (2000). Nipah virus. Infect Dis Rev. 2:53-54.

Lee KE, Umapathi T, Tan CB, Tjia HT, Chua TS, Oh HM, Fock KM, Kurup A, Das A, Tan AK, Lee WL (1999). The neurological manifestations of Nipah virus encephalitis, a novel paramyxovirus. Annals of Neurology. Sep; 46(3):428-432.

Lim CC, Sitoh YY, Lee KE, Kurup A, Hui F (1999). Meningoencephalitis caused by a novel paramyxovirus: an advanced MRI case report in an emerging disease. Singapore Medical Journal. May; 4095):356-358.

Lim CC, Sitoh YY, Hui F, Lee KE, Ang BS, Lim E, Lim WE, Oh HM, Tambyah PA, Wong JS, Tan CB, Chee TS (2000). Nipah viral encephalitis or Japanese encephalitis? MR finding in a new zoonotic disease. American Journal of Neuroradiology. Mar; 21(3):455-461.

Nor MNM, Gan CH, Ong BL (2000). Nipah virus infection of pigs in Peninsular Malaysia. Internet circulation of prepublication by USDA.

Paton NI, Leo Ys, Zaki SR, Auchus AP, Lee KE, Ling AE, Chew SK, Ang B, Rollin PE, Umapathi T, Sng I, Lee CC, Lim E, Ksiazek TG (1999). Outbreak of Nipah-virus infection among abattoir workers in Singapore. Lancet Oct 9;354 (9186):1253-1256.

Lecture Notes: porcine reproductive and respiratory syndrome virus (PRRSV)

History of PRRS. A mysterious and devastating swine disease, characterized by severe reproductive failure in sows, respiratory diseases in young pigs and influenza-like syndrome in grower-finisher pigs, was first noticed in 1987 in the U.S. The disease was often referred to as "Mystery Swine Disease” because of its unknown etiology. In late 1990, a similar disease was reported in Germany, and then rapidly recognized in most Western European countries. As the disease spread through the world, more and more names were acquired to describe this syndrome, such as porcine reproductive and respiratory syndrome (PRRS), swine infertility and respiratory syndrome (SIRS), porcine epidemic abortion and respiratory syndrome (PEARS), blue ear disease, Heko-Heko disease and so on. At the first international symposium on this disease at St. Paul, MN, PRRS was designated as the official name.

Etiology of PRRS. Initially, several pathogens were incriminated as the causative agents of PRRS, including swine encephalomyocarditis virus, hemagglutinating encephalomyelitis virus, porcine parvovirus, atypical swine influenza virus, porcine enterovirus, pseudorabies virus, porcine cytomegalovirus, etc. However, none of these suspected agents were proven to be the causative agent of PRRS. In 1991, a previously unrecognized virus, designated as the Lelystad virus (LV), was first isolated in the Netherlands from cases of PRRS by using swine alveolar macrophage (SAM). The LV caused PRRS in experimentally infected pigs and was demonstrated to be the causative agent of European PRRS. In the U.S., the PRRS virus (PRRSV) was first isolated in a continuous cell line in 1992, and subsequently demonstrated to be the causative agent of the U. S. PRRS. The LV and PRRSV are shown to be antigenically and structurally related.

Biochemical and Physical Characteristics of PRRSV. PRRSV is a small, enveloped, single positive-stranded RNA virus. Virus replication is inactivated after chloroform treatment. Electronmicrograph studies showed that PRRSV is pleomorphic, but mostly spherical, enveloped particles ranging from 50 to 80 nm in size. Its buoyant density is about 1.18 g/ml in cesium chloride gradient. The virus was heat labile in that it was inactivated at 37^oC in 24 hrs and at 56^oC in 20 minutes. However, the virus is stable at 4^oC for one month and at -70^oC for 4 months. The virus infectivity titers are reduced over 90% at pH levels < 5 or > 7. PRRSV does not hemagglutinate erythrocytes from many different species. PRRSV replicates in SAM and in at least three continuous cell lines, CL2621, MA-104 and CRL11171. The European PRRSV preferentially replicates in SAM cultures. Cytopathic effects (CPE) in SAM cultures began at about 24 to 36 hrs postinfection, and was characterized by clumping of the macrophages and cell lysis. The CPE in the three continuous cell lines, CL2621, MARC-145 and CRL11171, was slower to develop, appearing at about 2 to 3 days postinfection. The type of the CPEs in these cell lines was similar which is characterized by rounding and clumping of degenerating cells on top of the monolayers. Eventually, the degenerating cells will detach from the monolayers, which led to the disintegration of the monolayers. Immunofluorescence assay (IFA) showed that PRRSV replicates in the cytoplasm of these continuous cell lines.

Epidemiology of PRRSV. Retrospective studies by IFA indicated that the first positive cases of PRRS were detected in sera from Iowa collected in 1985, from Minnesota collected in 1986 and from east German herds in 1988 and 1989. PRRSV antibody was negative in all 1425 sera samples from Iowa collected in 1980. However, the earliest documented outbreaks of PRRS were in 1987. The incidence of clinical PRRS and seroprevalence increased rapidly since 1988. With the prevalence of seropositive herds in the U. S, the incidence of clinical cases, however, is decreasing. Pig movement and aerosols are both important in the transmission of PRRS. Airborne transmission is important in the local spread of PRRSV. High humidity, low temperature and low wind speed are ideal weather conditions for the airborne spread of PRRSV. Contact and intranasal route transmissions have been demonstrated. It has been shown that semen from viremic boars caused the inseminated gilts to seroconvert to PRRSV. Also, when semen from infected boars were inoculated into pigs intraperitoneally, the inoculated pigs seroconverted. There are no reports of human infection or diseases caused by PRRSV. It has also been shown that rodents are not susceptible to infection with PRRSV and are probably not a reservoir for PRRS. Since PRRSV infection also appears to be maintained in some all-in/all-out operations, it is possible that a non-porcine reservoir for PRRSV exists. Zimmermann et al. has demonstrated the susceptibility of four avian species (Mallard ducks, Muscovey ducks, Guinea fowl and Cornish-cross chickens) to PRRSV. PRRSV can be reisolated from these experimentally infected birds, but clinical signs were not detected and these birds did not seroconvert to PRRSV.

Genome Organization and Gene Expression of PRRSV. Meulenberg et al first cloned and sequenced the genome of LV, a European strain of PRRSV. Subsequently, Meng et al and Mardassi et al determined partial sequences of a U.S. strain and a Canadian strain of PRRSV. The genome of PRRSV is an ~ 15 kb positive strand RNA molecule that encodes eight overlapping open reading frames (ORFs) organized similarly to the ORFs of coronaviruses. ORFs 1a and 1b comprise about 80% of the viral genome and are predicted to encode the viral RNA polymerase. ORFs 2, 3 and 4 of PRRSV encode virion-associated proteins designated as GP2, GP3 and GP4, respectively. ORFs 5, 6 and 7 of PRRSV encode envelope (GP5), membrane (M) and nucleocapsid (N) proteins, respectively. The M protein is an unglycosylated protein which has the same hydrophobicity profile as that of equine arteritis virus (EAV) and LDV. The N protein is not N-glycosylated, although it contains 1 or 2 potential N-glycosylation sites. The order of PRRSV genes, 5'-viral polymerase (ORFs 1a/1b)-virion-associated proteins GP2 (ORF 2)-GP3 (ORF 3)-GP4 (ORF 4)-GP5 (ORF 5)-M (ORF 6)-N (ORF 7)-3', is the same as in EAV, LDV and simian hemorrhagic fever virus (SHFV). Therefore, PRRSV, along with EAV, LDV and SHFV, is now classified within a single genus Arterivirus in the family Arteriviridae in the order Nidovirales. The replication of PRRSV requires the production of at least six subgenomic mRNAs (sg mRNAs). These sg mRNAs, together with the genomic virion RNA, form a 3'-coterminal nested set. Each of these sg mRNAs contains a 5' common leader sequence of about 200 bp in size. The leader-mRNA junction sequence of PRRSV, in which the leader joins to the body of the sg mRNAs, is a conserved sequence motif of six nucleotides (UCAACC) or a highly similar sequence. It is generally believed that sg mRNAs are polycistronic, and only the ORF at the 5' end is translationally active and thus, each of the sg mRNAs is functionally monocistronic.

Genetic Variation and Genotypes. PRRSV is genetically heterogeneous. Extensive sequence variation was found between the European and the U. S. isolates as well as among U.S. isolates. The nucleotide sequence identity between LV and the U. S. isolates is 65-67% in ORF 2, 61-64% in ORF 3, 63-66% in ORF 4, and 61-63% in ORF 5. ORFs 6 and 7 genes are relatively conserved among the U.S. isolates or among the European isolates, but extensive genetic variation was observed in the ORFs 6 and 7 genes between European and U.S. isolates. It has been shown that the nucleotide sequence identity was 96-98% in ORF 2, 92-98% in ORF 3, 92-99% in ORF 4, and 90-98% in ORF 5 among six U.S. isolates. Kapur et al analyzed the nucleotide sequence of ORFs 2 to 7 of 10 U.S. PRRSV isolates, and found that the genetic distance ranges from 2.5 to 7.9% among these 10 U.S. isolates and is about 35% between LV and the U.S. isolates. The leader sequence of PRRSV strains also varies significantly. The 190 bp leader sequence of VR2332 strain is 31 bp shorter than that of LV, and has a sequence identity of 61% with that of LV. Like the leader sequence, the ORF1 gene sequence also differs extensively between the U.S. and the European strains. The ORF1a of VR2332 strain shares only about 55% nucleotide sequence identity with that of the LV strain. Considering the striking differences in the leader sequence and in all ORFs between European and North American strains, it is surprising that both European and North American strains cause a similar disease. Although the origin of PRRSV remains unknown, these data strongly suggest that the European and the North American strains have undergone divergent evolution on separate continents from a common ancestor. It has been speculated that European and North American PRRSV strains may have evolved from a LDV-like ancestor. However, the almost simultaneous emergence of PRRS in swine on two continents makes the theory of divergent evolution difficult to believe. More likely, the simultaneous emergence of PRRS on two continents might relate to global changes in commercial swine management and husbandry.

At least two distinct genotypes of PRRSV have been reported: the European genotype and the North American genotype. However, within each of the two major genotypes, several minor genotypes (or variants) were also identified. The N gene of PRRSV is relatively conserved. In contrast, the major envelope gene (GP5) of PRRSV exhibits greater genetic diversity. Interestingly, some PRRSV strains from Japan, China, Taiwan, Guatemala and three Danish strains are all clustered within the North American genotype. The three Danish strains, the Chinese strain (S1), a Canadian strain (PA8), and a strain from Nebraska (NE16244B) are all found to be closely related to the MLV vaccine and the vaccine strain VR2332. The three Danish strains of PRRSV are believed to originate from the MLV vaccine virus (VR2332) that was used in Danish swine herds. Phylogenetic trees confirm that these three Danish strains are most closely related to the MLV vaccine virus but are less related to other North American strains, indicating that the introduction of North American type of PRRSV in Denmark was due to the spread of vaccine virus VR2332. The origin of other strains that are closely related to the MLV and strain VR2332 is not known but it is possible that these strains also evolved from the MLV vaccine virus VR2332 as a result of large-scale vaccination programs in swine herds around the world.

Pathogenesis and Pathogenic Variation. The mechanism of PRRSV pathogenesis is poorly understood. It is generally believed that PRRSV initiates an infection in pigs via entry through nasal epithelial, tonsillar, and pulmonary macrophages. PRRSV replicates in these cells, causes viremia, and subsequently results in pneumonia, myocarditis, encephalitis, rhinitis, vasculitis, lymphadenopathy, etc. in target organs. It has been well documented that PRRSV causes persistent infections in pigs. Pigs persistently infected with PRRSV may appear clinically normal, but can still transmit virus to pigs in naive swine herds. Therefore, persistent infection of PRRSV plays an important role in PRRSV survival and transmission, and will likely pose a major obstacle in PRRS control programs. Despite the ample data documenting PRRSV persistence, little has been done to understand the mechanism of persistent infection or what clinical impact it may have. Marked differences in virulence among PRRSV strains have been observed in experimentally-infected pigs. Significant differences in severity of clinical respiratory disease, rectal temperatures, gross lung lesions and microscopic lung lesions were observed among nine different U. S. isolates of PRRSV. The European LV and the low virulence U.S. PRRSV isolate VR2431 (ISU3927) induced mild transient pyrexia, dyspnea and tachypnea, but several high virulence U.S. isolates induced labored respiration, pyrexia, lethargy, anorexia and patchy dermal cyanosis. Strains of PRRSV also vary in virulence for their ability to cause reproductive failure reported that the effects of PRRSV on reproductive performance are strain-dependent. In addition, apathogenic field isolates of PRRSV have been reported, indicating that field isolates of PRRSV differ in virulence. The recent outbreaks of severe atypical or acute PRRS further indicate that the recent atypical PRRSV strains circulating in the US swine herds are more virulent than those strains isolated earlier. Rossow et al reported that marked neurovirulence in neonatal pigs was found to be associated with infection by some field isolates of PRRSV.

Diagnosis. The diagnosis of PRRS was primarily based on clinical signs and histopathology before the successful isolation of PRRSV. Once the virus was isolated, a more definitive diagnosis of PRRSV infection can be made on the basis of serology, PCR and virus isolation. Clinical signs of PRRSV infection vary from pig to pig and from farm to farm. Therefore, diagnosis of PRRSV infection on the basis of clinical signs is difficult, especially when PRRSV infection was complicated by secondary bacterial infections. Various serological tests have been developed to detect PRRSV antibodies in swine sera. These tests include immunoperoxidase monolayer assay (IPMA), indirect fluorescent antibody test (IFA), serum virus neutralization test (SVN) and enzyme-linked immunosorbant assay (ELISA). The IPMA antibodies could be demonstrated as early as 6 days postinfection. The IFA test is similar to the IPMA and is extensively used in the U. S. However, both IFA and IPMA tests must rely on a subjective endpoint and can not be automated. Seroconversion, with sera samples taken pre and post-outbreak, is especially a good indicator for diagnosis of PRRSV infection. However, presence of sera antibodies in swine herds is no longer implicative of clinical diseases since the virus is now widespread in the world. The recent use of a modified-live vaccine for PRRSV would make it more difficult to interpret the results of these serological tests. Virus isolation is the definite diagnosis for PRRSV infection. Lung, spleen, lymph nodes are all appropriate samples for virus isolation. Serum and plasma are the best samples for virus isolation. However, PRRSV can not be isolated from autolyzed and mummified fetuses. RT-PCR, immunohistochemistry and in situ hybridization have also been used to detect and diagnose PRRSV infections. A PCR-RFLP test was developed to detect and differentiate vaccine strain from enzootic strains.

Prevention by Vaccination. Several PRRS vaccines are currently available; however, there are mixed results regarding the efficacy of these vaccines against the genetically diverse field strains of PRRSV. RespPRRS/Repro^TM (Boehringer Ingelheim, Inc.), an MLV, is recommended for use in 3-18 weeks old pigs and in non-pregnant females. The Prime Pac PRRS vaccine (Schering Plough Animal Health Corp) is also an MLV which has been shown to reduce the severity and duration of disease following challenge. However, it did not prevent infection of vaccinated pigs by a virulent heterologous strain. Suvaxn (Fort Dodge Animal health, Inc) is another newly introduced MLV, it showed good protections under experimental conditions but its efficacy in the field is not known yet. By using a restriction-site marker that is present in the vaccine virus (VR2332), Mengeling et al demonstrated that the marker was not detected in any of the 25 field strains of PRRSV isolated before use of the vaccine. However, the restriction-site marker was detected in 24 of 25 field strains isolated after the introduction of the vaccine, and these field strains were believed to be direct-line descendants of the vaccine virus. More importantly, these putative vaccine-related strains produced more pronounced pathological changes than did the vaccine virus alone. The use of MLVs in herds may lessen the clinical signs of PRRS following infection. However, the potential risk for reversion of MLVs to virulent phenotypes cannot be overlooked. The emergence and reemergence of viral infectious diseases is often influenced by the genetics of the viruses. Genetic heterogeneity of PRRSV could lead to the selection of virulent viruses and to the emergence or reemergence of new forms of PRRS. It is possible that virulent strains of PRRSV could be generated through RNA recombination between MLVs and enzootic field strains of PRRSV. The recent outbreaks of the atypical PRRS reflect the need to further study this virus to better understand its biology and develop more effective vaccines. Most of the herds affected by the atypical PRRS had been vaccinated with the current vaccines. It is possible that a mutant strain(s) of PRRSV may be responsible for the recent outbreaks of atypical PRRS. The heterogeneous nature of PRRSV suggests that complete elimination of the virus from the environment is unlikely. The observed genetic diversity among field isolates of PRRSV will continue to be the major obstacle for PRRS control. Therefore, the design for the next generation of vaccines will have to take into consideration the genetic heterogeneity of PRRSV, or PRRS will remain difficult to control.

PRRSV References

Benfield, D. A., Nelson, E., Collins, J. E., Harris, L., Goyal, S. M., Bobinson, D., Christianson, W. T., Morrison, R. B., Gorcyca, D., Chladek, D., 1992. Characterization of swine infertility and respiratory syndrome (SIRS) virus (isolate ATCC VR-2332). J. Vet. Diagn. Invest. 4, 127-133.

Collins, J. E., Benfield, D. A., Christianson, W. T., Harris, L., Hennings, J. C., Shaw, D. P., Goyal, S. M., McCullough, S., Morrison, R. B., Joo, H. S., 1992. Isolation of swine infertility and respiratory syndrome virus (isolate ATCC VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs. J. Vet. Diagn. Invest. 4, 117-126.

Conzelmann, K. K., Visser, N., Van Woensel, P., Thiel, H. J., 1993. Molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group. Virology 193, 329-39.

Halbur, P. G., Andrews, J. J., Huffman, E. L., Paul, P. S., Meng, X. J., Niyo, Y., 1994. Development of a streptavidin-biotin immunoperoxidase procedure for the detection of porcine reproductive and respiratory syndrome virus antigen in porcine lung. J. Vet. Diagn. Invest. 6, 254-257.

Halbur, P. G., Miller, L. D., Paul, P. S., Meng, X. J., Huffman, E. L., Andrews, J. J., 1995a. Immunohistochemical identification of porcine reproductive and respiratory syndrome virus (PRRSV) antigen in the heart and lymphoid system of three-week-old colostrum-deprived pigs. Vet. Pathol. 32, 200-204.

Halbur, P. G., Paul, P. S., Frey, M. L., Landgraf, J., Eernisse, K., Meng, X. J., Lum, M. A., Andrews, J. J., Rathje, J. A., 1995b. Comparison of the pathogenicity of two US PRRSV isolates with that of the Lelystad virus. Vet. Pathol. 32, 648-660.

Halbur, P. G., Paul, P. S., Frey, M. L., Landgraf, J., Eernisse, K., Meng, X. J., Andrews, J. J., Lum, M. A., Rathje, J. A., 1996a. Comparison of the antigen distribution of two US porcine reproductive and respiratory syndrome virus isolates with that of the Lelystad virus. Vet. Pathol. 33, 159-170.

Halbur, P. G., Paul, P. S., Meng, X. J., Lum, M. A., Andrews, J. J., Rathje, J. A., 1996b. Comparative pathogenicity of nine US porcine reproductive and respiratory syndrome virus (PRRSV) isolates in a five-week-old cesarean-derived, colostrum-deprived pig model. J. Vet. Diagn. Invest. 8, 11-20.

Haynes, J. S., Halbur, P. G., Sirinarumitr, T., Paul, P. S., Meng, X. J., Huffman, E. L., 1997. Temporal and morphologic characterization of the distribution of porcine reproductive and respiratory syndrome virus (PRRSV) by in situ hybridization in pigs infected with isolates of PRRSV that differ in virulence. Vet. Pathol. 34, 39-43.

Kapur, V., Elam, M. R., Pawlovich, T. M., Murtaugh, M. P., 1996. Genetic variation in porcine reproductive and respiratory syndrome virus isolates in the Midwestern United States. J. Gen. Virol. 77, 1271-1276.

Lager, K. M., Mengeling, W. L., Wesley, R. D., Halbur, P. G., Sorden, S. D., 1998. Acute PRRS. In 29th Annual Meeting of American Assoc Swine Practitioners. Des Moines, IA. pp. 449-453.

Madsen, K.G., Hansen, C.M., Madsen, E.S., Strandbygaard, B., Botner, A., Sorensen, K.J., 1998. Sequence analysis of porcine reproductive and respiratory syndrome virus of the American type collected from Danish swine herds. Arch. Virol. 143, 1683-1700.

Mardassi, H., Mounir, S., Dea, S., 1994. Identification of major differences in the nucleocapsid protein genes of a Quebec strain and European strains of porcine reproductive and respiratory syndrome virus. J. Gen. Virol. 75, 681-685.

Meng, X. J., Paul, P. S., Halbur, P. G., 1994. Molecular cloning and nucleotide sequencing of the 3' terminal genomic RNA of porcine reproductive and respiratory syndrome virus. J. Gen. Virol. 75, 1795-1801.

Meng, X. J., Paul, P. S., Halbur, P. G., Lum, M. A., 1995a. Phylogenetic analyses of the putative M (ORF 6) and N (ORF 7) genes of porcine reproductive and respiratory syndrome virus (PRRSV): implication for the existence of two genotypes of PRRSV in the U.S.A. and Europe. Arch. Virol. 140, 745-755.

Meng, X. J., Paul, P. S., Halbur, P. G., Morozov, I., 1995b. Sequence comparison of open reading frames 2 to 5 of low and high virulence United States isolates of porcine reproductive and respiratory syndrome virus. J. Gen. Virol. 76, 3181-3188.

Meng, X. J., Paul, P. S., Halbur, P. G., Lum, M. A., 1996a. Characterization of a high-virulence US isolate of porcine reproductive and respiratory syndrome virus in a continuous cell line, ATCC CRL11171. J. Vet. Diagn. Invest. 8, 374-381.

Meng, X. J., Paul, P. S., Morozov, I., Halbur, P. G., 1996b. A nested set of six or seven subgenomic mRNAs is formed in cells infected with different isolates of porcine reproductive and respiratory syndrome virus. J. Gen. Virol. 77, 1265-1270.

Meng, X.J., 2000. Heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development. Vet. Microbiol. 74:309-329.

Mengeling, W.L., Lager, K.M., Vorwald, A.C., 1998. Clinical consequences of exposing pregnant gilts to strains of porcine reproductive and respiratory syndrome (PRRS) virus isolated from field cases of "atypical" PRRS. Am. J. Vet. Res. 59, 1540-1544.

Mengeling, W.L., Vorwald, A.C., Lager, K.M., Clouser, D.F., Wesley, R.D., 1999b. Identification and clinical assessment of suspected vaccine-related field strains of porcine reproductive and respiratory syndrome virus. Am. J.Vet. Res. 60, 334-340.

Meulenberg, J. J., Hulst, M. M., De Meijer, E. J., Moonen, P. L., Den Besten, A., De Kluyver, E. P., Wensvoort, G., Moormann, R. J., 1993a. Lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (PEARS), is related to LDV and EAV. Virology 192, 62-72.

Morozov, I., Meng, X. J., Paul, P. S., 1995. Sequence analysis of open reading frames (ORFs) 2 to 4 of a U.S. isolate of porcine reproductive and respiratory syndrome virus. Arch. Virol. 140, 1313-1319.

Nelsen, C.J., Murtaugh, M.P., Faaberg, K.S., 1999. Porcine reproductive and respiratory syndrome virus comparison: Divergent evolution on two continents. J. Virol. 73, 270-

Rossow, K.D., Collins, J.E., Goyal, S.M., Nelson, E.A., Christopher-Hennings, J., Benfield, D.A., 1995. Pathogenesis of porcine reproductive and respiratory syndrome virus infection in gnotobiotic pigs. Vet. Pathol. 32, 361-373.

Wensvoort, G., Terpstra, C., Pol, J. M., Ter Laak, E. A., Bloemraad, M., De Kluyver, E. P., Kragten, C., Van Buiten, L., Den Besten, A., Wagenaar, F., 1991. Mystery swine disease in The Netherlands: the isolation of Lelystad virus. Vet. Q. 13, 121-130.

Lecture Notes: Swine Hepatitis E Virus

(swine HEV)

Human Hepatitis E Virus. Hepatitis E virus (HEV), the causative agent of hepatitis E, is the primary cause of enterically transmitted non-A, non-B hepatitis in many developing countries. Sporadic cases of acute hepatitis E have also been reported in the United States and other industrialized countries. Strains of HEV within large geographical regions tend to be closely related to each other and distinct from those in distant geographical areas. The disease generally affects young adults. Although the overall mortality rate associated with HEV infection has varied in different reports, it has been as high as 1%. The mortality rate of HEV infection in pregnant women is reportedly up to 20%. Transmission is thought to be predominately fecal-oral and often occurs through contaminated water. HEV was classified in the family Caliciviridae. Recent studies have indicated that the genomic organization of HEV is unique and thus, HEV has recently been declassified from the family Caliciviridae and designated as an unassigned genus “hepatitis E-like viruses”. HEV is a single-stranded RNA virus without an envelope. The positive sense viral RNA genome of about 7.2 kb contains 3 open reading frames (ORFs). ORF1 most likely encodes viral nonstructural proteins, ORF2 encodes the putative capsid protein and ORF3 encodes a cytoskeleton-associated phosphoprotein.

Evidence for the Existence of an Animal Reservoir(s) for HEV. HEV is an important public health problem in many developing countries in Asia and Africa. In industrialized countries where hepatitis E is non-endemic, however, HEV antibodies (anti-HEV) have also been detected in the general populations. According to the serological tests used, Thomas et al has reported that anti-HEV is detected in about 21% of the blood donors, 16% of homosexual men and 23% of injection drug users from Baltimore, MD. In Northern California, about 1.2 to 1.4% of blood donors are found to be positive for anti-HEV, and 31 to 38% of the seropositive individuals have no history of international travel. Paul et al has also found that about 1 to 2% of blood donors in the U.S. are positive for anti-HEV. Similar results have also been reported in other industrialized countries. The existence of a population of individuals in industrialized countries who are positive for anti-HEV has led to a hypothesis that an animal reservoir for HEV may exist.

Several animal species are believed to be infected by HEV or a closely related agent(s). Anti-HEV has been detected in 18 out of 55 domestic swine tested in Kathmandu Valley, Nepal where hepatitis E is endemic. In the U.S., anti-HEV has been detected in more than 80% of swine older than three months of age. In addition, anti-HEV has been detected in 77% of the rats from Maryland, 90% from Hawaii and 44% from Louisiana. Rats from urban as well as rural areas are both seropositive. The prevalence of anti-HEV increases in parallel with the estimated age of the rats. All three different species of wild rats (R. Norvegicus, R. Rattus and R. Exulans) are strongly positive for anti-HEV. In Vietnam where hepatitis E is endemic, anti-HEV has been detected in 44% of chickens, 36% of pigs, 27% of dogs and 9% of rats. Recently, CDC has reported that anti-HEV has been detected in 29 to 62% of cows from three HEV endemic countries (Somali, Tajikistan and Turkmenistan) and in about 12% of cows in a non-endemic country (Ukraine). In Turkmenistan, about 42 to 67% of the sheep and goats are also found to be positive for anti-HEV. Taken together, these data strongly suggest that these animals have been exposed to HEV or a related agent and that hepatitis E may be a zoonosis. However, until recently the source of seropositivity in these animals could not be definitively demonstrated since virus was either not recovered from these species or the recovered virus was not sequenced to confirm its identity.

Discovery of Swine HEV and Other Animal Strains of HEV. In a retrospective study of 15 commercial swine herds in the Midwestern U.S. in 1997, anti-HEV antibodies were found in pigs in all 15 herds tested and the majority of pigs over 3 months of age were seropositive. A prospective study was then conducted in one of the infected herds where the majority of the sows were seropositive. Maternal antibody in the suckling piglets born to seropositive sows waned by 8-9 weeks of age and most pigs seroconverted to swine HEV by 14-21 weeks of age. Clinical disease was not observed. No significant gross lesions were observed in four pigs necropsied during the early stages of infection. Microscopic examination revealed mild lymphoplasmacytic hepatitis and enteritis in the four pigs. A novel virus, designated as swine HEV, was genetically identified from a naturally-infected pig in Illinois by RT-PCR with human HEV degenerate PCR primers. Swine HEV is a ubiquitous agent in pigs in the U.S. Antibodies to swine HEV cross-react with capsid proteins from human strains of HEV. The genome organization of swine HEV is very similar to that of human strains of HEV and swine HEV is genetically related to, but distinct from, most human strains of HEV. Under experimental conditions, swine HEV is highly contagious: naïve swine quickly become infected through direct contact with experimentally inoculated swine. Swine HEV infection in swine is similar to that of human HEV infection in primates, although differences in the course of infection and in clinical and pathological manifestations were noted.

HEV infection in swine is likely common worldwide. Recently the prevalence of IgG anti-HEV in swine was assessed in two countries where HEV is endemic (China and Thailand) and two countries where it is non-endemic (Canada and Korea). It was found that swine herds in all four countries contain many swine that are seropositive for HEV, suggesting that HEV is enzootic in swine regardless of whether HEV is endemic in the respective human population. Chandler et al also found that about 30% of random samples from two piggeries, 92-95% of pigs by the age of 16 weeks from two other piggeries, and 17% of wild-caught pigs in Australia are seropositive for IgG anti-HEV. The swine HEV strain from pigs in Australia has not been genetically characterized yet.

Hsieh et al found that 37% of the pigs from Taiwan, an HEV non-endemic region, were seropositive for IgG anti-HEV. Subsequently, a novel strain of HEV was genetically identified from a pig and from a retired farmer with acute hepatitis E. The swine and human isolates of HEV identified in Taiwan share 97.3% nucleotide sequence identity and form a distinct branch divergent from all other known strains of HEV including the swine HEV identified from pigs in the U.S. More recently, Wu et al tested for HEV RNA from swine sera and hepatitis E patients in Taiwan with no history of travel to endemic regions, and reported the identification of yet another strain of HEV from pigs in Taiwan. Three pigs had detectable swine HEV RNA. Sequence analyses indicated that the swine and human HEV isolates in Taiwan formed a monophyletic group, distinct from other reported strains. The swine and human HEV strains in Taiwan share 84 to 95% nucleotide sequence identity compared to 72 to 79% identity between Taiwan HEV strains and strains from other regions.

Recently, a variant strain of HEV was genetically identified from tissue and fecal samples of wild-trapped rodents in Kathmandu Valley, Nepal. Phylogenetic analyses reveal that the HEV sequences recovered from rodents are most closely related to the HEV isolates from patients in Nepal. Genetic identification of HEV strains from swine and rodents further strengthens the argument that an animal reservoir(s) for HEV does exist. In addition to swine and rodents, a novel HEV-related virus, designated as big liver and spleen disease (BLS) virus (BLSV), was genetically identified from chickens in Australia. The BLS disease affects commercial broiler breeder flocks and causes decreased egg production and slight increase in mortality in broiler breeder flocks. Serological evidence of BLSV infection in flocks was also reported in the UK and U.S. A 523 bp genomic fragment has been amplified and sequenced from the nonstructural region of BLS virus. Sequence analyses indicated that BLSV shared 62% nucleotide sequence identity with human HEV in this region. Although HEV antibodies have been detected in chickens, it is not known if BLSV cross-reacts with HEV antibodies.

Novel Strains of HEV Identified from Hepatitis E Patients in the U.S. and Other Industrialized Countries. In the U.S., two cases of acute hepatitis E have recently been reported, one in Minnesota and one in Tennessee. The patient from Minnesota (US-1) has no history of travelling outside the U.S., whereas the patient from Tennessee (US-2) had traveled to Mexico prior to the diagnosis of hepatitis E. Surprisingly, the two U.S. strains of HEV (US-1 and US-2) are genetically distinct from known strains of HEV but are very similar to the swine HEV recovered from swine in Illinois. The two U.S. strains of human HEV are 92% identical to each other but only about 74% identical to the Burmese and Mexican strains at nucleotide sequence level. The two U.S. strains of human HEV share about 99% amino acid sequence identity with the swine HEV in ORF1, but only about 80% identity with other human strains of HEV worldwide. These data provide compelling evidence that a swine HEV, or one that is very similar to swine HEV, infects and causes hepatitis in humans.

Numerous novel strains of HEV have also been identified in other industrialized countries. In Taiwan, several novel isolates of HEV have been identified from patients with no history of travel to endemic regions. These novel Taiwanese isolates of HEV are distinct from other known strains of HEV but are most closely related to swine strains of HEV identified from pigs in Taiwan. Zanetti et al has reported the identification of a novel strain of HEV from an Italian patient who has no history of travelling to endemic regions. Sequence analyses reveal that this novel Italian strain of HEV has only about 79.5 to 85.8% nucleotide identity with other known strains of HEV. Schlauder et al has identified three more novel strains of HEV in patients from other non-endemic areas, two from Greece and one from Italy. The Greek and Italian strains are significantly divergent from other known strains of HEV and from each other. The two Greek strains are also significantly divergent from each other. Phylogenetic analyses indicate that at least six distinct genotypes of HEV exist worldwide. The source of these novel HEV strains identified from patients in industrialized countries is not clear, but given the genetic identification of novel HEV strains in pigs and rodents and the existence of several other potential animal reservoirs, a zoonotic infection seems plausible.

Evidence for Cross-species Infection by HEV. The genetic similarities between swine HEV and strains of human HEV identified in the same geographic area suggest that human infection with swine HEV may be possible. Meng et al has recently demonstrated that swine HEV can cross species barriers and infect non-human primates. Rhesus monkeys inoculated with swine HEV seroconverted to anti-HEV. Viremia and fecal excretion of swine HEV were detected in inoculated swine. Serum levels of isocitrate dehydrogenase and alanine aminotransferase were slightly elevated in the infected rhesus monkeys. Mild focal necroinflammatory changes, consistent with acute viral hepatitis, were observed in liver biopsies near the time of serum liver enzyme elevations. The chimpanzee inoculated with swine HEV also became infected.

Balayan et al has reported that Russian domestic swine are experimentally infected with a Central Asian strain of human HEV. However, others have failed to infect SPF swine with human strains of HEV. Meng et al was unable to infect crossbred SPF swine with two strains of human HEV: Mex-14 (Mexican) and Sar-55 (Pakistan). Similarly, Platt et al has failed to infect SPF swine experimentally with the Mexican strain of human HEV. It is possible that these epidemic strains may have a more limited host range than does swine HEV or closely-related HEV strains. In fact, when SPF swine were inoculated with the US-2 strain of human HEV, the inoculated swine become infected. The ability of swine HEV to infect across species may put pig handlers at potential risk of zoonotic infection. Recently, a limited number of pig handlers from two HEV endemic countries were tested for the prevalence of HEV antibodies, and 16 out of 18 samples are found to be positive for anti-HEV. Hsieh et al also tested the prevalence of anti-HEV in pig handlers in Taiwan, and they found that the seroprevalence of IgG anti-HEV in pig handlers (26.7%) is higher than that in control subjects (8%). The prevalence of HEV antibodies in individuals from industrialized countries could be explained by subclinical infection of humans with swine HEV or related strains. However, city dwellers in parts of the U.S. are also found positive for anti-HEV, and these individuals probably have no significant exposure to pigs, except possibly as food. Therefore, other animal species may also serve as reservoirs for HEV. Recently, Karetnyi et al tested anti-HEV prevalence in selected populations of Iowa including 204 patients with non-A, B, C hepatitis (non-A-C), 87 field staff members of the Department of Natural Resources (DNR) and 332 normal blood donors. Patients with non-A-C hepatitis (4.9%) and the healthy DNR field workers (5.7%) showed significantly higher prevalence of IgG anti-HEV compared to normal blood donor sera collected in 1998 (P< 0.05), suggesting that human populations with occupational exposure to wild animals have increased anti-HEV prevalence.

Interspecies transmission of HEV has also been demonstrated in other animal species. Usmanov et al has reported that lambs are experimentally infected with a pool of 10% human stool suspension containing HEV isolates Osh-225 and Osh-228. The inoculated lambs show clinical manifestation of acute hepatitis. Virus shedding in feces and the presence of HEV RNA in the parenchymal organs of the inoculated lambs has been detected. Rodents have also been shown to be susceptible to experimental infection with human HEV. Maneerat et al has inoculated Wistar rats with a human stool suspension containing infectious HEV. HEV RNA has been detected from the inoculated rats in feces and serum, and HEV antigens are detected in the liver and several other tissues of the inoculated rats.

Public Health Concerns: Zoonosis and Xenozoonosis. The potential for cross-species infection by HEV raises a public health concern. It has been shown that swine HEV can infect across species. Thus, individuals such as swine practitioners pig farmers and other pig handlers may be at risk for zoonotic HEV infection. However, the risk for zoonotic HEV infection is not limited to swine handlers as many domestic and farm animals were also found to be positive for HEV antibodies.

Xenotransplantation has the potential to offer a solution to the shortage of human organs for transplantation. However, xenozoonosis, the inadvertent transmission of pathogens from xenografts to human recipients, is of major concern in xenotransplantation. Swine are relatively easy to breed and maintain and have anatomic and metabolic characteristics similar to humans. Therefore, xenotransplantation with pig organs has received considerable attention and pigs may become the organ donors of choice. Swine HEV appears to be ubiquitous worldwide and has the ability to cross species barriers. Thus, xenozoonosis due to the transmission of swine HEV from pig xenografts to human recipients and the potential subsequent transmission of the virus to others (family members, healthcare professionals, etc.) are possible. Therefore, the proposed use of pig organs for human transplantation will have the potential to spread HEV in the general population and thus, should be considered as a potential public health risk. Although swine HEV appeared to cause only subclinical infection in pigs and primates under experimental conditions, it may become pathogenic in immunosuppressed xenotransplantation recipients, as a result of species-jumping or adaptation. Furthermore, pigs recovered from swine HEV infection may have a damaged liver (or other organs) which would limit its usefulness for xenotransplantation. Microscopic lesions of hepatitis were observed in pigs naturally-infected and in primates experimentally-infected by swine HEV. Therefore, it is important to develop sensitive and easy-to-perform assays to screen for swine HEV in xenograft donor pigs.

From a more positive perspective, swine HEV infection of pigs may provide a useful animal model to study HEV infection. Swine HEV may also prove to be useful in developing a vaccine against HEV infection of humans.

Swine HEV References

Chandler JD, Riddell MA, Li F, Love RJ, Anderson DA. Serological evidence for swine hepatitis E virus infection in Australian pig herds. Vet Microbiol 1999;68:95-105.

Erker JC, Desai SM, Schlauder GG, Dawson GJ, Mushahwar IK. A hepatitis E virus variant from the United States: molecular characterization and transmission in cynomolgus macaques. J Gen Virol 1999;80:681-90.

Hsieh SY, Yang PY, Ho YP, Chu CM, Liaw YF. Identification of a novel strain of hepatitis E virus responsible for sporadic acute hepatitis in Taiwan. J Med Virol 1998;55:300-304.

Hsieh SY, Meng XJ, Wu YH, Liu ST, Tam AW, Lin DY, Liaw YF. Identity of a Novel Swine Hepatitis E Virus in Taiwan Forming a Monophyletic Group with Taiwan Isolates of Human Hepatitis E Virus. J Clin Microbiol 1999;37:3828-3834.

Kabrane-Lazizi Y, Fine JB, Elm J, Glass GE, Higa H, Diwan A, Gibbs CJ, Meng XJ, Emerson SU, Purcell RH. Evidence for wide-spread infection of wild rats with hepatitis E virus in the United States. Am J Trop Med Hyg. 1999;61:331-335.

Kabrane-Lazizi Y, Meng XJ, Emerson SU, Purcell RH. Evidence that the genomic RNA of hepatitis E virus is capped. J. Virol. 1999; 000-000.

Kwo PY, Schlauder GG, Carpenter HA, Murphy PJ, Rosenblatt JE, Dawson GJ, et al. Acute hepatitis E by a new isolate acquired in the United States. Mayo Clin Proc 1997;72:1133-1136.

Meng XJ, Purcell RH, Halbur PG, Lehman JR, Webb DM, Tsareva TS, et al. A novel virus in swine is closely related to the human hepatitis E virus. Proc Natl Acad Sci USA 1997;94:9860-9865.

Meng XJ, Halbur PG, Haynes JS, Tsareva TS, Bruna JD, Royer RL, et al. Experimental infection of pigs with the newly identified swine hepatitis E virus (swine HEV), but not with human strains of HEV. Arch Virol 1998;143:1405-1415.

Meng XJ, Halbur PG, Shapiro MS, Govindarajan S, Bruna JD, Mushahwar IK, et al. Genetic and experimental evidence for cross-species infection by the swine hepatitis E virus. J Virol 1998;72:9714-9721.

Meng XJ, Dea S, Engle RE, Friendship R, Lyoo YS, Sirinarumitr T, et al. Prevalence of antibodies to the hepatitis E virus in pigs from countries where hepatitis E is common or is rare in the human population. J Med Virol 1999;58:297-302.

Meng XJ. Zoonotic and Xenozoonotic risks of hepatitis E virus. Infectious Dis. Rev. 2000;2:35-41.

Meng XJ. Novel strains of hepatitis E virus identified from human and other animal species: is hepatitis E a zoonosis? J. Hepatol. 2000.

Schlauder GG, Dawson GJ, Erker JC, Kwo PY, Knigge MF, Smalley DL, et al. The sequence and phylogenetic analysis of a novel hepatitis E virus isolated from a patient with acute hepatitis reported in the United States. J Gen Virol 1998;79:447-456.

Schlauder GG, Desai SM, Zanetti AR, Tassopoulos NC, Mushahwar IK. Novel hepatitis E virus (HEV) isolates from Europe: evidence for additional genotypes of HEV. J Med Virol 1999;57:243-51.

Wu JC, Chen CM, Chiang TY, Sheen IJ, Chen JY, Tsai WH, Huang YH, Lee SD. Clinical and epidemiological implications of swine hepatitis E virus infection. J Med Virol 2000;60:166-171.

Lecture 9 Sample Questions

1. What is the definition of xenozoonosis?

(a). The inadvertent transmission of pathogens from animal tissues and organs to human xenograft recipients.

(b). The transmission of zoonotic animal pathogens to humans due to direct or indirect contact with infected animals.

(c). Diseases acquired while traveling to foreign countries.

2. Which one of the following statements is NOT true regarding the swine hepatitis E virus?

(a). Because of the lack of a vaccine, the best preventive measure for potential swine HEV zoonosis is to wash hands after contacting potentially infected animals.

(b). Swine and rodents are potential animal reservoirs for the hepatitis E virus, and pig handlers are at high risk of zoonotic infection.

(c). Swine HEV infection in pigs is very common in the U.S.

3. Porcine reproductive and respiratory syndrome (PRRS) has been recognized for more than a decade. Why is the prevention and control of PRRS still difficult?

(a). Lack of appropriate diagnostic tests.

(b). Not much known about PRRS virus.

(c). Virus spread through international swine trading activities.

4. The recent emergence of the deadly Nipah virus in Malaysia raised important veterinary and medical public health concerns. Which one of the following sentences is NOT true?

(a). Nipah virus infects both pigs and humans, and human infections are due to direct contact with infected pigs.

(b). Humans infected by Nipah virus often develop febrile encephalitis.

(c). Human-to-human infection during the outbreaks is very common.