<i>In vivo</i> titration of white spot syndrome virus (WSSV) in specific pathogen-free <i>Litopenaeus vannamei</i> by intramuscular and oral routes

White spot syndrome virus (WSSV) is a devastating pathogen in shrimp aquaculture. Standardized challenge procedures using a known amount of infectious virus would assist in evaluating strategies to reduce its impact. In this study, the shrimp infectious dose 50% endpoint (SID50 ml-1) of a Thai isolate of WSSV was determined by intramuscular inoculation (i.m.) in 60 and 135 d old specific pathogen-free (SPF) Litopenaeus vannamei using indirect immunofluorescence (IIF) and 1-step polymerase chain reaction (PCR). Also, the lethal dose 50% endpoint (LD50 ml-1) was determined from the proportion of dead shrimp. The median virus infection titers in 60 and 135 d old juveniles were 10(6.8) and 10(6.5) SID50 ml-1, respectively. These titers were not significantly different (p >= 0.05). The titration of the WSSV stock by oral intubation in 80 d old juveniles resulted in approximately 10-fold reduction in virus titer compared to i.m. inoculation. This lower titer is probably the result of physical and chemical barriers in the digestive tract of shrimp that hinder WSSV infectivity. The titers determined by infection were identical to the titers determined by mortality in all experiments using both i.m. and oral routes at 120 h post inoculation (hpi), indicating that every infected shrimp died. The determination of WSSV titers for dilutions administered by i.m. and oral routes constitutes the first step towards the standardization of challenge procedures to evaluate strategies to reduce WSSV infection

Standardized white spot syndrome virus (WSSV) inoculation procedures for intramuscular or oral routes

In the past, strategies to control white spot syndrome virus (WSSV) were mostly tested by infectivity trials in vivo using immersion or per os inoculation of undefined WSSV infectious doses, which complicated comparisons between experiments. In this study, the reproducibility of 3 defined doses (10, 30 and 90 shrimp infectious doses 50% endpoint [SID50] of WSSV was determined in 3 experiments using intramuscular (i.m.) or oral inoculation in specific pathogen-free (SPF) Litopenaeus vannamei. Reproducibility was determined by the time of onset of disease, cumulative mortality, and median lethal time (LT50). By i.m. route, the 3 doses induced disease between 24 and 36 h post inoculation (hpi). Cumulative mortality was 100% at 84 hpi with doses of 30 and 90 SID50 and 108 hpi with a dose of 10 SID50. The LT50 of the doses 10, 30 and 90 SID50 were 52, 51 and 49 hpi and were not significantly different (p > 0.05). Shrimp orally inoculated with 10, 30 or 90 SID50 developed disease between 24 and 36 hpi. Cumulative mortality was 100% at 108 hpi with doses of 30 and 90 SID50 and 120 hpi with a dose of 10 SID50. The LT50 of 10, 30 and 90 SID50 were 65, 57 and 50 hpi; these were significantly different from each other (p 50 was selected as the standard for further WSSV challenges by i.m. or oral routes. These standardized inoculation procedures may be applied to other crustacea and WSSV strains in order to achieve comparable results among experiment

The effect of raising water temperature to 33°C in <i>Penaeus vannamei</i> juveniles at different stages of infection with white spot syndrome virus (WSSV)

This study investigated the effect of high water temperature (33°C) at different stages of infection with a highly virulent and low virulent white spot syndrome virus strain (WSSV Thai-1 and WSSV Viet) in Penaeus vannamei juveniles. Shrimp were inoculated intramuscularly with either a high dose (HD) or low dose (LD). Water temperature was kept either at continuously 27°C or switched from 27°C to 33°C at 0, 12 or 24 h post inoculation (hpi) for both strains and in addition at 48 or 96 hpi for WSSV Viet. The increased temperature 33°C was maintained till the end of the experiments (120–144 hpi with WSSV Thai-1 and 240 hpi with WSSV Viet). To determine the infection status at the moment of temperature increase, five shrimp that were kept continuously at 27 °C were euthanized at 0, 12, 24, 48 and 96 hpi with each dose of two strains. WSSV infections (viral antigen VP28) in dead and euthanized shrimp were demonstrated by indirect immunofluorescence.Shrimp inoculated with HD or LD of WSSV Thai-1 and kept continuously at 27°C till euthanasia were 100% viral antigen positive from 12 (HD) or 24 hpi (LD). Shrimp inoculated with WSSV Viet were 100% positive from 24 (HD) and 48 hpi (LD). Shrimp kept at 27°C, showed clinical signs from 24 (HD) or 24–36 hpi (LD) with both strains. Cumulative mortalities reached 100% with WSSV Thai-1 at 60 (HD) or 84–144 hpi (LD) and with WSSV Viet 100% at 216 hpi (HD) or 90% at 240 hpi (LD). Switch of temperature to 33°C from 0, 12 or 24 hpi was effective in reducing mortality of shrimp inoculated with the LD of both strains and with the HD of WSSV Viet. The switch to 33°C from 24 hpi with the Thai strain (HD) and from 48 and 96 hpi with the Viet strain (LD or HD) had no effect or even accelerated the mortality rate (80–100%). All shrimp were viral antigen positive at death and euthanasia (one shrimp LD WSSV Viet) when kept continuously at 27°C. All dead and euthanized shrimp kept at 33°C from 0 or 12 hpi were viral antigen negative. With 33°C from 24, 48 or 96 hpi, all dead shrimp were viral antigen positive and euthanized shrimp were negative.This study showed that 33°C is effective to prevent disease, reduce mortality and block WSSV replication, but only in the early stages of infection

Cross-protection against European swine influenza viruses in the context of infection immunity against the 2009 pandemic H1N1 virus : studies in the pig model of influenza

Pigs are natural hosts for the same influenza virus subtypes as humans and are a valuable model for cross-protection studies with influenza. In this study, we have used the pig model to examine the extent of virological protection between a) the 2009 pandemic H1N1 (pH1N1) virus and three different European H1 swine influenza virus (SIV) lineages, and b) these H1 viruses and a European H3N2 SIV. Pigs were inoculated intranasally with representative strains of each virus lineage with 6- and 17-week intervals between H1 inoculations and between H1 and H3 inoculations, respectively. Virus titers in nasal swabs and/or tissues of the respiratory tract were determined after each inoculation. There was substantial though differing cross-protection between pH1N1 and other H1 viruses, which was directly correlated with the relatedness in the viral hemagglutinin (HA) and neuraminidase (NA) proteins. Cross-protection against H3N2 was almost complete in pigs with immunity against H1N2, but was weak in H1N1/pH1N1-immune pigs. In conclusion, infection with a live, wild type influenza virus may offer substantial cross-lineage protection against viruses of the same HA and/or NA subtype. True heterosubtypic protection, in contrast, appears to be minimal in natural influenza virus hosts. We discuss our findings in the light of the zoonotic and pandemic risks of SIVs

Charting the Host Adaptation of Influenza Viruses

Four influenza pandemics have struck the human population during the last 100 years causing substantial morbidity and mortality. The pandemics were caused by the introduction of a new virus into the human population from an avian or swine host or through the mixing of virus segments from an animal host with a human virus to create a new reassortant subtype virus. Understanding which changes have contributed to the adaptation of the virus to the human host is essential in assessing the pandemic potential of current and future animal viruses. Here, we develop a measure of the level of adaptation of a given virus strain to a particular host. We show that adaptation to the human host has been gradual with a timescale of decades and that none of the virus proteins have yet achieved full adaptation to the selective constraints. When the measure is applied to historical data, our results indicate that the 1918 influenza virus had undergone a period of preadaptation prior to the 1918 pandemic. Yet, ancestral reconstruction of the avian virus that founded the classical swine and 1918 human influenza lineages shows no evidence that this virus was exceptionally preadapted to humans. These results indicate that adaptation to humans occurred following the initial host shift from birds to mammals, including a significant amount prior to 1918. The 2009 pandemic virus seems to have undergone preadaptation to human-like selective constraints during its period of circulation in swine. Ancestral reconstruction along the human virus tree indicates that mutations that have increased the adaptation of the virus have occurred preferentially along the trunk of the tree. The method should be helpful in assessing the potential of current viruses to found future epidemics or pandemics

Vomiting and wasting disease associated with hemagglutinating encephalomyelitis viruses infection in piglets in jilin, china

One coronavirus strain was isolated from brain tissues of ten piglets with evident clinical manifestations of vomiting, diarrhea and dyskinesia in Jilin province in China. Antigenic and genomic characterizations of the virus (isolate PHEV-JLsp09) were based on multiplex PCR and negative staining electron microscopy and sequence analysis of the Hemagglutinin-esterase (HE) gene. These piglets were diagnosed with Porcine hemagglutinating encephalomyelitis virus (PHEV)

Detection of Group 1 Coronaviruses in Bats in North America

Bats of 2 species harbor group 1 coronaviruses